Ca men So ia Ped o dos San os
Disse a ion p esen ed o ob ain he Ph.D deg ee in
Biology
Ins i u o de Tecnologia Química e Biológica An ónio Xa ie |
Uni e sidade No a de Lisboa
Oei as, Janua y, 2017
Genomic app oaches o unde s and he gene ic
esponse o Phy oph ho a cinnamomi Rands in
Cas anea spp.
II
Wo k pe o med a :
Molecula Biology Labo a o y
UEISSAFSV - Ins i u o Nacional de In es igação Ag á ia e Ve e iná ia, I.P.
A . da República, 2780-157 Oei as, Po ugal
Plan Cell Bio echnology Labo a o y
Ins i u o de Tecnologia Química e Biológica An ónio Xa ie
Uni e sidade No a de Lisboa
A . da República, 2780-157 Oei as, Po ugal
PhD Supe iso s:
Doc o Ri a Lou enço Cos a
Head o labo a o y, Molecula Biology Labo a o y -UEIS Sis emas Ag á ios
Flo es ais e Sanidade Vege al, Ins i u o Nacional de In es igação Ag á ia e
Ve e iná ia, I.P.
P o esso Ped o Fe e ei o
Head o labo a o y, Plan Cell Bio echnology Labo a o y, Ins i u o de
Tecnologia Química e Biológica An ónio Xa ie , Uni e sidade No a de
Lisboa.
Assis an P o esso , Depa amen o de Biologia Vege al, Faculdade de
Ciências da Uni e sidade de Lisboa
II
III
‘(...) o u o dos u os, o único que ao mesmo empo alimen a e simboliza, cai de
umas á o es al as, imensas, cen ená ias, que, pu as como es ais, pa ecem
enca na a i gindade da p óp ia paisagem.
Só em No emb o as agi a uma inquie ação unda, dolo osa, que as az lança
ao chão lág imas (...). Ab indo-as, essas lág imas e içadas de espinhos deixam
e numa camada o a a ma a ilha singula de que alo, ão desa ec ada que a é
no p óp io nome é doce e modes a – a cas anha.’
Miguel To ga (‘Reino Ma a ilhoso’)

IV
V
Table o con en s
Acknowledgmen s/Ag adecimen os
VII
Lis o abb e ia ions
XI
Summa y
XIII
Sumá io
XVII
Chap e I: Gene al in oduc ion
1
Chap e II: Pheno yping Cas anea hyb ids o Phy oph ho a
cinnamomi esis ance
43
Chap e III: Cas anea oo ansc ip ome in esponse o
Phy oph ho a cinnamomi in ec ion
73
Chap e IV: Exp ession analysis o genes associa ed wi h
Cas anea - Phy oph ho a cinnamomi in e ac ion
139
Chap e V: Gene ic mapping o esis ance o Phy oph ho a
cinnamomi in in e speci ic p ogenies o Cas anea species
167
Chap e VI: Conclusions and u u e pe spec i es
203
Funding acknowledgmen
211
VI
Acknowledgmen s/Ag adecimen os
VII
Acknowledgmen s/Ag adecimen os
This hesis ep esen s no only my wo k a he keyboa d, i is a miles one in
hese las yea s o wo k wi h ches nu . A he end o his jou ney, I would like
o exp ess my g a i ude o he people who di ec ly o indi ec ly con ibu ed o
his hesis:
• Ri a Lou enço Cos a, my supe iso , i s o gi ing me he oppo uni y o
de elop his PhD wo k in ches nu , he plan o which I am passiona e. I
am so p oud o ha e wo ked unde he b eeding p og am ha you
ini ia ed. I am uly g a e ul o all supe ision, guidance, suppo ,
dedica ion and ad ices, which we e p ecious in all s ages o his wo k and
allowed me o imp o e a bo h p o essional and pe sonal le el. Thank you
so much o you con inuous op imism and en husiasm wi h his wo k,
which encou aged me h oughou he las yea s. I am also hank ull o
ca e ul and e icien way on e ise his manusc ip .
• Ped o Fe e ei o, my co-supe iso , o his pe inen ques ions and c i ical
ision ha helped me o look o he esul s in di e en pe spec i es, which
made me g ow scien i ically. I am also hank ul o igo ous eading and
commen ing his manusc ip .
• Helena Machado, he scien is ha mos augh me abou Phy oph ho a
and how o deal wi h his challenging pa hogen. Thank you o all
uncondi ional suppo and encou agemen , you ha e been a ue iend o
me.
• Dana Nelson, my Ame ican supe iso , o you knowledgeable ad ices
on mapping app oaches. I owe my deepes g a i ude o you o ha ing
welcomed me so well in you daily li e in USA. Thank you o ou didac ic
and cul u al con e sa ions du ing he ips o he lab and back home, you
we e he bes hos e e !
Summa y
XIV
assays, was s ongly and nega i ely co ela ed wi h he days o su i al
eco ded a e oo inocula ions. The e o e, he excised shoo inocula ion es
e ealed be a eliable app oach o sc eening he me ics o esis ance o
ches nu geno ypes o P. cinnamomi. Mo eo e , a se o esis an geno ypes
was selec ed, cons i u ing a aluable sou ce o new gene ic esou ces,
essen ial o add ess he sho comings o he Po uguese and Eu opean
ches nu ma ke .
The associa ion be ween geno ype and pheno ype enabled he iden i ica ion
o unique QTLs o P. cinnamomi esis ance. Ten QTLs we e mapped on i e
linkage g oups o he Eu opean x Japanese ches nu map. The p esence o
QTLs on linkage g oup E was consis en wi h a p e ious pilo s udy o
iden i ica ion o QTLs in backc oss amilies (Chinese ches nu x Ame ican
ches nu hyb id), sugges ing ha di e en Cas anea species migh sha e
esis an haplo ypes, and he e o e, common esis ance mechanisms.
Conce ning he ansc ip omic app oach, candida e genes o P. cinnamomi
esis ance we e iden i ied om he oo ansc ip ome o Eu opean and
Japanese ches nu inocula ed and non-inocula ed wi h he pa hogen. Those
genes a e in ol ed, in bo h species, in he egula ion o plan immune
esponse and s ess adap a ion and eco e y. The exp ession le els o eigh
o he candida e genes we e quan i ied by digi al PCR, using Eu opean and
Japanese ches nu and ou hyb id geno ypes showing di e en le els o
suscep ibili y o he disease. RNA-seq and gene exp ession analysis
sugges ed ha bo h species ecognize he pa hogen a ack, which may
igge esis ance signaling pa hways and cell wall modi ica ion, as well as,
he p oduc ion o an i- ungal p o eins. Howe e , he esis an species may
in ol e basal de ense mechanisms, being p o ec ed in ad ance o he
in ec ion.
Addi ionally, new molecula ma ke s we e de eloped om he sequences o
candida e genes iden i ied by ansc ip ome sequencing. Fo y-one
mic osa elli e showing polymo phism and high ans e abili y wi hin and

Summa y
XV
among ches nu species we e used o geno yping Eu opean x Japanese
ches nu popula ions. Two o hem we e mapped wi hin he iden i ied QTL
in e als, being s ong candida es o u he alida ion and ma ke -assis ed
selec ion.
The knowledge acqui ed in his p ojec is a majo b eak h ough in
unde s anding he Cas anea-P. cinnamomi in e ac ions and may con ibu e
o he de elopmen o s a egies o con ol ink disease. Fu he mo e, his
p ojec de eloped a c ucial deli e able o a me s and socie y, since he
geno ypes wi h imp o ed esis ance o he pa hogen ha e been p opaga ed,
o be eleased o he ma ke as oo s ocks, in he nea u u e.
XVI
Sumá io
XVII
Sumá io
O cas anhei o é uma á o e poli alen e, com impo an e impac o económico,
ecológico e cien í ico. O cas anhei o Eu opeu (Cas anea sa i a) p oduz as
cas anhas mais ap eciadas e alo izadas no mundo. No en an o, a á ea de
sou os e cas inçais es á a diminui na Eu opa de ido a doenças e p agas,
p incipalmen e a doença da in a. Es a doença al amen e des u i a é
causada pelo oomice a Phy oph ho a cinnamomi, di undido po odo o
mundo. A in ecção po P. cinnamomi oco e nas aízes causando a sua
pod idão e le ando à mo e em espécies suscep í eis. No en an o, o ní el
suscep ibilidade a ia en e cas anhei os, sendo as espécies asiá icas as
mais esis en es ao pa ógeno.
A in es igação desen ol ida du an e es a ese oi ealizada em
descendências que seg egam pa a a ca ac e ís ica de in e esse, ob idas a
pa i do p og ama de melho amen o es abelecido há 10 anos. Es e
p og ama é baseado em c uzamen os con olados en e o cas anhei o
japonês esis en e e o cas anhei o eu opeu suscep í el ao agen e
pa ogénico. Fo am implemen adas abo dagens de mapeamen o e de
ansc ip ómica isando comp eende os di e en es mecanismos de
espos a do cas anhei o à doença. A é ao momen o o am ob idas 155
descendências, que o am geno ipadas e eno ipadas de o ma a mapea as
egiões genómicas que con olam a esis ência a P. cinnamomi (Quan i a i e
T ai Loci-QTLs).
Usando ma cado es molecula es (mic osa éli es e SNPs) de i ados de
ansc ip omas ob idos, p e iamen e e du an e es e abalho, oi ealizada a
geno ipagem dos p ogeni o es e espec i as descendências. Os dados de
seg egação ob idos o am analisados pa a a cons ução do p imei o mapa
gené ico de cas anhei o Eu opeu x cas anhei o Japonês. O mapa gené ico
in e especí ico con ém 283 ma cado es molecula es, mapeados em 15
Sumá io
XVIII
g upos de ligação e ab angendo um o al de 714,8 cM, o que co esponde a
ce ca de 96% do mapa de e e ência de cas anhei o Chinês.
A eno ipagem oi ealizada a a és da a aliação das mé icas de espos a
à doença, ob idas pa a odas as descendências, após a inoculação de aízes
e/ou de es acas excisadas das plan as-mãe, com P. cinnamomi. A axa de
p og essão da lesão obse ada nos ensaios de inoculação ealizados em
es acas oi o emen e e nega i amen e co elacionada com os dias de
sob e i ência egis ados após a inoculação das aízes. Assim, o es e de
inoculação em es aca demons ou possui igo pa a a alia a esis ência à
doença da in a em di e en es genó ipos de cas anhei o. Além disso, o am
selecionados um conjun o de genó ipos com esis ência melho ada a P.
cinnamomi, cons i uindo no os ecu sos gené icos essenciais pa a colma a
o ele ado dé ice de ma e ial ege al melho ado no me cado an o em
Po ugal e na Eu opa.
A associação en e genó ipo e enó ipo pe mi iu a iden i icação de QTLs
elacionados com a esis ência a P. cinnamomi pela p imei a ez em
cas anhei o. Dez QTLs o am mapeados em cinco g upos de ligação do
mapa gené ico de cas anhei o Eu opeu x cas anhei o Japonês. A p esença
de QTLs em de e minados g upos de ligação oi consis en e com um es udo
pilo o ealizado an e io men e pa a a iden i icação de QTLs em amílias do
p og ama de melho amen o Ame icano, suge indo que as di e en es
espécies do géne o Cas anea podem pa ilha hapló ipos e mecanismos de
esis ência.
Em elação à abo dagem de ansc ip ómica, os genes candida os pa a a
esis ência a P. cinnamomi o am seleccionados a pa i do ansc ip oma de
aízes de cas anhei o eu opeu e japonês espe i amen e inoculadas e não
inoculadas com o pa ogénio. Pa a ambas as espécies, es es genes es ão
en ol idos na egulação da espos a imune das plan as e na adap ação e
ecupe ação do s ess bió ico. Os ní eis de exp essão de oi o genes o am
quan i icados po PCR digi al, em aízes de cas anhei o Eu opeu e Japonês
Sumá io
XIX
e de qua o genó ipos híb idos mos ando di e en es ní eis de
suscep ibilidade à doença. A análise de exp essão génica e da
sequenciação do ansc ip oma, suge e que ambas as espécies
econhecem o a aque de pa ogénio, podendo desencadea ias de
sinalização de esis ência que podem esul a na a modi icação da pa ede
celula e/ou na p odução de p o eínas an i úngicas. No en an o, as espécies
esis en es pa ecem en ol e mecanismos de de esa basal, encon ando-se
p o egidas an ecipadamen e à in ecção.
Adicionalmen e, o am desen ol idos no os ma cado es molecula es a
pa i das sequências de genes candida os, iden i icados na sequenciação
dos ansc ip omas. Qua en a e um mic ossa éli es mos ando polimo ismo
e al a ans e ibilidade, den o e en e as di e en es espécies de cas anhei o,
o am u ilizados pa a a geno ipagem das populações híb idas de
cas anhei o Eu opeu x cas anhei o Japonês. Dois dos ma cado es
desen ol idos o am mapeados den o de in e alos dos QTLs iden i icados,
po isso cons i uem-se como o es candida os pa a alidação adicional e
seleção assis ida po ma cado es molecula es.
O conhecimen o adqui ido nes e es udo cons i uiu um g ande a anço na
comp eensão da in e ação en e Cas anea e P. cinnamomi, podendo
con ibui pa a o desen ol imen o de es a égias de con olo da doença da
in a. Além disso, es e p ojec o desen ol eu um alioso p odu o pa a os
ag icul o es e pa a a sociedade, uma ez que os genó ipos com esis ência
melho ada ao pa ógenio que es ão a se p opagados, se ão lançados no
me cado, como os po a-enxe os, num u u o p óximo.

XX
Gene al in oduc ion
1
Chap e I
Gene al In oduc ion
Chap e I
2
The ches nu : since ancien imes o he p esen
The ches nu is a mul ipu pose ee ha has a e y ancien his o y and
adi ion, as well as an impo an economic and en i onmen al ole.
Ches nu s we e his o ically dis ibu ed only h oughou he no he n
hemisphe e, bu due o an h opogenic in luences ha e been in oduced in o
Chile, A gen ina, Aus alia and New Zealand, being cu en ly widely
cul i a ed all o e he empe a e egions (Conede a and K ebs, 2008;
Pe ei a-Lo enzo e al. 2012).
Since he Middle Ages, he nu s o Eu opean ches nu , a noble ha dwood,
and also o Japanese and Chinese ches nu s, p o ided an essen ial ood
sou ce, esul ing in di e se ypes o use: esh consump ion, long- e m
s o age, d ying, lou and animal eed (Bounous and Ma inoni 2005; Bounous
2009). In No h Ame ica, he Ame ican ches nu , known as a o es gian ,
was a dominan species along he Appalachian ange. The Ame ican
ches nu ees played a dominan ole o Ame ican people, since he wood
was ex ensi ely used o building houses and u ni u e o used as uelwood.
Nu s we e pa o he human die and also, hey had an impo an ole o
wildli e in he o es s (Anagnos akis, 2012; Jacobs e al. 2015).
Nowadays, he ches nu con inues o ha e an impo an ole in many
ag o o es y sys ems. Al hough hey a e no longe a subsis ence ood,
ches nu s a e cu en ly an app ecia ed p oduc o an inc easingly la ge
ma ke sec o . The nu s, wi h bo h mode n and adi ional me hods o s o age
and p ocessing, mee he demand o consume s, who a e p og essi ely
seeking o nu i ious and heal hy oods.
Impac o ches nu in economy and en i onmen
The cul i a ion o ches nu has been mainly ela ed wi h he abundan and
e sa ile uses o he p oduc s ha can be ob ained: edible nu s, imbe o
building and o o he woody p oduc s, associa ed mush ooms p oduc ion
and ex ac ed annins o anning lea he o pha maceu ical pu poses.
Gene al in oduc ion
3
Mo eo e , he ches nu ecosys ems always con ibu ed o biodi e si y
p ese a ion, ep esen ing an impo an cul u al he i age (Paille 2002;
Bounous 2005; Bounous and Ma inoni 2005; Conede a and K ebs 2008;
Bounous 2009).
Ches nu s a e one o he mos impo an nu c ops in he empe a e zone.
They ha e a delicious as e, being consumed in esh, d ied o p ocessed.
P ocessing is necessa y o inc ease he a ailable p oduc s and o ex end he
use o he p oduc along he yea (Bounous and Ma inoni 2005). They ha e
many culina y uses, anging om i s o main cou se dishes (used as side
o se ed whole, boiled o oas ed) as well as ege able dishes (as soups,
ches nu pu ées), desse s (ma ons glacés, cakes, ice-c eams and sy ups)
and pas ies (as ches nu c eams, mousse). I is also possible o p epa e
be e ages om ches nu s such as liqueu s, bee s and non-alcoholic d inks.
Some examples o ches nu uses a e shown in Figu e 1. F om a nu i ional
poin o iew, ches nu s a e a e y heal hy, balanced and high-ene gy ood.
F esh ches nu s a e high in calo ie con en , low in a and sodium, ee o
choles e ol and glu en, wi h a mode a e bu high-quali y p o ein con en and
a a o able amino-acid a io (Pe ei a-Lo enzo e al. 2006; De Vasconcelos
e al. 2010).
Many coun ies a ound he wo ld ha e sui able edaphoclima ic condi ions o
ches nu plan a ion. The Eu opean, Chinese and Japanese ches nu s a e he
main species cul i a ed o ui , due o hei la ge nu size. Ne e heless,
Eu opean ches nu (ma one ypes) a e conside ed he mos aluable o nu
p oduc ion (Pe ei a-Lo enzo e al. 2012). In e speci ic hyb ids which ha e
eme ged om disease esis ance s udies a e also used o nu p oduc ion
di ec ly o as oo s ocks.
Chap e I
10
The i s unambiguous pollen da a showing e idence o Eu opean ches nu
ees sp eading due o human ac i i ies da e back o a ound 2100-2050 B.C.
(Conede a e al. 2004). Cu en ly, C. sa i a is commonly ound in Eu ope
be ween 400 and 1 000 m abo e sea le el depending on he la i ude. The
lowes ele a ions a e ecommended o he highes la i udes and ice e sa
(Mellano e al. 2012).
The Eu opean ches nu ge mplasm is e y ex ensi e; he e a e hund eds o
cul i a names used o ches nu s, many o which a e synonyms o
homonyms (Bo a e al. 2001). In 2013, I aly was he la ges ches nu
p oduce in he Eu opean Union (FAOSTAT, 2016, aos a . ao.o g), and led
he wo ld in p oducing p ocessed ches nu p oduc s such as ma on glacé
(Bounous 2009). Howe e , he in oduc ion o gall wasp has dec eased he
nu p oduc ion (Ba is i e al. 2014). I alian ma on ype cul i a s a e ‘Chiusa
Pesio’, ‘Luse na’, ‘Val Susa’, ‘Cas el del Rio’, ‘Ma adi’ and ‘Fio en ino’.
Wi hin Eu ope, F ance is he la ges ches nu impo e , mos ly buying om
I aly, bu also om Spain and Po ugal. Some C. sa i a adi ional F ench
cul i a s include ‘Bouche Rouge’, ‘Ve dale’, ‘A izinca’, ‘Toumi e’, ‘Belle
Epine’, ‘Sa oye’, ‘Châ aigne de Laguepie’, ‘Sa donne’, ‘Rouse de Nay’, and
‘Do ée de Lyon’ (Mellano e al. 2012).
In Ibe ian Peninsula, i seems ha cul i a di e si ica ion was a esul o
dis inc geno ypes being ela ed ia hyb idiza ion and mu a ion, ega dless
o whe he hey sha ed he same name o no (Pe ei a-Lo enzo e al. 2011).
In Spain, he main cul i a s a e ‘Lou a’, ‘Ga ida’, and ‘Pa ede’, being
‘Ga ida’ mo e sui ed o indus ial pu poses, conse a ion and gene ic
s udies and Eu opean b eeding p og ams (Pe ei a-Lo enzo e al. 2001;
Blanco Sil a and Fe nández-López, 2005; Pe ei a-Lo enzo e al. 2006;
Míguez-So o and Fe nández-López 2012; Fe nández-López and
Fe nández-C uz, 2015; Míguez-So o and Fe nández-López 2015;
Fe nández-C uz and Fe nández-López, 2016).

Gene al in oduc ion
11
In Po ugal, mo e han 25 cul i a s a e known, hey showed g ea gene ic
a iabili y (each cul i a includes di e en geno ypes) indica ing hei
polyclonal o igin (Cos a e al. 2008). One o he mos ances al cul i a , ha
is dis ibu ed h oughou he en i e Ibe ian Peninsula, is ‘Longal’ ha has
been p omo ed as he bes cul i a o indus y (Pe ei a-Lo enzo e al. 2011).
‘Judia’ and ‘Ma aínha’, due o hei la ge nu size, a e usually p e e ed o
he esh ma ke .
In Po ugal, ches nu is dis ibu ed mainly in he No heas (T ás-os-Mon es,
Minho and Bei a Li o al) bu also is ound in he cen e eas , especially in
Ma ão egion (Cos a e al. 2008). Acco dingly, ou egions o P o ec ed
Designa ion o O igin (POD) we e c ea ed o p ese e he Po uguese
cul i a s: Cas anha da Te a F ia, Cas anha da Pad ela, Cas anha dos
Sou os da Lapa and Cas anha do Ma ão.
Rep oduc i e biology and hyb idiza ion
Cas anea species is a monoecious species ha gene ally lowe s om June
o July depending on he species; Asian species show p ecocious
blossoming when compa ed o Eu opean species (Bo a e al. 1995). C.
sa i a ha e been epo ed o begin lowe ing a e 8-10 yea s, bu lowe ing
ime can be sho ed hough g a ing. P oduc ion is egula and high (Gomes-
La anjo e al. 2009).
Female lowe s a e pollina ed by wind (mo e usual in case o d y wea he
du ing lowe ing) o insec s (domina ing in we wea he condi ions).
In lo escence male lowe s a e ga he ed in ca kins ha can occu in wo
ypes: bisexual ca kins ha bea one o mo e emale lowe s a he base and
male lowe s owa d he ip; and unisexual male ca kins, also called
s amina e ca kins (Me and Soylu 2006). Female in lo escence gene ally
con ains h ee lowe s ha a e p o ec ed by a g een, scaled w apping ha is
des ined o o m he cupule ha de elops in o he ches nu bu . Usually
emale in lo escences a e posi ioned a he base o he male ones, in he
Chap e I
12
uppe pa o he cu en yea ’s shoo s (Heba d e al. 2014a). Fe iliza ion
p oduces ypically h ee la ge and b ownish nu s encapsula ed in a spiny bu .
The bu s p o ec he seeds un il hey a e ipe and hen open widely, making
he nu s eadily a ailable. Nu s ipen ea ly in Sep embe o No embe
(Heba d e al. 2014a). The nu s o Cas anea genus a y g ea ly among
species and cul i a s. Ne e heless, in a e age Eu opean ches nu s show
he la ges size in he genus and can weigh mo e han 30 g (Figu e 3).
Chinese and Japanese ches nu s ha e simila size and he Ame ican
ches nu s a e much smalle (Figu e 3).
Figu e 3. Ches nu s mo phology depending on
he species. Pho og aph by D . Paul Sisco.
The ches nu is e y o en sel -incompa ible, he e o e c oss-pollina ion is
compulso y (Me and Soylu 2006). Ve y li le is s ill known abou he gene ic
sys em con olling ma ing and he sel -incompa ibili y sys em in ches nu ,
al hough i is conside ed o be o game ophy ic ype (Zou e al. 2014).
In e speci ic hyb idiza ions be ween all Cas anea species a e possible. Main
p oblems a e ela ed wi h he di e en lowe ing ime be ween species. In all
in e species c osses, ch omosome pai ing exis s among Cas anea species,
bu he p esence o seg ega ion dis o ion in some mapping popula ions
(Casasoli e al. 2001; Kubisiak e al. 1997; Kubisiak e al. 2013) sugges ha
signi ican ch omosomal di e ences such as ansloca ions and/o in e sions
may occu .
Fo b eeding pu poses, a i icial con olled c osses ha e been pe o med
(Cos a e al. 2011; Takada e al. 2012; Nishio e al. 2013; Nelson e al. 2014;
Fe nández-C uz 2015): dono pollen is easily collec ed om he ca kins;
ecep o emale lowe s a e isola ed a oiding unknown pollina ion; male
lowe s om he ecep o ee mus be also emo ed (emascula ion); manual
pollina ion is pe o med by placing dono pollen o e he pis ils; pollina ion
Gene al in oduc ion
13
bags a e used o co e ing emale lowe s a oiding pollen con amina ion;
pollina ion bags a e emo ed and eplaced by ne bags in o de o collec he
bu s con aining he nu s.
Genomic esou ces
Genomic esea ch on o es ees has been mo i a ed by he need o suppo
gene ic b eeding p og ams and de elop ools o conse a ion, es o a ion
and managemen o na u al popula ions (Neale and K eme 2011). Impo an
genomic esou ces such as ESTs molecula ma ke s and gene ic maps ha e
been de eloped o ches nu and in eg a ed in a Web-based esou ce o he
Cas anea gene ics/genomics communi y (Fagaceae Genomic Da abase:
www. agaceae.o g). Mo eo e , he whole genome sequencing p ojec o C.
mollissima is unde way (www.ha dwoodgenomics.o g). Based on low
cy ome ic analysis, genome size appea s o be ai ly conse ed among
species: he es ima ed 1C genome size o C. sa i a is 777Mb, o C. c ena a
and C. mollissima is 794 Mb and o C. den a a is 803 Mb (K eme e al. 2007).
The e o e, he genome size o Cas anea species is only i e imes la ge han
A abidopsis and less han wice he size o he popla genome. The
manageable genome size and abundan gene ic and genomic esou ces
make Cas anea a good candida e as model o Fagaceae amily in he nea
u u e.
A la ge componen o ches nu genomic esou ces is ocused on he
ansc ip omes ob ained o C. mollissima, C. den a a, C. sa i a and C.
c ena a (Ba aka e al. 2009; Sebas iana e al. 2009; Nishio e al. 2011;
Ba aka e al. 2012; Se azina e al. 2015, Chap e III). La ge EST da abases
a e being c ea ed wi h signi ican numbe s o sequence con igs showing
simila i y o p edic ed p o eins in woody plan s. Resis ance candida e genes
o ches nu bligh and ink disease (Ba aka e al. 2009; Ba aka e al. 2012;
Se azina e al. 2015, Chap e III). ha e been iden i ied in EST sequence
da a, as well as candida e genes o o he ai s (Sebas iana e al. 2009;
Chap e I
14
Nishio e al. 2011). Fu he mo e, a g ea numbe o molecula ma ke s ha e
been de eloped om hose sequences mainly Simple Sequence Repea s
(SSRs) o mic osa elli es and Single Nucleo ide Polymo phism (SNPs)
(Nishio e al. 2011; Kubisiak e al. 2013; San os e al. 2015, Chap e V).
Beyond molecula ma ke s de i ed om ESTs da abases, smalle se s o
SSR ma ke s we e ea lie de eloped om en iched genomic lib a ies o
Eu opean ches nu (Ma inoni e al. 2003; Buck e al. 2003), Japanese
ches nu (Yamamo o e al. 2003) and Chinese ches nu (Inoue e al. 2009).
Mic osa elli e and SNP ma ke s a e highly in o ma i e, ans e able ac oss
ela ed axa, ha ing g ea p e alence in he genome and amenabili y o
au oma ed high- h oughpu analysis. The e o e, he molecula ma ke s
de eloped so a o ches nu a e an in aluable esou ce o he scien i ic
communi y in e es ed in all aspec s o he gene ics, b eeding and
bio echnology. Fo b eeding pu poses, he molecula ma ke s ha e been
mapped on gene ic maps cons uc ed o he ou main Cas anea species
(Kubisiak e al. 1997; Casasoli e al. 2001; Sisco e al. 2005; Kubisiak e al.
2013; Nishio e al. 2013 and Chap e V). The gene ic map cons uc ed o C.
mollissima (Kubisiak e al. 2013) was accep ed as he ches nu e e ence
map and was in eg a ed wi h he physical map ob ained by sequencing o
BAC lib a ies (Fang e al. 2013). Quan i a i e ai loci (QTLs) ela ed wi h
ches nu bligh and adap i e ai s ha e been iden i ied and con i med
(Kubisiak e al. 1997; Casasoli e al. 2004; Kubisiak e al. 2013). Mo eo e ,
syn enic egions ha e been iden i ied be ween he ches nu physical map
and some genomes a ailable o o he ela ed axa, e ealing syn enic
egions be ween QTLs o eis ance o ches nu bligh diasese and QTLs o
esis ance o o he ungal pa hogens in P unus spp. (S a on e al. 2015).
Howe e , only an explo a o y s udy iden i ied QTLs o P. cinnamomi
esis ance (Zheben yaye a e al. 2014) un il o he p esen s udy.
Gene al in oduc ion
15
Majo diseases a ec ing ches nu
The mos damaging diseases o ches nu a e he ink disease, caused by he
oomyce e Phy oph ho a cinnamomi (and P. cambi o a) and ches nu bligh
caused by he ascomyce e ungus Ch yphonec ia pa asi ica. Eu opean and
he Ame ican ches nu a e highly suscep ible o hese pa hogens whe eas,
Asian Cas anea species show g ea esis ance o he diseases (C andall e
al. 1945).
Ink disease
Ink disease, also known as oo o , is he mos des uc i e disease a ec ing
Eu opean ches nu . In mos cases, Phy oph ho a cinnamomi is he causal
agen o ink disease, whils Phy oph ho a cambi o a is less equen and
agg essi e (Gou eia 2004). P. cinnamomi is a soilbo ne pa hogen ha
pa asi es ine oo s causing oo and colla o ha ex ends o unk and
b anches o young and ma u e ees, and consequen ly causes dea h.
Typical symp oms include chlo osis and wil ing o oliage, dieback o
b anches and c own (Robin e al. 2001; Ve aino e al. 2001; Ha dham 2005;
Kamoun e al. 2014). I is a silen disease since when i s symp oms become
isible in he c own he des uc ion o he ine oo sys em is al eady in an
ad anced s age.
The geog aphical o igin o P. cinnamomi is no clea ly es ablished, howe e
he e a e e idences o an Asian o igin and i was sp ead ac oss he Paci ic
o La in Ame ica (Ko e al. 1978; Zen mye 1988; Zhang e al. 1994). Ink
disease on Cas anea was i s epo ed in Po ugal in 1838 (Ve aino e al.
2001), and in he USA in 1825 (Rhoades e al. 2003). Since hen, P.
cinnamomi ha e sp ead o e Eu ope and No h Ame ica and hei na i e
plan species we e no adap ed and he e o e hey a e o en highly
suscep ible. Cu en ly, P. cinnamomi is he mos widely dis ibu ed
Phy oph ho a species (Figu e 4). Fu he mo e, clima e change is p edic ed
o ha e a significan impac on he in ensi y and dis ibu ion o P. cinnamomi

Chap e I
16
(Thompson e al. 2014). The pa hogen in ec s mo e han 3000 hos species
causing g ea economic impac s in o es y and ho icul u e, and in he
nu se y indus y (Ha dham 2005). Beyond Cas anea species, P. cinnamomi
a ec s mos o he empe a e o ui ees such as Pe sea, Que cus,
E icaceae, Eucalyp us, Cinnamomum, Coni e ales, Fagus, Juglans and
many o namen al ees and sh ubs (Ha dham 2005; Robin e al. 2012).
Like o he Phy oph ho a spp., P. cinnamomi has a numbe o s a egies o
su i al, p opaga ion and dissemina ion. I is an oomyce e and no a ungus,
al hough e e y hing abou i s biology and li e cycle is ungus-like, such as
mycelial g ow h habi . Fea u es ha di e oomyce es om ungi include he
p oduc ion o bi lagella e he e okon zoospo es, he occu ence o cellulose
a he han chi in in he cell walls and diploid soma ic cells (Ha dham e al.
1994; Ha dham 2005).
Figu e 4. Phy oph ho a cinnamomi dis ibu ion wo ldwide (in g ey), adap ed om
Eu opean and Medi e anean Plan P o ec ion O ganiza ion, Global Da abase
(2016).
P. cinnamomi is able o su i e unde unsui able en i onmen al condi ions
o e se e al yea s in he soil o in in ec ed oo issue, as do man es ing
spo es: chlamydospo es, which a e he mos common o oospo es,
p oduced when di e en s ains ma e. Al hough sexual ep oduc ion o P.
cinnamomi is poo ly unde s ood, i is known ha he pa hogen is
Gene al in oduc ion
17
he e o hallic, equi ing he p esence o opposi e ma ing ypes, designa ed A1
and A2, o o m oospo es (Hübe li e al. 1997; Ha dham 2005). Ne e heless,
in he mos cases, P. cinnamomi has an asexual spo ula ion, h ough
de elopmen o mul inuclea e spo angia (Ha dham 2005) (Figu e 5). When
condi ions a ou g ow h p e ails (high soil mois u e, soil empe a u e
supe io o 10°C) he es ing spo es ge mina e and soma ic hyphae o m
mul inuclea e spo angia ha clea e and elease mo ile, bi lagella e and wall-
less zoospo es in o he soil wa e (Figu e 5). These zoospo es a e
chemo ac ically a ac ed by young ine oo exuda es, a he con ac momen
he zoospo es encys , o ming walled cys s ha ge mina e and pene a e he
issue. P. cinnamomi is able o g ow in e - and in acellula showing ypical
co alloid o i egula and non-sep a e hyphae. Wi hin 2-3 days in a
suscep ible hos , spo angia will o m on he plan su ace. The asexual cycle
may be epea ed million o imes in quick succession, apidly ampli ying he
inoculum po en ial in he in ec ed a ea (Ha dham e al. 1994; E win and
Ribei o. 1996; Ha dham 2005; Jung e al. 2013; Oßwald e al. 2014).
Figu e 5. Li e cycle o
soilbo ne Phy oph ho a
cinnamomi (adap ed
om Ha dham e al.
2005). Sexual and
asexual spo ula ion a e
shown.
Chap e I
18
On a local scale, he pa hogen can be mo ed na u ally by soil-splash, by
wind-blown soil o deb is, o by wa e mo emen and un-o in
d ainage/i iga ion di ches. The mos likely sou ce o mo e dis an mo emen
is in con amina ed soil o plan deb is. P opagules can also be ca ied on
machine y used o cul i a ion/ha es ing (Ha dham 2005; Robin e al. 2012).
Cul u al con ol measu es include eli ing o high soil mois u e le els and
imp o ing ae a ion by inc easing d ainage, and a en ion o mine al nu i ion.
Ches nu bligh
C yphonec ia pa asi ica, a ilamen ous ascomyce e ungus, is a
nec o ophic pa hogen ha inci es he ches nu bligh disease. The
des uc ion o he Ame ican ches nu by C. pa asi ica, was he g ea es
disas e in he his o y o o es pa hology. I is hough o ha e been impo ed
on seedlings om Asia and i was i s disco e ed in 1904, on in ec ed
Ame ican ches nu ees a he B onx Zoological Pa k in New Yo k
(Anagnos akis, 1987; Anagnos akis, 2001). By 1950, he disease had sp ead
h oughou i s na u al ange, and by 1960 had killed an es ima ed 4 billion
ees. In Eu ope, C. pa asi ica was i s eco ded in 1938 in I aly and was
apidly sp ead o he su ounding coun ies. Ches nu bligh became one o
he majo pa hogens ha a acked ches nu ees and cons i u ed a se ious
damage o Eu opean ches nu (Anagnos akis 1987; Robin and Heinige
2001; Jacobs e al. 2015). Cu en ly, C. pa asi ica is dis ibu ed along
Eu ope, Uni ed S a es, wes Asian and Aus alia (Figu e 6).
The pa hogen in ec s p ima ily h ough wounds on s em issues and kills he
abo e g ound po ions o ees by gi dling he cambium. Once es ablished
as ge mina ing conidia (single-celled spo es, p oduced asexually) o
ascospo es, he ungus g ows apidly h ough he ba k and colonizes he
cambial zone. Resis an eac ions slow his g ow h, main aining he ungus
in a supe icial canke , whe eas suscep ible eac ions con inue de elopmen
Gene al in oduc ion
19
unimpeded, enci cling he s em and causing ascula dys unc ion, esul ing
in dea h o dis al issues and s em dieback (Anagnos akis, 2012).
Figu e 6. C yphonec ia pa asi ica dis ibu ion wo ldwide (in g ey), adap ed om
Eu opean and Medi e anean Plan P o ec ion O ganiza ion, Global Da abase
(2016).
Majo pes s a ec ing ches nu
Mo e han 50 species o insec s a e known o damage ches nu , including
he D yocosmus ku iphilus (Yasuma su), which is he mos se e e insec
pes wo ldwide a ec ing ches nu . D. ku iphilus a acks he ege a i e buds
o ches nu s and o ms a gall, dis up ing wig g ow h and educing ui ing.
Se e e in es a ions may esul in he decline and dea h o ches nu ees.
This insec is endemic in China, and was acciden ally in oduced in o Japan
(1941), Ko ea (1963), and he USA (1974) (Abe e al. 2007). In 2002, gall
wasp was epo ed o he i s ime in Eu ope in no hwes I aly (B ussino e
al. 2002). F om hen, ches nu gall wasp has been sp ead h oughou
Eu ope, being p esen in many coun ies (Eu opean and Medi e anean
Plan P o ec ion O ganiza ion, Global Da abase). Beyond Eu opean
ches nu , D. ku iphilus a acks Asian ches nu species, he Ame ican
ches nu and hei hyb ids.
Chap e I
26
ep og amming he hos cell o accommoda e he needs o he pa hogen.
In acellula disease esis ance p o eins media e ecogni ion o e ec o s
en e ing he hos cell and elici e ec o - igge ed immuni y (ETI) (Jones and
Dangl 2006; S ael e al. 2015). Besides local immune esponses, PTI and
ETI ac i a e long-dis ance de ense eac ions, such as sys emic acqui ed
esis ance (SAR) (Du an and Dong 2004). Plan s also ha e he abili y o
de end hemsel es agains di e en pa hogens also by egula ing
ansc ip ional ac i i y, induc ion o ailo ed de ense esponses including
callose deposi ion, cell wall hickening and p oduc ion o eac i e oxygen
species (ROS) (Jones and Dangl 2006; S ael e al. 2015; He e a-Vásquez
e al. 2015).
Plan ho mone balance also play a key ole in de e mining he ou come o
plan –pa hogen in e ac ions. The bes cha ac e ized de ense ho mones
include salicylic acid (SA), jasmonic acid (JA), absisic acid (ABA) and
e hylene (Spoel and Dong 2008; Ba i and Jones 2009; Pie e se e al. 2009).
I all de ense esponses ail o inhibi pa hogen ing ess, he plan cell unde
a ack can unde go hype sensi i e cell dea h (Kamoun e al. 1999; Mu e al.
2008; Choupina e al. 2014). Ne e heless, Phy oph ho a species ha e
e ol ed a ange o coun e -de ense mechanisms ha can inhibi all hos
de ense p ocesses men ioned (Ha dham and Blackman 2010). Despi e all
he knowledge acqui ed, molecula mechanisms in ol ed in woody plan s
esis ance o Phy oph ho a species a e poo ly unde s ood. This esea ch is
challenging because no genome da a is a ailable o many wood species
and so, iden i ica ion and cha ac e iza ion o Phy oph ho a esis ance genes
is necessa y. Ne e heless, s a egies o a ack and de ense in plan s-
oomyce e in e ac ions we e ecen ly e iewed (Oßwald e al. 2014; Fawke e
al. 2015). Apa om he Cas anea genus, he mos cha ac e ized
in e ac ions in Fagaceae amily a e Que cus sube - P. cinnamomi (Coelho
e al. 2006; Coelho e al. 2011; Ebadzad and C a ado 2014) and Fagus
syl a ica - P. ci icola (Po z e al. 2011; Schlink, 2010). Q. sube - P.

Gene al in oduc ion
27
cinnamomi in e ac ions ha e been s udied by cloning and cha ac e iza ion o
a se o candida e esis ance genes (Coelho e al. 2006; Coelho e al. 2011;
Ebadzad and C a ado 2014). Mo eo e , a hypo he ical mechanisms model
was p oposed o i e o hose genes o which exp ession was inc eased
24hpi (Coelho e al. 2011). Molecula in e ac ions obse ed be ween F.
syl a ica and P. ci icola was cha ac e ized by ansc ip ional changes a e
in ec ion. Resul s indica ed ha P. ci icola escapes he main ecogni ion
sys ems and/o supp esses he hos 's esponse (Schlink 2010). As a i s
s ep o iden i y ansc ip s in ol ed in he Cas anea - P. cinnamomi
in e ac ion, ou esea ch g oup iden i ied and cha ac e ized oo
ansc ip omes exp essed sequence ags (ESTs) di e en ially exp essed in
Eu opean ches nu (Cas anea sa i a) and Japanese ches nu (Cas anea
c ena a), in esponse o inocula ion wi h P. cinnamomi (Se azina e al. 2015,
Chap e III). Ne e heless, he pa hogenic p ocess should comp ise a
ne wo k o molecula signaling and in e ac ion e en s in di e en ime poin s
a e P. cinnamomi in ec ion ha we e no ye achie ed in Cas anea spp.
Resea ch objec i es and hesis layou
The gene al aim o he wo k he e desc ibed was o p o ide new insigh s
abou he Cas anea esis ance mechanisms o P. cinnamomi in ec ion using
di e en app oaches: genomics, phenomics and ansc ip omics.
The ou comes o his p ojec cons i u e an essen ial con ibu ion o he
unde s anding o ches nu esponse o P. cinnamomi based on an eli e plan
ma e ial c ea ed om he b eeding p og am, ha seg ega es o he ai o
esis ance. The de elopmen o imp o ed ches nu geno ypes wi h inc eased
esis ance o pa hogens and he p oduc ion o genomic esou ces o u u e
molecula assis ed selec ion will also cons i u e an asse o he imp o emen
and adap a ion o woody plan s, mainly belonging o Fagaceae amily, o
bio ic s esses.
Chap e I
28
The speci ic objec i es o his wo k we e:
1. Pe o m new con olled c osses be ween C. sa i a and C. c ena a in o de
o inc ease he hyb id ches nu popula ion ob ained in 2006 and 2009;
2. Pheno ype C. sa i a x C. c ena a (SC) hyb id p ogenies ob ained
p e iously and ob ained om new c osses. Pheno ype a small popula ion
om C. sa i a x C. mollissima (SM) con olled c osses o compa e le els
o esis ance among p ogenies wi h di e en dono s o esis ance;
3. Cons uc he i s in e speci ic gene ic map o C. sa i a x C. c ena a
popula ion h ough geno yping o pa en s and p ogenies wi h molecula
ma ke s: mic osa elli es o Simple Sequence Repea s - SSRs and Single
Nucleo ide Polymo phism - SNPs;
4. Pe o m DNA ma ke : ai associa ion analysis o iden i y genomic egions
ha explains he pheno ypic a ia ion in he SC popula ion, by
iden i ica ion o Quan i a i e T ai Loci.
5. Iden i y candida e genes ela ed wi h he esis ance o P. cinnamomi by
compa ing he oo ansc ip p o iles o esis an and suscep ible species,
be o e and a e inocula ion;
6. E alua e he exp ession o genes po en ially in ol ed in he esis ance o
P. cinnamomi in pa en al geno ypes (C. sa i a and a C. c ena a), as well
as, in hyb id geno ypes wi h di e en esponses o P. cinnamomi.
7. Localize in he gene ic map he di e en ial exp essed genes by
de eloping molecula ma ke s on sequences ob ained om he oo
ansc ip omes.
This hesis p esen s all he wo k o ganized in scien i ic a icles, om Chap e
II o V, and he wo k ollowed he s eps desc ibed in Figu e 7. Final
conclusions and u u e pe spec i es a e discussed in Chap e VI.
Gene al in oduc ion
29
Figu e 7. Gene al o ganiza ion o he esea ch and hesis, highligh ing he main app oaches and echniques used du ing hesis
s udies.
Chap e I
30
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42

Pheno yping Cas anea hyb ids o P. cinnamomi esis ance
43
Chap e II
Pheno yping Cas anea hyb ids o Phy oph ho a
cinnamomi esis ance
Pa o he wo k p esen ed in his chap e was published in he ollowing
esea ch publica ion:
San os C., Machado H., Co eia I., Gomes F., Gomes-La anjo J. and Cos a
R. (2015) Pheno yping Cas anea hyb ids o Phy oph ho a cinnamomi
esis ance. Plan Pa hol. 64, 901–910. doi: 10.1111/ppa.12313
In his esea ch pape Ca men San os pa icipa ed in he expe imen al
design, labo a o y expe imen s, esul s in e p e a ion and pape w i ing.
Chap e II
44
Abs ac
Cas anea sa i a is suscep ible o Phy oph ho a spp., a se ious oo pa hogen
causing ink disease, while C. c ena a and C. mollissima show esis ance o
in ec ion. In e speci ic con olled c osses we e p oduced o in og ess
esis ance genes om he esis an species in o he suscep ible C. sa i a,
and h ee mapping popula ions we e c ea ed. Phy oph ho a cinnamomi
esis ance o he p ogenies C. sa i a x C. c ena a and C. sa i a x C.
molissima we e e alua ed by oo and/o excised shoo inocula ion es s. The
numbe o days o su i al a e oo inocula ion was he bes disc imina o
o esis ance o P. cinnamomi while he pe cen age o shoo s wi h in e nal
lesions was he symp om mos associa ed wi h su i al. The lesion
p og ession a e in he excised shoo inocula ion es was s ongly and
nega i ely co ela ed wi h su i al in he oo inocula ion es . The excised
shoo inocula ion es appea s o be a eliable app oach o sc eening he
esis ance o ches nu geno ypes o P. cinnamomi. The e o e, a ecen ly
ob ained p ogeny (in 2015) was pheno yped using he excised shoo
inocula ion es . S ong gene ic co ela ions we e ob ained be ween su i al
and ink disease symp oms and among symp oms, indica ing ha common o
linked genes migh in luence esis ance o P. cinnamomi. The mos esis an
geno ypes selec ed om his s udy will be es ed o o he comme cial
a iables, such as ease o ege a i e p opaga ion and s ock–scion
compa ibili y.
Keywo ds: Cas anea hyb ids, he i abili y, pheno ypic and gene ic
co ela ions, Phy oph ho a cinnamomi
In oduc ion
The genus Cas anea belongs o Fagaceae, a plan amily ha domina es
much o he climax ha dwood o es s o he No he n Hemisphe e (Manos e
Pheno yping Cas anea hyb ids o P. cinnamomi esis ance
45
al. 2008). The Eu opean ches nu (Cas anea sa i a) is conside ed o be he
only na i e species in Eu ope. Ches nu s a e mul ipu pose ees being used
in he ood indus y, o i s edible nu s, in he wood indus y, as imbe and
also o ecological and landscaping pu poses, ha ing a majo economic
impo ance in he Medi e anean egion.
Ches nu ui p oduc ion has declined conside ably in sou hwes e n Eu ope
due o social changes and cul u al de elopmen , and pa icula ly o he
eme gence o hea ily damaging diseases. Ink disease, caused by
Phy oph ho a spp. is one o he mos des uc i e diseases a ec ing
Cas anea sa i a. Phy oph ho a cinnamomi is an agg essi e oo pa hogen,
o iginally om he sou heas Asian opics (Ha dham 2005). Nowadays, P.
cinnamomi is widesp ead and con inues o be des uc i e in o es s o
Medi e anean coun ies, Aus alia, sou heas USA, sou he n Cali o nia and
mo e ecen ly i was ecognized as a dange o o es s in wes e n No h
Ame ica (Robin e al. 2012).
Phy oph ho a cinnamomi has an excep ionally wide hos ange, being able
o in ade mo e han 3 000 plan species a ound he wo ld (Ha dham 2005;
Cahill e al. 2008). Cu en ly, i is he mos impo an Phy oph ho a pa hogen
o o es ees; besides ches nu , P. cinnamomi causes oo diseases in
eucalyp us, oaks, pines and membe s o he E icaceae amily, as well as,
se e al ag icul u al c ops (Robin e al. 2012).
Disease symp oms in ches nu a e simila o o he species: ink disease
causes oo o , wi h nec osis o ap oo , which ex ends o he la e al oo s
and he colla . P. cinnamomi in ec ion induces nec osis o he cambial and
xylem issues, causing in e e ence wi h anspi a ion om oo s o shoo s,
and consequen ly causes wil ing o lea es and dieback o young shoo s
(Ma çais and Dupuis 1996; Robin e al. 2001; Vannini and Ve aino 2001;
Ha dham 2005; Gomes-La anjo e al. 2009).
The pa hogen sp eads slowly h ough oo - o- oo con ac and mo e apidly
in p esence o wa e . Human ac i i ies ha mo e soil and he plan ing o
Chap e II
46
in es ed nu se y s ock in ensi y pa hogen sp ead (Robin e al. 2012). Wi h
changing clima es, P. cinnamomi is expec ed o expand i s a ea o
des uc ion, mainly in Eu ope and No h Ame ica (Robin e al. 2012;
Thompson e al. 2014).
In Po ugal, ink disease has become widesp ead, since P. cinnamomi was
i s eco ded, in 1838. Despi e he p o ec ion measu es aken, i is s ill a
g ea h ea o ches nu o cha ds, as ui and imbe p oduc ion is nega i ely
impac ed. Ches nu p oduc ion is an impo an sou ce o income o u al
popula ions and so new plan ings ha e been ca ied ou while old o cha ds
a e being es o ed. In Eu ope, ches nu b eeding o ink esis ance began
wi h he in oduc ion o he Asian ches nu ge mplasm, which is esis an o
he main diseases: ink and bligh (caused by C yphonec ia pa asi ica).
Japanese and Chinese species (Cas anea c ena a and Cas anea
mollissima, espec i ely) we e in oduced since 1917 in se e al sou he n
Eu opean coun ies (Elo ie a, 1949). Howe e , he low alue o he Asian
species as imbe and ui p oduce s was no able. They also p esen ed low
compa ibili y o g a ing wi h local swee ches nu a ie ies (Elo ie a, 1949).
In Po ugal, he i s in e speci ic hyb idiza ions we e ini ia ed in 1948 by
Be na dino Ba os Gomes o in oduce esis ance o ink disease in C. sa i a
(Gue ei o, 1948; Gue ei o, 1957). The objec i es o hese p og ams we e
o b eed o esis ance o ink disease, as well as o p oduce oo s ock o
a ie ies selec ed o ea ly nu p oduc ion o be e wood p oduc ion as
compa ed wi h Asian species (Fe nández-López 2011).
In 2006, in e speci ic con olled c osses we e pe o med be ween C. sa i a
and C. c ena a (SC) and be ween C. sa i a and C. mollissima (SM) in o de
o in og ess he esis ance om Asian species in o he Eu opean (Cos a e
al. 2011). The main goal o ou ongoing p og am was o p oduce a hyb id
seg egan popula ion o pe o m DNA ma ke -pheno ype associa ion
analysis o iden i y genomic egions ela ed wi h he ink disease esis ance
(Quan i a i e T ai Loci, QTL). Fo his pu pose, i is c ucial o de e mine
Pheno yping Cas anea hyb ids o P. cinnamomi esis ance
47
accu a ely bo h geno ype and pheno ype o each hyb id p ogeny. Howe e ,
he e a e some limi a ions o he de e mina ion o he esis ance o Cas anea
spp. o P. cinnamomi.
Di e en me hods ha e been used used o sc eening he esis ance o
ches nu o Phy oph ho a spp. by di e en au ho s.: i) oo inocula ion using
seedlings (Ve aino e al. 2001; San ini e al. 2003; Robin e al. 2006; Je e s
e al. 2009); ii) oo inocula ion using cu ings (Mi anda-Fon aiña e al. 2007)
o iii) plan le s om mic op opaga ion (Cuenca e al. 2009) and i ) di ec
inocula ion on he op o excised o in ac s em/shoo om seedlings o
clones selec ed in he ield (Guedes-La a gue & Salesses 1999; Fe nández-
López e al. 2001; Ve aino e al. 2001b; Robin e al. 2006; Mi anda-Fon aíña
e al. 2007; Cuenca e al. 2009). The e a e ad an ages and d awbacks o
each me hod; in pa icula , shoo inocula ion is easy o achie e and enables
he sc eening o a high numbe o indi iduals a low cos (Fe nández-López
2011), bu a c i icism o his me hod is ha P. cinnamomi is a oo pa hogen.
The objec i es o he p esen s udy we e (i) o selec he bes esis ance
disc imina o s om oo and excised shoo s inocula ion es s and cla i y hei
co ela ions; and (ii) o assess he esis ance o P. cinnamomi and e alua e
i s he i abili y in p ogenies o h ee Cas anea sp. seg egan ing popula ions.
Ma e ial and Me hods
Plan ma e ial
Th ee ull-sib p ogenies we e ob ained om a i icial con olled c osses: C.
sa i a (cul i a A elei a) x C. c ena a2 (SC), C. sa i a (cul i a A elei a) x C.
mollissima (SM) and C. sa i a (cul i a Bá ia) x C. c ena a1 (BC) in 2006,
2009, 2012 and 2015. C. sa i a emale lowe s we e isola ed be o e
pollina ion season by placing pollina ion bags on he b anches and cu ing
o he close ca kins. The pa en al line o he C. sa i a (cul i a A elei a) was
he same o c osses wi h C. c ena a2 and C. mollissima. Ca kins om C.
c ena a and C. mollissima we e collec ed and d ied he day be o e

Chap e II
48
pollina ion. On he day o pollina ion, pollen was emo ed om ca kins and
il e ed. Then, pollen was placed on he s igmas using a pain b ush o a piece
o glass (only he pollen g ains a e adhe ed o he glass). Pollina ed lowe s
we e co e ed wi h pape o polyes e pollina ion bags o and kep un il he
end o pollina ion season. Subsequenl y, pollina ion bags we e eplaced by
ne bags in o de o collec he seeds. The c osses we e pe o med a he
ge mplasm bank o Uni e sidade de T ás-os-Mon es e Al o Dou o, Vila Real
o SC and SM c osses, and in a p i a e o cha d in Ma ão o BC c osses,
bo h in Po ugal.
A o al o 142 F1 geno ypes we e es ed o P. cinnamomi in ec ion by ei he
oo inocula ion es o excised shoo inocula ion es s o by bo h (Table 1).
Fo oo inocula ion es , 137 plan le s we e p oduced om 20 geno ypes by
in i o p opaga ion om buds o mo he plan s. A he ime o inocula ion,
plan le s we e di e en ages, as de e mined by he numbe o days a e
acclima iza ion, bu we e mos equen ly 80 days old; ae ial pa s we e 16.66
cm on a e age.
Table 1. Numbe o indi iduals om Cas anea sa i a (cul i a A elei a) x C. c ena a2
(SC), C. sa i a (cul i a A elei a) x C. mollissima (SM) and C. sa i a (cul i a Bá ia)
x C. c ena a1 (BC) c osses es ed by oo inocula ion and excised shoo inocula ion.
Tes me hod
SC
SM
BC
To al
Roo inocula ion
16
4
0
20
Excised shoo inocula ion ( o al)
45
18
76
139
Excised shoo inocula ion (sp ing)
30
17
0
47
Excised shoo inocula ion (au umn)
42
18
76
136
To al
48
18
76
142
The excised shoo inocula ion es s we e ca ied ou in sp ing and au umn o
2012 o a o al o 63 SC and SM p ogenies. Fo BC p ogenies, he excised
shoo inocula ion es s was pe o med in au umn o 2016 (Table 1). A o al
numbe o 1034 shoo s we e collec ed om he mo he plan s. Bo h
Pheno yping Cas anea hyb ids o P. cinnamomi esis ance
49
expe imen s we e pe o med in a con olled chambe wi h empe a u es
anging be ween 18 and 22ºC, pho ope iod 16h ligh /8h da k and 65% o
ela i e humidi y.
Inoculum o P. cinnamomi
In all expe imen s, he same isola e o P. cinnamomi was used (IMI 340340),
which was selec ed, as he mos i ulen , ollowing es s using se e al
isola es (Ab eu e al. 1999). The high pa hogenici y o his isola e in
Eu opean ches nu s was also con i med by Dinis e al. (2011).
Fo he oo inocula ion es , he P. cinnamomi inoculum was p epa ed by
g owing mycelia on s e ilized mille seeds (Ponicium mileaceum), which we e
ho oughly mois ened wi h V8 medium b o h [20% ( / ) wi h 3 g/L o CaCO3].
A e wa ds, his mix u e was incuba ed o 3 weeks in da kness a 24°C.
Fo he excised shoo inocula ion es , P. cinnamomi was g own on po a o
dex ose aga o 6 days in da kness a 22ºC.
Roo inocula ion es
Fou expe imen s o oo inocula ion we e ca ied ou using clonal plan le s
placed in s e ile subs a e. Fo each expe imen , one o wo plan le s o each
geno ype, we e used as a con ol, wi hou inocula ion. Fo oo inocula ion,
P. cinnamomi-in ec ed mille seed inoculum was ca e ully placed in o he
subs a e (600 mL) o each po , a a concen a ion o 5% ( / ). Mos ly, eigh
eplica es pe geno ype we e inocula ed, bu , due o limi a ions o in i o
p opaga ion, his was no alwalys possible. The e o e, he mean numbe o
plan s pe geno ype was 6.85.
Inocula ed plan s and con ols we e placed sepa a ely in di e en ays and
each po was looded o 1h, h ee imes a week, o s imula e zoospo e
elease and o p omo e disease de elopmen .
P. cinnamomi was eco e ed om wa e collec ed om looding p ocess,
using a modi ied bai ing echnique adap ed om Jung e al. (1996).
Chap e II
50
The expe imen al design was adap ed om Mi anda-Fon aíña e al. (2007).
Fo each indi idual, he days o su i al a e inocula ion we e eco ded, un il
100 days a e inocula ion (dai). A e dea h, plan le s we e emo ed om he
soil and he oo s we e gen ly washed o obse e and eco d ink disease
symp oms.The le el o oo o was assessed on a scale om 1 o 6,
acco ding o Mi anda-Fon aíña e al. (2007), whe e 1 indica es he leas
se e e le el o oo o and 6 indica es he mos se e e le el. The pe cen age
was hen used o a e he le el o oo colla o on a scale o 1-6 (1, no o ;
2, 0.1-9.9% o ; 3, 10-19.9% o ; 4, 20-29.9% o ; 5, 30-49.9% o and 6,
>50% o ). Shoo in e nal and ex e nal lesions we e eco ded as he
pe cen age leng h o in e nal and ex e nal lesion o he longes shoo ,
espec i ely. Biomass pa ame e s we e also e alua ed o each plan le : lea
and shoo d y weigh (g) and oo d y weigh (g). In o de o de e mine he
d y weigh , lea es and shoo s we e sepa a ed om oo s and bo h pa s we e
d ied a 60ºC o 2 days.
A he end o each expe imen , plan le s ha did no die du ing he es we e
analysed wi hou des uc ion and we e ansplan ed o new po s ha we e
placed in a g eenhouse wi h con olled condi ions (20-25ºC). In he sp ing o
he nex yea , he numbe o plan le s ha showed budbu s was eco ded.
Excised shoo s inocula ion es
Excised shoo inocula ion es o SC and SM popula ions ook place in
sp ing, using 47 geno ypes (eigh geno ypes we e common o he oo
inocula ion es ), and in au umn, using 60 geno ypes (16 common o he oo
inocula ion es ). The 76 p ogenies om BC c oss we e sc enned in au umn,
2015 (Table 1). Excised shoo s, he majo i y wi h a leng h o 15 cm, we e
collec ed om each mo he plan . The mean numbe o excised shoo s
inocula ed pe geno ype was 7.94 in sp ing and 4.86 in au umn. All apa
om wo uppe lea es we e emo ed o educe e apo anspi a ion. The
diame e o he op o each excised shoo was eco ded be o e inocula ion.
Pheno yping Cas anea hyb ids o P. cinnamomi esis ance
51
Mycelial plugs o P. cinnamomi we e hen placed on he op o he shoo s
and we e co e ed wi h an aluminium shee o a oid desicca ion. Replica es
we e dis ibu ed andomly in h ee ays wi h pe li e and wa e , in an
en i onmen al con olled chambe . Fi e days a e inocula ion, he aluminum
shee s we e emo ed om each shoo , when coloniza ion by he pa hogen
had occu ed. Resis ance o P. cinnamomi was e alua ed by measu ing he
isible ex e nal lesion leng h (LL) a 5, 7, 9, 12 and 14 dai. The lesion
p og ession a e (cm/day) was calcula ed o each geno ype, using he
ollowing o mula:
( 𝐿𝐿5 𝑑𝑎𝑖
5)+( 𝐿𝐿7 𝑑𝑎𝑖 − 𝐿𝐿5 𝑑𝑎𝑖
2)+( 𝐿𝐿9 𝑑𝑎𝑖 − 𝐿𝐿7 𝑑𝑎𝑖
2)+( 𝐿𝐿12 𝑑𝑎𝑖 − 𝐿𝐿9 𝑑𝑎𝑖
3)+( 𝐿𝐿14 𝑑𝑎𝑖 − 𝐿𝐿12 𝑑𝑎𝑖
2)
5
S a is ical analysis
Analysis o a iance (ANOVA) was conduc ed o oo and shoo a iables
using linea mixed e ec s models o he gene al o m y = Xβ + Zγ + ε, whe e
y is he ec o o obse a ions; X and Z a e design ma ices o he pa ame e s
associa ed o ixed and andom e ec s, espec i ely; β and γ a e ec o s o
ixed e ec s (including he gene al mean) and andom e ec s, espec i ely;
and ε is he ec o o esidual e o s. In p elimina y da a analyses, esis ance
om SC and SM c osses was e alua ed sepa a ely o each c oss by
speci ying a wo-le el ‘Family’ e ec (SC and SM). Because no signi ican
di e ences we e ound be ween he wo ull-sib amilies, he amily e ec was
d opped om he model. Fo he oo inocula ion es da a, Age, Geno ype,
Inocula ion, Inocula ion Da e and Geno ype x Inocula ion Da e we e i ed as
ixed e ec s (β ec o ); o he excised shoo inocula ion es da a, Leng h,
Geno ype, Season and Geno ype x Season we e ea ed as ixed e ec s. F-
es s we e used o es he signi icance o he ixed e ec s and o geno ype
mean compa isons, he la e implemen ing he Tukey adjus men and a
ma ching le e display (adap ed om Piepho, 2012). I signi ican , he
Chap e II
58
lesion was lowe han shoo in e nal lesion, anging om 4.16% (SM901) o
26.63% (SC918) (Table 3).
The pheno ypic and gene ic co ela ion coe icien s we e es ima ed o all he
a iables eco ded on inocula ed plan le s. Bo h co ela ion coe icien s
showed a g ea simila i y among pai s o a iables, in ega d o di ec ion and
o magni ude (Table 4).
The pheno ypic co ela ions showed ha su i al had highly signi ican
nega i e co ela ions wi h h ee o he ou ink disease symp oms analysed:
le el o oo colla o , shoo in e nal lesion and shoo ex e nal lesion. Bo h
pheno ypic and gene ic co ela ions showed ha shoo in e nal lesion was
he main symp om nega i ely associa ed o su i al. The le el o oo o was
he leas impo an symp om associa ed o su i al, wi h nonsigni ican
pheno ypic co ela ion (Table 4).
The pheno ypic co ela ions e alua ed be ween su i al and he biomass
pa ame e s we e posi i e and highly signi ican (Table 4). Co ela ions we e
lowe o lea es and shoo s han o oo s, while he symp oms and biomass
pa ame e s we e no s ongly co ela ed. The pheno ypic co ela ion
coe icien s obse ed be ween symp oms we e posi i e and highly
signi ican , especially le el o oo colla o wi h shoo ex e nal lesion,
ollowed by shoo ex e nal lesion wi h shoo in e nal lesion. The weakes
co ela ed symp oms we e le el o oo o wi h shoo ex e nal lesion.
The highes gene ic co ela ions we e ound among symp oms and among
biomass pa ame e s. The gene ic co ela ion coe icien s among symp oms
anged om 0.95 o 1.00 (le el o oo colla o wi h shoo ex e nal lesion),
in ag eemen wi h pheno ypic co ela ions.

Pheno yping Cas anea hyb ids o P. cinnamomi esis ance
59
Days o
su i al
Lea and
shoo s d y
weigh
Roo d y
weigh
Le el o oo
colla o
Le el o oo
o
Shoo
ex e nal
lesion
Shoo
in e nal
lesion
Age
0.31***
0.38***
0.60***
-0.01
-0.02
-0.05
-0.22*
Days o su i al
0.43***
0.50***
-0.42***
-0.16
-0.44***
-0.69***
Lea and shoo s d y
weigh
0.51
0.75***
0.02
0.18
-0.13
-0.26*
Roo d y weigh
0.64
0.99
0.02
0.18
-0.10
-0.24*
Le el o oo colla o
-0.88
-0.08
-0.23
0.49***
0.76***
0.61***
Le el o oo o
-0.86
-0.05
-0.21
1.00
0.40***
0.44***
Shoo ex e nal lesion
-0.90
-0.13
-0.29
1.00
1.00
0.66***
Shoo in e nal lesion
-0.97
-0.36
-0.50
0.96
0.95
0.97
Table 4. Pheno ypic (abo e he diagonal) and gene ic (below he diagonal) co ela ion coe icien s de e mined be ween pai s
o he a iables measu ed in oo inocula ion es (N=108 o 137).
Pheno ypic co ela ions we e analysed wi h he Spea man’s co ela ion coe icien . Gene ic co ela ions we e adjus ed o he co a ia e Age
e ec (Age=80 days a e acclima iza ion). Signi icance is indica ed by as e isks: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.
Chap e II
60
The he i abili y alues o ches nu esis ance o P. cinnamomi a ied
be ween 0.34 and 0.90, wi h low s anda d e o s. Su i al showed he
highes he i abili y (0.90 ± 0.04) wi h low esidual a iance and hus he
highes po en ial o be inhe i ed. Among symp oms, he highes he i abili y
alue was ob ained o shoo in e nal lesion whe eas shoo ex e nal lesion
showed he lowes alue, wi h he lowes a iance explained by bo h
componen s, gene ic and esidual (Table 5).
Table 5. Na ow-sense he i abili ies (h2) and hei s anda d e o s (in pa en heses)
es ima ed o he a iables ( oo inocula ion es ).
Va iables
Va iance componen s
Na ow-sense
he i abili ies (h2)
Addi i e gene ic (σa
2)
Residual (σe
2)
Days o su i al
0.92 (0.34)
0.10 (0.01)
0.90 (0.04)
Le el o oo o
0.03 (0.02)
0.04 (0.01)
0.46 (0.16)
Le el o oo colla o
0.25 (0.11)
0.22 (0.03)
0.54 (0.11)
Shoo in e nal lesion
0.45 (0.19)
0.15 (0.02)
0.75 (0.09)
Shoo ex e nal lesion
0.01 (0.01)
0.03 (0.00)
0.34 (0.14)
n=108-137.
Excised shoo s inocula ion
A 5 dai in bo h seasons, P. cinnamomi had induced isible nec o ic lesions
o a ying leng h, depending on he geno ype. A 14 dai a e y low
pe cen age (0.36%) o shoo s did no show any lesion and 8.80% o shoo s
showed o al nec osis (100% o lesion leng h).
Analysis o a iance e ealed ha lesion leng h was signi ican ly a ec ed by
Geno ype, which had he s onges e ec , and also by Season and he
in e ac ion o bo h. In con as o diame e , he Ini ial Shoo Leng h had a
signi ican e ec on he lesion leng h, and he e o e was used as a co a ia e
e ec in he analysis o SC and SM da a.
Fu he analysis showed ha di e ences obse ed be ween geno ypes o
lesion leng h we e e y high o all ime poin s o measu emen s and in bo h
Pheno yping Cas anea hyb ids o P. cinnamomi esis ance
61
seasons, when applicable. Mo eo e , di e ences obse ed be ween
geno ypes (SC and SM p ogenies) o lesion leng h we e maximal a 5 dai
(F=19.80, P < 0.001) in sp ing (F=33.11, P < 0.001) a e bud bu s .
Di e en esponses o P. cinnamomi we e obse ed in he p ogenies: a
con inuous ange o esis ance-suscep ibili y le els among geno ypes was
obse ed. Fo he majo i y o geno ypes, he lesion leng h in he shoo s
inc eased o e ime. In addi ion, o he mos esis an geno ypes he lesion
leng h s opped a a gi en ime poin , un il he end o he expe imen .
The e o e, he lesion p og ession a e (cm/day) was calcula ed o each
geno ype. Geno ype mean alues (SC and SM), es ima ed ac oss he wo
seasons and adjus ed o he co a ia e Ini ial Leng h, a e shown in Figu e 2.
The lesion p og ession a e a ied om 0.15 o 1.13 cm pe day ac oss
geno ypes and seasons; SC57 was he mos esis an geno ype while SC915
he geno ype wi h he g ea es lesion p og ession a e (i.e. mos suscep ible).
Conce ning BC p ogenies, lesion p og ession a es ob ained in au umn 2015
we e also anged om he mos suscep ible (BCC01) o he mos esis an
(BDC40) (Supplemen a y ma e ial 1). This popula ion seems o be mo e
esis an o P. cinnamomi han SC and SM, since lesion p og ession a e
a ied om 0.11 o 0.89 cm pe day.
Co ela ions be ween inocula ion es s
The es ima ion o pheno ypic and gene ic co ela ions be ween he oo
inocula ion es and excised shoo s inocula ion es was possible using he
mean esponse o common geno ypes in bo h es s (n=17). In his way, some
di e ences we e ob ained in he pheno ypic and gene ic co ela ions
be ween su i al and symp oms (Table 4 and 6).
Chap e II
62
Figu e 2. Mean alues in lesion
p og ession a e, adjus ed o
he co a ia e Ini ial Leng h, o
63 geno ypes es ed wi h
excised shoo inocula ion. The
geno ypes we e anked by
lesion p og ession a e, om
he mos esis an o he mos
suscep ible. SC915 geno ype
showed he highes
suscep ibili y o Phy oph ho a
cinnamomi whe eas SC57 was
he mos esis an . Ba s
ep esen s anda d e o s.
Pheno yping Cas anea hyb ids o P. cinnamomi esis ance
63
Pheno ypic co ela ions ob ained among all a iables om bo h inocula ion
es s showed ha lesion p og ession a e was he pa ame e wi h he
s onges co ela ion wi h su i al ( = -0.85, P < 0.001). Shoo in e nal lesion
was co ela ed mode a ely wi h lesion p og ession a e, as well as le el o
oo colla o (Table 6). Gene ic co ela ion coe icien s we e in ag eemen
wi h pheno ypic co ela ions and he he i abili y o lesion p og ession a e
was 0.67 ± 0.04.
Table 6. Pheno ypic (abo e he diagonal) and gene ic (below he diagonal)
co ela ion coe icien s de e mined be ween pai s o he a iables measu ed in oo
inocula ion es : Days o su i al, Le el o oo colla o , Le el o oo o , Shoo
ex e nal lesion and Shoo in e nal lesion; and be ween he a iables measu ed in
oo inocula ion es and Lesion p og ession a e om excised shoo inocula ion es
(n=17).
Days o
su i al
Le el o
oo
colla o
Le el o
oo o
Shoo
ex e nal
lesion
Shoo
in e nal
lesion
Lesion
p og ession
a e
Days o su i al
-0.63**
-0.36
-0.47
-0.74**
-0.85***
Le el o oo colla o
-0.67
0.59*
0.58*
0.56*
0.62*
Le el o oo o
-0.56
0.82
0.09
0.36
0.44
Shoo ex e nal lesion
-0.45
0.62
-0.05
0.75***
0.45
Shoo in e nal lesion
-0.87
0.44
0.39
0.66
0.67**
Lesion p og ession
a e
-0.83
0.62
0.73
0.34
0.75
Signi icance is indica ed by as e isks: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.
Discussion
This s udy add esses wo ypes o inocula ion es used o de e mine he
esponse o indi idual ches nu plan le s om h ee mapping popula ions o
inocula ion wi h P. cinnamomi. Lesion p og ession a e was he a iable
selec ed o pe o m DNA ma ke – a iable associa ion, o QTL iden i ica ion
(Chap e V).

Chap e II
64
Al hough mo e han one isola e o P. cinnamomi is commonly used in his
ype o s udy, only one isola e was used in he p esen in es iga ion in o de
o maximize he numbe o eplica es o each ches nu geno ype sc eened,
hus making he analysis mo e obus and accu a e. Se e al p e ious s udies
o P. cinnamomi on ches nu (Ab eu e al. 1999; Dinis e al. 2011) enabled
he mos i ulen isola e o be selec ed o he p esen in es iga ion.
F amp on e al. (2013) also used a single isola e o P. cinnamomi in soil
inocula ion o Abies spp. seedlings. Mo eo e , Fe nández-Lóopez e al.
(2001) and Mi anda-Fon aiña e al. (2007) obse ed ha he e was no
signi ican in e ac ion be ween isola es and geno ypes in he o symp oms
e alua ed, indica ing no speci ici y o hose isola es in ches nu .
In p e ious s udies, he o igin and physiological condi ions o plan ma e ial,
eplica e numbe , ime poin o lesion measu emen s and es condi ions
a ied o bo h inocula ion es s (Ve aino e al. 2001a,b; Robin e al. 2006;
Mi anda-Fon aiña e al. 2007; Cuenca e al. 2009). The e o e, in he p esen
s udy, he expe imen s we e designed in o de o ob ain he mos eliable
esul s possible: a high numbe o clonal plan le s pe geno ype and he use
o he same con olled en i onmen al condi ions o bo h inocula ion es s.
Clonal es ing o p ogeny om mapping popula ions is he mos e icien way
o minimize he e ec o en i onmen al a ia ion and ob ain be e es ima es
o he pheno ypic alue (B adshaw & Fos e 1992). Minimizing en i onmen al
a ia ion and he e o e inc easing he i abili y, inc eases he obus ness and
he abili y o QTL de ec ion.
In he oo inocula ion es , con ol plan le s g ew mo e han inocula ed
plan le s, as expec ed. The lesions in oo s and shoo s caused by P.
cinnamomi may hinde wa e and nu ien abso p ion, and as a consequence,
cause a educ ion in he pho osyn hesis a e and g ow h. Robin e al. (2006)
and Mi anda-Fon aiña e al. (2007) also epo ed a educ ion in g ow h o
ches nu plan s inocula ed wi h P. cinnamomi. Cahill e al. (1989) obse ed
Pheno yping Cas anea hyb ids o P. cinnamomi esis ance
65
ha P. cinnamomi inocula ion s opped oo g ow h in a g oup o plan species
wi hin 24–48 h.
P e ious s udies ha e indica ed ha days o su i al should be he main
disc imina o o Phy oph ho a spp. esis ance in ches nu (Ve aino e al.
2001a), in Abies spp. (F amp on e al. 2013) and in Eucalyp us spp. (S ukely
& C ane, 1994). Howe e , o he au ho s ha e conside ed he le el o oo o
colla o as he main indica o o esis ance o Phy oph ho a spp. in ches nu
(Robin e al. 2006; Mi anda-Fon aiña e al. 2007; Cuenca e al. 2009). The
p esen s udy showed ha a iable ‘Days o su i al’ was he mos impo an
indica o o esis ance o P. cinnamomi because di e ences in esponse
be ween geno ypes we e maximized; he p esence o high le els o oo and
colla o in almos all inocula ed plan le s indica ed ha hese symp oms
we e no good disc imina o s o esis ance. Mi anda-Fon aiña e al. (2007)
also epo ed high le els o oo and colla o in a high pe cen age o
ches nu plan s. Likewise, Cuenca e al. (2009) obse ed oo o in 60% o
he esis an C. c ena a plan s. Su i al has no been conside ed as he main
desc ip o o esis ance o P. cinnamomi in ches nu , mainly because o he
high mo ali y o con ol ches nu plan s be o e and du ing he expe imen ,
due o bio ic o abio ic ac o s o c oss con amina ion (Mi anda-Fon aiña e
al. 2007; Cuenca e al. 2009). In his s udy, all con ol plan le s su i ed un il
he end o he expe imen , indica ing ha P. cinnamomi c oss con amina ion
and o he bio ic and abio ic s esses, such as looding, did no occu . In
u u e, simila s udies should include p e en i e measu es, such as he use
o s e ile subs a es and a oiding excess looding ime du ing he
expe imen s. Twen y o hi y minu es looding is su icien o P. cinnamomi
elease zoospo es and cys s o ge mina e (Ha dham, 2005). In summa y,
long su i al was conside ed e idence o high esis ance and hus se en
geno ypes (35%) we e selec ed as he mos esis an .
Chap e II
66
In his s udy, shoo in e nal lesion was e alua ed o he i s ime as a
pa ame e o assess ches nu esis ance o P. cinnamomi. I was chosen
because i indica es he sp ead o he pa hogen om he oo s and colla o
he ae ial ascula sys em. This is impo an o de e mining he deg ee o
plan esis ance, as he apid in asion o he pa hogen in o he phloem and
xylem may a ec wa e and nu ien mo emen h ough he shoo s, causing
dea h.
Wi h ega d o biomass pa ame e s, he pheno ypic co ela ion was s onge
be ween su i al and oo d y weigh han wi h lea and shoo d y weigh .
Cuenca e al. (2009) also obse ed a good co ela ion be ween esh oo
weigh and su i al. A heal hy and de eloped oo sys em is an impo an
ac o o esis ance o P. cinnamomi.
The lesion caused by he inocula ion o excised shoo s is conside ed o be
an indi ec measu e o Phy oph ho a spp. esis ance. The leng h o he lesion
is nega i ely p opo ional o esis ance o he pa hogen (Fe nández-López e
al. 2001). The esul s showed ha , simila o oo -inocula ed plan s, he
esis ance o P. cinnamomi in he shoo s is ela ed o he con inemen o he
lesion o poin o inocula ion. Fo he mos esis an geno ypes, he
su ounding issues d ied, limi ing he p og ession o he lesion.
In he p esen s udy, di e ences obse ed be ween geno ypes o lesion
leng h we e e y high o all ime poin s o measu emen and in bo h seasons.
Ne e heless, he esul s e ealed ha sp ing was he be e season o
pe o m excised shoo inocula ion es s. A e budbu s (in sp ing), plan s
ha e good physiological condi ions ha may allow a be e esis ance
esponse. I was ound ha he bes ime o ake measu emen s in u u e
in es iga ions would be 5 dai, when di e ences in lesion leng hs be ween
geno ypes we e maximized.
The p esen s udy has shown ha he s onges and mos signi ican
pheno ypic and gene ic co ela ions we e ob ained o lesion p og ession
Pheno yping Cas anea hyb ids o P. cinnamomi esis ance
67
a e and su i al; he e o e, hese would be he bes a iables o measu e in
u u e in es iga ions.
The es ima ion o he i abili ies and gene ic co ela ions (gene ic pa ame e s)
is an impo an s a egy o plan b eeding. Pheno ypic a iables wi h highe
he i abili ies (in his s udy: su i al, shoo in e nal lesion and lesion
p og ession a e) ha e he po en ial o be inhe i ed o a ying deg ees in
popula ions exposed o di e en ial na u al selec ion p essu es in dis inc
en i onmen s (Whi e e al. 2007).
Resis ance o P. cinnamomi is a polygenic and quan i a i e ai (I win e al.
1995) ha was he e e alua ed by measu ing se e al a iables. The s ong
gene ic co ela ions obse ed be ween su i al and symp oms sugges
common gene ic de e minan s. Simila ly, su i al had a s ong gene ic
co ela ion wi h lesion p og ession a e in he excised shoo inocula ion es .
The assessmen o su i al by oo inocula ion es ing is expensi e and
labo ious and canno always be de e mined in a popula ion. The s ong
a ou able gene ic co ela ion obse ed be ween he wo a iables sugges s
ha indi ec selec ion could be made by lesion p og ession a e, which is
easily measu ed. The e o e, he C. sa i a x C. c ena a popula ion ob ained
in 2015 (BC) was pheno yped using he excised shoo inocula ion es in
au umn. P elimina y esul s indica e a highe le el o esis ance when
compa ed wi h SC and SM popula ions. Howe e , excised shoo inocula ion
es shall be epea ed in sp ing o ob ain lesion p ogession a es ac oss bo h
seasons. Then, mo he plan s will be oo -inocula ed wi h P. cinnamomi o
iden i y he mos esis an geno ypes (su i o s o he inocula ion). Finally,
hose geno ypes will be es ablished o in i o cul u e o u he pheno ype
alida ion by oo inocula ion es .
Acknowledgmen s
We acknowledge Helen Bel ame and Pa ícia Figuei edo o he
es ablishmen , mul iplica ion, oo ing and acclima iza ion o ches nu
Chap e III
74
Abs ac
The Eu opean ches nu , an impo an o es species o he economy o
Sou he n Eu ope, co e s an a ea o 2.53 million hec a es, including almos
110 000 hec a es de o ed o ui p oduc ion. Cas anea sa i a is declining
due o ink disease caused by Phy oph ho a cinnamomi. To elucida e
ches nu de ense mechanisms o ink disease we compa ed he oo
ansc ip ome o he suscep ible species C. sa i a and he esis an species
C. c ena a a e P. cinnamomi inocula ion. Fou cDNA lib a ies we e
cons uc ed, wo o hem included oo samples om C. sa i a, inocula ed
and non-inocula ed and he o he wo lib a ies comp ised samples om C.
c ena a a iden ical condi ions.
Py osequencing p oduced 771 030 eads and assembly se up 15 683
con igs o C. sa i a and 16 828 o C. c ena a. GO anno a ion e ealed e ms
ela ed o s ess as ‘ esponse o s imulus’, ‘ ansc ip ion ac o ac i i y’ o
‘signaling’ o bo h ansc ip omes. Di e en ial gene exp ession analysis
e ealed ha C. c ena a in ol ed mo e genes ela ed wi h bio ic s ess upon
pa hogen inocula ion han C. sa i a. Those genes o bo h species a e
in ol ed in egula ion o plan immune esponse and s ess adap a ion and
eco e y. Fu he mo e, i is sugges ed ha bo h species ecognize he
pa hogen a ack; howe e , he esis an species may in ol e mo e genes in
he de ense esponse han he suscep ible species. RNA-seq enabled he
selec ion o candida e genes o ink disease esis ance in Cas anea. The
p esen da a is a aluable con ibu ion o he a ailable Cas anea genomic
esou ces and cons i u es he basis o u he s udies.
Keywo ds: Cas anea sa i a; Cas anea c ena a; Phy oph ho a cinnamomi;
RNA-seq; di e en ially exp essed genes; bio ic s ess.

Cas anea oo ansc ip ome in esponse o P. cinnamomi
75
In oduc ion
Eu opean ches nu (swee ches nu , Cas anea sa i a Mille ) has g ea
economic alue due o ui p oduc ion, and ecological alue including o es
di e si y and soil s abili y. In he las 100 yea s, ink disease caused by he
soil oomyce e Phy oph ho a cinnamomi Rands has con ibu ed o a d as ic
educ ion o C. sa i a dis ibu ion a ea in Eu ope. Oomyce es (euka yo ic
he e okon s) show s a egies o plan in ec ion ha a e simila o many ungal
pa hogens (La ijnhouwe s e al. 2003). Swee ches nu g o es ha e also
been a ec ed since he 1930’s by he ches nu bligh ungus [C yphonec ia
pa asi ica (Mu il) Ba ], causal agen o he Ame ican ches nu [Cas anea
den a a (Ma shall) Bo kh.] decima ion.
Ink disease was in oduced o Eu ope om he USA h ough he Azo es
islands (Fe nandes 1955; Anagnos akis 2001). The i s eco ds on i s
appea ance in no he n Po ugal da e om 1838. I has since been epo ed
in many Eu opean coun ies, including Spain, I aly, F ance and he Uni ed
Kingdom. The p og ession o he disease in g o e a eas wi h high humidi y
has limi ed he es ablishmen o new g o es and impeded he conse a ion
o old ones (Vannini and Ve aino 2001). P esen ly, he g ea es impac o
ink disease is limi ed o he wa m sou hwes e n and sou he n egions o
cen al Eu ope [ e iewed by (B asie and Jung 2006)]. In he 19 h cen u y, ink
disease was pa ially esponsible o a decline o C. den a a in he
Sou heas e n USA (Anagnos akis 2001), p io o i s b oad decima ion by
ches nu bligh . Ink disease is cu en ly e-eme ging in he USA and
cons i u es a se ious h ea o he Ame ican ches nu ein oduc ion (Jacobs
e al. 2013).
Common woody hos s o P. cinnamomi include Eucalyp us, Que cus,
Juglans, Be ula and Cas anea, and he mycelia also pe sis sap ophy ically
in soil. In he p esence o wa e , oospo es and chlamydospo es di e en ia e
spo angia ha o m and elease zoospo es. Zoospo es a e mo ile and a e
able o pene a e non-ligni ied oo issue and he base o s ems o unks:
Chap e III
76
bo h scena ios esul in local issue o . G ow h, ep oduc ion and
dissemina ion o he pa hogen a e a o ed unde compac ed and wa e
sa u a ed soils wi h poo ae a ion. Symp oms on he adul ees include lea
chlo osis, hinning o he c own and he pe sis ence o imma u e ui s on he
ees a e lea - all. La ge oo s a e mainly a ec ed, p oducing a black
exuda e which inc eases du ing sp ing and all. In ec ed seedlings unde go
a apid o g adual lea wil ing, depending on he se e i y o he in ec ion. The
oo sys em su e s ex ensi e nec osis o he ap oo ha ex ends o he
la e al oo s and up he lowe s em (Vannini and Ve aino 2001). Oßwald e
al (Oßwald e al. 2014) explain he p ima y physiological, biochemical and
molecula eac ions desc ibed on in ec ed oo s o suscep ible Phy oph ho a-
hos in e ac ion, summa ized as ollows: 1) The pa hogen eleases elici ins
in o he hizosphe e, acili a ing oo pene a ion; 2) Down- egula ion o
de ense genes in he hos , acili a ing pa hogen g ow h; 3) Des uc ion o
oo s and impai men o wa e and nu ien up ake; 4) Inc ease o he abscisic
acid phy oho mone in oo s; 5) Dec ease in lea wa e po en ial; 6) S oma a
closu e and dec ease in pho osyn hesis; 7) P obable elease o oxins and
e ec o s in o he hos issue du ing bio ophic g ow h o he pa hogen and
anspo in o he canopy ia xylem sap low; 8) Up- egula ion o genes o he
e hylene pa hway and elease o he phy oho mone by lea es; 9) Dec ease
in cy okinin con en in oo s du ing he nec o ophic g ow h o he pa hogen;
10) Chlo osis and wil ing o lea es esul ing om he changed wa e and
ho monal s a us o he hos caused by oo in ec ion.
P og ession o ink disease depends on en i onmen al condi ions, pa hogen
i ulence and plan suscep ibili y. One s a egy o con ol he disease is
h ough b eeding wi h esis an species. Soon a e he in oduc ion o Asian
ches nu s o Eu ope i was e i ied ha C. c ena a (Cas anea c ena a Siebold
& Zucc., he Japanese ches nu ) has a high le el o esis ance o
Phy oph ho a (Vannini and Ve aino 2001). Since he 1950’s, b eeding
p og ams wi h he Eu opean and Japanese ches nu we e es ablished in
Cas anea oo ansc ip ome in esponse o P. cinnamomi
77
Po ugal, F ance and Spain o ob ain hyb ids ole an o ink disease, while
main aining ui p oduc ion and quali y ai s o sa is y comme cial demands
(Vannini and Ve aino 2001; Ma ins e al. 2009). Howe e , ui quali y
p oduced by hese hyb ids is below cu en ma ke s anda ds, so he e is
demand om bo h esea che s and p oduce s, o de eloping genomic ools
o unde s and esis ance mechanisms agains P. cinnamomi. Ba aka e al
(Ba aka e al. 2009; Ba aka e al. 2012) desc ibed he gene a ion o mo e
han 1,5 million cDNA sequences o he Ame ican and Chinese ches nu s
ha ha e been used o analyse ches nu esis ance o C. pa asi ica. The da a
a e a ailable h ough he Fagaceae Genomics Web
(h p://www. agaceae.o g/) and ep esen he i s public esou ce on
ches nu ansc ip omes. The da a we p esen he e con ibu e o his
esou ce by iden i ying Japanese and Eu opean ches nu genes in ol ed in
he eac ion o ink disease, ano he c i ical h ea o Cas anea.
To compa e he esponse o he esis an Japanese ches nu wi h he
esponse o he suscep ible Eu opean ches nu o P. cinnamomi in ec ion,
ou cDNA lib a ies o C. sa i a (Cs) and C. c ena a (Cc) oo issues,
inocula ed (i) and non-inocula ed (n) wi h he pa hogen we e p epa ed o
454 py osequencing. Con ig anno a ion and analysis o ansc ip abundance
suppo ed he quan i ica ion o ansc ip exp ession on inocula ed and non-
inocula ed oo s in each species, as well as, iden i ying di e en ially
exp essed genes upon pa hogen inocula ion. This allowed a compa ison o
each species’ esponse o he pa hogen and he selec ion o candida e genes
o esis ance o ink disease.
Ma e ials and Me hods
Plan ma e ial and pa hogen inocula ion
The TRAGSA nu se y (G upo TRAGSA-SEPI, Maceda, Spain) p o ided 36
mic op opaga ed plan s a i e yea s o age, 18 o C. sa i a (Cs, suscep ible)
and 18 o C. c ena a (Cc, esis an ). Fou ea men s we e se , co esponding
Chap e III
78
o C. sa i a and C. c ena a inocula ed and non-inocula ed wi h P. cinnamomi
(Supplemen a y ma e ial 1). Plan s we e dis ibu ed in 15 L po s wi h pea .
A hype i ulen isola e o P. cinnamomi (IMI 340340) p o ided by T ás-os-
Mon es and Al o Dou o Uni e si y was g own a 22°C on Po a o Dex ose
Aga . Fo soil in es a ion P. cinnamomi inoculum was p epa ed by g owing
mycelia on s e ilized mille seeds (Ponicium mileaceum), which we e
ho oughly mois ened wi h ege able juice (V8®) b o h [20% ( / ) wi h 3 g/L
o CaCO3]. The mix u e was incuba ed o h ee weeks in da kness a 24°C.
A he ime o inocula ion (0 h), P. cinnamomi was ca e ully added o each
con aine subs a e a a concen a ion o 5% ( / ), in o de o minimize oo
dis u bance and wounding. No pa hogen was added o non-inocula ed
plan s. A e inocula ion all con aine s we e looded o 3h o s imula e
zoospo e elease and o p omo e disease de elopmen . A 2, 4 and 7 days
a e inocula ion, 6 plan s pe ea men (3 o C. sa i a and 3 o C. c ena a,
Supplemen a y ma e ial 1) we e emo ed om con aine s and oo samples
we e collec ed. A e insing, oo s we e ozen in liquid ni ogen and s o ed
a -80°C.
RNA isola ion
To al RNA om oo issue was isola ed based on Le P o os e al (Le
P o os e al. 2007). RNA in eg i y and pu i y was de e mined wi h a 2100
Bioanalyse wi h he RNA 6000 Pico ki (Agilen Technologies, Palo Al o, CA,
USA). In o de o compa e gene exp ession be ween he wo ches nu
species a e pa hogen inocula ion, ou RNA pools we e p epa ed, based on
he expe imen al design desc ibed by Ba aka e al (Ba aka e al. 2009;
Ba aka e al. 2012): Cci, Ccn, Csi and Csn (i: inocula ed; n: non-inocula ed).
Each pool included he RNA om nine plan s, 3 biological eplica es collec ed
a 3 ime poin s a e inocula ion (2, 4 and 7 days, Supplemen a y ma e ial
1).
Cas anea oo ansc ip ome in esponse o P. cinnamomi
79
Poly(A) RNA en ichmen , cDNA lib a y cons uc ion and py osequencing
The p ocedu es desc ibed in his sec ion we e p o ided by he Nex Gen
Sequencing Uni a Biocan (Can anhede, Po ugal).
The in eg i y o all RNA pools was e i ied on a 2100 Bioanalyse as abo e
and he quan i y assessed by luo ome y wi h he Quan -iTRiboG een RNA
ki (In i ogen, CA, USA). Poly(A)RNA was en iched om o al RNA using
wo ounds o he Mic oPoly(A) Pu is Ki (Applied Biosys ems, Ambion, CA,
USA), acco ding o he manu ac u e ’s ins uc ions. The RNA quali y was
again assessed on a 2100 Bioanalyse and he quan i y de e mined by
luo ome y as desc ibed abo e.
A ac ion o 200 ng o Poly(A)+ RNA o each isola e was used as s a ing
ma e ial o cDNA lib a y cons uc ion using Mul iplex Iden i ie s (MIDs)
acco ding o he cDNA Rapid Lib a y P epa a ion Me hod Manual, ‘GS FLX
Ti anium Se ies, Oc obe 2009’ (Roche-454 Li e Sciences, B and o d, CT,
USA). The ou dscDNA lib a ies we e quan i ied by luo escence, pooled in
equimola amoun s and py osequenced in a single pla e wi h GS FLX
Ti anium chemis y (Roche-454 Li e Sciences, B and o d, CT, USA),
acco ding he s anda d manu ac u e s’ p ocedu es.
T ansc ip assembly and unc ional anno a ion
A e 454 sequencing, he aw eads we e p ocessed o emo e sequences
wi h less han 100 nucleo ides and low quali y egions. Ribosomal,
mi ochond ial and chlo oplas eads we e iden i ied h ough BLASTx agains
he non- edundan NBCI da abase and any hi s wi h an E alue o 0.0 we e
emo ed om he da a se . All emaining eads we e hen assembled in o
con igs using 454 Newble 2.6 (Roche, B an o d, CT, USA) wi h he de aul
pa ame e s (40 bp o e lap and 90% iden i y). A h ee s ep analysis was
ca ied ou o iden i y genes. Fi s , he ansla ion ame o each con ig was
assessed h ough BLASTx sea ches agains Swissp o (E alue<1E-6) and
he co esponding amino acid sequence was ansla ed using an in-house

Chap e III
80
sc ip . Then any con igs wi hou ansla ion we e submi ed o F ameDP
(Gouzy e al. 2009) so wa e wi h de aul pa ame e s. Finally, all emaining
con igs we e analysed wi h ESTScan (Lo az e al. 2003) wi h de aul
pa ame e s. T ansc ip s iden i ied by F ameDP o ESTScan we e sea ched
using BLASTp agains he non- edundan NBCI da abase (E alue<1E-2) o
ansla e pu a i e p o eins. The unc ional anno a ion o all ansla ed amino
acid sequences was p edic ed h ough assignmen in o p o ein amilies and
iden i ica ion o p o ein domains using In e P oScan e sion 4.6 (Hun e e
al. 2009). Gene On ology (GO) e ms iden i ied by In e P oScan esul s o
each ansla ed amino acid sequence we e addi ionally e ie ed and added
o classi y each ansc ip p oduc . The p ocedu es abo e desc ibed we e
p o ided by he Nex Gen Sequencing Uni a Biocan .
All con igs we e axonomy anno a ed in o de o sepa a e he sequences
belonging o he S ep ophy a phylum o u he analysis. To ob ain he
axonomical assignmen s we uploaded he con igs o MG-RAST (Meye e
al. 2008) (wi h de aul pa ame e s), an au oma ed analysis pla o m o
me agenomes based on sequence simila i y o bo h p o ein and nucleo ide
da abases.
Iden i ica ion o di e en ially exp essed genes ela ed o P. cinnamomi
esis ance
Di e en ially exp essed genes we e iden i ied as genes showing signi ican
highe /lowe exp ession le els in inocula ed oo issue e sus non-
inocula ed oo issue. The numbe o eads mapping o each ansc ip
(con ig) in he wo ea men s (inocula ed and non-inocula ed) was coun ed
and used as an app oxima e es ima ion o gene exp ession le el in he
co esponding issues. Fi s , he con igs om he di e en samples we e
clus e ed a 90% simila i y and 95% iden i y by CD-Hi 454 (Niu e al. 2010)
o elimina e edundan sequences and gene a e e e ence con igs. The
eads om each sample we e hen mapped o hose e e ences wi h Newble
Cas anea oo ansc ip ome in esponse o P. cinnamomi
81
mapping 2.6 (Roche, B an o d, CT, USA) using he de aul pa ame e s, and
he numbe o eads con ibu ed by each sample coun ed. Reads wi h
mul iple hi s we e disca ded. The numbe o eads pe e e ence con ig pe
sample was used o build a con ingency able, which was analysed wi h he
My na s a is ical analysis package (Langmead e al. 2010), wi h he
no maliza ion ac o se o 95 h pe cen ile. S a is ical signi icance o he
di e en ial exp ession was e alua ed using a linea eg ession model based
on a Gaussian dis ibu ion, and using only con igs wi h a minimum o eigh
mapped eads. All esul s we e compiled in o a SQL da abase de eloped as
an in o ma ion managemen sys em. The p ocedu es desc ibed abo e we e
p o ided by he Nex Gen Sequencing Uni a Biocan .
Fo he selec ion o Di e en ially Exp essed Genes (DEGs), con igs wi h a P
alue <1E-03 we e conside ed. P alue desc ibes he p obabili y ha
di e ences in coun s be ween he wo se s in compa ison a e due o chance
(Langmead e al. 2010). Fold exp ession changes we e calcula ed o he
inocula ed s. non-inocula ed compa isons, Csi-Csn and Cci-Ccn. Con igs
wi h a old change g ea e han 1 we e classi ied as up- egula ed genes and
con igs wi h a old change less han o 1 we e classi ied as down- egula ed
genes. Fo u he analysis o DEGs (P. cinnamomi esis ance- ela ed
genes), he log2 o old change>ǀ1ǀ c i e ia was applied.
DEGs we e also analysed o he wo inocula ed species in he compa ison
Csi-Cci (P alue<1E-03) o e eal he genes ha we e signi ican ly induced
in bo h species a e pa hogen challenge. On he o he hand, he compa ison
o he wo non-inocula ed species Csn-Ccn (P alue<1E-03) e ealed he
cons i u i e genes in bo h species whi hou inocula ion.
The applica ion Blas 2GO (Conesa and Gö z 2008), namely he En ichmen
Analysis, was used o s a is ically analyse GO anno a ion in he compa isons
Csi-Csn, Cci-Ccn, Csi-Cci and Csn-Ccn o DEGs. I employs a Fishe 's
exac es wi h mul iple es ing co ec ion o FDR (Benjamini and Hochbe g).
Upon selec ion o a single es and P alue <5E-3, all GO e ms we e es ed
Chap e III
82
i hey a e en iched in he DEGs g oup when compa ed o a e e ence g oup
(all con igs in he compa ison).
454 sequencing alida ion by eal- ime PCR
The ela i e exp ession o a subse o genes was achie ed by quan i a i e
eal- ime PCR (qRT-PCR) o alida e RNA-sequencing (RNA-seq). The ou
RNA pools used o sequencing (Cci, Ccn, Csi and Csn) we e p epa ed o
qRT-PCR as ollows: RNA was ea ed wi h DNase (Tu bo DNase- ee ki
Ambion, Inc., USA), acco ding o manu ac u e 's ins uc ions. cDNA was
syn hesized using Re e Aid H Minus Re e se T ansc ip ase (The mo Fishe
Scien i ic, Wal ham, USA) acco ding o manu ac u e 's ins uc ions. Gene
speci ic p ime s we e designed o six a ge genes (Supplemen a y ma e ial
2) using P ime Exp ess ( e sion 1.0, Applied Biosys ems, Sou ce o ge,
USA). Ac in-7 was selec ed as a e e ence gene a e e i ying a simila
numbe o eads o all cDNA lib a ies and used o no maliza ion o
exp ession. A inal concen a ion o 0,2 μM o each p ime was used in 25 μL
eac ions, oge he wi h cDNA as empla e and Maxima SYBR G een/ROX
qPCR Mas e Mix (Fe men as, On a io, Canada), on a S epOne™ Real-Time
PCR sys em (Applied Biosys ems, Fos e Ci y, CA, USA). The mal cycling
o all genes s a ed wi h a dena u a ion s ep a 95°C o 10 min ollowed by
40 cycles o dena u a ion a 95°C o 15 s and annealing empe a u es o 30
s. Th ee echnical eplica es we e used pe eac ion se , including empla e
and no empla e con ols. Non-speci ic PCR p oduc s we e analysed by
dissocia ion cu es. The ela i e exp ession alue and mean absolu e
de ia ion alues we e calcula ed o he pool compa isons Cci-Ccn and Csi-
Csn acco ding o he ΔΔCT me hod (Li ak and Schmi gen 2001).
Resul s
454 sequencing and assembly summa y
Cas anea oo ansc ip ome in esponse o P. cinnamomi
83
Two Japanese ches nu cDNA lib a ies we e cons uc ed, one om a RNA
pool o inocula ed oo issue (Cci) and he o he om a RNA pool o non-
inocula ed oo issue (Ccn). A hal pla e o sequencing was used, esul ing
in 220 412 eads o Cci and 182 314 eads o Ccn, wi h an a e age ead
leng h o 350 n (Table 1). App oxima ely 77 and 64 megabases o cDNA
we e gene a ed o Cci and Ccn espec i ely. A e assembly, 8 528 con igs
we e gene a ed o Cci and 8 300 con igs we e gene a ed o Ccn, wi h an
a e age leng h o 885 n . 2 712 Cci con igs and 2 214 Ccn con igs had mo e
han 1000 n , co esponding o 32% and 27% o all espec i e con igs.
Table 1. Summa y o 454 sequencing o Cas anea c ena a and Cas anea sa i a
oo ansc ip omes.
cDNA lib a y
Cci
Ccn
Csi
Csn
Roo s sampled
C. c ena a
inocula ed
C. c ena a
non-inocula ed
C. sa i a
inocula ed
C. sa i a non-
inocula ed
No. o pla es
¼
¼
¼
¼
No. o eads
220 412
182 314
181 384
186 920
A e age ead
leng h (n )
350
350
357
367
No. o bp
77 175 000
63 823 300
64 884 000
68 672 896
No. o con igs
8 528
8 300
7 208
8 475
A e age con ig
leng h (n )
915
854
856
823
No. o la ge
con igsa
2 712
2 214
1 943
2 065
No. o pu a i e
p o eins
8 149
7 969
6 852
8 073
AA sequences
assigned o
In e P o e ms
6 373
6 279
5 350
6 213
AA sequences
assigned o GO
e ms
4 885
4 790
4 090
4 691
a G ea e han 1000 n
Chap e III
90
Figu e 4. Dis ibu ion o Cas anea c ena a and Cas anea sa i a di e en ially
exp essed genes (DEGs) in o unc ional sub-ca ego ies o Gene On ology. En iched
Analysis was applied sepa a ely o up egula ed and down egula ed DEGs and
compa ed wi h he e e ence se s o all con igs in he compa ison, o ob ain
signi ican GO e ms. The selec ed P alue on Fishe ’s Exac Tes was lowe han
5E−3.
P. cinnamomi esis ance- ela ed genes
We conside ha he mos signi ican candida e genes o esis ance o P.
cinnamomi a e C. c ena a ( esis an species) genes up- egula ed a leas 2
imes a e inocula ion ha a e no p esen among C. sa i a DEGs and a e
pu a i ely ela ed o s ess esponse. A selec ion o C. c ena a candida e
genes was in e ed om he Cci-Ccn DEGs lis in Supplemen a y ma e ial 3
and assigned o he unc ional ca ego ies in Table 2.

Cas anea oo ansc ip ome in esponse o P. cinnamomi
91
Table 2 Cas anea c ena a (Japanese, esis an ) candida e genes o Phy oph ho a cinnamomi esis ance. Genes we e
associa ed in o he unc ional ca ego ies in he le column (Bold).
Chap e III
92
Cas anea oo ansc ip ome in esponse o P. cinnamomi
93
Chap e III
94
Especially no ewo hy a e he kinase ecep o genes ha may be in ol ed in
pa hogen ecogni ion (as he P obable LRR ecep o -like se ine/ h eonine-
p o ein kinase) (Dié a and Cla k 2003) and genes co esponding o TF
in ol ed in he egula ion o hos esponse a e pa hogen pe cep ion (as
WRKY TF) (Yang e al. 2009). The pu a i e in ol emen o JA and salicylic
acid (SA) signaling pa hways was in e ed om he ca ego ies ‘Regula ion o
hos esponse a e pa hogen pe cep ion’ and ‘Lipid signaling’. Two genes in
he ‘Regula ion o plan immune esponse’ ca ego y a e esponsi e o he
plan ho mone e hylene (e.g. Ocs elemen -binding ac o 1) (Zhang and
Singh 1994).
Ce ain C. c ena a up- egula ed genes may p e en pa hogen p og ess, such
as he p ecu so o Ca ionic pe oxidase 1 (Reime s e al. 1992) [ca ego y
‘Hype sensi i e esponse’ (HR)], and Pec ines e ase 2 (Wen e al. 2013)
(ca ego y ‘Cell wall s eng hening’). Th ee genes, e.g. P obable glu a hione
S- ans e ase, we e associa ed in he ca ego y ‘HR eco e y’ (Ryu e al.
2009). Genes in ol ed in ‘An i- ungal me aboli e syn hesis’ (such as UDP-
glycosyl ans e ase 85A2) (Woo e al. 2007) and ‘An i- ungal enzymes’ (such
as P obable ca boxyles e ase 120) (Ma shall e al. 2003) may ake pa in
he hos esponse o enhance he de ense o P. cinnamomi.
Genes in he ca ego ies ‘Regula ion o d ough s ess’, ‘Response o d ough
s ess’ and ‘S ess eco e y’ a e also ep esen ed in he C. c ena a candida e
lis , such as NAC domain-con aining p o ein 72 (Singh e al. 2013),
Phosphop o ein ECPP44 (Tan and Kamada 2000) and Lon p o ease
homolog 2 pe oxissomal (Linga d and Ba el 2009), espec i ely.
C. sa i a DEGs a e P. cinnamomi inocula ion a e p esen ed in
Supplemen a y ma e ial 4. In o de o iden i y he suscep ible species’
esponse o he pa hogen we selec ed up- egula ed genes in inocula ed C.
sa i a (a leas wo imes) ha a e no p esen among C. c ena a DEGs and
a e pu a i ely ela ed o s ess esponse. Those genes we e dis ibu ed in a
se ies o unc ional ca ego ies in Table 3. In he ‘Regula ion o plan immune
Cas anea oo ansc ip ome in esponse o P. cinnamomi
95
esponse’ ca ego y we emphasize he gene REF/SRPP-like p o ein
A 1g67360 (Taki e al. 2005), induced by a p ecu so o JA. C. sa i a also
in es s in genes ela ed o ‘S ess eco e y’ (e.g. Aminophospholipid lippase
9) (López-Ma qués e al. 2012) and ‘HR eco e y’ (e.g. 4-
hyd oxyphenylpy u a e dioxygenase) (Peal e al. 2011). Genes in ol ed in
‘An i- ungal me aboli e syn hesis’ (e.g. Fla onoid 3-hyd oxylase) (Sha ma e
al. 2012) and ‘Cell wall s eng hening’ (e.g. UPF0497 memb ane p o ein
A 3g06390) (Roppolo e al. 2011) may p e en P. cinnamomi p oli e a ion in
he hos . Finally, C. sa i a up- egula ed genes linked o d ough s ess
egula ion, such as he TF Homeobox-leucine zippe p o ein HAT5
(Hen iksson e al. 2005) may play a ole in hos eco e y om pa hogenici y.
Valida ion o RNA-seq
The RNA-seq app oach allowed o he quan i ica ion o gene exp ession
le els by sequence ead dep h. DEGs we e iden i ied by es ima ing he a io
be ween eads in inocula ed lib a ies and non-inocula ed con ols (Cci-Ccn,
Csi-Csn). To alida e he di e en ial exp ession le els obse ed by RNA-
seq, qRT-PCR was used o ob ain he exp ession le el o DEGs in inocula ed
lib a ies (Cci, Csi) ela i e o non-inocula ed lib a ies (Ccn, Csn). The
selec ed DEGs (Figu e 5) a e pu a i ely ela ed o Cas anea esponse o he
pa hogen and o hos eco e y, and include: E hylene- esponsi e TF 4,
Disease esis ance p o ein A 4g27190, E hylene- esponsi e TF ABR1,
P ecu so o glucan 1,3-be a-glucosidase ( amily 5), Pec ines e ase 2 and
C2 domain-con aining p o ein A 1g53590.
The di e en ial gene exp ession o he compa isons Cci-Ccn and Csi-Csn
acqui ed wi h he 454 sequencing was compa ed wi h he ela i e exp ession
le els ob ained wi h qRT-PCR o he selec ed DEGs.

Chap e III
96
Table 3 Cas anea sa i a (Eu opean, suscep ible) up- egula ed genes upon Phy oph ho a cinnamomi inocula ion ela ed o
s ess esponse. Genes we e associa ed in o he unc ional ca ego ies in he le column (Bold).
Cas anea oo ansc ip ome in esponse o P. cinnamomi
97
Chap e III
98
Cas anea oo ansc ip ome in esponse o P. cinnamomi
99
The esul s p esen ed in Figu e 5 e eal di e ences in he exp ession le els
o C. c ena a ansc ip s upon inocula ion when compa ed o C. sa i a
ansc ip s upon inocula ion. Those di e ences a e in acco dance wi h ead
da a ob ained by RNA-seq and may e lec Cas anea oo ansc ip ome in
esponse o P. cinnamomi.
Discussion
Func ional anno a ion
GO anno a ion compa ison o exp essed genes a e inocula ion be ween
Japanese (C. c ena a, esis an o pa hogen) and Eu opean ches nu (C.
sa i a, suscep ible o pa hogen) e ealed a co ela ion o gene on ology,
sugges ing a con e gen esponse a e pa hogen inocula ion. Howe e ,
among DEGs, GO anno a ion e ealed di e ences ha sugges dis inc hos
suscep ibili y o he pa hogen as well as a ia ions in gene exp ession and
iming. C. c ena a inocula ed wi h he pa hogen up- egula ed genes wi h he
unc ional GO anno a ion ‘Oxida ion educ ion p ocess’ (BP), disclosing
genes in ol ed in he syn hesis o an i- ungal seconda y me aboli es (6 in
27) and in s ess eco e y (10 in 27). Examples a e Squalene
monooxygenase (Belchí-Na a o e al. 2013) and P olyl 4-hyd oxylase
subuni alpha-2 (Vlad e al. 2007), espec i ely. On o he hand, ‘Sequence-
speci ic DNA binding ansc ip ion ac o ac i i y’ (MF) poin o genes coding
o TF ela ed o pa hogen ecogni ion and bio ic s ess egula ion (10 in 14,
examples in Table 2).
Con as ing wi h C. c ena a, GO anno a ion o inocula ed C. sa i a e ealed
ha he down- egula ed genes in ‘Ca aly ic ac i i y’ (MF) we e in ol ed in he
syn hesis o seconda y me aboli es, p o ein kinases and ecep o -like p o ein
kinases (24 in 84). In he same e m a e also included genes ela ed o s ess
eco e y (9 in 84).
Chap e III
106
deca boxylase 1 codes o oo -speci ic calcium/calmodulin- egula ed GAD1,
which plays a majo ole in GABA syn hesis in plan s esponding o s ess,
he eby helping main ain plan homeos asis (Bouché and F omm 2004).
Thus he ep ession o GAD1 may a ec hos eco e y om pa hogen a ack.
Glu a edoxins a e candida es o media ing edox egula ion o
ansc ip ional egula o s ha a ge genes associa ed wi h de oxi ica ion and
pa hogen de ense (Ndamukong e al. 2007). The Glu a edoxin-C9 gene was
highly up- egula ed in Japanese ches nu ; i s down- egula ion in Eu opean
ches nu may also a ec hos eco e y.
Compa ison be ween Japanese and Eu opean ches nu esponse
DEGs anno a ion analyses e ealed ha Japanese and Eu opean ches nu s
show many common ea u es in hei esponses o P. cinnamomi. Howe e ,
his e alua ion mus be ca e ully ega ded as i is no suppo ed by ansc ip
p o iling o unc ional analysis. Upon P. cinnamomi inocula ion, bo h species
up- egula ed genes in ol ed in HR/HR eco e y, genes ela ed o he
egula ion o JA pa hway and genes induced by JA ela ed o an i- ungal
me aboli e syn hesis and an i- ungal enzymes. The p esence o HR and JA
signaling upon pa hogen inocula ion indica es ha bo h hos species we e
able o ecognize he pa hogen a ack. Acco ding o Thomma (Thomma
1998), he JA-dependen de ense esponse pa hway is equi ed o
esis ance o nec o ophic pa hogens. Esh agui e al (Esh aghi e al. 2014)
sugges ha a P. cinnamomi challenge ac i a es JA- ela ed plan de ense
esponses in lea es o A. haliana Col-0. HR is sugges ed o be associa ed
wi h all o ms o esis ance o Phy oph ho a (Kamoun e al. 1999) and is
belie ed o cons i u e one o he p ima y mechanisms o esis ance o plan
pa hogens. Induc ion o HR is o en associa ed wi h syn hesis o an imic obial
compounds and cell wall hickening (Hammond-Kosack and Jones 1996).
We iden i ied C. c ena a and C. sa i a DEGs ela ed o cell wall s eng hening
and an i- ungal me aboli e syn hesis. HR also induces se e al genes

Cas anea oo ansc ip ome in esponse o P. cinnamomi
107
in ol ed in cellula p o ec ion (Jabs e al. 1996). We also iden i ied DEGs
ela ed o HR eco e y in bo h species. The sugges ed occu ence o HR in
inocula ed Eu opean ches nu , he suscep ible species, po en ially poin s o
pa ial esis ance wi hin his geno ype. Pa ial esis ance o Phy oph ho a
in es ans is common in wild Solanum species, which may e eal HR-like
nec o ic eac ions and, occasionally, la e o ailing HR. This sugges s a weak
R gene-A gene in e ac ion o a gene-dosage e ec esul ing in ine ec i e
HR and pa ly esis an pheno ypes (Kamoun e al. 1999).
Japanese ches nu esis ance o ink disease may in pa esul om a se o
up- egula ed genes du ing P. cinnamomi a ack in ol ed in pa hogen
ecogni ion, egula ion o hos esponse a e pa hogen pe cep ion, and
signaling h ough lipids. When compa ed o Japanese ches nu , Eu opean
ches nu up- egula ed much less genes in hose unc ional ca ego ies.
Cu en knowledge desc ibes he plan immune esponse as s a ing wi h he
ecogni ion o pa hogen elici o s by plan ecep o s, ollowed by induc ion o
esis ance genes (R genes) ha ini ia e signal ansduc ion cascades leading
o: a) HR and apid cell dea h and b) he ac i a ion o phy oho mone signaling
pa hways [ e iewed in (Ba i and Jones 2009)]. In ou s udy inocula ed
Japanese ches nu induced DEGs in ol ed in he SA pa hway egula ion
(e.g. Calcium-dependen p o ein kinase iso o m 3) (Chung e al. 2004) as
well as DEGs induced by SA ela ed o he egula ion o plan immune
esponse (e.g. Sul a e anspo e 3,1) (Ma solais e al. 2007) and HR (e.g.
A ginine deca boxylase) (Nakane e al. 2003). Ga cía-Pineda e al (2009)
obse ed ha SA inhibi ed a ocado oo coloniza ion in he in e ac ion
be ween Pe sea ame icana and P. cinnamomi. In he complex web o
de ense esponses JA, SA, E hylene and Abscisic Acid a e essen ial playe s
(Ba i and Jones 2009). SA is ac i a ed du ing and ollowing HR (Jabs e al.
1996) and is gene ally in ol ed in he ac i a ion o de ense esponses
agains bio ophic and hemi-bio ophic pa hogens, as well as in he
es ablishmen o sys emic acqui ed esis ance (Ba i and Jones 2009).
Chap e III
108
Vleeshouwe s e al (Vleeshouwe s e al. 2000) s udied he P. in es ans-
Solanum in e ac ion using wild species and epo ed ha in ully esis an
geno ypes, he HR was as e and esul ed in smalle lesions han in pa ially
esis an clones. The au ho s sugges ha he di e ence be ween
compa ibili y (non- esis an hos esponse) and incompa ibili y ( esis an hos
esponse) is quan i a i e a he ha quali a i e. In ou s udy, Japanese
ches nu egula ed a highe numbe o genes in ol ed in bio ic s ess upon
P. cinnamomi inocula ion when compa ed o he Eu opean ches nu . The
iden i ied DEGs a e no only ela ed o HR bu also wi h cell wall
s eng hening, an i- ungal me aboli e syn hesis and an i- ungal enzyme
syn hesis, and may accoun o he Japanese ches nu ’s adequa e
esis ance o ink disease.
Cas anea esponse o P. cinnamomi and C. pa asi ica: b ie compa ison
The epo s o Ba aka e al. (2009, 2012) p o ided he i s insigh s in o
ches nu esis ance o C. pa asi ica using high- h oughpu RNA-seq. The
esponse o ches nu o C. pa asi ica and P. cinnamomi may be compa able,
as ungi and Oomyce es sha e simila in ec ion mechanisms (La ijnhouwe s
e al. 2003). When compa ing Chinese and Ame ican ches nu esponses o
C. pa asi ica wi h he Japanese and Eu opean ches nu esponses o P.
cinnamomi, we ound simila DEGs ha all in he ollowing unc ional
ca ego ies: a) Regula ion o bio ic s ess esponse (ATPase anspo e ,
Py idine nucleo ide-disulphide oxido educ ase), b) HR and cell wall
ligni ica ion (Pe oxidase), c) HR eco e y (A ginine deca boxylase,
Manganese supe oxide dismu ase), d) An i- ungal enzymes (Thauma in-like
p o ein, β-1,3-glucanase, Chi inase), e) An i- ungal me aboli e syn hesis
( amily 1 Cy och ome P450 glycosyl ans e ase, Abscisic acid 8’-
hyd oxylase, Squalene monooxygenase, UDP-glucosyl ans e ase), ) Cell
wall syn hesis (β-expansin), and g) S ess eco e y (ABC anspo e amily,
Glyce aldehyde 3-phospha e dehyd ogenase). O he sha ed Cas anea
Cas anea oo ansc ip ome in esponse o P. cinnamomi
109
esponses o bo h pa hogens include DEGs ela ed o kinase genes in ol ed
in pa hogen ecogni ion and JA pa hway ac i a ion, gene egula ion by Myb
TF and E hylene- esponsi e TF, and genes o he 26S p o easome
egula o y uni . The esponse o all ou species o bo h pa hogens u he
includes genes om he la onoid pa hway ha p omo e phy oalexin
syn hesis.
In summa y, he DEG analysis o C. sa i a and C. c ena a oo
ansc ip omes a e P. cinnamomi inocula ion e ealed simila i ies among
he ou Cas anea species esponse o bo h pa hogens, namely genes
ela ed o sys emic acqui ed esis ance, HR ha may p e en pa hogen
sp ead and he pu a i e in ol emen o JA pa hway. Some o hese DEGs
may also p omo e cell wall s eng hening h ough ligni ica ion and syn hesis
o la onoids as an i- ungal me aboli es.
Final conside a ions
RNA-seq using 454 pla o m was adequa e o compa ing he oo
ansc ip omes o wo Fagaceae species, Cas anea sa i a and Cas anea
c ena a when ei he inocula ed o non-inocula ed wi h he pa hogen
Phy oph ho a cinnamomi. The ou sequenced ansc ip lib a ies allowed a
d a compa ison o bo h species’ esponses o he pa hogen in e ms o gene
egula ion and pa hways, oge he wi h he selec ion o candida e genes o
hos esis ance o P. cinnamomi. Al hough u he esea ch is equi ed on
gene exp ession a speci ic ime poin s a e inocula ion, in silico analysis has
shown ha Japanese and Eu opean ches nu , despi e he associa ion o
exp essed genes in simila unc ional ca ego ies, di e in he dis ibu ion o
DEGs a e pa hogen inocula ion. The mos no ewo hy esul om DEG
analysis was he o e all down- egula ion o genes in suscep ible C. sa i a,
which may acili a e he pa hogenici y o P. cinnamomi. On he o he hand,
in he esis an C. c ena a he e was he egula ion o a highe numbe o
genes ela ed wi h bio ic s ess when compa ed o C. sa i a, mos ly up-
Chap e III
110
egula ed. Analysis o homology and unc ional anno a ion e ealed
associa ions be ween many o hose up- egula ed genes wi h pa hogen
esponse in o he plan species, and sugges s in ol emen in pa hogen
ecogni ion, egula ion o he hos immune esponse, signaling,
hype sensi i e esponse, cell wall s eng hening and encoding o enzymes
and syn hesis o me aboli es agains Oomyce es and ungal pa hogens. The
egula ion o DEGs in C. c ena a and many o he speci ic ansc ip s we
iden i ied may accoun o he adequa e esis ance le el o his species o P.
cinnamomi.
SSR ma ke s we e also de eloped om he sequences o hese candida e
genes in o de o imp o e he mapping app oach o iden i ica ion o QTLs
ela ed o pa hogen esis ance in Japanese and Eu opean ches nu (Cos a
e al. 2011 and Chap e V). In Chap e V we will analyse i he candida e
genes map o disease esis ance QTLs, which will p o ide u he suppo o
a majo ole in ches nu esis ance o he pa hogen.
Acknowledgmen s
We acknowledge D . Bea iz Cuenca (TRAGSA-SEPI) o p o iding he plan
ma e ial used in his s udy. The au ho s a e also g a e ul o D . And eia
Figuei edo (BioFIG) and D . Filipa Mon ei o (BioFIG) o help on qRT-PCR,
D . Conceição Egas (Nex Gen Sequencing Uni , Biocan ) o submi ing aw
da a o NCBI, D . Dana Nelson (USDA Fo es Se ice, MS, Uni o Fo es
gene ics and Ecosys ems Biology) o a angemen s on submi ing da a o
Fagaceae.o g, and P o . William Powell and And ew Newhouse (SUNY
College o En i onmen al Science and Fo es y, NY, Depa men o
En i onmen al and Fo es Biology) o c i ical e iew o he manusc ip .
Da a A chi ing S a emen
Raw da a iles can be accessed in he Sho Read A chi e a NCBI
(h p://www.ncbi.nlm.nih.go ) wi h he e e ence PRJNA215368. Nucleo ide
Cas anea oo ansc ip ome in esponse o P. cinnamomi
111
and aminoacid sequences a e publicly a ailable in he Fagaceae Genomics
Web (h p://www. agaceae.o g/).
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