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Scien i ic RepoR s | 7:43766 | DOI: 10.1038/s ep43766
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Gene Dele ion in Ba ley Media ed
by LTR- e o ansposon BARE
Yi Shang1,*, Fei Yang2,3,*, Alan H. Schulman4,5, Jinghuan Zhu1, Yong Jia3, Junmei Wang1,
Xiao-Qi Zhang3, Qiaojun Jia6, Wei Hua1, Jianming Yang1 & Chengdao Li2,3
A poly- ow b anched spike (p bs) ba ley mu an was ob ained om soaking a wo- owed ba ley
in lo escence in a solu ion o maize genomic DNA. Posi ional cloning and sequencing demons a ed
ha he p bs mu an esul ed om a 28 kb dele ion including he in lo escence a chi ec u e gene
H RA2. Sequence anno a ion e ealed ha he H RA2 gene is lanked by wo LTR (long e minal epea )
e o ansposons (BARE) sha ing 89% sequence iden i y. A ecombina ion be ween he in eg ase (IN)
gene egions o he wo BARE copies esul ed in he o ma ion o an in ac BARE and loss o H RA2. No
maize DNA was de ec ed in he ecombina ion egion al hough he lanking sequences o H RA2 gene
showed o e 73% o sequence iden i y wi h epe i i e sequences on 10 maize ch omosomes. I is s ill
unknown whe he he in e ac ion o e o ansposons be ween ba ley and maize has esul ed in he
ecombina ion obse ed in he p esen s udy.
The a chi ec u e o b anched in lo escences in g asses depends on he de elopmen al a e o p imo dia and axis
o ien a ion1. The ice (O yza sa i a L.) panicle gene a es se e al p ima y and seconda y b anches on which spike-
le s a e p oduced. So ghum and maize male in lo escences sha e a s uc u e simila o ha o ice. In ba ley
(Ho deum ulga e L.) spikes, howe e , spikele s a e bo ne di ec ly on he main axis, he achis, and he e a e no
pedicels. A diagnos ic ea u e o ba ley is he possession o h ee one- lowe ed spikele s a each achis node2,3.
Based on la e al spikele size and e ili y, ba ley is classi ied in o wo- owed and six- owed ypes. Two- owed
ba ley only has a cen al e ile spikele wi h small and in e ile la e al spikele s while he six- owed ba ley has
h ee ully-de eloped e ile spikele s.
The majo genes ha con ol ow- ype a ia ion in ba ley a e V s14, In -c5 and V s46. The ba ley domes ica-
ion gene V s1, loca ed on he long a m o ch omosome 2H, encodes a homeodomain-leucine zippe (HD-Zip)
ansc ip ion ac o ha supp esses he de elopmen o la e al spikele s in wo- owed ba ley. Mu an s1 esul s
in a well-de eloped six- owed pheno ype4. In -c, loca ed on ch omosome 4H, is an o holog o he maize (Zea
mays. L.) domes ica ion gene, Teosin e b anched 1 (TB1), a membe o he TCP gene amily encoding pu a i e
basic helix-loop-helix DNA-binding p o eins5. The In -c gene modi ies la e al spikele e ili y in ba ley, and can
in luence he pheno ypic e ec o he V s1 locus7.
V s4 con ols ow- ype and spikele de e minacy in ba ley; an induced mu a ion, s4, can con e he
wo- owed o a six- owed pheno ype5,8. V s4 is an o holog o he maize in lo escence a chi ec u e gene
RAMOSA2 (RA2), which encodes a ansc ip ional egula o ha con ains he la e al o gan bounda ies (LOB)
domain. Exp ession analyses by mRNA in si u hyb idiza ion and mic oa ay app oaches showed ha V s4 is
exp essed e y ea ly du ing in lo escence de elopmen and con ols he ow- ype pa hway h ough V s1 by
nega i ely egula ing he la e al spikele e ili y in ba ley. Mo eo e , he V s4 gene is an impo an modi ie o
in lo escence de elopmen . He e, we epo on a new mu an , poly- ow and b anched spike (p bs) ob ained by
soaking a wo- owed ba ley in lo escence in maize genomic DNA om a single c oss hyb id9,10, and cha ac e ize
i s gene ics, epo i s posi ional cloning, and analyze i s o igin.
1Na ional Ba ley Imp o emen Cen e, Ins i u e o C op and Nuclea Technology U iliza ion, Zhejiang Academy
o Ag icul u al Science, Hangzhou 310021, China. 2Depa men o Gene ics and Cell Biology, Yang ze Uni e si y,
Jingzhou, Hubei 434023, China. 3Wes e n Ba ley Gene ics Alliance, Mu doch Uni e si y, 90 Sou h S ee , Mu doch
WA 6150, Aus alia. 4Luke/BI Plan Genomics Lab, Ins i u e o Bio echnology and Viikki Plan Science Cen e,
Uni e si y o Helsinki, FIN-00014 Helsinki, Finland. 5G een Technology, Na u al Resou ces Ins i u e Finland (Luke),
Viikinkaa i 1, FIN-00790 Helsinki, Finland. 6College o Li e Sciences, Zhejiang Sci-Tech Uni e si y, Hangzhou 310018,
China. *These au ho s con ibu ed equally o his wo k. Co espondence and eques s o ma e ials should be
add essed o J.Y. (email: [email p o ec ed]) o C.L. (email: [email p o ec ed])
Recei ed: 23 Augus 2016
Accep ed: 27 Janua y 2017
Published: 02 Ma ch 2017
OPEN
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Scien i ic RepoR s | 7:43766 | DOI: 10.1038/s ep43766
Resul s
Mu an p bs esul ed om dele ion o he V s4 gene. The poly- ow and b anched spike (p bs) ba -
ley mu an was ob ained by soaking a wo- owed ba ley in lo escence in maize genomic DNA solu ion9,10. The
mu an p bs no only changes wo- owed ba ley in o a poly- owed o m bu also adds a spikele ow, o ming
i egula poly- ow and b anched spikes (Fig.1). Gene ic analysis indica ed ha he mu an pheno ype was caused
by a ecessi e gene, which has an epis a ic e ec on V s111. The p bs was ini ially mapped o he cen ome e o he
sho a m o ch omosome 3H11,12, a loca ion simila o ha o s4. Fu he mo e, he imma u e spikes o he p bs
mu an unde s e eoscope a e akin o he scanning elec on mic oscopy images o he V s4 imma u e spikes12.
Th ee molecula ma ke s (DQ327702, Cbic43, and Cbic44), closely linked wi h he V s4 gene, co-seg ega ed
wi h he p bs gene in he p bs/Kunlun12 RIL and p bs/Zangqing 320 F2 popula ions (Supplemen a yFig.1). No
agmen was ampli ied in he mu an plan s using hese h ee molecula ma ke s. Simila ly, h ee o he p ime s
co e ing he V s4 gene sequence also ailed o ampli y a speci ic DNA agmen om ei he he p bs mu an o
i s p ogeny 11R258-95. Exp ession analyses e ealed ha he exp ession o V s4 was no de ec ed and he exp es-
sion o V s1 was signi ican ly down- egula ed in imma u e spikes a lemma p imo dium s age o he p bs mu an
(Fig.2). These esul s indica ed ha he p bs mu an may ha e esul ed om a la ge dele ion a ound he V s4 gene.
Iden i ica ion o dele ion egion in p bs mu an . To iden i y he dele ion egion in he p bs mu an , a
Mo ex BAC clone was iden i ied ha con ains he V s4 gene. PCR p ime s we e designed a 2 kb in e als om
14 kb ups eam o 22 kb downs eam o he V s4 gene and we e es ed on he p bs mu an , 11R258-95, Pudamai-2,
Figu e 1. Mo phology o de eloping and ma u e spikes. (a) no mal spikes; (b,c and d) b anching spikes;
(e) b anches o (d, ) mu an p bs; (g) wild pa en Pudamai-2.
Figu e 2. Rela i e exp ession le el o V s4 and V s1 de e mined by quan i a i e RT-PCR in imma u e
spikes a lemma p imo dium s age o he p bs mu an and wild pa en Pudamai-2. Cons i u i ely exp essed
H Ac in was used o no maliza ion.
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and Mo ex. PCR p ime s loca ed in he egion om 3 kb ups eam o 10 kb downs eam o he V s4 gene ailed
o ampli y a speci ic DNA agmen in he p bs mu an and 11R258-95 bu ampli ied a single band in Pudamai-2
and Mo ex ins ead. Sequencing e ealed ha amplicons ep esen ed a single p oduc in Pudamai-2 and Mo ex.
P ime s designed om 3 o 13 kb ups eam and 10 o 21 kb downs eam o he V s4 gene ampli ied a single band
in all es ed plan s, bu he amplicons ep esen ed mul iple p oduc s when sequenced. These esul s did no sup-
po hese egions a ising om a single dele ion e en in he p bs mu an . Howe e , an addi ional p ime pai ,
Cbic123, ma ching a si e 14 kb ups eam o V s4, ampli ied a single band in all es ed plan s; he PCR p oduc
had 100% sequence iden i y among he p bs mu an , 11R258-95, Pudamai-2, and Mo ex. Ano he p ime pai
Cbic119, 22 kb downs eam o V s4, also ampli ied a single band in all es ed plan s (sequencing was iden ical in
all es ed lines). These esul s e ealed ha he dele ion sequence was om ~13 o 36 kb in he p bs mu an .
A e ailu e o ampli y a single DNA agmen using many PCR p ime s in he a ge egion, long- ange PCR
was used o isola e he sequence co e ing he p bs mu a ion. Based on he abo e PCR es esul s, PCR p ime s
Cbic131 and Cbic132 we e designed o his pu pose; he o wa d p ime was nea he si e o p ime Cbic123 and
he e e se p ime nea he si e o p ime Cbic119, as bo h ha e been con i med o ampli y a single copy o DNA
om he con ol a ie ies and mu an s. A 15 kb agmen was success ully ampli ied om bo h he p bs mu an
and 11R258-95 (Fig.3), whe eas he con ol PCRs using DNA om Pudamai-2 and Mo ex as a empla e ailed o
ampli y. The ampli ica ion p oduc om p bs was 14,715 bp (accession numbe KU758926).
We iden i ied a 48,951 bp sequence om Mo ex using he 14,715 bp p bs sequence as que y in BLASTN
sea ches o he Mo ex genome da abase (h p://webblas .ipk-ga e sleben.de/ba ley/ i oblas .php). Alignmen
o hese wo sequences showed ha a 27,804 bp sequence in Mo ex, ex ending om n 15,680 o n 43,769 and
con aining he en i e V s4 gene, is dele ed in p bs (Fig.4). Sequence analysis demons a ed ha he V s4 gene in
Mo ex is lanked by wo long e minal epea (LTR) e o ansposons loca ed, espec i ely, a n 10,967 o 19,828
ups eam and n 38,791 o n 47,684 downs eam (Fig.4). A sea ch o he T i iceae Repe i i e Elemen s (TREP)
Figu e 3. PCR-ampli ica ion o he 15-kb ecombina ion sequence agmen in he p bs mu an and RIL
11R258-95.
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da abase e ealed high simila i y o he e o ansposon RLC BARE1 B consensus-1 (TREPACC = TREP3133)
wi h 90% and 98% o sequence iden i y, espec i ely, o he wo elemen s. We e e o he e o ansposon
ups eam o V s4 as BARE up, and he one downs eam o V s4 as BARE down. The wo sha e 90% iden i y, a e
bound by 1.8 kb LTRs, and con ain he ull-leng h open eading ame encoding Gag, aspa ic p o einase (AP),
in eg ase (IN), e e se ansc ip ase (RT), and RNaseH (RH) expec ed o canonical BARE1 elemen s13 (Fig.4).
We u he sequenced pa o he dele ion om he wild pa en Pudamai-2, which con ains a comple e V s4
gene. A phylogene ic ee was cons uc ed using he MEGA6 p og am14 wi h he minimum e olu ion me hod.
The esul s showed ha he V s4 haplo ype in Pudamai-2 was simila o haplo ype 8 (Supplemen a yFig.2) which
was cha ac e ized by an inse ion o TA bases in he 5′ UTR o he gene6 and was mainly dis ibu ed in Asia. Thus,
he p bs mu an esul ed om a dele ion o he en i e unc ional gene H RA2, a ba ley o holog o he maize
in lo escence a chi ec u e gene RAMOSA2 (RA2), which he eby ans o med a wo- ow ba ley in o a poly- ow
b anched s uc u e.
Recombina ion be ween he in eg ase genes o wo BAREs o med p bs. Sequence analysis o
he 15 kb egion om p bs iden i ied a single BARE elemen o he canonical 8.9 kb in leng h. Alignmen e ealed
ha he 5′ pa o he p bs (n 1 o 4,714) BARE was iden ical o BARE up, whe eas he 3′ pa (4,972 o 8,918 bp)
was he same as BARE down (Fig.5). The join be ween he wo hal es is be ween n 4,715 and n 4,971 in he
Figu e 4. Schema ic compa ison he Mo ex and p bs mu an sequences. The V s4 gene is lanked by wo
BARE e o ansposons which a e bounded by 1.8 kb long e minal epea s (LTRs) and con ains coding
domains o GAG, in eg ase (IN), e e se ansc ip ase (RT), and RNaseH in Mo ex. The wo BAREs sequences
a e highly conse ed and sha e 89% o sequence iden i y. The p bs mu an has 28 kb dele ion including he
en i e V s4 gene and pa s o he wo e o ansposons. The non- e o ansposon sequence in he wild pa en
Pudamai 2 sha e 100% o sequence iden i y wi h Mo ex.
Figu e 5. Alignmen o he h ee LTR e o ansposons: BARE up, BARE down, BARE 3, shows
ecombina ion egion. Red capi al le e s ma ked Indel si es and black lowe le e s ma ked consensus
sequences. Yellow backg ound show BARE up sequences and hei homologous sequences in BARE 3. G een
backg ound show BARE down sequences and hei homologous sequences in BARE 3. Pu ple ed show he
ecombina ion egion decided by he alignmen .
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p bs BARE (Fig.5), wi hin he in eg ase (IN) domain, co esponding o posi ion n 9,402–9,690 in he cloned
p bs agmen (accession numbe KU758926). Re o ansposon in eg a ion gene a es a di ec - epea a ge -si e
duplica ion (TSD) lanking he indi idual elemen , as a consequence o epai o he s agge ed cu made by he
in eg ase15. BARE up is lanked by impe ec CCAAG TSDs and BARE down by a pe ec pai o CTGAA mo i s.
The BARE in p bs is lanked by CCAAG and CTGAA, suppo ing he o igin o his BARE by ecombina ion.
Mo eo e , he ups eam and downs eam sequences su ounding he single BARE in p bs co espond, espec-
i ely, o ha ups eam o BARE up and downs eam o BARE down. Thus, he H RA2 gene, lanked by wo BARE
elemen s in Pudamai-2, was dele ed by ecombina ion be ween hem, he eby gene a ing he p bs mu a ion.
Sea ch he maize genome o sequence simila i y. To in es iga e he possible ole o maize DNA in
o ming he p bs mu a ion, we used he egion spanning he ecombina ion zone in he BARE elemen s (9,402–
9,690 bp) o ca y ou a BLASTn sea ch he MaizeGDB B73 e e ence genome sequence. No maize-speci ic
sequence was ound in he egion. Hence, i appea s ha no maize DNA has been inse ed in o he p bs egion
and ha he p bs pheno ype esul s solely om he V s4 gene dele ion.
As an al e na i e o inse ion, he maize DNA may ha e played a ole h ough sequence simila i y a he
ecombina ion poin . The egion o ecombina ion in p bs co esponds o he mos conse ed pa o he in e-
g ase gene, which is he co e domain ha includes he D-D-35-E ac i e si e mo i 16. The ecombina ion i sel ook
place in he egion be ween he second Asp and he Glu o he ac i e si e; a BLASTn sea ch o his egion agains
he MaizeGDB B73 e e ence genome sequence ound mo e han 120 ma ches be ween 73% and 80% iden i y,
con aining mul iple s e ches o ~10 n pe ec iden i y, dispe sed o e all maize ch omosomes (Table1). Gi en
hei numbe s, i is highly likely ha hese BLAST ma ches co espond o membe s o he Copia supe amily in
he maize genome, which comp ises ~425–485 Mb o he maize genome17, he uni e sal p esence o in eg ase
in in ac , au onomous LTR e o ansposons. Fu he esea ch is equi ed whe he o eign DNA may induce
ecombina ion h ough sequence simila i y, especially when he o eign DNA exis in high concen a ion o high
copy numbe s.
Discussion
Ba ley is classi ied as wo- owed o six- owed based on la e al spikele size and e ili y. Two- owed ba ley has
a cen al e ile spikele and wo in e ile la e al spikele s, and six- owed ba ley has h ee ully-de eloped e ile
spikele s. V s4 is an o holog o he maize (Zea mays. L.) in lo escence a chi ec u e gene RAMOSA2 (RA2), which
encodes a LOB-domain-con aining ansc ip ional egula o 18–20. V s4 con ols ow- ype a ia ion and modi ies
in lo escence de elopmen in ba ley (Ho deum ulga e. L)6. Exp ession analyses o mRNAs by in si u hyb idi-
za ion and mic oa ay analysis e ealed ha V s4 is exp essed e y ea ly du ing in lo escence de elopmen and
con ols he ow- ype pa hway in ba ley h ough V s1, a nega i e egula o o la e al spikele e ili y.
The p bs mu an was ob ained om Pudamai-2, which has he no mal V s4 gene and a wo- owed pheno ype,
by soaking he ba ley in lo escence in maize genomic DNA solu ion. In he mu an , V s4 is dele ed h ough
a ecombina ion be ween wo BARE e o ansposons on ei he side o he V s4 gene. LTR e o ansposons
a e known o ecombine; he ecombina ion be ween wo LTRs o a single elemen , which esul s in dele ion
o he in e nal domain o he e o ansposon and gene a es a solo LTR, has been s udied16. The p ocess o e -
o ansposon eplica ion gene a es LTRs ha a e iden ical a he momen o in eg a ion21; he accumula ion o
mu a ions in he LTRs a he neu al a e a e ha allows o he es ima ion o he age o he in eg a ed ele-
men 22. Genome-wide analyses show ha he a e age hal -li e o a e o ansposon in he Copia supe amily,
which includes BARE, is 859,000 yea s, o a a e o 1.16 × 10−6 e en s pe elemen pe gene a ion, in he g ass
B achypodium dis achyon, which loses e o ansposons h ough ecombina ion ela i ely apidly23. In ba ley
and o he plan s, he a e o solo LTR o ma ion a ies conside ably be ween e o ansposon amilies and also
be ween ch omosomes and egions20,23,24. Analyses o he equency o ecombina ion e en s be ween in e nal
e o ansposon domains, such as he gene a ed p bs epo ed he e, ha e no been made and a e di icul o iden-
i y in he absence o no el pheno ypes.
Recombina ion be ween he LTRs o wo di e en elemen s can gene a e a conca ena ed s uc u e comp ising
wo in e nal domains lanking a single, ecombinan LTR, which esul s in he loss o he in e ening genomic
sequence, including any gene ha happens o be he e. A quan i a i e PCR su ey o he ba ley genome o such
s uc u es wi h h ee LTRs and wo in e nal domains showed ha hei p esence in abou 4.3 × 103 copies pe
haploid genome23. While his indica es he po en ial o gene loss h ough ecombina ion o e o ansposons
lanking a gene, especially gi en ha he gene islands25 a e lanked by e o ansposon “seas” which inc eases he
Mo ex-s a Mo ex-end Leng h Highes pe cen iden i y Highes e alue Hi numbe De ail alignmen esul s
115345 16193 848 73.01 1.484e-75 926 TableS2, Fig.S3
43155 43887 732
216423 18000 1577 72.61 2.035e-121 1019 TableS3, Fig.S4
44227 45804 1577
318046 18087 41 97.62 3.38E-12 879 TableS4, Fig.S5
45850 45891 41
4 27390 27543 153 81.1 5.543e-169 35 TableS5, Fig.S6
5 25868 26648 780 88.2 3.106e-45 1 TableS6, Fig.S7
Table 1. Sequence alignmen o he Mo ex dele ion wi h he maize genome sequence.
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in e ening dis ance be ween ecombining e o ansposon sequences and appea s o be co ela ed wi h dec eas-
ing ecombina ion equency20.
The ques ion a ises as o whe he he maize DNA soaking p ocedu e is connec ed o he ecombina ion ha
gene a ed he p bs mu an . Ou p ocedu e and he o he me hods in oduce o eign DNA in o he megagame o-
phy e be o e e iliza ion26,27. Whe he o no any o eign DNA is in eg a ed, he p esence o ex a ch omosomal
o cy oplasmic DNA igge s a ange o de ense esponses in animals28,29, media ed by DNA ecogni ion by p o-
eins including STING (also called MITA, MPYS, TMEM173, o ERIS)30, speci ic oll-like ecep o s (TLRs)31,
Z-DNA binding p o eins (ZBP-1, DLM-1, o DAI)32, and M e11 (meio ic ecombina ion 11)33. M e11 is pa icu-
la ly in iguing because, oge he wi h RAD50 and NBS1, is a pa o he MRN complex and has been shown o
play a i al ole in double-s and b eak (DSB) epai 34 in plan s, which is an in e media e s ep in ecombina ion.
The maize genome con ains 404,000 Copia supe amily e o ansposons35,36; he in eg ase domains o hese
a e e y simila o he in eg ase co e domain o BARE ha unde wen ecombina ion in p bs. We specula e
whe he he homologous maize and ba ley in eg ase sequences may ha e in e ac ed wi h each o he , media -
ing he ecombina ion. Exp ession analyses, in si u hyb idiza ion and mic oa ays e ealed ha V s4 is ac i ely
exp essed du ing in lo escence de elopmen 6, co esponding o he s age a which he ba ley in lo escence was
soaked in maize genomic DNA o gene a e he p bs mu an . Due o i s ansc ip ional ac i i y, his egion is likely
o ha e an open ch oma in con o ma ion, which could p o ide an oppo uni y o maize DNA o in e ac a he
BARE in eg ase domains and p omo e he ecombina ion. The high concen a ion o conse ed e o ansposon
sequences would make binding and ecombina ion in a e o ansposon sequence mo e likely han elsewhe e.
While ecombina ions be ween endogenous e o i uses (ERVs)—which a e s uc u ally iden ical o LTR e -
o ansposons—ha e caused genic dele ions h ough ecombina ion37, o ou knowledge, he e has no been an
ea lie demons a ion o his in plan s.
Ho izon al gene ans e (HGT) is well documen ed in p oka yo ic genome e olu ion. I is ela i ely clea
ha he e a e se e al HGT pa hways, including ans o ma ion, conjuga ion, and ansduc ion. In euka yo es,
di ec DNA exchanges may occu du ing g a ing38, symbiosis39,40, pa asi ism41, pa hogenesis42, and epiphy e
o en ophy e43. Some ec o s, such as pollen43, ungi44, bac e ia45, i uses46, plasmids38, insec s47 and anspos-
ons43, may also be in ol ed in HGT. T ansposable elemen s (TEs) ha e been ecognized as impo an ec o s
o he ho izon al mo emen o genes be ween euka yo ic genomes48–51. T ansposons, wi h hei inhe en abil-
i y o mobilize, can p oli e a e and in eg a e in o genomic DNA and gene a e HGTs wi h ease52. T ansposons
ha e also cap u ed and ansduced genomic DNA sequences in bo h Daphnia pulex53 and D osophila species52.
The ans e o Mu-like ansposons be ween Se a ia and ice has been documen ed48,54. LTR e o ansposons
can p oduce i us-like pa icles, which may wo k as mo e equen ec o s o HGT52,55. Such cases ha e been
demons a ed in LTR- e o ansposon RIRE1 wi hin he genus O yza56 and he LTR- e o ansposon Rou e66
in Poaceae50. Wi h he inc easing a ailabili y o euka yo ic genome sequences, mo e e idence will be a ailable
ha plan s a e also likely o unde go HGT. Howe e , he esul s ha e been based on incong uences in molecula
phylogene ic ees. On he o he hand, he e a e nume ous epo s in he li e a u e ha ha e di ec ly in oduced
o eign DNA by injec ing exogenous DNA o di ec ly DNA soaking o pollen ube pa hway in o ice, ba ley,
whea , so ghum, maize, co on, oa s, ye, cucumbe , pumpkin, kidney bean and soybean o c ea e new gene ic
a ia ions (Supplemen a yTable7). RAPD, AFLP and SSR molecula ma ke s ha e been used o es DNA ans-
e s be ween species in se e al s udies57–59. Howe e , no s udy has demons a ed how he exogenous DNA causes
gene ic a ia ion in o he species. Ou s udy p o ides p elimina y e idence ha LTR- e o ansposon-media ed
gene dele ion/inse ion may play a ole in di ec gene ans e be ween di e en species.
In addi ion o ac as po en ial ec o s o ho izon al gene ans e , ansposable elemen s a e also esponsi e
and suscep ible o en i onmen al changes. I is well documen ed ha s esses could ac i a e TE o gene a e new
gene ic di e si y60. This is especially ue o LTR- e o ansposons as he LTR is su icien in i sel o ac i a e TE
ansc ip ion in esponse o s ess. I is possible ha soaking he ba ley spike in he maize DNA solu ion c ea ed
s ess condi ions o he de eloping spikele s, which ac i a ed LTR-TE media ed ecombina ion. In his scena io,
he maize genomic DNA may be no essen ial o he mu a ion. Fu he esea ch is equi ed o es his assump-
ion by soaking he de eloping ba ley spikes in wa e o sal solu ion o p o ide simila s esses o iden i ica ion
o new mu an s.
Ma e ials and Me hods
Plan ma e ials. A poly- ow b anched spike (p bs) ba ley mu an was ob ained by soaking a wo- owed ba -
ley in lo escence (c . Pudamai-2) in maize genomic DNA solu ion61. The me hod ollowed ha desc ibed ea lie
o whea 62. Flowe ing ba ley spikes we e soaked in o al maize DNA a 1.6 ug/ul in 0.1 × SSC o 24 hou s. A e
soaking, he head was mo ed om he solu ion and ai -d ied unde ambien condi ions. Plan s we e sel -polli-
na ed and seeds ha es ed. The mu an was iden i ied a lowe ing o he nex gene a ion plan s.
Gene ic mapping was conduc ed in wo popula ions: one ecombinan inb ed line (RIL, F2:6) popula ion con-
sis ing o 207 plan s de i ed om a c oss be ween he p bs mu an and a six- owed ba ley cul i a Kunlun 12,
and an F2 popula ion consis ing o 285 spike mu an plan s de i ed om a c oss be ween he p bs mu an and
a six- owed ba ley cul i a Zangqing 320. The p bs mu an , RIL 11R258-95 wi h a b anched spike pheno ype,
Pudamai-2, and a . Mo ex we e used o DNA sequence analysis.
Genomic DNA ex ac ion and geno ype analysis. Genomic DNA was ex ac ed om lea es o indi-
idual plan s and hei pa en s using a modi ied CTAB me hod63. DNA samples we e quan i ied using a Unican
UV300 UV/Vis spec ome e (The mo Elec on Co po a ion, Camb idge, UK), and hen adjus ed o 25 ng/μ l.
Because a DQ327702 ma ke associa ed wi h he mu an is closely linked wi h he V s4 gene6, new molecula
ma ke s Cbic43 and Cbic44 we e designed a ound he V s4 gene using he ba ley genome sequence14 om he
IPK Ba ley BLAST se e (h p://webblas .ipk-ga e sleben.de/ba ley/ i oblas .php). P ime pai s speci ic o he
www.na u e.com/scien i ic epo s/
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Scien i ic RepoR s | 7:43766 | DOI: 10.1038/s ep43766
V s4 gene (AS12, AS34, and AS56)6 we e designed o V s4 haplo ype analysis. P ime s we e syn hesized by
Shanghai Sunny Bio echnology (Shanghai, China). PCR eac ions we e pe o med in 10 μ L olumes con ain-
ing app oxima ely 25 ng genomic DNA, 0.2 μ M o each p ime , and 5 μ L 2 × Taq Mas e Mix (Gene Solu ion,
Shanghai, China) using he ollowing p og am: 94 °C o 3 min, 32 cycles o 94 °C o 30 sec, 55 °C o 45 sec, 72 °C
o 1 min, and 72 °C o 5 min. PCR p oduc s we e sepa a ed on 8% polyac ylamide gels.
Cloning o he dele ion in mu an p bs. BAC sequences we e iden i ied by blas ing he V s4 agains
he In e na ional Ba ley Genome Sequencing Conso ium da abase (unpublished da a). PCR p ime pai s we e
designed a 2 kb in e als in he egion nea he V s4 locus using he P ime -Blas ool (h p://www.ncbi.nlm.nih.
go / ools/p ime -blas /). PCR eac ions we e desc ibed as abo e. Annealing empe a u es we e op imized o
each p ime pai (Supplemen a yTable1). PCR p oduc s we e examined by elec opho esis on 1% aga ose gels.
Long- ange PCRs we e pe o med in 50 μ L eac ions con aining 1 × bu e , 5 μ L empla e DNA, 0.4 μ M o each
p ime , 400 μ M each deoxy ibonucleo ide, and 2.5 U LA Taq DNA polyme ase (Taka a, Dalian, China) using he
ollowing p og am: 94 °C o 3 min, 32 cycles o 98 °C o 10 sec, 68 °C o 15 min, and 68 °C o 20 min. PCR p od-
uc s we e examined by elec opho esis using a 0.8% aga ose gel, analyzed by Bio-Red Quan i y One gel image
analysis sys em and sequenced by Shanghai RuiDi Biological Technology (Shanghai, China).
Quan i a i e RT-PCR. RNA was ex ac ed om imma u e spikes a lemma p imo dium s age o he p bs
mu an and wild pa en Pudamai-2 using Spin Column Plan o al RNA Pu i ica ion Ki (Sanggon Bio ech
(Shanghai) Co.,L d). cDNA was p epa ed om 1 ug RNA using AMV Fi s S and cDNA Syn hesis Ki (Sanggon
Bio ech (Shanghai) Co., L d). qPCR eac ions we e pe o med using SYBR G een (SG Fas qPCR Mas e Mix
(HighRox), BBI ) and he Applied Biosys ems S epone plus Real- ime PCR Sys em. The Real- ime PCR assays
we e pe o med in iplica e o each cDNA sample. V s46, V s14 and H Ac in6 p ime sequences used o quan i-
a i e RT-PCR. The H Ac in gene was used as e e ence gene o no maliza ion.
Sequence analysis. Alignmen s o mu an p bs and ba ley genomic sequences we e cons uc ed using
MEGA 6.014 and BLASTN 2.3.0+ 64. The p edic ion o ansposable elemen s was iden i ied h ough LTR Finde
1.05 (h p:// li e. udan.edu.cn/l _ inde /)65 and BLAST66 agains he T i iceae Repe i i e Elemen (TREP) da a-
base (h p://whea .pw.usda.go /GG2/blas .sh ml). Sea ches o sequence homology o maize was conduc ed wi h
MaizeGDB agains he sequence da abase B73 Re Gen_ 3 (MGSC), using he BLAST p og am BLASTN63 wi h
an E- alue cu o < 1e-50.
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Acknowledgemen s
We a e g a e ul o P o esso Jing Zhang, Ins i u e o C op Ge mplasm Resou ces, Chinese Academy o
Ag icul u al Science, o p o iding he mu an p bs, and o D Nils S ein Leibniz Ins i u e o Plan Gene ics
and C op Plan Resea ch (IPK) o accessing he un-published ba ley BAC sequence. This wo k was pa ially
suppo ed by he Na u al Science Founda ion o Zhejiang P o ince (LY15C130004), China Ag icul u e Resea ch
Sys em (CARS-05), he Na ional Na u e Science Founda ion o China (31501309) and he Academy o Finland,
P ojec 266430.
www.na u e.com/scien i ic epo s/
9
Scien i ic RepoR s | 7:43766 | DOI: 10.1038/s ep43766
Au ho Con ibu ions
C.L. and J.Y. concei ed, designed and supe ised he s udy; Y.S. and F.Y. a e p incipal in es iga o s; A.H.S.,
F.Y.W.Z. and Y.J. da a analysis; J.Z., J.W., W.H. pheno yping; N.S., X.Z. and Q.J. DNA sequencing; Y.S., F.Y. A.S.
and C.L. w o e he pape .
Addi ional In o ma ion
Supplemen a y in o ma ion accompanies his pape a h p://www.na u e.com/s ep
Compe ing In e es s: The au ho s decla e no compe ing inancial in e es s.
How o ci e his a icle: Shang, Y. e al. Gene Dele ion in Ba ley Media ed by LTR- e o ansposon BARE.
Sci. Rep. 7, 43766; doi: 10.1038/s ep43766 (2017).
Publishe 's no e: Sp inge Na u e emains neu al wi h ega d o ju isdic ional claims in published maps and
ins i u ional a ilia ions.
This wo k is licensed unde a C ea i e Commons A ibu ion 4.0 In e na ional License. The images
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use s will need o ob ain pe mission om he license holde o ep oduce he ma e ial. To iew a copy o his
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