

Ci a ion: F anco-Dua e, R.; ˇ
Cadež,
N.; Ri o, T.; D umonde-Ne es, J.;
Dominguez, Y.R.; Pais, C.; Sousa,
M.J.; Soa es, P. Whole-Genome
Sequencing and Anno a ion o he
Yeas Cla ispo a san aluciae Re eals
Impo an Insigh s abou I s
Adap a ion o he Vineya d
En i onmen . J. Fungi 2022,8, 52.
h ps://doi.o g/10.3390/jo 8010052
Academic Edi o : B ian Monk
Recei ed: 6 Decembe 2021
Accep ed: 4 Janua y 2022
Published: 5 Janua y 2022
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
published maps and ins i u ional a il-
ia ions.
Copy igh : © 2022 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
Fungi
Jou nal o
A icle
Whole-Genome Sequencing and Anno a ion o he Yeas
Cla ispo a san aluciae Re eals Impo an Insigh s abou I s
Adap a ion o he Vineya d En i onmen
Rica do F anco-Dua e 1,2,* , Neža ˇ
Cadež 3, Te esa Ri o 1,2 , João D umonde-Ne es 4,
Yazmid Reyes Dominguez 5, Célia Pais 1,2 , Ma ia João Sousa 1,2 and Ped o Soa es 1,2
1CBMA, Cen e o Molecula and En i onmen al Biology, Depa men o Biology, Uni e si y o Minho,
4710-057 B aga, Po ugal; [email p o ec ed] (T.R.); [email p o ec ed] (C.P.);
[email p o ec ed] (M.J.S.); ped osoa [email p o ec ed] (P.S.)
2
Ins i u e o Science and Inno a ion o Bio-Sus ainabili y (IB-S), Uni e si y o Minho, 4710-057 B aga, Po ugal
3Depa men o Food Science and Technology, Bio echnical Facul y, Uni e si y o Ljubljana, 101,
1000 Ljubljana, Slo enia; [email p o ec ed]
4IITAA—Ins i u e o Ag icul u al and En i onmen al Resea ch and Technology, Uni e si y o Azo es,
9700-042 Ang a do He oísmo, Po ugal; d [email p o ec ed]
5Laimbu g Resea ch Cen e, Laimbu g 6, 39052 Vadena, I aly; Yazmid.Reyes-Dominguez@laimbu g.i
*Co espondence: [email p o ec ed] o [email p o ec ed]
Abs ac :
Cla ispo a san aluciae was ecen ly desc ibed as a no el non-Saccha omyces yeas species,
isola ed om g apes o Azo es ineya ds, a Po uguese a chipelago wi h pa icula en i onmen al
condi ions, and om I alian g apes in ec ed wi h D osophila suzukii. In he p esen wo k, he genome
o i e Cla ispo a san aluciae s ains was sequenced, assembled, and anno a ed o he i s ime, using
obus pipelines, and a combina ion o bo h long- and sho - ead sequencing pla o ms. Genome
compa isons e ealed speci ic di e ences be ween s ains o Cla ispo a san aluciae e lec ing hei
isola ion in wo sepa a e ecological niches—Azo ean and I alian ineya ds—as well as mechanisms
o adap a ion o he in ica e and a duous en i onmen al ea u es o he geog aphical loca ion
om which hey we e isola ed. In pa icula , ele an di e ences we e de ec ed in he numbe o
coding genes (sha ed and unique) and ansposable elemen s, he amoun and di e si y o non-
coding RNAs, and he enzyma ic po en ial o each s ain h ough he analysis o hei CAZyome.
A compa a i e s udy was also conduc ed be ween he Cla ispo a san aluciae genome and hose o
he emaining species o he Me schnikowiaceae amily. Ou phylogene ic and genomic analysis,
comp ising
126 yeas
s ains (alignmen o 2362 common p o eins) allowed he es ablishmen o a
obus phylog am o Me schnikowiaceae and de ailed incong uencies o be cla i ied in he u u e.
Keywo ds:
genomics; phylogenomics; unc ional gene analysis; Me schnikowiaceae; Azo es; wine
yeas s; bio echnology; adap a ion
1. In oduc ion
In ou p e ious su eys on he yeas di e si y o Azo ean ineya ds, in 2009 and
2010 [
1
–
3
], we desc ibed a new yeas species Cla ispo a san aluciae [
4
], isola ed om g apes.
I was cha ac e ized on he basis o he sequences o he in e nal ansc ibed space (ITS)
egion (ITS1-5.8S–ITS2), he sequences o he D1/D2 domain o he la ge subuni (LSU)
RNA gene, and pa icula physiological cha ac e is ics. Tha s udy also desc ibed his
species as being isola ed om g apes in ec ed wi h D osophila suzukii in I aly. This showed
iden ical D1/D2 sequences and e y simila ITS egions ( i e nucleo ide subs i u ions) o
he Azo ean s ains. The new species was ob ained om pa icula i icul u al en i on-
men s, ypical o he Azo es a chipelago, which esul om he in e ac ion be ween speci ic
clima ic condi ions, au och honous g ape ine cul i a s, and local i icul u al p ac ices.
Pheno ypic cha ac e iza ion o his new species e ealed some in e es ing ea u es ha
J. Fungi 2022,8, 52. h ps://doi.o g/10.3390/jo 8010052 h ps://www.mdpi.com/jou nal/jo
J. Fungi 2022,8, 52 2 o 18
posi ioned i apa om he closely ela ed ones, such as he inabili y o g ow a empe a-
u es abo e
35 ◦C
, p oduc ion o ace ic acid, and he capaci y o assimila e s a ch. The ull
bio echnological po en ial o his new species emains o be explo ed, as does he unde -
s anding o he genomic ea u es associa ed wi h he adap a ion o i s en i onmen . The
occu ence o bio echnologically impo an ea u es associa ed wi h species o his clade is
no uncommon. (Candida)in e media, a xylose-u ilizing species o he Me schnikowiaceae
amily, belonging o he Cla ispo a clade, displays a high-capaci y xylose anspo sys-
em [
5
,
6
]. Due o hese cha ac e is ics, his species has been ca ego ized as an a ac i e
species o p oduce e hanol om lignocellulosic biomass [7].
Ou p e ious s udy was one o he ew o epo he p esence o yeas species belonging
o he Cla ispo a clade in ineya ds, e en hough some a e epo s ha e al eady associa ed
hese yeas s wi h winemaking, as de ailed below. We highligh ed he a i y o hese
occu ences, in a e iew o he associa ion o non-Saccha omyces yeas s wi h i icul u e and
winemaking [
8
]. In ha s udy, we sys ema ized 80 yea s o he li e a u e desc ibing non-
Saccha omyces yeas species isola ed om g apes and/o g ape mus s and compiled a lis o
293 species. Only wo species belonging o he Cla ispo a clade we e iden i ied—Cla ispo a
uc us and Cla ispo a lusi aniae. E en hough he e is no s ong associa ion be ween
Cla ispo a species and wine, some epo s ha e al eady desc ibed he possible ad an ages o
hese yeas s in he p oduc ion o wines wi h al e na i e senso y cha ac e is ics, albei wi h
some associa ed disad an ages, such as he p esence o an abno mally high concen a ion
o ace aldehyde [
9
]. In Azo ean ineya ds, no o he yeas o his genus was ound wi hin
he 2910 isola es iden i ied in ou p e ious wo k [
1
,
2
], bu a single species o his amily
(Me schnikowia pulche ima) was ound in ou di e en islands in wo consecu i e sampling
yea s. The use o his species in wine bio echnology was ecen ly e iewed [
10
], wi h he
au ho s concluding ha i s e sa ili y lies in i s abili y o e men mus in combina ion
wi h o he yeas species (mainly o ci cum en i s low e men a i e powe ), as well as
modula ing he syn hesis o seconda y e men a ion me aboli es o imp o e and di e si y
he senso y p o ile o he wine.
The phylog am ob ained in ou p e ious s udy [
4
], based on conca ena ed sequences
o he D1/D2 domain o he LSU RNA gene, and o he ITS egion, placed Cla ispo a
san aluciae s ains nea closely he ela ed species Cla ispo a uc us, (Candida)aspa agi,
(Candida) i iphila, (Candida)phyllophila, (Candida)ca ajalis, and Cla ispo a lusi aniae. The
desc ip ion o his new species was conside ed as an impo an add-on in unde s anding
he phylogene ic ela ionships wi hin he Cla ispo a clade and cla i ying hei biodi e si y
and ecology. P e iously, se e al yeas s ha now belong o he Cla ispo a genus, we e placed
in he anamo phic genus Candida, due o hei inabili y o o m sexual spo es [
11
]. Wi h
ecen phylogene ic analysis based on DNA sequences, in combina ion wi h physiological
e idence, he ela ionships be ween he species o Candida and he genus Cla ispo a began
o be cla i ied, leading o a new classi ica ion o his g oup by Daniel e al. in 2014 [
11
],
wi h 40 species o Candida assigned o he genus Cla ispo a, based on sequences o he
LSU D1/D2 domain, ITS egion and ou coding genes (ACT1,TEF1,MCM7, and RPB2).
In 2018, Ku zman e al. [
12
] desc ibed ou new species o Me schnikowia and p oposed
o ans e se en addi ional Candida species o bo h Me schnikowia and Cla ispo a gene a.
Ku zman e al. concluded ha he axonomy o he Cla ispo a clade could only be cla i ied
by whole-genome compa isons, which s ill need o be pe o med. Rega ding he Cla -
ispo a genus, only wo species ha e been genome sequenced and anno a ed: Cla ispo a
lusi aniae (
11.9–12.1 Mb
, 8 ch omosomes, genome accession ASM167369 2 ), and Cla ispo a
uc us (11.4 Mb, NCBI genome accession ASM370779 1). In 2016, a phylogeny o he
Me schnikowiaceae amily was p esen ed by Lachance e al. [
13
], combining d a genomes
o 55 s ains and iden i ying 3016 o hologues, 1061 o which exis in all he analyzed
s ains. E en hough he Cla ispo a lusi aniae genome was used as a que y o compa e
be ween all he genomes, no species belonging o he Cla ispo a genus we e conside ed,
wi h he analysis ocused on only he Me schnikowia genus. Mo e ecen ly, Shen e al. [
14
]
a emp ed o econs uc he phylogeny o 300 budding yeas species, ocusing mainly on
J. Fungi 2022,8, 52 3 o 18
he di e si y o Saccha omyco ina. In ha wo k, whole genomes we e used o pa ially
desc ibe he phylogeny o Me schnikowiaceae clade conside ing only 22 Me schnikowia
species. This analysis used only he ype s ains o each species, which lack in a-species
di e si y, and he genome anno a ion was no di ec ed o he analysis o his clade. Thus,
a deep, b oad, and ocused analysis using obus ly anno a ed genomes o he phyloge-
ne ic ela ions be ween Cla ispo a species (including also he ecen ly assigned Candida
species) and he sis e genus Me schnikowia is lacking. Wi h his in mind, he objec i e o
he p esen wo k was o sequence, assemble and anno a e whole genomes o he di e en
Cla ispo a san aluciae s ains, using a combina ion o bo h long- and sho - ead sequencing
pla o ms, in o de o ob ain high-quali y sequences o compa a i e genomics. We used
he assembled genomes o u he elucida e he molecula mechanisms unde lying he
adap a ion o his species o he pa icula en i onmen al cha ac e is ics om which i
was isola ed. In pa icula , he main goal was o un a el he genomic ea u es ha can
explain he pheno ypic cha ac e is ics p e iously obse ed wi hin isola es o Cla ispo a
san aluciae, as well as o p edic hei bio echnological po en ial. In addi ion, all species
om he Me schnikowiaceae amily whose comple e genome was publicly a ailable we e
conside ed and combined in phylogene ic and genomic analysis, o help cla i y he phyloge-
ne ic placemen o Cla ispo a yeas s wi hin his la ge g oup o impo an non-Saccha omyces
yeas s. We plan o use ou esul s o cla i y he posi ioning o (Candida) species in ela ion
o he sis e genus Me schnikowia and econs uc Me schnikowiaceae amily phylogeny
using comple e genomes.
2. Ma e ials and Me hods
2.1. Cell Cul u e, Sample Collec ion, and DNA Ex ac ion
The ype s ain o Cla ispo a san aluciae (A1.18
T
= CBS 16465
T
), oge he wi h he h ee
s ains isola ed om g apes o Azo ean ineya ds (A1.5, A1.7, and A1.19), and one addi-
ional s ain isola ed om g apes in ec ed wi h D osophila suzukii in I aly (
LB-NB-3.3
) [
4
],
we e g own in YPD b o h (yeas ex ac , 1% w/ ; pep one, 1% w/ ; glucose 2% w/ ), in
50 mL conical lasks, o 48 h a 28
◦
C, 220 pm. Genomic DNA was isola ed acco ding
o he p o ocol published by Schwa z and She lock [
15
], wi h a ew adap a ions o he
isola ion o DNA om non-Saccha omyces yeas s. A e washing in 0.9 M so bi ol solu-
ion, he cells we e incuba ed by adding 20
µ
L o Ly icase (30 mg/mL, Sigma-Ald ich,
S . Louis
, MO, USA) o he cells. The incuba ion ime was a leas 4 h a 37
◦
C. Following
phenol/chlo o o m (Millipo e, Bu ling on, MA, USA) ex ac ion, he DNA was p ecipi a ed
using 40
µ
L o 3 M sodium ace a e (pH 5.5) and 1 mL o absolu e e hanol and esuspended
in 200 µL o TE bu e .
2.2. Genome Sequencing and Assembly
The genomes o all he Cla ispo a san aluciae s ains we e sequenced by using a combi-
na ion o he long-and sho - ead sequencing echnologies o PacBio and Illumina, espec-
i ely. A e DNA ex ac ion, lib a y p epa a ion and PacBio/Illumina sequencing we e
pe o med a No ogene acili ies (No ogene Company LTD, Camb idge, Uni ed Kingdom).
Low-quali y eads and adap e s we e emo ed by No ogene, and sequencing quali y was
accessed using Fas QC so wa e (h p://www.bioin o ma ics.bbs c.ac.uk/p ojec s/ as qc/;
accessed on 1 Augus 2021). The sequencing da a a e a ailable a NCBI BioP ojec ID
PRJNA784374.
Long- eads ob ained om PacBio sequencing we e de no o assembled using Canu
.1.9 [
16
] wi h de aul pa ame e s. Illumina pai ed-end eads we e hen used o imp o e
assembly quali y, using Masu ca so wa e .4.0.5, in pa icula he Polca package [
17
,
18
].
Finally, RagTag so wa e .2.1.0 [
19
] was used o assemble all he sca olds in o longe eads,
using ch omosome in o ma ion om he closely ela ed species Cla ispo a lusi aniae and
(Candida)in e media. Genome assembly quali y me ics, a ailable in Table 1, we e compu ed
using QUAST .5.0.2 [20].
J. Fungi 2022,8, 52 4 o 18
Table 1. Genome assembly s a is ics o Cla ispo a san aluciae s ains.
A1.18TA1.5 A1.7 A1.19 LB-NB-3.3
Canu assemble
Assembly leng h (bp) 11,088,431 11,018,248 10,921,443 10,861,576 11,019,028
Numbe o sca olds 43 13 46 86 30
N50 (bp) 315,943 802,369 355,153 1,329,122 494,470
L50 7 3 6 14 7
Numbe o N’s pe 100 Kb 0 0 0 0 0
Numbe o sca olds > 5000 bp 29 11 28 53 23
To al leng h > 5000 bp 10,780,215 10,974,595 10,557,056 10,118,395 10,856,688
Masu ca assemble
Subs i u ion e o s e ised 42 8 141 428 70
Inse ion/Dele ion e o s e ised 1686 536 2825 6144 1607
Assembly leng h (bp) 11,089,145 11,018,616 10,922,446 10,863,639 11,019,715
Numbe o sca olds 43 13 46 86 30
N50 (bp) 532,329 1,048,728 654,799 218,696 650,701
L50 7 3 6 14 7
Numbe o N’s pe 100 Kb 0 0 0 0 0
Numbe o sca olds >5000 bp 29 11 28 53 23
To al leng h >5000 bp 10,780,920 10,974,959 10,558,055 10,120,373 10,857,358
RagTag
assemble
Assembly leng h (bp) 11,092,545 11,019,016 10,925,846 10,870,339 11,021,815
Numbe o sca olds/ch omosomes
9 9 12 19 9
Numbe o N´s pe 100 Kb 3065 3.63 31.12 61.64 19.05
Numbe o sca olds >5000 bp 4 8 4 3 7
To al leng h >5000 bp 11,025,073 11,000,234 10,766,609 10,606,285 10,966,127
Ploidy haploid haploid haploid haploid haploid
GC con en (%) 49.66 49.70 49.73 49.66 49.76
To de e mine ploidy, we used nQui e so wa e [
21
] o align sequencing eads o he
ype s ain genome assembled a e RagTag and de e mine base equency dis ibu ions
be ween equencies 20 and 80. Assessmen o each genomes’ comple eness was pe o med
using Benchma king Uni e sal Single-Copy O hologs (BUSCO) so wa e .5.2.2 [
22
]. A -
e age nucleo ide iden i y (ANI) was calcula ed using he O hoANIu web ool [
23
] in
pai wise mode, o compa e he nucleo ide con en o genomes.
2.3. Genome Anno a ion
Anno a ion o Cla ispo a san aluciae genome assemblies was pe o med using AU-
GUSTUS so wa e .3.4.0 [
24
,
25
], conside ing 11 di e en p e- ained models, chosen
as belonging o he Ascomyco a phyla Saccha omyces ce e isiae S288c, Candida albicans,
Meye ozyma (Candida)guillie mondii, Candida opicalis, Deba yomyces hansenii, E emo he-
cium gossypii, Kluy e omyces lac is, Lodde omyces elongispo us, Sche e somyces (Pichia) s ipi is,
Schizosaccha omyces pombe, and Ya owia lipoly ica. Resul s we e manually e iewed o selec
he anno a ion wi h he highe numbe o p edic ed coding genes, which was ob ained
using Lodde omyces elongispo us as he p e- ained model, o all he Cla ispo a san aluciae
s ains. The po en ial coding egions (nucleo ide sequences) epo ed by AUGUSTUS we e
ex ac ed om he comple e genomes o FASTA iles.
CMsea ch [
26
] and S uc RNA inde [
27
] we e used o sc eening he p esence o
non-coding RNA (ncRNA). The R am da abase [
28
] was employed o ncRNA sea ching,
using an e- alue o 0.01.
Func ional genomic anno a ion was pe o med wi h eggNOG-mappe .2 [
29
] by
conside ing p o eins p edic ed by AUGUSTUS and choosing only o hologs ha we e
in e ed om he expe imen al e idence. The esul s we e desc ibed conside ing clus e s
o o hologous g oups (COGs) wi h hei associa ed unc ional ca ego ies [
30
], and also
conside ing he Kyo o Encyclopedia o Genes and Genomes (KEGG) pa hways, in pa ic-
ula he KEGG O hology (KO) desc ip o s [
31
,
32
]. Gene unc ion p edic ions we e also
accomplished by assessing he Ca bohyd a e-Ac i e EnZymes (CAZymes) da abase [33].
J. Fungi 2022,8, 52 5 o 18
Final genome anno a ions o all Cla ispo a san aluciae s ains a e a ailable in
Supplemen a y Da a S1.
2.4. Homology Analysis, Compa a i e Genomics, and Phylogenomics
To compa e he genome o Cla ispo a san aluciae ype s ain A1.18 wi h ha o he
emaining s ains, do plo s we e p oduced using he Re-Do -Able ool (h ps://www.
bioin o ma ics.bab aham.ac.uk/p ojec s/ edo able/). In e -species di e ences be ween
membe s o he amily Me schnikowiaceae we e e alua ed by downloading all he comple e
genomes publicly a ailable a NCBI (121 s ains belonging o 48 di e en species). When
mo e han one s ain was a ailable o a ce ain species, all s ains we e conside ed. The
excep ion was (Candida)au is o which only he ep esen a i e genome was used since he
hund eds o s ains wi h genome sequence a ailable would ha e inc eased edundancy.
KEGG Mappe was used as a collec ion o KEGG mapping ools o linking genes
and p o eins o me abolic pa hways [
32
,
34
]. In pa icula , KO gene anno a ions, ob ained
om eggnog-mappe , we e used o assess pa hway comple eness using KEGG Mappe –
Recons uc web ool (www.genome.jp/kegg/mappe / econs uc .h ml). Resul s we e
applied in he cons uc ion o a hea map using Mic oso Excel®.
A da abase was p epa ed by conside ing all 126 comple e genomes (121 s ains o
Me schnikowiaceae amily plus he 5 Cla ispo a san aluciae isola es). To a oid inconsis-
ency, he 121 Me schnikowiaceae genomes we e anno a ed using Augus us wi h he same
p e- ained model as was applied o he anno a ion o he Cla ispo a san aluciae genomes.
BLASTP analysis was pe o med using he ull p o eome o he Cla ispo a san aluciae ype
s ain A1.18
T
as a que y agains he o al da abase. An E- alue cu o o 10
−6
was used o
exclude alse esul s, and a pipeline adap ed om [
35
] was used o pe o m compa a i e
genomics be ween all isola es. The BLASTP esul s we e il e ed whe e ep esen a i e
p o eins we e de ec ed in he o he 121 isola es. Each se o p obable homologous p o eins
(con aining he que y and he espec i e esul s) we e mul iple aligned using he MAFFT
algo i hm in FasPa se (h ps://gi hub.com/Sun-Yanbo/FasPa se ) [
36
]. All p o eins om
a gi en o ganism we e conca ena ed using he alignmen esul s o ob ain he co e con-
se ed aligned p o eome con aining mos ly essen ial genes no ela ed o speci ic biological
ai s o each species. This alignmen was hen used o phylogene ic econs uc ion by
conside ing he maximum likelihood in IQ-TREE (www.iq ee.o g) [
37
], wi h he JTT model
o amino acid e olu ion and gamma-dis ibu ed a es ( ou a es) wi h 500 boo s ap epli-
ca es. Two ou g oups we e conside ed: Lipomyces lipo e and Cybe lindne a jadinii. FigT ee
.1.4.4 (h p:// ee.bio.ed.ac.uk/so wa e/ ig ee/) was used o isualize and edi he ee.
The second ound o BLASTP analysis, using he p o eomes o he i e Cla ispo a san aluciae
s ains as que ies, allowed building Venn diag ams o schema ize he numbe o genes
common be ween he i e genomes using he a e age esul s be ween all pai s o be ween
g oups o h ee, ou , o in all i e s ains.
3. Resul s and Discussion
3.1. Sequencing, De No o Assembly, and Anno a ion o Cla ispo a San aluciae Genome
Genome sequencing o Cla ispo a san aluciae s ains A1.18
T
, A1.5, A1.7, A1.19, and
LB-NB-3.3 was pe o med using a combina ion o long- and sho - ead sequencing pla -
o ms. Be ween 27,903 and 44,977 eads we e ob ained wi h long- ead sequencing, wi h a
maximum ead leng h o 110,418 base pai s (bp). Sho - ead sequencing was used o e ine
long- ead sequencing esul s. An a e age alue o 3
×
10
6
pai ed-end eads, wi h 250 bp
each, was ob ained o each s ain. The i s ound o assembly was pe o med using Canu
and Masu ca assemble s (sequencing s a is ics a e p esen ed in Table 1), and hen RagTag
so wa e assembled he sca olds in o pu a i e ch omosomes. By using h ee assemble s
we we e able o assemble long and sho - ead sequences in o ull ch omosomes o h ee o
he s ains, including he ype s ain A1.18
T
and s ains A1.5 and LB-NB-3.3. The emain-
ing wo s ains, possibly due o lowe sequencing dep h, we e only assembled in o la ge
sca olds. The a ained haploid genome size (10.8 Mb o 11.1 Mb) was compa able wi h

J. Fungi 2022,8, 52 6 o 18
he p e iously published genomes o Cla ispo a yeas s, in pa icula wi h he
11.9–12.1 Mb
o Cla ispo a lusi aniae (8 ch omosomes) [
38
,
39
], o wi h he 11.4 Mb o Cla ispo a uc us
(NCBI genome accession ASM370779 1).
The high-quali y-assembled genomes allowed he p edic ion o be ween 6015 and 6092
p o ein-coding genes o he i e Cla ispo a lusi aniae s ains using AUGUSTUS so wa e
(Table 2, Supplemen a y Da a S1). These alues a e among he highes epo ed o yeas s o
he Cla ispo a clade, and a e compa able only o he anno a ion o one ((Candida)in e media
s ain YCC 4715), o which 6082 coding genes we e p edic ed [
40
], bu co esponding o a
g ea e genome leng h o 13.08Mb.
Table 2. Cla ispo a san aluciae genome anno a ion s a is ics.
A1.18TA1.5 A1.7 A1.19 LB-NB-3.3
P o ein coding genes
To al numbe 6092 6034 6067 6015 6038
Range o p o ein leng hs (aa) 66–4974 63–4974 57–4974 60–4974 66–5293
A e age p o ein leng h (aa) 557.6 556.6 550.9 543.5 518.3
Non-coding RNAs
mic oRNAs (miRNAs) 32 32 33 31 21
small RNAs (sRNA) 20 21 22 20 23
nuclea RNAs (snRNA) 7 7 6 7 7
nucleola RNAs (snoRNA) 93 91 99 94 98
long noncoding RNAs (lncRNA) 8 8 9 8 12
ibosomal RNAs ( RNA) 96 63 42 69 124
ans e RNAs ( RNA) 276 259 279 299 248
O he 29 32 32 35 32
BUSCO O hologs
Ascomyco a odb10 da abase
Genome Comple eness (%) 93.5 94.4 93.4 90.7 93.6
Comple e BUSCOs 1595 1611 1594 1547 1597
F agmen ed BUSCOs 17 14 18 21 4
Missing BUSCOs 94 81 94 138 94
Saccha omyce es odb10 da abase
Genome Comple eness (%) 98.0 99.1 98.0 95.1 98.2
Comple e BUSCOs 2094 2118 2094 2032 2099
F agmen ed BUSCOs 14 11 12 17 13
Missing BUSCOs 29 8 31 88 25
Eggnog-mappe unc ional anno a ion
Genes wi h KO assigned 3130 (51.4%) 3129 (51.9%) 3125 (51.6%) 3130 (52.0%) 3101 (51.4%)
Genes wi h COG assigned 4180 (68.6%) 4171 (69.1%) 4166 (68.7%) 4119 (68.5%) 4141 (68.6%)
CAZymes unc ional anno a ion
Numbe o genes anno a ed 120 121 118 112 117
The unusually high numbe o p edic ed p o eins in he genome o Cla ispo a san-
aluciae was likely no ela ed, in ou opinion, o any peculia i y o his yeas
´
s genome
bu a he o he use o ad anced sequencing echnologies, oge he wi h an imp o ed
anno a ion pipeline. The lowes numbe o p edic ed coding sequences was de e mined o
s ain A1.19. This could be a ibu ed o lowe sequencing dep h. This was also he sho es
genome o he i e, he one wi h he lowes N50 alues (Table 1), and he one wi h lowe
BUSCO genome comple eness sco es, bo h in Ascomyco a and Saccha omyce es da abases
(Table 2).
The highes numbe o p edic ed p o eins was desc ibed in he anno a ion o he
genome o he ype s ain A1.18
T
, wi h 6092 coding sequences (Table 2). The a e age leng h
o he p edic ed p o eins was sligh ly lowe in LB-NB-3.3, al hough he la ges p o ein o
5293 amino acids (aa) was anno a ed in his s ain. This la ge open eading ame encodes
he p o ein midasin (Mdn1), an ATPase o 560 kDa ha is essen ial o cell iabili y. I
was iden i ied in all Cla ispo a san aluciae s ains and epo ed in o he yeas s, such as
in he gene a Saccha omyces and Schizosaccha omyces, as well as in dis an o ganisms as
J. Fungi 2022,8, 52 7 o 18
D osophila and A abidopsis [
41
]. The lowes coding sequence anno a ed (57 aa) co esponds
o a hypo he ical p o ein no ye cha ac e ized in he Me schnikowiaceae (da a no shown)
bu iden i ied as a mi ochond ial ATP syn hase
ε
chain-domain-con aining p o ein in he
Te ezia cla e yi myco hizal ungus (NCBI accession KAF8454923.1). The ac ha we ound
no p o eins below his size, which could co espond o he anno a ion o alse posi i es,
highligh s he high anno a ion quali y ob ained wi h he compu a ional pipeline and he
sequencing echnology applied.
The o al numbe o non-coding RNAs (ncRNA) p edic ed using s uc RNA inde
and he P am da abase was simila among he i e Cla ispo a san aluciae s ains (Table 2,
Supplemen a y Da a S2). The e was a high simila i y be ween s ains o he majo i y o
he ncRNA anno a ed, wi h he excep ion o ibosomal and ans e RNAs ( RNA and
RNA, espec i ely), whose quan i ies showed ele an in e -s ain a ia ion no di ec ly
co ela ed wi h he numbe o p edic ed coding sequences o wi h he genome size. Many
sequencing p ojec s igno e he compa ison o ncRNA be ween s ains, bu by de ailing
hei analysis, i may be possible o unde s and pa icula and in ica e mechanisms o
adap a ion o he en i onmen .
3.2. Compa a i e Genomics o Cla ispo a San alucieae S ains
To compa e s uc u al a ia ions be ween he genomes o he Cla ispo a san aluciae
s ains pai wise, do plo s we e ob ained (Figu e 1A). Resul s showed a s iking pa e n
o conse a ion o mos s ains, wi h a high deg ee o mac osyn eny mainly be ween he
ype s ain and s ains A1.19 and LB-NB-3.3. On he o he hand, s ains A1.5 and A1.7
showed some di e en ia ion, in pa icula by he p esence o se e al dele ions in pa s o
he genome, as ep esen ed by ansloca ions (“jumps” in he do plo ) away om he main
diagonal. In pa icula , s ain A1.5 seems o ha e mesosyn eny wi h he ype s ain, since
we can gene ally obse e conse a ion o he gene con en . Howe e , in some pa s o he
genome, many in e sions (blue lines) and ansloca ions we e de ec ed. This obse a ion
is no conco dan wi h he simila i ies obse ed in he ITS and D1/D2 egions [
4
], which
showed ha s ain A1.5 is mos closely ela ed o he ype s ain.
J. Fungi 2022, 8, x FOR PEER REVIEW 8 o 18
o he ou Azo ean s ains (Supplemen a y Da a S1). Acco ding o ou p e ious wo k [42]
on he cha ac e iza ion o isogenic isola es o wine S. ce e isiae yeas s, ansposable ele-
men s seem o be ela ed o he adap a ion o yeas s o he luc ua ing en i onmen al con-
di ions ound in he ha sh en i onmen o he Azo es a chipelago, and hese gene ic ea-
u es a e ela ed wi h impo an pheno ypic cha ac e is ics ha de e mine he s ains bi-
o echnological po en ial [43,44].
Figu e 1. Compa a i e genomics o Cla ispo a san aluciae genomes: (A) whole-genome do -plo com-
pa ison be ween he sequenced s ains in pai wise mode. Homologous egions a e plo ed as do s.
Red lines link pa allel homologous pai s, and blue lines link an i-pa allel pai s; (B) Venn diag am
indica ing he numbe o sha ed coding genes among Cla ispo a san aluciae s ains.
3.3. Func ional Anno a ion o Cla ispo a San aluciae P o eome
Fo his analysis, eggNOG-mappe unc ionally anno a ed he p edic ed open ead-
ing ames o Cla ispo a san aluciae, p o iding impo an insigh s in o hei biological sig-
ni icance (Table 2, Figu e 2). Be ween 3101 and 3103 genes we e assigned o a KO ca ego y,
co esponding o an a e age o 51.7% o all he anno a ed genes. A o al o 4180 genes o
Cla ispo a san aluciae ype s ain A1.18T (68.6% o he o al genes) we e clus e ed in o 24
COGs using eggNOG-mappe (Figu e 2A), which we e hen classi ied in o h ee main
unc ional ca ego ies (Figu e 2). This analysis e ealed low a ia ion be ween he i e
s ains which is in acco dance wi h he emaining anno a ion s a is ics shown be o e. O
no e is he ac ha he numbe o unc ionally anno a ed genes ob ained in all s ains
a ied be ween 68.5 and 69.1% (Table 2) and is a he low, as indica ed by he high num-
be o genes wi h “unknown unc ion” in panel B o Figu e 2 (g ay ba s; be ween 20.7 and
20.9%). Howe e , hese alues a e lowe han hose ob ained o o he species (Figu e 2C,
and ca ego y S in panel D), such as Cla ispo a lusi aniae, wi h 24%, (Candida in e media),
wi h 25%, and Me schnikowia eukau ii, wi h 24%, o e en o Saccha omyces ce e isiae (22%)
o To ulaspo a delb ueckii (22%), as shown in ou p e ious wo k [35]. This low numbe o
genes wi h “unknown unc ion” is a consequence o an imp o emen in he sequencing
and anno a ion pipelines no mally used o anno a e yeas genomes.
Func ional anno a ion o Cla ispo a san aluciae e ealed ha he highes pe cen age
o anno a ed genes (Figu e 2B) was ela ed o “me abolism” (be ween 27.3 and 27.7%),
ollowed by “cellula p ocesses and signaling” (26.4–26.6%). This esul is in ag eemen
wi h ha o o he yeas s o Me schnikowiaceae (Figu e 2, panels C and D), al hough his
Figu e 1.
Compa a i e genomics o Cla ispo a san aluciae genomes: (
A
) whole-genome do -plo
compa ison be ween he sequenced s ains in pai wise mode. Homologous egions a e plo ed
as do s. Red lines link pa allel homologous pai s, and blue lines link an i-pa allel pai s; (
B
) Venn
diag am indica ing he numbe o sha ed coding genes among Cla ispo a san aluciae s ains.
J. Fungi 2022,8, 52 8 o 18
A o al o 5564 coding genes we e ound o be sha ed be ween he i e Cla ispo a
san aluciae s ains, co esponding o he pangenome o he species (Figu e 1B). S ain NB-
LB-3.3 showed a su p isingly high numbe o unique genes (298), no sha ed by any o
he o he s ains, e lec ing i s adap a ion o a di e en ecological niche, as his s ain was
isola ed om I alian g apes in ec ed wi h D osophila suzukii. On he o he hand, 283 genes
we e sha ed only by he s ains isola ed om Azo ean ineya ds, indica ing adap a ion
mechanisms o he in ica e and a duous en i onmen al condi ions o he geog aphical
loca ion om which hey we e isola ed. Addi ionally, and o pa icula no e, is he ac ha
no ansposable elemen was iden i ied in he genome o s ain LB-NB-3.3, unlike he o he
ou Azo ean s ains (Supplemen a y Da a S1). Acco ding o ou p e ious wo k [
42
] on he
cha ac e iza ion o isogenic isola es o wine S. ce e isiae yeas s, ansposable elemen s seem
o be ela ed o he adap a ion o yeas s o he luc ua ing en i onmen al condi ions ound
in he ha sh en i onmen o he Azo es a chipelago, and hese gene ic ea u es a e ela ed
wi h impo an pheno ypic cha ac e is ics ha de e mine he s ains bio echnological
po en ial [43,44].
3.3. Func ional Anno a ion o Cla ispo a San aluciae P o eome
Fo his analysis, eggNOG-mappe unc ionally anno a ed he p edic ed open eading
ames o Cla ispo a san aluciae, p o iding impo an insigh s in o hei biological signi i-
cance (Table 2, Figu e 2). Be ween 3101 and 3103 genes we e assigned o a KO ca ego y,
co esponding o an a e age o 51.7% o all he anno a ed genes. A o al o 4180 genes
o Cla ispo a san aluciae ype s ain A1.18
T
(68.6% o he o al genes) we e clus e ed in o
24 COGs using eggNOG-mappe (Figu e 2A), which we e hen classi ied in o h ee main
unc ional ca ego ies (Figu e 2). This analysis e ealed low a ia ion be ween he i e
s ains which is in acco dance wi h he emaining anno a ion s a is ics shown be o e. O
no e is he ac ha he numbe o unc ionally anno a ed genes ob ained in all s ains
a ied be ween 68.5 and 69.1% (Table 2) and is a he low, as indica ed by he high numbe
o genes wi h “unknown unc ion” in panel B o Figu e 2(g ay ba s; be ween 20.7 and
20.9%). Howe e , hese alues a e lowe han hose ob ained o o he species (Figu e 2C,
and ca ego y S in panel D), such as Cla ispo a lusi aniae, wi h 24%, (Candida in e media),
wi h 25%, and Me schnikowia eukau ii, wi h 24%, o e en o Saccha omyces ce e isiae (22%)
o To ulaspo a delb ueckii (22%), as shown in ou p e ious wo k [
35
]. This low numbe o
genes wi h “unknown unc ion” is a consequence o an imp o emen in he sequencing
and anno a ion pipelines no mally used o anno a e yeas genomes.
Func ional anno a ion o Cla ispo a san aluciae e ealed ha he highes pe cen age
o anno a ed genes (Figu e 2B) was ela ed o “me abolism” (be ween 27.3 and 27.7%),
ollowed by “cellula p ocesses and signaling” (26.4–26.6%). This esul is in ag eemen
wi h ha o o he yeas s o Me schnikowiaceae (Figu e 2, panels C and D), al hough his
no el yeas species has a highe pe cen age o genes ela ed o me abolism, which poin s
o a supe io bio echnological po en ial o his species. The impo ance o his alue is
e en mo e e iden i we compa e i wi h he unc ional anno a ions o yeas s om o he
amilies, o which usually “in o ma ion s o age and p ocessing” is he mos ep esen ed
ca ego y, as is he case o T. delb ueckii and S. ce e isiae, as p e iously shown [
35
]. The
mos abundan COG ca ego y in he genome o Cla ispo a san aluciae A1.18
T
(panel A)
was “ ansla ion, ibosomal s uc u e, and biogenesis” (333 genes, ep esen ing 8% o he
anno a ed genes), ollowed closely by “pos ansla ional modi ica ion, p o ein u no e ,
chape ones” (328/7.8%). The leas abundan ca ego ies we e “ex acellula s uc u es”,
wi h only wo associa ed genes.
J. Fungi 2022,8, 52 9 o 18
J. Fungi 2022, 8, x FOR PEER REVIEW 10 o 18
Figu e 2. Func ional anno a ion o Cla ispo a san aluciae genome: (A) p o eome classi ica ion in o 23
unc ional ca ego ies, co esponding o clus e s o o hologous g oups (COGs): A, RNA p ocessing
and modi ica ion; B, ch oma in s uc u e and dynamics; C, ene gy p oduc ion and con e sion; D,
cell cycle con ol and mi osis; E, amino acid me abolism and anspo ; F, nucleo ide me abolism
and anspo ; G, ca bohyd a e me abolism and anspo ; H, coenzyme me abolism; I, lipid me ab-
olism; J, ansla ion; K, ansc ip ion; L, eplica ion and epai ; M, cell wall/memb ane/en elop bio-
genesis; O, pos ansla ional modi ica ion, p o ein u no e , chape one unc ions; P, ino ganic ion
anspo and me abolism; Q, seconda y S uc u e; S, unc ion unknown; T, signal ansduc ion; U,
in acellula a icking and sec e ion; Y, nuclea s uc u e; Z, cy oskele on; (B) classi ica ion o he
anno a ed genes in o ou la ge unc ional ca ego ies; (C) compa ison be ween he i e Cla ispo a
san aluciae s ains and o he ele an yeas species in p opo ions o he la ge unc ional ca ego ies;
(D) compa ison be ween ele an yeas species classi ica ion o he anno a ed genes in o 23 COG
ca ego ies; (E) pe cen age o CAZymes in he i e sequenced genomes o Cla ispo a san aluciae and
o he ele an yeas s, showing he dis ibu ion o p edic ed p o eins in o majo amilies.
Func ional anno a ion o Cla ispo a san aluciae was also accomplished using KEGG
Mappe —Recons uc Pa hway ool [32,34]. This ool comple ed KO-based mapping
Figu e 2.
Func ional anno a ion o Cla ispo a san aluciae genome: (
A
) p o eome classi ica ion in o
23 unc ional
ca ego ies, co esponding o clus e s o o hologous g oups (COGs): A, RNA p ocessing
and modi ica ion; B, ch oma in s uc u e and dynamics; C, ene gy p oduc ion and con e sion; D,
cell cycle con ol and mi osis; E, amino acid me abolism and anspo ; F, nucleo ide me abolism and
anspo ; G, ca bohyd a e me abolism and anspo ; H, coenzyme me abolism; I, lipid me abolism;
J, ansla ion; K, ansc ip ion; L, eplica ion and epai ; M, cell wall/memb ane/en elop biogenesis;
O, pos ansla ional modi ica ion, p o ein u no e , chape one unc ions; P, ino ganic ion anspo
and me abolism; Q, seconda y S uc u e; S, unc ion unknown; T, signal ansduc ion; U, in acellula
a icking and sec e ion; Y, nuclea s uc u e; Z, cy oskele on; (
B
) classi ica ion o he anno a ed genes
in o ou la ge unc ional ca ego ies; (
C
) compa ison be ween he i e Cla ispo a san aluciae s ains and
o he ele an yeas species in p opo ions o he la ge unc ional ca ego ies; (
D
) compa ison be ween
ele an yeas species classi ica ion o he anno a ed genes in o 23 COG ca ego ies;
(E) pe cen age
o
CAZymes in he i e sequenced genomes o Cla ispo a san aluciae and o he ele an yeas s, showing
he dis ibu ion o p edic ed p o eins in o majo amilies.
J. Fungi 2022,8, 52 16 o 18
Re e ences
1.
D umonde-Ne es, J.; F anco-Dua e, R.; Lima, T.; Schulle , D.; Pais, C. Associa ion be ween g ape yeas communi ies and he
ineya d ecosys ems. PLoS ONE 2017,12, e0169883. [C ossRe ] [PubMed]
2.
D umonde-Ne es, J.; F anco-Dua e, R.; Lima, T.; Schulle , D.; Pais, C. Yeas biodi e si y in ineya d en i onmen s is inc eased
by human in e en ion. PLoS ONE 2016,11, e0160579. [C ossRe ]
3.
D umonde-Ne es, J.; F anco-Dua e, R.; Viei a, E.; Mendes, I.; Lima, T.; Schulle , D.; Pais, C. Di e en ia ion o Saccha omyces
ce e isiae popula ions om ineya ds o he Azo es A chipelago: Geog aphy s. Ecology. Food Mic obiol.
2018
,74, 151–162.
[C ossRe ] [PubMed]
4.
D umonde-Ne es, J.; ˇ
Cadež, N.; Domínguez, Y.R.; Gallme ze , A.; Schulle #, D.; Lima, T.; Pais, C.; F anco-Dua e, R. Cla ispo a
san aluciae .a., sp. no ., a no el ascomyce ous yeas species isola ed om g apes. In . J. Sys . E ol. Mic obiol.
2020
,70, 6307–6312.
[C ossRe ]
5.
Gá donyi, M.A.; Ös e be g, M.A.; Rod igues, C.; Spence -Ma ins, I.; Hahn-Häge dal, B. High capaci y xylose anspo in
Candida in e media PYCC 4715. FEMS Yeas Res. 2003,3, 45–52. [C ossRe ]
6.
Geije , C.; Fa ia-Oli ei a, F.; Mo eno, A.D.; S enbe g, S.; Mazu kewich, S.; Olsson, L. Genomic and ansc ip omic analysis o
Candida in e media e eals he gene ic de e minan s o i s xylose-con e ing capaci y. Bio echnol. Bio uels
2020
,13, 48. [C ossRe ]
[PubMed]
7.
Mo eno, A.D.; Tomás-Pejó, E.; Olsson, L.; Geije , C. Candida in e media CBS 141442: A no el glucose/xylose co- e men ing
isola e o lignocellulosic bioe hanol p oduc ion. Ene gies 2020,13, 5363. [C ossRe ]
8.
D umonde-Ne es, J.; Fe nandes, T.; Lima, T.; Pais, C.; F anco-Dua e, R. Lea ning om 80 yea s o s udies: A comp ehensi e
ca alogue o non-Saccha omyces yeas s associa ed wi h i icul u e and winemaking. FEMS Yeas Res.
2021
,21, oab017. [C ossRe ]
9.
Mingo ance-Cazo la, L.; Clemen e-Jiménez, J.M.; Ma ínez-Rod íguez, S.; Las He as-Vázquez, F.J.; Rod íguez-Vico, F. Con i-
bu ion o di e en na u al yeas s o he a oma o wo alcoholic be e ages. Wo ld J. Mic obiol. Bio echnol.
2003
,19, 297–304.
[C ossRe ]
10.
Mo a a, A.; Loi a, I.; Esco , C.; del F esno, J.M.; Bañuelos, M.A.; Suá ez-Lepe, J.A. Applica ions o Me schnikowia pulche ima in
wine bio echnology. Fe men a ion 2019,5, 63. [C ossRe ]
11.
Daniel, H.M.; Lachance, M.A.; Ku zman, C.P. On he eclassi ica ion o species assigned o Candida and o he anamo phic
ascomyce ous yeas gene a based on phylogene ic ci cumsc ip ion. An onie Leeuwenhoek 2014,106, 67–84. [C ossRe ] [PubMed]
12.
Ku zman, C.P.; Robne , C.J.; Basehoa , E.; Wa d, T.J. Fou new species o Me schnikowia and he ans e o se en Candida
species o Me schnikowia and Cla ispo a as new combina ions. An onie Leeuwenhoek
2018
,111, 2017–2035. [C ossRe ] [PubMed]
13.
Lachance, M.-A.; Hu ado, E.; Hsiang, T. A s able phylogeny o he la ge-spo ed Me schnikowia clade. Yeas
2016
,33, 261–275.
[C ossRe ] [PubMed]
14.
Shen, X.X.; Opulen e, D.A.; Kominek, J.; Zhou, X.; S eenwyk, J.L.; Buh, K.V.; Haase, M.A.B.; Wiseca e , J.H.; Wang, M.;
Doe ing, D.T
.; e al. Tempo and Mode o Genome E olu ion in he Budding Yeas Subphylum. Cell
2018
,175, 1533–1545.e20.
[C ossRe ]
15.
Schwa z, K.; She lock, G. P epa a ion o yeas DNA sequencing lib a ies. Cold Sp ing Ha b. P o oc.
2016
,2016, 871–876. [C ossRe ]
16.
Ko en, S.; Walenz, B.P.; Be lin, K.; Mille , J.R.; Be gman, N.H.; Phillippy, A.M. Canu: Scalable and accu a e long- ead assembly ia
adap i e κ-me weigh ing and epea sepa a ion. Genome Res. 2017,27, 722–736. [C ossRe ] [PubMed]
17. Zimin, A.V.; Ma çais, G.; Puiu, D.; Robe s, M.; Salzbe g, S.L.; Yo ke, J.A. The MaSuRCA genome assemble . Bioin o ma ics 2013,
29, 2669–2677. [C ossRe ] [PubMed]
18.
Zimin, A.V.; Salzbe g, S.L. The genome polishing ool POLCA makes as and accu a e co ec ions in genome assemblies. PLoS
Compu . Biol. 2020,16, e1007981. [C ossRe ] [PubMed]
19.
Alonge, M.; Soyk, S.; Ramak ishnan, S.; Wang, X.; Goodwin, S.; Sedlazeck, F.J.; Lippman, Z.B.; Scha z, M.C. RaGOO: Fas and
accu a e e e ence-guided sca olding o d a genomes. Genome Biol. 2019,20, 224. [C ossRe ]
20.
Mikheenko, A.; P jibelski, A.; Sa elie , V.; An ipo , D.; Gu e ich, A. Ve sa ile genome assembly e alua ion wi h QUAST-LG.
Bioin o ma ics 2018,34, i142–i150. [C ossRe ]
21.
Weib, C.L.; Pais, M.; Cano, L.M.; Kamoun, S.; Bu bano, H.A. nQui e: A s a is ical amewo k o ploidy es ima ion using nex
gene a ion sequencing. BMC Bioin o m. 2018,19, 122. [C ossRe ]
22.
Seppey, M.; Manni, M.; Zdobno , E. BUSCO: Assessing Genome Assembly and Anno a ion Comple eness. In Me hods in Molecula
Biology; Humana: New Yo k, NY, USA, 2019; Volume 1962, pp. 227–245. ISBN 9781493991730.
23.
Yoon, S.-H.; Ha, S.-M.; Lim, J.; Kwon, S.; Chun, J. A la ge-scale e alua ion o algo i hms o calcula e a e age nucleo ide iden i y.
An onie Leeuwenhoek 2017,110, 1281–1286. [C ossRe ] [PubMed]
24.
S anke, M.; Schö mann, O.; Mo gens e n, B.; Waack, S. Gene p edic ion in euka yo es wi h a gene alized hidden Ma ko model
ha uses hin s om ex e nal sou ces. BMC Bioin o m. 2006,7, 62. [C ossRe ] [PubMed]
25.
S anke, M.; Mo gens e n, B. AUGUSTUS: A web se e o gene p edic ion in euka yo es ha allows use -de ined cons ain s.
Nucleic Acids Res. 2005,33, W465–W467. [C ossRe ]
26.
Cui, X.; Lu, Z.; Wang, S.; Jing-Yan Wang, J.; Gao, X. CMsea ch: Simul aneous explo a ion o p o ein sequence space and s uc u e
space imp o es no only p o ein homology de ec ion bu also p o ein s uc u e p edic ion. Bioin o ma ics
2016
,32, i332–i340.
[C ossRe ]

J. Fungi 2022,8, 52 17 o 18
27.
A ias-Ca asco, R.; Vásquez-Mo án, Y.; Nakaya, H.I.; Ma acaja-Cou inho, V. S uc RNA inde : An au oma ed pipeline and web
se e o RNA amilies p edic ion. BMC Bioin o m. 2018,19, 55. [C ossRe ] [PubMed]
28.
G i i hs-Jones, S.; Moxon, S.; Ma shall, M.; Khanna, A.; Eddy, S.R.; Ba eman, A. R am: Anno a ing non-coding RNAs in comple e
genomes. Nucleic Acids Res. 2005,33, D121–D124. [C ossRe ]
29.
Can alapied a, C.P.; He nández-Plaza, A.; Le unic, I.; Bo k, P.; Hue a-Cepas, J. eggNOG-mappe 2: Func ional Anno a ion,
O hology Assignmen s, and Domain P edic ion a he Me agenomic Scale. Mol. Biol. E ol. 2021,38, 5825–5829. [C ossRe ]
30.
Ta uso , R.L.; Galpe in, M.Y.; Na ale, D.A.; Koonin, E.V. The COG da abase: A ool o genome-scale analysis o p o ein unc ions
and e olu ion. Nucleic Acids Res. 2000,28, 33–36. [C ossRe ]
31. Kanehisa, M. KEGG: Kyo o Encyclopedia o Genes and Genomes. Nucleic Acids Res. 2000,28, 27–30. [C ossRe ] [PubMed]
32.
Kanehisa, M.; Sa o, Y.; Kawashima, M. KEGG mapping ools o unco e ing hidden ea u es in biological da a. P o ein Sci.
2021
.
[C ossRe ] [PubMed]
33.
Can a el, B.I.; Cou inho, P.M.; Rancu el, C.; Be na d, T.; Lomba d, V.; Hen issa , B. The Ca bohyd a e-Ac i e EnZymes da abase
(CAZy): An expe esou ce o glycogenomics. Nucleic Acids Res. 2009,37, D233–D238. [C ossRe ] [PubMed]
34.
Kanehisa, M.; Sa o, Y. KEGG Mappe o in e ing cellula unc ions om p o ein sequences. P o ein Sci.
2020
,29, 28–35.
[C ossRe ] [PubMed]
35.
San iago, C.; Ri o, T.; Viei a, D.; Fe nandes, T.; Pais, C.; Sousa, M.J.; Soa es, P.; F anco-Dua e, R. Imp o emen o o ulaspo a
delb ueckii genome anno a ion: Towa ds he exploi a ion o genomic ea u es o a bio echnologically ele an yeas . J. Fungi
2021
,
7, 287. [C ossRe ]
36. Sun, Y.B. FasPa se : A package o manipula ing sequence da a. Zool. Res. 2017,38, 110–112. [C ossRe ]
37.
Nguyen, L.T.; Schmid , H.A.; Von Haesele , A.; Minh, B.Q. IQ-TREE: A as and e ec i e s ochas ic algo i hm o es ima ing
maximum-likelihood phylogenies. Mol. Biol. E ol. 2015,32, 268–274. [C ossRe ]
38.
Du ens, P.; Klopp, C.; Bi eau, N.; Fi on-Ouhabi, V.; Demen hon, K.; Accocebe y, I.; She man, D.J.; Noël, T. Genome Sequence o
he Yeas Cla ispo a lusi aniae Type S ain CBS 6936. Genome Announc. 2017,5, 30–31. [C ossRe ]
39.
Kannan, A.; Asne , S.A.; T achsel, E.; Kelly, S.; Pa ke , J.; Sangla d, D. Compa a i e Genomics o he Elucida ion o Mul id ug
Resis ance in Candida lusi aniae. MBio 2019,10, e02512-19. [C ossRe ]
40.
Mo eno, A.D.; Tellg en-Ro h, C.; Sole , L.; Daina , J.; Olsson, L.; Geije , C. Comple e Genome Sequences o he Xylose-Fe men ing
Candida in e media S ains CBS 141442 and PYCC 4715. Genome Announc. 2017,5, e00138-17. [C ossRe ]
41.
Ga ba ino, J.E.; Gibbons, I.R. Exp ession and genomic analysis o midasin, a no el and highly conse ed AAA p o ein dis an ly
ela ed o dynein. BMC Genom. 2002,3, 18. [C ossRe ]
42.
F anco-Dua e, R.; Bigey, F.; Ca e o, L.; Mendes, I.; Dequin, S.; San os, M.A.S.; Pais, C.; Schulle , D. In as ain genomic and
pheno ypic a iabili y o he comme cial Saccha omyces ce e isiae s ain Zyma lo e VL1 e eals mic oe olu iona y adap a ion o
ineya d en i onmen s. FEMS Yeas Res. 2015,15, o 063. [C ossRe ] [PubMed]
43.
F anco-Dua e, R.; Umek, L.; Zupan, B.; Schulle , D. Compu a ional app oaches o he gene ic and pheno ypic cha ac e iza ion
o a Saccha omyces ce e isiae wine yeas collec ion. Yeas 2009,26, 675–692. [C ossRe ]
44.
F anco-Dua e, R.; Umek, L.; Mendes, I.; Cas o, C.C.; Fonseca, N.; Ma ins, R.; Sil a-Fe ei a, A.C.; Sampaio, P.; Pais, C.;
Schulle , D
. New in eg a i e compu a ional app oaches un eil he Saccha omyces ce e isiae pheno-me abolomic e men a i e
p o ile and allow s ain selec ion o winemaking. Food Chem. 2016,211, 509–520. [C ossRe ]
45.
Da ies, G.J.; Glos e , T.M.; Hen issa , B. Recen s uc u al insigh s in o he expanding wo ld o ca bohyd a e-ac i e enzymes.
Cu . Opin. S uc . Biol. 2005,15, 637–645. [C ossRe ]
46.
Piombo, E.; Sela, N.; Wisniewski, M.; Ho mann, M.; Gullino, M.L.; Alla d, M.W.; Le in, E.; Spada o, D.; D oby, S. Genome
sequence, assembly and cha ac e iza ion o wo Me schnikowia uc icola s ains used as biocon ol agen s o pos ha es diseases.
F on . Mic obiol. 2018,9, 593. [C ossRe ]
47.
Fe nandes, T.; Sil a-Sousa, F.; Pe ei a, F.; Ri o, T.; Soa es, P.; F anco-Dua e, R.; Sousa, M.J. Bio echnological Impo ance o
To ulaspo a delb ueckii: F om he Obscu i y o he Spo ligh . J. Fungi 2021,7, 712. [C ossRe ]
48. Sahay, S. Wine enzymes: Po en ial and p ac ices. Enzym. Food Bio echnol. P od. Appl. Fu u . P ospec . 2018, 73–92. [C ossRe ]
49.
Daenen, L.; Saison, D.; S e ckx, F.; Del aux, F.R.; Ve ach e , H.; De delinckx, G. Sc eening and e alua ion o he glucoside
hyd olase ac i i y in Saccha omyces and B e anomyces b ewing yeas s. J. Appl. Mic obiol.
2008
,104, 478–488. [C ossRe ]
[PubMed]
50.
S eenwyk, J.L.; Opulen e, D.A.; Kominek, J.; Shen, X.X.; Zhou, X.; Labella, A.L.; B adley, N.P.; Eichman, B.F.; ˇ
Cadež, N.;
Libkind, D
.; e al. Ex ensi e loss o cell-cycle and DNA epai genes in an ancien lineage o bipola budding yeas s. PLoS Biol.
2019,17, e3000255. [C ossRe ] [PubMed]
51.
Dujon, B.; She man, D.; Fische , G.; Du ens, P.; Casa egola, S.; La on aine, I.; de Mon igny, J.; Ma ck, C.; Neu église, C.; Talla, E.;
e al. Genome e olu ion in yeas s. Na u e 2004,430, 35–44. [C ossRe ] [PubMed]
52. Scannell, D.R.; Bu le , G.; Wol e, K.H. Yeas genome e olu ion— he o igin o he species. Yeas 2007,24, 929–942. [C ossRe ]
53.
Gómez, S.; Be dugo, S.; Mena, R. Occu ence o indigenous a buscula myco hizal ungi associa ed wi h he hizosphe e o he
naidípalm in Colombia. Cienc. Tecnol. Ag opecu. 2020,21, e1275.
54.
Res epo-Co ea, S.; Pineda-Meneses, E.; Rios-Oso io, L. Mechanisms o ac ion o ungi AND bac e ia used as bio e ilize s in
ag icul u al soils: A sys ema ic e iew. Co poica. Tecnol. Ag opecu. 2017,18, 335–351.
J. Fungi 2022,8, 52 18 o 18
55.
Youdkes, D.; Helman, Y.; Bu dman, S.; Ma an, O.; Ju ke i ch, E. Po en ial con ol o po a o so o disease by he obliga e
p eda o s bdello ib io and like o ganisms. Appl. En i on. Mic obiol. 2020,86, e02543-19. [C ossRe ] [PubMed]
56.
Leon-T acca, B.; A é alo-Ga dini, E.; Bouchon, A.S. Sudden dea h o Theob oma cacao L. caused by Ve icillium dahliae Kleb. In
Pe u and i s in i o biocon ol. Cienc. Tecnol. Ag opecu. 2019,20, 133–148. [C ossRe ]
57.
López-He nández, F.; Co és, A.J. Las -Gene a ion Genome–En i onmen Associa ions Re eal he Gene ic Basis o Hea Tole ance
in Common Bean (Phaseolus ulga is L.). F on . Gene . 2019,10, 954. [C ossRe ] [PubMed]
58.
Blai , M.W.; Co és, A.J.; Fa me , A.D.; Huang, W.; Ambachew, D.; Va ma Penme sa, R.; Ca asquilla-Ga cia, N.; Asse a, T.;
Cannon, S.B. Une en ecombina ion a e and linkage disequilib ium ac oss a e e ence SNP map o common bean (Phaseolus
ulga is L.). PLoS ONE 2018,13, e0189597. [C ossRe ] [PubMed]
59.
Co és, A.J.; López-He nández, F.; Oso io-Rod iguez, D. P edic ing The mal Adap a ion by Looking In o Popula ions’ Genomic
Pas . F on . Gene . 2020,11, 564515. [C ossRe ]
60.
Co és, A.J.; López-He nández, F. Ha nessing c op wild di e si y o clima e change adap a ion. Genes
2021
,12, 783. [C ossRe ]