Whole-genome sequencing and annotation of the yeast Clavispora santaluciae reveals important insights about its adaptation to the vineyard environment

  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. 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