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Angustifolius lupine (NLL, Lupinus angustifolius L.) chirimwa cheleguminous chinoshandiswa kugadzira chikafu nekuvandudza ivhu.Kuwedzera kwepasi rose kweNLL sechirimwa kwakwezva akawanda pathogenic fungi, kusanganisira lupine anthracnose, iyo inokonzeresa chirwere cheanthracnose.Maviri alleles, Lanr1 neAnMan, ayo anopa kuwedzera kuramba, akashandiswa mukuberekesa kweNLL, asi ari pasi pemamorekuru maitiro anoramba asingazivikanwe.Muchidzidzo ichi, mamaki eLanr1 neAnMan akashandiswa kuongorora European NLL samples.Kuongororwa kwejekiseni munzvimbo inodzorwa kwakasimbisa kushanda kwevanopa vanopa.Differential gene expression profiling yakaitwa pamitsetse isingachinjiki uye mitsara inotapukira.Kupokana neAnthracnose kwakabatana nekuwedzeredza kweiyo gene ontology mazwi "GO:0006952 Defence Response", "GO:0055114 Redox process", uye "GO:0015979 Photosynthesis".Mukuwedzera, iyo Lanr1 (83A: 476) mutsara wakaratidza zvakakosha transcriptome reprogramming nokukurumidza mushure meinoculation, nepo mamwe mitsetse yakaratidza kunonoka mumhinduro iyi nemaawa anenge 42.Mhinduro dzekudzivirira dzinosanganiswa neTIR-NBS, CC-NBS-LRR uye NBS-LRR majini, mapuroteni gumi anobatanidzwa mupathogenesis, lipid transfer proteins, endoglucan-1,3-β-glucosidase, glycine-rich cell wall proteins, uye majini kubva kune reactive nzira yeokisijeni.Mhinduro dzekutanga ku83A: 476, kusanganisira kudzvanyirira kwemajini ane hukama nephotosynthesis, yakabatana nekubudirira kuchengetedzwa panguva yekukura kwezvinomera chikamu chefungal biology, zvichiratidza kuti chinhu chinokonzera kusadzivirirwa.Maitiro eMandeloop anodzikiswa, sezvakaita kukweva kwese kwakatwasuka.
Lupine ine mashizha nhete (NLL, Lupinus angustifolius L.) ipuroteni yakakwira cereal inobva kumadokero kwedunhu reMediterranean1,2.Parizvino chirimwa sechirimwa chekudya kumhuka nevanhu.Inoonekwawo semupfudze wegirinhi mumasisitimu ekutenderera kwezvirimwa nekuda kwekugadziriswa kwenitrogen nesymbiotic nitrogen inogadzirisa mabhakitiriya uye kuvandudzwa kwese kwevhu.NLL yakaita hurongwa hwekukurumidza hwekuchengetwa kwemhuri muzana remakore rapfuura uye ichiri pasi pekudzvinyirirwa kwepamusoro3,4,5,6,7,8,9,10,11,12.Nekurima kwakapararira kweNLL, kutevedzana kwepathogenic fungi kwakagadzira niches nyowani dzekurima uye kwakakonzera zvirwere zvitsva zvinoparadza zvirimwa. Chakanyanya kushamisa kune varimi ve lupine nevarimi kwaive kutaridzika kweanthracnose, yakakonzerwa neiyo pathogenic fungus, Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13. Chakanyanya kushamisa kune varimi ve lupine nevarimi kwaive kutaridzika kweanthracnose, yakakonzerwa neiyo pathogenic fungus, Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13. Наиболее примечательным для фермеров и селекционеров люпина было появление антракноза, вызванного патогенным грибком Nitrorihum Colletotrichum (3) Chinonyanya kukosha kuvarimi velupine nevarimi kwaive kubuda kweanthracnose yakakonzerwa neiyo pathogenic fungus Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13.对于羽扇豆农民和饲养者來说，最引人注目的是炭疽病的起现，它是由病原真菌Colletotrichum lupini).对于羽扇豆农民和饲养者來說，最引人注目的是炭疽病的出现，它是由病原真菌Colletotrichum lupini). Наиболее поразительным для фермеров и селекционеров люпина является появление антракноза, вызываемого патогенгеным гриtricбком Colpile . Chinonyanya kushamisa kuvarimi nevarimi ve lupine kubuda kwe anthracnose inokonzerwa nechirwere chepathogenic fungus Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13.Mishumo yekutanga yechirwere ichi yakabva kuBrazil neUnited States, iine zviratidzo zvinoonekwa muna 1912 na1929 zvakateerana.Zvisinei, pashure pemakore anenge 30, hosha yacho yakanzi Colletotrichum gloeosporioides (Penz.) Penz. & Sacc., teleomorph Glomerella cingulata (Stoneman) Spauld. & Sacc., teleomorph Glomerella cingulata (Stoneman) Spauld. & Sacc., телеоморф Glomerella cingulata (Stoneman) Spauld. & Sacc., teleomorph of Glomerella cingulata (Stoneman) Spauld. & Sacc.，有目的形态的Glomerella cingulata (Stoneman) Spauld. & Sacc.，有目的形态的Glomerella cingulata (Stoneman) Spauld. & Sacc., Glomerella cingulata (Stoneman) Spauld в Целенаправленной морфологии. & Sacc., Glomerella cingulata (Stoneman) Spauld in Targeted Morphology. & H. Schrenk,. & H. Schrenk, .uye H. Schrenk. & H.施伦克，. & H.施伦克，.uye H. Schlenk, .Chirwere chekutanga che phenotyping chakaitwa pakati pezana remakore rechi20 chakaratidza kupikisa muNLL uye yellow lupine (L. luteus L.) accessions, asi zvose zvitsvuku zve lupine (L. albus L.) zvakaongororwa zvakaedzwa zvakanyanya zvakanyanya15,16.Zvidzidzo zvakaratidza kuti kukura kweanthracnose kunobatanidza nekuwedzera kwemvura (kunyorova kwemhepo) uye kutonhora (muchikamu che12-28 ° C), zvichiita kuti kutyora kwekupikisa pakakwirira kwekushisa17, 18. Kutaura zvazviri, nguva yaidiwa kuti conidia imere uye chirwere chacho chitange , yakanga iri nguva ina shoma pa 24 ° C (12 ° C) (12 maawa) pasi pe9 (12 maawa).Saka, kudziya kwepasirese kuri kuitika kwakakonzera kupararira kweanthracnose.Nekudaro, chirwere ichi chakaonekwa muFrance (1982) neUkraine (1983) sechiratidzo chekutyisidzira kwaiuya, asi sezviri pachena chakafuratirwa neindasitiri yelupine panguva iyoyo20,21.Makore mashoma gare gare, chirwere chinoparadza ichi chakapararira pasi rose uye chakabatawo nyika huru dzinogadzira lupine dzakaita seAustralia, Poland neGermany22,23,24.Kutevera kubuda kweanthracnose pakati pemakore ekuma1990, kuongororwa kwakadzama kwakaguma nekuzivikanwa kwevanopa vakati wandei vanopokana mumasamples eNLL19.NLL kuramba anthracnose inodzorwa neakasiyana maviri akasimba alleles anowanikwa mune akasiyana germplasm masosi: Lanr1 mu cultivar Tanjil uye Wonga uye AnMan mukurima.Mandalay 25, 26. Aya alleles anozadzisa mamorekuru anocherekedza anotsigira kusarudzwa kwejemisi rekudzivirira muchirongwa chekuberekesa25,26,27,28,29,30.Mutsara wekuberekesa unoramba 83A: 476 wakatakura Lanr1 allele wakayambutswa nemutsara wemusango weP27255 kuti uwane huwandu hwevanhu veRIL vachipatsanura kurwisa anthracnose, izvo zvakaita kuti zvikwanise kupa Lanr1 locus kuchromosome NLL-1131, 32, 32, 33. framework, NLL yakaratidza nzvimbo yeese matatu alleles pachromosome imwechete (NLL-11), asi munzvimbo dzakasiyana29,34,35.Nekudaro, nekuda kwenhamba diki yeRILs uye yakakura genetic chinhambwe pakati pemakaki uye anoenderana alleles, hapana mhedziso dzakavimbika dzinogona kutorwa pamusoro pemajeni awo ari pasi.Nekune rumwe rutivi, kushandiswa kwereverse genetics mu lupins kwakaoma nekuda kwekuderera kwavo kwekuzvarwa patsva, izvo zvinoita kuti genetic manipulation iwande37.
Kuvandudzwa kwejemusi remumba rakatakura aleli inodiwa muhomozygous state, se83A:476 (Lanr1) neMandelup (AnMan), yakavhura musuwo wekudzidza kuramba kweanthracnose pamberi pekuvapo kwekupokana kusanganiswa kwealleles muvanhu vesango.Mikana yemakemikari maitiro.Enzanisa mhinduro dzekudzivirira dzinogadzirwa neakananga genotypes.Chidzidzo ichi chakaongorora mhinduro yepakutanga yezvinyorwa zveNLL kune C. lupini vaccination.Chekutanga, yeEuropean NLL germplasm panel ine mitsara ye215 yakaongororwa pachishandiswa mamorekuru anomaka Lanr1 uye AnMan alleles.Anthracnose phenotyping yakazoitwa pamitsetse ye50 NLL, yakambosarudzirwa mamorekuru mamaki, pasi pemamiriro akadzorwa.Zvichienderana nezviyedzo izvi, mitsara mina inosiyana mukuramba kweanthracnose uye Lanr1/AnMan allelic composition yakasarudzwa kusiyanisa yekudzivirira geni kutaura profiling vachishandisa nzira mbiri dzinopindirana: yakakwirira-kuburikidza RNA sequencing uye chaiyo-nguva PCR quantification.
Kutariswa kweseti yeNLL germplasm (N = 215) ine mamaki Lanr1 (Anseq3 uye Anseq4) uye AnMan (Anseq4) uye AnMan (AnManM1) yakaratidza kuti mutsara mumwe chete (95726, padhuze neSalamanca-b) unokurisa "kupokana" allele kune ese mamarker, nepo "Presusables" zvese zvinowanikwa mu58 marker. (~ 73.5%) mitsetse.Mitsetse gumi nenhatu yakaburitsa maviri "resistant" alleles eLanr1 marker, uye mitsetse misere yakagadzira "resistant" alleles yeLanr1.marker.Iyo "kupokana" allele yeAnMan marker (Supplementary Tafura S1).Mitsetse miviri yaive heterozygous yeAnseq3 marker uye imwe heterozygous yeAnManM1 marker.Mitsetse ye42 (19.5%) yakatakura zvikamu zvakapesana zveAnseq3 neAnseq4 alleles, zvichiratidza kuwanda kwehuwandu hwekubatanidza pakati peidzi loci mbiri.Anthracnose phenotypes pasi pemamiriro ezvinhu akadzorwa (Supplementary Table S2) yakaratidza kusiyana-siyana mukupikisa kwakaedzwa genotypes, iyo yakaratidzwa mukuoma kweanthracnose.Misiyano yezvibodzwa zvakaenzana kubva ku1.8 (inopikisa zvine mwero) kuenda ku6.9 (inobatika) uye misiyano yehuremu hwechirimwa kubva pa0.62 (inobatika) kusvika ku4.45 g (inodzivirirwa). Ikoko kwakanga kune kuwirirana kwakakosha pakati pezviyero zvakaonekwa mukudzokorora kuviri kwekuedza (0.51 yezvirwere zvakakomba, P = 0.00017 uye 0.61 yehuremu hwemiti, P <0.0001) pamwe chete nepakati pezvikamu zviviri izvi (- 0.59 uye - 0.77, P0100). Pakanga paine kuwirirana kwakakosha pakati pezviyero zvakacherechedzwa mukudzokorora kuviri kwekuyedza (0.51 yezvirwere zvekuomarara zvibodzwa, P = 0.00017 uye 0.61 yehuremu hwechirimwa, P <0.0001) pamwe nepakati pezviviri izvi paramita (- 0.59 uye - 0.0.0.0. Выявлена ​​достоверная корреляция между значениями, наблюдаемыми в двух повторностях эксперимента (0,51 для баллов тяжезд00, 1 = массы растения, P < 0,0001), а также между этими двумя параметрами (-0,59 и -0,77, Р < 0,0001) 0,0001). Kubatana kwakakosha kwakawanikwa pakati pehukoshero hwakacherechedzwa mukudzokorora kuviri kwekuyedza (0.51 yezvirwere zvekuomarara zvibodzwa, P = 0.00017 uye 0.61 yehuremu hwechirimwa, P <0.0001), pamwe nepakati pezviviri izvi paramita (- 0.59 uye -0.77, P <0 0.0).在两次重复实验中观察到的值之间存在显着相关性（疾病严重程度评分為0.51,P = 70.000. 0.0001）以及這两个参数之间（- 0.59 和- 0.77，P <0.0001).在 两 次 重复 实验 中 观察 的值 之间 存在 相关性 （疾病 严重 程度 程度 评分0為 0 1 = 0為 0 1 = 0 1 = 0 1 = 0 1 = 0 1 = 1 7 ， 植物 為 為 0.61 , p <0.0001） 以及 两 个 参数 之间 （（（- 0.59 和– 0.59 和– 0.59 和– 0.50 ,–0 0 0 7) .– 0 0 0 7 .– 0 0 7 .– 0 0 7 . Наблюдалась значительная корреляция между значениями, наблюдаемыми в двух повторностях (оценка тяжести заболевания 0,51, 60, 70, 10 = P <0,0001), и между этими двумя параметрами (-0,59 и -0,0001) 0,77, P <0,0001. Paive nekudyidzana kwakakosha pakati pehunhu hwakacherechedzwa muduplicate (chirwere chekuomarara mamakisi 0.51, P = 0.00017 uye uremu hwechirimwa 0.61, P <0.0001) uye pakati pemaparamita maviri aya (-0.59 uye -0 .0001) 0.77, P <0.00. ).Zviratidzo zvinowanzoonekwa mumiti inotapukira zvinosanganisira kinking uye kumonyoroka kwedzinde rakafanana ne "uta hwemufudzi" chimiro, chinoteverwa neoval maronda ane orange / pink sporozoites (Supplementary Fig. 1).Makwikwi eAustralia akatakura Lanr1 (83A:476 uye Tanjil) uye AnMan (Mandelup) majini anopikisa zvine mwero, 0.0331 uye 0.0036).Mimwe mitsetse inotakurawo "resistant" Lanr1 uye/kana AnMan alleles inoratidza zviratidzo zvechirwere.
Sezvineiwo, mitsetse mishoma yeNLL inoshaya chero "resistant" marker allele yakaratidza huwandu hwepamusoro hwekuramba kweanthracnose (inofananidzwa kana yakakwirira kupfuura yeLanr1 kana AnMan genotypes), yakadai seBoregine (P kukosha <0.0001 kune ese ma paramita), Bojar (P kukosha <0.000000000 kukosha kweP9 / P9) kukosha kwePo4 / P9. 0.0001 yezvibodzwa uye isina-yakakosha kurema). Sezvineiwo, mitsetse mishoma yeNLL inoshaya chero "resistant" marker allele yakaratidza huwandu hwepamusoro hwekuramba kweanthracnose (inofananidzwa kana yakakwirira kupfuura yeLanr1 kana AnMan genotypes), yakadai seBoregine (P kukosha <0.0001 kune ese ma paramita), Bojar (P kukosha <0.000000000 kukosha kweP9 / P9) kukosha kwePo4 / P9. 0.0001 yezvibodzwa uye isina-yakakosha kurema). Интересно, что несколько линий NLL, лишенных какого-либо резистентного е высокий, чем для генотипов Lanr1 или AnMan), таких как Boregine (значение P <0,0001 для обоих параметров), Bojar (значение P < 0,0001 0,0001 стения) и популяции B-549/79b (значение P <0,0001 для оценки и незначимо для массы). Sezvineiwo, mitsetse yakati wandei yeNLL isina chero 'resistant' marker allele yakaratidza huwandu hwepamusoro hwekupokana neanthracnose (inofananidzwa kana yakakwirira kupfuura yeLanr1 kana AnMan genotypes), yakadai seBoregine (P kukosha <0.0001 kune ese ma paramita), Bojar (P kukosha <0.0001 yehuwandu hwevanhu uye 7 b-9 huremu hwe0b-9) . P kukosha <0.0001 yekuongorora uye kwete yakakosha kune uremu).有趣的是，一些缺乏任何“抗性”标记等位基因的NLL 系显示出高水平的炭疽病抗性（帎ManAn1更高），例如Boregine（两个参数的P 值< 0.0001）、Bojar（P 值<得分为0.0001，植物重和量為0.0001（9得（9得（9)值< 0.0001, 重量不显着）. Zvinonakidza kuti mamwe maNLL masisitimu asina chero "antigenic" mamakisi anoratidza yakakwira yakatwasuka kupikisa (yakaenzana neLanr1 kana AnMan genes kana yakakwirira), yakadai seBoregine (zvose parameters P <0.0001), Bojar (P kukosha <0.0001, simiti inorema 0.001) uye 4 90 inokosha < 0 , 000 , 9 strain , 9b inokosha , 0 000 . Интересно, что некоторые линии NLL, лишенные каких-либо маркерных аллелей «резистентности», показали высокие уровни устойчерных аллелей «резистентности» чем у генотипов Lanr1 или AnMan), такие как Boregine (значение P для обоих параметров <0,0001), Bojar (значение P <0,0001, масса раст00лц1b/9) и 9 енка P-значение <0,0001, масса незначительна). Sezvineiwo, mamwe mitsara yeNLL inoshaya chero 'resistance' marker alleles yakaratidza mazinga akakwirira ekupikisa anthracnose (inofananidzwa kana yakakwirira kupfuura Lanr1 kana AnMan genotypes), yakadai seBoregine (P kukosha kwezvikamu zvose <0.0001) , Bojar (P-value <0.000/501,0001,0001,0001, 2001, P-value <0.000.70101,0001,0001, 2001, P-value). <0.0001, uremu husina kukosha).Chiitiko ichi chinoratidza mukana weinoveli genetic sosi yekuramba, ichitsanangura kucherechedzwa kushaikwa kwehukama pakati pemarker genotypes uye chirwere phenotypes (P kukosha kubva ~ 0.42 kusvika ~ 0.98).Saka, bvunzo yeKolmogorov-Smirnov yakaratidza kuti data pamusoro pekuramba anthracnose yaiwanzo kugovaniswa kune zvibodzwa (P-values ​​0.25 uye 0.11) uye kukura kwechirimwa (P-values ​​0.47 uye 0.55), zvichiratidza kuti ndinofungidzira kuti mamwe alleles kupfuura Lanr1 uye AnMan anobatanidzwa.
Kubva pamigumisiro ye anthracnose resistance screening, mitsara ye 4 yakasarudzwa kuti iongorore transcriptome: 83A: 476, Boregine, Mandelup, uye Population 22660. Iyi mitsara yakadzoserwa kupikisa kweanthrax mukuedzwa kweinoculation neRNA sequencing, kunze kwekuti yakanga yakafanana neyokuedzwa kwekare.Zvibodzwa zvibodzwa zvaive zvinotevera: Boregin (1.71 ± 1.39), 83A: 476 (2.09 ± 1.38), Mandelup (3.82 ± 1.42) uye huwandu hwevanhu 22660 (6.11 ± 1.29).
Iyo Illumina NovaSeq 6000 protocol yakawana avhareji ye40.5 Mread pairs per sample (29.7 kusvika 54.4 Mreads) (Supplementary Table S3).Alignment zvibodzwa mukutevedzana kwereferensi zvakabva pa75.5% kusvika 88.6%.Avhareji yekubatana kwekuverenga kuverenga data pakati pekuyedzwa kwakasiyana pakati pezvinyorwa zvipenyu zvakabva pa0.812 kusvika 0.997 (zvinoreva 0.959). Kunze kweiyo 35,170 majini akaongororwa, 2917 yakaratidza hapana kutaura, uye mamwe 4785 majini akaratidzirwa padanho risingagoneki (base zvinoreva <5). Kunze kweiyo 35,170 majini akaongororwa, 2917 yakaratidza hapana kutaura, uye mamwe 4785 majini akaratidzirwa padanho risingagoneki (base zvinoreva <5). Из 35 170 проанализированных генов 2917 не проявляли экспрессии, а остальные 4785 генов экспрессировались на ненезнабрессии <br><br> Pakati pemajeni e35,170 akaongororwa, 2917 haana kuratidza kutaura, uye mamwe mazamu e4785 akasara akaratidzirwa pamwero usingakoshi (chigadziko chinoreva <5).在分析的35,170 个基因中，2917 个沒有表达，其他4785 个基因的表达可以忽略不计（基本平均值。35,170 Из 35 170 проанализированных генов 2917 не экспрессировались, а остальные 4785 генов имели незначительную экспрессировались, а остальные 4785 генов имели незначительную экспрессировались. Pakati pemajeni e35,170 akaongororwa, 2917 haana kuratidzwa uye akasara 4785 genes aiva nekutaura kusina basa (base zvinoreva <5).Nokudaro, nhamba yemajini inofungidzirwa yakaratidzwa (chigadziko chinoreva ≥ 5) panguva yekuedza yaiva 27,468 (78.1%) (Supplementary Table S4).
Kubva panguva yekutanga, mitsara yose yeNLL yakapindura kune inoculation yeC. lupini (strain Col-08) nekugadzirisa zvakare transcriptome (Table 1), zvisinei, kusiyana kukuru kwakaonekwa pakati pemitsara.Nokudaro, mutsara wekupikisa 83A: 476 (inotakura Lanr1 gene) yakaratidza zvakakosha transcriptome reprogramming panguva yekutanga (6 hpi) ne 31-69-fold kuwedzera kwenhamba yepamusoro-uye pasi-genes yakaenzaniswa nedzimwe nguva nguva panguva ino.Mukuwedzera, iyi yepamusoro yaiva yenguva pfupi, sezvo kutaura kwemajini mashomanana chete kwakaramba kwakashandurwa zvakanyanya panguva yechipiri (12 hpi).Sezvineiwo, Boregine, iyo yakaratidzawo yakakwira mwero wekupokana muyedzo yegraft, haina kuita yakakura kudaro transcriptional reprogramming panguva yekuyedza.Zvisinei, nhamba yemajini akasiyana-siyana (DEG) akanga akafanana neBoregine uye 83A: 476 pa12 HPI.Zvose Mandelup uye vanhu 22660 vakaratidza DEG peaks panguva yekupedzisira (48 l / s), zvichiratidza kunonoka kunonoka mumhinduro dzekudzivirira.
Nemhaka yokuti 83A: 476 yakawana hukuru hwezvinyorwa zvinyorwa zvekare mukupindura C. lupini pa6 HPI yakaenzaniswa nemimwe mitsara yose, ~ 91% yeDEGs yakaonekwa panguva ino yakanga iri mutsara-wakananga (Fig. 1).Nekudaro, pakanga paine kupindirana mumhinduro dzekutanga pakati pemitsara yekudzidza, se68.5%, 50.9%, uye 52.6% DEG muBoregine, Mandelup, uye huwandu hwevanhu 22660, zvichiteerana, zvakapindirana neaya anowanikwa mu83A:476 mune mamwe mapoinzi nenguva.Zvisinei, maDEG aya akaverengera chikamu chiduku chete (0.97-1.70%) yeDEGs yose ikozvino yakawanikwa uchishandisa 83A: 476.Mukuwedzera, 11 DEGs kubva mumitsara yose yakanga yakabatana panguva ino (Supplementary Tables S4-S6), kusanganisira zvikamu zvakajairika zvezvidziviriro zvemiti: lipid transfer protein (TanjilG_32225), endoglucan-1,3-β-glucoside enzyme (TanjilG_23384), maviri stress-inducible TaM2_13 mapuroteni SAMG513 TanjilG251 uye TanjilG23384 protein. 31), basic latex protein (TanjilG_32352), uye maviri glycine-rich structural cell wall mapuroteni (TanjilG_19701 uye TanjilG_19702).Paivawo nekupindirana kwakanyanya mumhinduro dzetranscriptome pakati pe83A:476 neBoregine pa24 HPI (yakazara 16-38% DEG) uye pakati peMandelup nePopulation 22660 pa48 HPI (yakazara 14-20% DEG).
Venn dhayagiramu inoratidza nhamba yemajini akasiyana-siyana (DEG) mumitsetse yakatsetseka yelupine (NLL) inoculated neColletotrichum lupini (strain Col-08 yakawanikwa kubva kuminda yelupine muWierzhenice, Poland, 1999).Mitsara yeNLL yakaongororwa yaive: 83A: 476 (inopikisa, inotakura Lanr1 allele), Boregine (inodzivirira, genetic background isingazivikanwe), Mandelup (inopikisa zvine mwero, inotakura AnMan allele) uye huwandu hwevanhu 22660 (inotapukira zvakanyanya).Chidimburidzo chekuti hpi chinomirira maawa mushure mekubayiwa.Zero tsika dzakabviswa kuti girafu rive nyore.
Seti yemajini akawandisa pa6 hpi akaongororwa kuvepo kwecanonical R gene domains (Supplementary Table S7).Ichi chidzidzo chakaratidza transcriptome induction yemhando yemhando yekudzivirira zvirwere neNBS-LRR domains chete pa83A:476.Seti iyi yaive negeneti rimwe reTIR-NBS-LRR (tanjilg_05042), shanu CC-NBS-LRR genes (tanjilg_06165, tanjilg_06162, tanjilg_22773, tanjilg_22640, uye tanjilg_16162R, Four NBS_16162), uye Four NBS-L1 RRE (tanjilg_16162) pamwe ne Four NBS-Lrr (tanjilg_16162) uye Four NBS-LRR (TANJILG_16162).Ese aya majini ane canonical domains akarongwa mumitsetse yakachengetedzwa.Pamusoro peiyo NBS-LRR domain genes, akati wandei eRLL kinase akabatidzwa pa6 hpi, inova imwe muBoregine (TanjilG_19877), maviri muMandelup (TanjilG_07141 neTanjilG_19877) uye muhuwandu 22660 (TanjilG_068G3777 A7 uye maviri: 6.
Majini ane mazwi akashandurwa zvakanyanya mukupindura inoculation neC. lupini (strain Col-08) akaiswa pasi peGene Ontology (GO) enrichment analysis (Supplementary Table S8). Iyo yakanyanya kunyanya kumiririrwa yebiological process term yaive "GO: 0006952 mhinduro yekudzivirira" iyo yakaonekwa mu6 kunze kwe16 (nguva × mutsara) kusanganiswa nekukosha kwepamusoro (P kukosha <0.001) (Fig. 2). Iyo yakanyanya kunyanya kumiririrwa yebiological process term yaive "GO: 0006952 mhinduro yekudzivirira" iyo yakaonekwa mu6 kunze kwe16 (nguva × mutsara) kusanganiswa nekukosha kwepamusoro (P kukosha <0.001) (Fig. 2). Наиболее часто чрезмерно представленным термином биологического процесса был «GO: 0006952 защитный отвенным термином биологического процесса был «GO: 0006952 защитный ответ», который песни комбинаций с высокой значимостью (значение P <0,001) (рис. 2). Iyo yainyanya kumiririrwa yebiological process term yaive 'GO:0006952 defence response', iyo yakaonekwa mu6 ye16 (nguva × mutsara) misanganiswa ine kukosha kwepamusoro (P kukosha <0.001) (Fig. 2).最常被过度代表的生物过程术语是“GO:0006952 防御反应”着性 (P 值< 0.001) (图2). Iyo inonyanya kumiririra biological process term ndeye "GO: 0006952 kudzivirira mhinduro", iyo inowanikwa mu6 ye16 (时间× 线) misanganiswa, ine kukosha kukuru (P kukosha <0.001) (图2) . Наиболее часто представленным термином биологического процесса был «GO: 0006952 Defence Response», который появлялся 10 × 6 излим с высокой значимостью (значение P <0,001) (рис. 2). Iyo inonyanya kumiririrwa yebiological process term yaive 'GO:0006952 Defense Response', iyo yakaonekwa mu6 kubva mu16 misanganiswa (nguva × mutsetse) ine kukosha kwepamusoro (P kukosha <0.001) (Fig. 2).Iri izwi raimiririrwa zvakanyanya panguva mbiri mu83A: 476 uye Boregine (6 uye 24 hpi) uye pane imwe nguva nzvimbo muMandelup uye Population 22660 (12 uye 6 hpi, zvichiteerana).Uyu ndiwo mugumisiro unotarisirwa, kuratidza mhinduro ye antifungal yemitsara inopikisa.Mukuwedzera, 83A: 476 yakapindura C. lupini nekukurumidza kupinza majini ane chokuita nekuputika kweoxidative inomiririrwa neshoko rokuti "GO: 0055114 redox process", zvichiratidza mhinduro chaiyo yekudzivirira, nepo Boregine yakaratidza mhinduro dzakananga dzekudzivirira, dzine chokuita neshoko rokuti 'GO'.:0006950 Stress mhinduro”.Vagari 22660 vakamisikidza mhinduro yakachinjika yekupikisa inosanganisira yechipiri metabolites, ichiratidza huwandu hwakawandisa hwematemu "GO: 0016104 Maitiro e triterpene biosynthesis" uye "GO: 0006722 Maitiro e triterpene metabolism" (matemu ese ari maviri akafanana seti yemajini ), tichifunga nezve mhedzisiro yePopure 2 2660. Pamusoro pezvo, kuita kwekutanga 83A:476 (6 hpi) uye kunonoka kuita Mandelup nePopulation 22660 inosanganisira izwi rokuti GO:0015979 'photosynthesis' nemamwe ane chekuita nebiological process.
Matemu ebioprocess gene ontology akasarudzwa mutsanangudzo yemajini anoratidzwa zvakasiyana panguva yemhinduro dze transcriptome ye narrow-leaved lupine (NLL) yakabaiwa neanthrax lupine (Col-08 strain yakawanikwa kubva kuminda yelupine kuWierzhenice, Poland, muna 1999) yakawedzeredzwa zvakanyanya.Mitsara yeNLL yakaongororwa yaiva: 83A: 476 (inopikisa, inotakura homozygous Lanr1 allele), Boregine (inodzivirira, isingazivikanwe genetic background), Mandelup (inopikisa zvine mwero, inotakura homozygous AnMan allele) uye vanhu 22660 (inobatwa).
Nekuti chidzidzo ichi chakanangana nekuona majini anobatsira mukuramba anthracnose, majini akaiswa kune mazwi GO "GO: 0006952 Defensive responses" uye "GO: 0055114 Redox maitiro" akaongororwa nekucheka-kubvisa sezvo yekutanga inoreva ≥ 30 ine kanenge mutsara mumwe chete.× poindi nenguva kusanganisa manhamba akakosha eiyo log2 (peta shanduko).Huwandu hwemajini akasangana nemaitiro aya aive makumi matanhatu neshanu eGO:0006952 uye 524 yeGO:0055114.
83A: 476 yakaratidza mbiri mbiri dzeDEG dzakatsanangurwa neizwi GO: 0006952, yekutanga pa6 genes painch (64 majini, kumusoro uye pasi regulation) uye yechipiri pamakumi maviri nemana majini painch (15 majini, kumusoro mutemo chete).Boregine yakaratidzawo kuti GO: 0006952 yakakwira panguva imwe chete, asi neDEG shoma (11 uye 8) uye yakasarudzika activation.Mandeloop yakaratidza nhongonya mbiri dzeGO: 0006952 pa 12 uye 48 HPI, ese akatakura gumi nemaviri majini (yekutanga aine majene e activating, uye yechipiri aine magene anodzvinyirira chete), nepo huwandu hwe22660 pa6 HPI (13 majini) yaive nehukuru hwekuwedzera kwepamusoro.mutemo.Zvinofanira kucherechedzwa kuti 96.4% yeGO: 0006952 DEG mumatunhu aya aive nemhando imwechete yekupindura (kumusoro kana pasi), zvichiratidza kupindirana kwakakosha mumhinduro dzekudzivirira kunyangwe kusiyana kwehuwandu hwemajini anobatanidzwa.Iro boka rakakura kwazvo remateedzana ane hukama neizwi GO:0006952 encodes iyo Nzara Stress-Yakabatana Mharidzo Protein 22 (SAM22-yakafanana), inova yekirasi gumi pathogenesis-yakabatana protein (PR-10) protein clade uye yakakosha protein latex.yakafanana (MLP-like) protein) protein (Fig. 3).Mapoka maviri aya aisiyana pachimiro chekutaura uye negwara remhinduro.Majini encoding SAM22-akaita mapuroteni airatidza kuenderana uye kwakakosha induction panguva dzekutanga nguva (6 kana 12 hpi) uye kazhinji aisapindura pakupera kwekuyedza (48 hpi), nepo MLP-senge mapuroteni airatidza kurongeka pa6 hpi.hpi.83A: 476 uye Mandelup pa48 hp/in, anenge ese mamwe mapoinzi edata akanga asingateereri.Pamusoro pezvo, misiyano yekutaura profiles yeSAM22-yakafanana neprotein genes yakateedzera kusiyanisa mukupokana kweanthracnose, sezvo mitsetse yakawanda inopokana yaive neakawanda mapoinzi enguva zvakanyanya kukonzeresa majini aya kupfuura mamwe magene anotapukira.Imwe LlR18A/B-yakafanana nePR-10 gene yakaratidza chimiro chekutaura chakafanana kune SAM22-yakafanana neprotein gene.
Izvo zvakakosha zveiyo biological process term "GO: 0006952 Defense Response" uye mataurirwo emhando dzemajini eLanr1 uye AnMan alleles akaonekwa.Iyo Log2 chiyero inomiririra log2 kukosha (kupeta shanduko) pakati peinoculated (Colletotrichum lupini, strain Col-08, yakawanikwa kubva kuminda lupine, Wizhenica, Poland, 1999) uye kutonga (sham-inoculated) zvirimwa panguva imwe chete.Mitsara ye lupine inotevera yakaongororwa: 83A: 476 (inodzivirira, inotakura homozygous Lanr1 allele), Boregine (inopikisa, genetic background isingazivikanwe), Mandelup (inopikisa zvine mwero, inotakura homozygous AnMan allele), uye Population 22660 (inobatwa).
Pamusoro pezvo, iwo maprofiles eRNA-seq mumiriri wemajini Lanr1 (TanjilG_05042) uye AnMan (TanjilG_12861) akaongororwa (Fig. 3).Iyo TanjilG_05042 gene yakaratidza mhinduro yakakosha (activation) pa83A: 476 chete panguva yekutanga (6 hpi), nepo TanjilG_12861 yaive yakakosha muMandeloop chete panguva mbiri: 6 hpi (pasi mutemo) uye 24 hpi (6 hpi).With.).inochinjika)).
Majeni akawandisa zvikuru muzwi rokuti GO: 0055114 "redox process" yaiva majini encoding cytochrome P450 mapuroteni uye peroxidase (Fig. 4).Kune masampuli akaparadzaniswa kubva ku83A: 476 pa6 HPI, yakakwira kana yakaderera log2 (kupeta shanduko) tsika (ye86.6% yemajini) yaiwanzoonekwa pakati pezvinomera uye kudzora zvirimwa, zvichiratidza mhinduro yepamusoro yeiyi genotype kune inoculating bonde.83A: 476 yakaratidza iyo inonyanya kukosha GO: 0055114 DEG pa6 hpi (503 majini), nepo mamwe mitsetse ari 48 hpi (Boregine, 31 majini; Mandelup, 85 majini; uye Population 22660, 78 majini)).Mune akawanda majini eGO: 0055114 mhuri, marudzi maviri emhinduro kune vaccination (activation uye inhibition) akaonekwa.Sezvineiwo, kusvika ku97.6% yeDEGs yakacherechedzwa izwi rekuti GO: 0055114 muMandelupe pa48 hp Aya maratidziro anoratidza kuti, kunyangwe chiyero chidiki diki (kureva, huwandu hweakachinja redox genes, 85 kupesana ne503), maitiro ekunonoka transcriptome mhinduro kune 7 mandeloup mhinduro dze6 mandeloup dzakafanana ne6A8.MuBoregine uye Population 22660, kusangana uku kwakaderera pa51.6% uye 75.6%, zvichiteerana.
Maitiro ekutaura kwezvikamu zvikuru zvezwi reiyo biological process "GO: 0055114 Redox process" yakaratidzwa.Iyo Log2 chiyero inomiririra log2 kukosha (kupeta shanduko) pakati peinoculated (Colletotrichum lupini, strain Col-08, yakawanikwa kubva kuminda lupine, Wizhenica, Poland, 1999) uye kutonga (sham-inoculated) zvirimwa panguva imwe chete.Mitsara ye lupine inotevera yakaongororwa: 83A: 476 (inodzivirira, inotakura homozygous Lanr1 allele), Boregine (inopikisa, genetic background isingazivikanwe), Mandelup (inopikisa zvine mwero, inotakura homozygous AnMan allele), uye Population 22660 (inobatwa).
83A: 476 Mhinduro dzeTranscriptomic kune inoculation neC. lupini (strain Col-08) yaisanganisira kunyarara kwakarongeka kwemajini anonzi GO: 0015979 "photosynthesis" uye mamwe maitiro ehupenyu ane hukama (FIG. 5).Iyi GO: 0015979 DEG set yaive ne 105 genes yakatsikiswa zvakanyanya pa6 hpi pa83A: 476.Muchikamu ichi, 37 majini akaderedzwa zvakare muMandelup pa48 HPI uye 35 panguva imwe chete muhuwandu hwe22660, kusanganisira gumi nemapfumbamwe DEGs akajairika kune ese genotypes.Hapana maDEG ane hukama neshoko GO: 0015979 akashandiswa zvakanyanya mune chero musanganiswa (mutsara x nguva).
Maitiro ekutaura kwezvikamu zvikuru zvezwi reiyo biological process "GO: 0015979 Photosynthesis" yakaratidzwa.Iyo Log2 chiyero inomiririra log2 kukosha (kupeta shanduko) pakati peinoculated (Colletotrichum lupini, strain Col-08, yakawanikwa kubva kuminda lupine, Wizhenica, Poland, 1999) uye kutonga (sham-inoculated) zvirimwa panguva imwe chete.Mitsara ye lupine inotevera yakaongororwa: 83A: 476 (inodzivirira, inotakura homozygous Lanr1 allele), Boregine (inopikisa, genetic background isingazivikanwe), Mandelup (inopikisa zvine mwero, inotakura homozygous AnMan allele), uye Population 22660 (inobatwa).
Zvichienderana nemhedzisiro yekusiyanisa kutaura kwekuongorora uye pamwe inobatanidzwa mumhinduro dzekudzivirira kurwisa pathogenic fungi, iyi seti yemajini manomwe akasarudzwa kuti averengerwe ekutaura profiles nechaiyo-nguva PCR (Supplementary Tafura S9).
Iyo putative protein gene TanjilG_10657 yakanyudzwa zvakanyanya mumitsara yese yakadzidzwa uye mapoinzi enguva zvichienzaniswa nekudzora (kutevedzera) zvirimwa (Supplementary Tables S10, S11).Pamusoro pezvo, chimiro chekutaura cheTanjilG_10657 chakaratidza kuwedzera kwemaitiro mukati meyedzo yemitsara yese.Population 22660 yakaratidza kunzwisiswa kwepamusoro kweTanjilG_10657 kune inoculation ne 114-fold activation uye yepamusoro-soro yehuwandu hwekutaura (4.4 ± 0.4) pa 24 HPI (Fig. 6a).Iyo PR10 LlR18A protein gene TanjilG_27015 yakaratidzawo activation pamitsara yese uye mapoinzi enguva, nekukosha kwenhamba pane dzakawanda data data (Fig. 6b).Zvakafanana neTanjilG_10657, iyo yepamusoro-soro yekutaura nhanho yeTanjilG_27015 yakaonekwa mu22660 inoculated vanhu pa24 HPI (19.5 ± 2.4).Asidhi endochitinase gene TanjilG_04706 yakanyatsogadziriswa mumitsara yose uye panguva dzose kunze kweBoregine 6 hpi (Fig. 6c).Yakasimbiswa zvakasimba panguva yekutanga (6 HPI) pa83A: 476 (ne10.5 nguva) uye yakawedzera zvishoma mune mamwe mitsara (ne6.6-7.5 nguva).Munguva yekuedza, kutaura kweTanjilG_04706 kwakaramba kuri pamatanho akafanana mu83A: 476 uye Boregine, nepo muMandelup nePopulation 22660 yakawedzera zvakanyanya, ichisvika pahutsika hwepamusoro (5.9 ± 1.5 uye 6.2 ± 1.5, zvichiteerana).Endoglucan-1,3-β-glucosidase-like gene TanjilG_23384 yakaratidza kushandiswa kwepamusoro panguva mbiri dzekutanga (6 uye 12 hpi) mumitsara yose kunze kwevanhu 22660 (Fig. 6d).Mamiriro epamusoro ekutaura eTanjilG_23384 akaonekwa panguva yechipiri (12 hpi) muMandelup (2.7 ± 0.3) uye 83A:476 (1.5 ± 0.1).Pa24 HPI, TanjilG_23384 kutaura kwaive kwakadzikira mumitsara yese yakadzidzwa (kubva pa0.04 ± 0.009 kusvika 0.44 ± 0.12).
Mataurirwo emaprofayiri emajini akasarudzwa (ag) anoratidzwa nehuwandu hwePCR.Nhamba 6, 12 uye 24 inomiririra maawa mushure mekudzivirira.Iyo LanDExH7 neLanTUB6 majini akashandiswa kuita zvakajairika uye LanTUB6 yaishandiswa kuita inter-series calibration.Mabhawa ekukanganisa anomiririra kutsauka kwakajairwa kwakavakirwa pazvinyorwa zvitatu zvebhayoloji, imwe neimwe iri avhareji yeatatu ehunyanzvi replicate. Kukosha kwehuwandu hwekusiyana kwematanho ekutaura pakati peinoculated (Colletotrichum lupini, strain Col-08, yakawanikwa muna 1999 kubva kumunda welupine muWierzenica, Poland) uye kutonga (kunyomba-inoculated) zvinomera zvinoratidzwa pamusoro pe data points (*P kukosha <0.05, **P kukosha ≤0 0.0 P value ≤0 0.0). Kukosha kwehuwandu hwekusiyana kwematanho ekutaura pakati peinoculated (Colletotrichum lupini, strain Col-08, yakawanikwa muna 1999 kubva kumunda welupine muWierzenica, Poland) uye kutonga (kunyomba-inoculated) zvinomera zvinoratidzwa pamusoro pe data points (*P kukosha <0.05, **P kukosha ≤0 0.0 P value ≤0 0.0). Статистическая значимость различий в уровнях экспрессии между инокулированными (Colletotrichum lupini, штам Col-08, уровнях экспрессии между инокулированными) (Colletotrichum lupini, штам Col-08, получен в 1999 в 1999) а) и контрольными (ложно инокулированными) растениями отмечена над точками данных (*значение P < 0,05, **значение P ≤ 0,01, ***значение 0, 0. Zvikamu zvakasiyana-siyana muhuwandu hwekutaura pakati peinoculated (Colletotrichum lupini, strain Col-08, yakawanikwa muna 1999 kubva kumunda we lupine muWierzhenice, Poland) uye kutonga (sham-inoculated) zvinomera zvinoonekwa pamusoro pe data points (*P kukosha <0.05, **P-value ≤P-00 0.01, ***P-value ≤ 0.0 01, *** ≤ 0.01 ≤ 0.01.接种（Colletotrichum lupini，Col-08株，1999年从波兰Wierzenica的羽扇豆田获得）和对照（模拟接种种种）椋异的统计学显着性标记在数据点上方（*P值< 0.05, **P 值≤ 0.01, ***P 值≤ 0.001).接种 （colletorichum lupini , color-08 株 ， 1999 年 波兰 波兰 wierzenica 的羽扇 获得） 和对照 鼈接种 对照 鼈接种 对照 对照 鼈接种 內 容內种 波兰 波兰的 统计学 显着性 标记 数据点 上方*p 值 <0.05, **P ≤ 0.01, ***P ≤ 0.001). Статистически значимые различия в уровнях экспрессии между инокулированными (Colletotrichum lupini, штамм Col-08, полученный с полей люпи, 9, 90 10 г. Zvikamu zvakasiyana-siyana mumazinga ekutaura pakati peinoculated (Colletotrichum lupini, strain Col-08, yakawanikwa kubva kuminda ye lupine muVerzhenice, Poland, muna 1999) uye kutonga (sham-inoculated) zvinomera zvinoonekwa pamusoro pe data points (*P kukosha <0.05 , **P-value ≤ 0-10 ≤ 0-11 ≤ 0.Mitsara yeNLL yakaongororwa yaiva: 83A: 476 (inopikisa, inotakura homozygous Lanr1 allele), Mandelup (inopikisa zvine mwero, inotakura homozygous AnMan allele), Boregine (inodzivirira, isingazivikanwe genetic background) uye vanhu 22660 (inobatwa).
Mumiriri wejini TanjilG_05042 paLanr1 locus yakaratidza yakasarudzika nzira yekutaura kubva kumaprofayiri akawanikwa kubva kuRNA-seq zvidzidzo (Fig. 6e).Kushandiswa kwakakosha kwejeni iri kwakaonekwa muMandelup uye 22660 vanhu (kusvika 39.7 uye 11.7 nguva, zvichiteerana), zvichikonzera mazinga ekutaura akanyanya (kusvika 1.4 ± 0.14 uye 7.2 ± 1.3, zvichiteerana).83A: 476 yakaratidzawo kumwe kukwidziridzwa kweTanjilG_05042 gene (kusvika ku3.8-fold), zvisinei, nhanho dzekutaura dzakawanikwa (0.044 ± 0.002) dzaive dzakaderera zvakapetwa makumi matatu pane dzakaonekwa muMandelup uye 22660 vanhu.yakaongororwa neqPCR yakaratidza kusiyana kukuru muhuwandu hwekutaura pakati pe genotypes mu mock-vaccinated (control) zvakasiyana-siyana, kusvika kune 58-fold musiyano pakati pevanhu 22660 uye 83A: 476, uyewo pakati pehuwandu hwevanhu 22660 uye 22660. Musiyano wakapetwa kaviri wakawanikwa pakati peBoregine neMandalup.
Jeni remunhu paAnMan locus, TanjilG_12861, rakaitwa mukupindura kubaiwa mu83A:476 neMandelup, rakanga risina kwarakarerekera muhuwandu hwe22660, uye rakadzikiswa muBoregine (Fig. 6f).Chirevo chetauro cheTanjilG_12861 gene chaive chepamusoro-soro mune inoculated 83A: 476 (0.14±0.01).The 17.4 kDa kirasi I heat shock shock protein gene TanjilG_05080 HSP17.4 yakaratidza yakaderera hama yekutaura mwero mumhando dzese dzakadzidzwa uye mapoinzi enguva (Fig. 6g).Kukosha kwepamusoro kwakaonekwa pa24 HPI muhuwandu hwe22660 (0.14 ± 0.02, kuwedzera kasere pakupindura kubayiwa).
Kuenzanisa kwemajini ekutaura profiles (Fig. 7) kwakaratidza kuwirirana kwepamusoro pakati peTanjilG_10657 nemamwe majini mana: TanjilG_27015 (r = 0.89), TanjilG_05080 (r = 0.85), TanjilG_05042 (r = 0.80G = 0.80 G_9), uye r = 0.80 G_9.Migumisiro yakadaro inogona kuratidza kuwirirana kwemajini aya panguva yemhinduro dzekudzivirira.Iyo TanjilG_12861 uye TanjilG_23384 majini airatidza akasiyana ekutaura profiles ane yakaderera Pearson correlation coefficient values ​​​​(kubva pa0.08 kusvika 0.43 uye -0.19 kusvika 0.28, zvichiteerana) zvichienzaniswa nemamwe majini.
Kuwirirana pakati pemageneti ekutaura profiles kwakaonekwa pachishandiswa kuwanda kwePCR.Mitsara yelupine inotevera yakaongororwa: 83A: 476 (inodzivirira, inotakura homozygous Lanr1 allele), Mandelup (inopikisa zvine mwero, inotakura homozygous AnMan allele), Boregine (inodzivirira, genetic background isingazivikanwe), uye Population 22660 (inobatwa).Nguva nhatu dzenguva dzakaverengerwa (6, 12 uye 24 maawa mushure mekudzivirira), kusanganisira inoculated (Colletotrichum lupini, strain Col-08, yakawanikwa kubva kuminda lupine muWierzhenice, Poland, mu1999) uye kutonga (sham-inoculated) zvirimwa.Chikero chinoratidza kukosha kwePearson correlation coefficient.
Kubva pane data yakawanikwa pa6 horsepower per inch, WGCNA yakaitwa pa9981 DEG yakaonekwa nekuenzanisa inoculated uye kudzora zvirimwa kuti zvitarise pamhinduro dzekutanga dzekudzivirira (Supplementary Table S12).Makumi maviri nemaviri gene modules (masumbu) akawanikwa aine correlated (yakanaka kana yakaipa) maratidziro ekutaura pakati pe genotypes uye misiyano yekuedza. Paavhareji, mazinga ekutaura kwemajini aive achidzika mukurongeka 83A:476 > Mandelup > Boregine > Population 22660 (mune maviri akasiyana, zvisinei, maitiro aya aive akasimba mukudzora zvirimwa). Paavhareji, mazinga ekutaura kwemajini aive achidzika mukurongeka 83A:476 > Mandelup > Boregine > Population 22660 (mune maviri akasiyana, zvisinei, maitiro aya aive akasimba mukudzora zvirimwa). В среднем уровни экспрессии генов снижались в порядке 83A:476 Mandelup > Boregine > Population 22660 (в обоих вариантах, однако, этальбинценцев, однако, этальбенденый растений). Paavhareji, mazinga ekutaura kwemajini akadzikira muhurongwa 83A:476 > Mandelup > Boregine > Population 22660 (mune maviri akasiyana, zvisinei, maitiro aya aive akasimba mukudzora zvirimwa).平均而言，基因表达水平按83A:476 > Mandelup > Boregine > Population 22660 的顺序下降（然而，在两种中种中变中变给區更强）.平均 而 言 , 基因 水平 按 83a: 476> mandelup> boregine> vanhu 22660 的顺序 下降 （，在 种 中 中 在 种 中 中 在 种 中 中 。 ........ ........ В среднем уровни экспрессии генов снижались в ряду 83A:476 Mandelup > Boregine > Population 22660 (однако в обоих вариантах эта тенденция тенденция систеный систены в обоих). ний). Paavhareji, mazinga ekutaura kwemajini akadzikira munhevedzano 83A:476 > Mandelup > Boregine > Population 22660 (zvisinei, mune ese ari maviri akasiyana, maitiro aya aive akasimba mukudzora zvirimwa).Vaccination yakakonzera kukwidziridzwa kwekutaura kwemajini, kunyanya mumamodule 18, 19, 14, 6 uye 1 (mukudzika kwekuita), mutemo wakashata (semuenzaniso mamodule 9 uye 20) kana neasina kwazvakarerekera (eg modules 11, 22, 8 uye 13).GO term enrichment analysis (Supplementary Table S13) yakaratidza "GO: 0006952 Protective responses" yeinoculated module (18) ine yakanyanya activation, kusanganisira majini akaongororwa neqPCR (TanjilG_04706, TanjilG_23384, TanjilG_1065G mafoto akawanda as sunjil 10657 yakawanda as TanjilG_1065G) synthesis modules (9).Module 18 concentrator (Fig. 8) yakaonekwa seTanjilG_26536 gene encoding PR-10-like LlR18B protein, uye module 9 concentrator yakaonekwa seTanjilG_28955 gene encoding the photosystem II PsbQ protein.A candidate anthracnose resistance gene Lanr1, TanjilG_05042, yakawanikwa mumodule 22 (Fig. 9) uye inobatanidzwa nemashoko okuti “GO:0044260 Cellular macromolecular metabolic process” uye “GO:0006355 Transcriptional regulation, DNA templating1G01 Tap.iyo gene inosimbisa kupisa kushushikana kwekunyora chinhu A-4a (HSFA4a).
Weighted network ongororo yejini co-kutaura kwemamodule ane yakanyanyisa kumiririra biological process mazwi "GO: 0006952 Defense mhinduro".Ligation yakaitwa nyore kuratidza iwo majini mana akaongororwa neqPCR (TanjilG_04706, TanjilG_23384, TanjilG_10657 uye TanjilG_27015).
Weighted network wongororo yegene co-expression yemodule ine yakawandisa biological process term "GO: 0006355: Transcriptional regulation, DNA templating" uye kutakura mumiriri weanthracnose resistance gene Lanr1 TanjilG_05042.Ligation yakareruka kuti isiyanise TanjilG_05042 gene nepakati TanjilG_01212 gene.
Kuongororwa kweAnthracnose resistance yakaunganidzwa muAustralia kwakaratidza kuti mizhinji yembesa dzakatanga kuburitswa dzaive nenjodzi;Kalya, Coromup neMandelup vakatsanangurwa sevanopikisa zviri pakati nepakati, ukuwo Wonga, Tanjil ne83A:476 vakatsanangurwa sevasingamirire zvakanyanya26,27,31.yaive neyakafanana yekupikisa allele, yakasarudzwa Lanr1, uye Coromup neMandelup vaive neimwe allele, yakasarudzwa AnMan10, 26, 39, uku Kalya akapfuudza imwe allele., Lanr2.Kuongororwa kweanthracnose kuramba muGermany kwakaguma nekuzivikanwa kwemutsara unopokana Bo7212 ine mumiriri allele kunze kweLanr1, yakasarudzwa LanrBo36.
Chidzidzo chedu chakaratidza yakaderera frequency (inenge 6%) yeLanr1 allele mune yakaedzwa germplasm.Kucherechedza uku kunopindirana nemigumisiro yekuongorora East European germplasm uchishandisa Anseq3 uye Anseq4 zviratidzo, izvo zvakaratidza kuti Lanr1 allele iripo mumitsara miviri yeBelarusian chete.Izvi zvinoratidza kuti iyo Lanr1 allele haisati yashandiswa zvakanyanya nezvirongwa zvekupfuya zvemuno, kusiyana neAustralia, uko iri imwe yemakiyi e alleles yekucheresa-kubatsirwa kubereka.Izvi zvinogona kunge zvakakonzerwa neiyo yakaderera nhanho yekupikisa yakapihwa neLanr1 allele muEuropean field mamiriro zvichienzaniswa nereAustralia shumo.Mukuwedzera, zvidzidzo zveanthracnose munzvimbo dzinonaya mvura yakawanda muAustralia zvakaratidza kuti mhinduro dzekupikisa dzakagadziriswa neLanr1 allele dzinogona kusashanda mumamiriro ekunze anofarira kukura uye kukurumidza kukura kwepathogen19,42.Kutaura zvazviri, mune zvidzidzo zvemazuva ano, zvimwe zviratidzo zveanthracnose zvakaonekwawo mune genotypes inotakura Lanr1 allele, zvichiratidza kuti kushorwa kunogona kupera pasi pemamiriro ezvinhu akanaka ekugadzirwa kweC lupini.Mukuwedzera, kududzirwa kwenhema kwakanaka kwekuvapo kweAnseq3 uye Anseq4 mamaki, ayo anenge 1 cm kubva kuLanr1 locus, anogoneka 28,30,43.
Chidzidzo chedu chakaratidza kuti 83A: 476, inotakura Lanr1 allele, yakapindura kuC. lupini inoculation nehurukuro yakakura yezvinyorwa zvekare panguva yekutanga yakaongororwa nguva (6 hpi), apo muMandelup, inotakura AnMan allele, mhinduro dzezvinyorwa zvakaonekwa gare gare.(kubva pa24 kusvika ku48 hp).Izvi zvakasiyana-siyana zvenguva mumhinduro dzekudzivirira zvinosanganiswa nekusiyana kwezviratidzo zvechirwere, zvichiratidza kukosha kwekuzivikanwa kwekutanga kwepathogen kuti ibudirire mhinduro yekupikisa.Kutapura matishu echirimwa, anthrax spores anofanirwa kupfuura nematanho akati wandei ekukura panzvimbo inotambira, kusanganisira kumera, kupatsanurwa kwesero, uye kuumbwa kweappressorium.An appendage chimiro chinotapukira chinonamira pamusoro pemugamuchiri uye chinogonesa kupinda mukati mematishu anotambira.Nokudaro, spores yeC. gloeosporioides mumucheka wepea yakaratidza kupatsanurwa kwekutanga kweiyo nucleus mushure memaminitsi 75-90 ekudzivirira, kuumbwa kwehutachiwana hwehutachiwana mushure me90-120 maminitsi, uye kudzvinyirira mushure memaawa mana 45.Mango C. gloeosporioides yakaratidza kudarika 40% kumera kweconidial mushure memaawa matatu ekufukidzira uye inenge 20% kuumbwa kweappressors mushure memaawa mana.The virulence-associated CAP20 gene yeC. gloeosporioides yakaratidza kuita kwekunyora muepiphyte-forming conidia mushure memaawa 3.5 incubation muavocado surface wax ine high concentration yeCAP20 protein mushure me4 h 46 min.Saizvozvowo, basa re melanin biosynthesis genes muC trifolii rakakonzerwa munguva ye2-hour incubation yakateverwa nekuumbwa kwe appressorium mushure meawa 1.Zvidzidzo zvemashizha emashizha zvakaratidza kuti strawberries inoculated neC. acutatum inotanga kudzvinyirirwa pa8 hpi, nepo tomato inoculated neC. coccodes inotanga kudzvinyirira pa 4 hpi48,49.zvakanyanya kuenderana nechiyero chenguva cheColletotrichum spp.kutapukira.Kukurumidza kudzivirira mhinduro ku83A: 476 inoratidzira kubatanidzwa kwekudyara kwemiti uye effector-triggered immunity (ETI) genes mumutsara uyu, nepo Mandelup akanonoka mhinduro dzinotsigira micro-associated molecular pattern-triggered immunity (MTI) hypothesis 50. Mhinduro dzepakutanga ku83A: 476 uye Mandelup.Kupindirana kwechikamu pakati pemajini ekumusoro-kana pasi-anogadziriswa mukupindura kunonokerwa kunotsigirawo pfungwa iyi, sezvo ETI inowanzoonekwa seyekukurumidza uye yakagadziridzwa MTI mhinduro inoguma nehurongwa hwekufa kwesero panzvimbo yehutachiona, inozivikanwa seanaphylactic shock 51,52.
Mazhinji emajini anonzi akanyanya kumiririrwa izwi reGene Ontology GO: 0006952 "Defense Response" ndiwo gumi nerimwe homologues e-stress-induced fasting message 22 protein (yakafanana neSAM22) uye manomwe makuru latex protein-likes (MLPs).-kufanana nemapuroteni 31, 34, 43 uye 423 akaratidza kutevedzana kufanana.SAM22-yakafanana nemajeni yakaratidza kushandiswa kwakakosha kwakagara kwenguva refu, kuratidza kuwedzera kwemazinga ekudzivirira anthracnose (83A: 476 uye Boregine).Nekudaro, majini akafanana neMLP akaderedzwa chete mumitsara inotakura mukwikwidzi kuramba allele (83A: 476/Lanr1 pa6 hpi uye Mandelup/AnMan pa24 hpi).Izvo zvinofanirwa kucherechedzwa kuti ese anozivikanwa SAM22-akafanana homologues anobva kune geno cluster inosvika 105 kb, nepo MLP-senge majini anobva kumatunhu akasiyana ejenome.Coordinated activation yeakadaro SAM22-like genes akawanikwawo mukudzidza kwedu kwekare kweNLL kushorwa neDiaporthetoxica inoculation, zvichiratidza kuti vanobatanidzwa muzvikamu zvakadzika zvemhinduro yekudzivirira.Iyi mhedziso inotsigirwawo nemishumo yemhinduro yakanaka yeSAM22-yakafanana nemajeni kukuvadza kana kurapwa ne salicylic acid, fungal inducers, kana hydrogen peroxide.
MLP-semajini akaratidzwa kuti anopindura kune dzakasiyana siyana abiotic uye biotic kushushikana, kusanganisira mabhakitiriya, hutachiona uye pathogenic fungal utachiona mumhando zhinji dzezvirimwa55.Mirayiridzo yemhinduro kune kumwe kudyidzana pakati pezvirimwa neutachiona hwakasiyana kubva pakuwedzera kwakasimba (kureva, panguva yekuzadzwa kwedonje neVerticillium dahliae) kusvika pakuderera zvakanyanya (kureva, mushure mekutapukirwa kwemuapuro neAlternaria spp.)56,57.Kuderera kwakakosha kweMLP-like 423 gene yakaonekwa panguva yekudzivirira kweavocado kuF. niger utachiona uye panguva yehutachiona hwemuti weapuro Botryosphaeria berengeriana f.cn.piricola neAlternaria alternata maapuro pathotypes58,59.Pamusoro pezvo, apple calli yainyanya kuratidza iyo MLP-yakaita 423 gene yaive nekushomeka kwemajini ane hukama uye ainyanya kutapukirwa nehutachiona59.Kutevera Fusarium oxysporum f, iyo MLP-yakaita 423 gene yakadzvanyirirwa mune isingamirire bhinzi germplasm.cn.Bhinzi chirwere 60.
Dzimwe nhengo dzemhuri yePR-10 dzakaonekwa mune yedu RNA-seq yekudzidza yaive LlR18A uye LlR18B majini mukupindura kukwidziridzwa, pamwe neiyo yakakwira (1 gene) kana yakadzikira (3 majini) gene reiyo lipid kutamisa protein DIR1..Pamusoro pezvo, WGCNA inosimbisa iyo LlR18B gene sehubhu mune ino module, iyo inotapukirwa nejekiseni uye inotakura akati wandei ekudzivirira ekudzivirira.Majini eLlR18A neLlR18B akaitwa mumashizha e-yellow lupine achipindura mabhakitiriya epathogenic, uyewo muNLL stems mushure meD. toxica inoculation, nepo rice homologue yemajini aya, RSOsPR10, yakakurumidza kukonzerwa nehutachiona hwefungal hunofungidzirwa kuti hwakabatanidzwa mujasmonic acid, 562, DIFIDE, DIFIDE62, DIFIDE62 nzira, DIFIDE62, DIFIDE62, DIFIDE62, DIFIDE62 nzira, DIFIDE62. lipid kutakura mapuroteni anodiwa kuti atange systemic akawana kupikisa (SAR).Nekuvandudzwa kwemaitiro ekudzivirira, puroteni yeDIR1 inotakurwa kubva pakatarisana nehutachiona kuburikidza nephloem kuti iite SAR munhengo dziri kure.Sezvineiwo, iyo TanjilG_02313 DIR1 gene yakanyudzwa zvakanyanya panguva yekutanga mumitsara 84A:476 uye Population 22660, asi kuramba kweanthracnose kwakabudirira chete mumutsara 84A:476.Izvi zvinogona kuratidza kumwe kusashanda kweDIR1 gene muNLL, sezvo vatatu vakasara vakapindura kune inoculation chete mu83A: 476 mutsara pa6 hpi, uye mhinduro iyi yakananga pasi.
Muchidzidzo chedu, zvikamu zvakajairika zvinoenderana neiyo biological process inonzi "GO:0055114 Redox process" yaive cytochrome P450 protein, peroxidase, linoleic acid 9S-/13S-lipoxygenase, uye 1-aminocyclopropane-1-carboxylic acid oxidase.Pamusoro pezvo, yedu WGCNA inotsanangura iyo HSFA4a homologue sehubhu inotakura mamodule akadai seLanr1 resistance gene candidate TanjilG_05042.HSFA4a inhengo yeredox-inotsamira kudzora kwekunyora kwenyukireya muzvirimwa.
Cytochrome P450 mapuroteni maoxidoreductases anokonzeresa NADPH uye/kana O2-inotsamira hydroxylation reactions muprimary and secondary metabolism, kusanganisira the metabolism of xenobiotics, pamwe nemahomoni, fatty acids, sterols, cell wall components, biopolymers, uye biosynthesis yeprotectoral compounds In 50 tsvakurudzo yakaderedzwa In cytochrome 60 mukudzidza kwedu 60. 10.6 log2 (kupeta shanduko) kusvika ku5.7 nekuda kwenhamba huru yakashandurwa homologues (37) uye misiyano yemhinduro mapatani pakati pemajini chaiwo, inoratidzira kudzokororwa kwepamusoro..Kushandisa chete RNA-seq data kujekesa iyo putative biological function yeNLL majini mune yakakura kudaro protein superfamily zvingave zvekufungidzira zvakanyanya.Zvisinei, zvakakosha kucherechedza kuti mamwe ma cytochrome P450 genes anobatanidzwa nekuwedzera kupikisa kune pathogenic fungi kana mabhakitiriya, kusanganisira mupiro kune allergic reactions69,70,71.
Kirasi III peroxidases maenzayimu ezvirimwa anoshanda akawanda anosanganisirwa mumhando dzakasiyana dzemetabolism panguva yekukura kwechirimwa uye kukura, pamwe nekupindura kushushikana kwezvakatipoteredza senge salinity, kusanaya kwemvura, kusimba kwechiedza, uye pathogen kurwisa72.Peroxidases inobatanidzwa mukudyidzana kwemarudzi akawanda emiti neAnthracis, kusanganisira Stylosanthes humilis uye C. gloeosporioides, Lens culinaris uye C. truncatum, Phaseolus vulgaris uye C. lindemuthianum, Cucumis sativus uye C. lagenarium73,764,75.Mhinduro yacho inokurumidza zvikuru, dzimwe nguva kunyange pa4 HPI, fungus isati yapinda mumuti wezvirimwa73.Iyo peroxidase gene yakapindurawo kune D. toxica NLL inoculation.Pamusoro pemaitiro avo akajairika kudzora oxidative kuputika kana kubvisa oxidative kusagadzikana, peroxidase inogona kukanganisa kukura kwepathogen nekugadzira zvipingamupinyi zvemuviri zvinoenderana nesero madziro ekusimbisa panguva ye lignification, subunit kana kuyambuka-kubatanidza kweakananga makomisheni.Iri basa rinogona kuverengerwa musilico kune TanjilG_03329 gene encoding a putative lignin-forming anion peroxidase iyo yakanyanya kukwidziridzwa muchidzidzo chedu mu83A:476 inopikisa mutsetse pa6 HPI, asi kwete mune mamwe madhiri uye nguva mapoinzi asina kupindura.
9S-/13S-lipoxygenase ye linoleic acid ndiyo nhanho yekutanga muoxidative nzira ye lipid biosynthesis78.Zvigadzirwa zvenzira iyi zvine mabasa akawanda mukudzivirira kwemiti, kusanganisira kusimba kwemadziro emadziro kuburikidza nekugadzirwa kwe callose uye pectin deposits, nekugadzirisa kwekunetseka kweoxidative kuburikidza nekugadzirwa kweiyo reactive oxygen species79,80,81,82,83.Muchidzidzo chemazuva ano, kutaura kwe linoleic acid 9S-/13S-lipoxygenase kwakashandurwa mumatambudziko ose, asi muhuwandu hwehuwandu hwe22660, kukwidziridzwa kwakakunda panguva dzakasiyana-siyana, apo mumatambudziko anotakura Lanr1 uye AnMan allele, inosimbisa kusiyanisa kwe oxylipinthrection ye oxylipinnotype muzvikamu zve oxylipinnotype.
Iyo 1-aminocyclopropane-1-carboxylate oxidase (ACO) homologue yakanyanya kukwidziridzwa (9 majini) kana kuderedzwa (2 majini) painoculated ne lupine.Nezvisizvo zviviri, mhinduro dzese idzi dzakaitika pa6 hp.pa83A:476.Iyo enzymatic reaction inomiririrwa neACO mapuroteni ndiyo nhanho-yekumisa nhanho mukugadzirwa kwe ethylene uye saka inodzorwa zvakanyanya84.Ethylene ihomoni yechirimwa inoita mabasa akasiyana-siyana mukugadzirisa kukura kwezvirimwa uye kupindura kune abiotic uye biotic kusagadzikana mamiriro.Kuiswa kweACO kunyorwa uye kushandiswa kweiyo ethylene chiratidzo nzira inobatanidzwa mukuwedzera kushorwa kwemupunga kune hemibiotrophic fungus oryzae oryzae nekugadzirisa kugadzirwa kweiyo reactive oxygen marudzi uye phytoalexins.Kunyanya kufanana kwehutachiona hwemashizha kunowanikwa pakati peM. oryzae neC. lupini88,89, kunopesana nemashure ekugadzirisa kwakakosha kweACO homologues mu83A: 476 mutsara wakataurwa muchidzidzo ichi, inoshandura mukana wekupa kushorwa kweNLL anthracnose Ethylene chiratidzo chepakati chepakati munzira dzema molecular.
Muchidzidzo chezvino, kudzvinyirirwa kwakakura kwemajini akawanda ane chekuita nephotosynthesis kwakaonekwa pa6 hpi mu83A: 476 uye pa48 hpi muMandeloop uye nehuwandu hwe22660.Kukura uye kufambira mberi kweshanduko idzi kunoenderana nedanho.Anthracnose kuramba yakaonekwa muchiyedzo ichi.Munguva pfupi yapfuura, kudzvinyirirwa kwakasimba uye kwekutanga kwezvinyorwa zvine chokuita nephotosynthesis kwakataurwa mumhando dzinoverengeka dzekudyidzana kwezvirimwa-pathogen, kusanganisira pathogenic bacteria uye fungi.Kukurumidza (kubva ku2 HPI mune kumwe kupindirana) uye kudzvinyirirwa kwepasi rose kwemajini ane chokuita nephotosynthesis mukupindura utachiona hunogona kukonzera hutachiona hwemiti hunobva pakutumirwa kwemhando dzeokisijeni dzakagadziriswa uye kushamwaridzana kwavo ne salicylic acid nzira yekuyananisa maitiro allergic 90,94.
Mukupedzisa, nzira dzekudzivirira dziviriro dzakarongedzerwa kune yakanyanya kuramba mutsara (83A: 476) inosanganisira kukurumidza kuzivikanwa kwepathogen neiyo R gene (zvichida TIR-NBS-LRR TanjilG_05042) uye allergic reaction-mediated salicylic acid uye ethylene siginecha, inoteverwa nekugadzwa kwerefu-range SAR.chiito chinotsigirwa neDIR-1 protein.Zvinofanira kuonekwa kuti nguva yebiotrophic yeC. lupini yehutachiona ipfupi kwazvo (anenge mazuva maviri), inoteverwa necrotic growth95.Shanduko iri pakati pematanho aya inogona kuve yakabatana necrosis uye kutaura kweethylene-inducible mapuroteni anoita seakonzeresa maitiro e hypersensitivity mumiti inotambira.Nokudaro, hwindo renguva yekubudirira kubatwa kweC. lupini pabiotrophic stage yakanyanya kutetepa.Kugadziriswa kwemajini ane chekuita neredox uye photosynthesis akaonekwa mu83A: 476 pa6 hpi inopindirana nekufambira mberi kwefungal hyphae uye inozivisa kuvandudzwa kwemhinduro yekudzivirira inobudirira padanho rebiotrophic.The transcriptomic responses of Mandelup and the 22660 population may be too delayed to catch the fungus before switching to necrotic growth, zvisinei, Mandelup inogona kuva yakanyatsoshanda kudarika 22660 vanhu nokuti mutemo unokurumidza kukurumidza wePR-10 protein inokurudzira kupikisa kwakakomberedzwa.
ETI, inofambiswa neiyo canonical R gene, inoratidzika seyakajairwa nzira yekuramba bhinzi kune anthracnose.Nokudaro, mumuenzaniso we legume Medicago truncatula, kupikisa anthracnose kunopiwa neRCT1 gene, nhengo yeTIR-NBS-LRR97 plant R gene class.Iri jeni rinopawo yakakura-spectrum anthracnose kuramba mu alfalfa kana yaendeswa kune zvinomera zvinotapukira.Mubhinzi inojairika (P. vulgaris), anopfuura makumi maviri nemaviri ekudzivirira anthracnose akaonekwa kusvika nhasi.Mamwe emajini aya anowanikwa mumatunhu asina chero canonical R genes, zvisinei mamwe akawanda ari kumucheto kwemakromosomes anotakura NBS-LRR gene cluster, kusanganisira TIR-NBS-LRRs99.Iyo genome-wide SSR kudzidza yakasimbisawo kubatanidzwa kweNBS-LRR gene ine anthracnose kuramba mubhinzi yakajairika.Iyo canonical R gene yakawanikwa zvakare mudunhu re genomic yakatakura hombe anthracnose kuramba locus muchena lupine 101.
Basa redu rinoratidza kuti kukurumidza kupikisa kuita, kunoitwa padanho rekutanga rehutachiona hwechirimwa (zvichida kwete mushure me12 hpi), inochengetedza yakatetepa-inosiya lupine kubva kune anthracnose inokonzerwa neiyo pathogenic fungus Collelotrichum lupini.Tichishandisa kutevedzana kwepamusoro, takaratidza mutsauko wekutaura profiles yeanthracnose kuramba majini muNLL zvidyarwa zvakamiririrwa neLanr1 uye AnMan resistance genes.Kubudirira kudzivirira kunosanganisira kunyatsogadzira majini emapuroteni ane chekuita neredox, photosynthesis, uye pathogenesis mukati memaawa echirimwa chatanga kusangana nechirwere ichi.Kuita kwakafanana kwekudzivirira, asi kunonokerwa nekufamba kwenguva, hakuna kunyanya kushanda mukudzivirira zvirimwa kubva kuzvirwere.Anthrax kuramba inoyananiswa neLanr1 gene inofanana neyakajairwa kukurumidza mhinduro yeR gene (effector-triggered immunity), nepo AnMan gene inowanzopa mhinduro yakachinjika (kusadzivirirwa kunokonzerwa nemicrobe-yakabatana molecular pattern), ichipa mwero wepakati wekusimba.
Mitsetse mazana maviri negumi neshanu yeNLL yaishandiswa kuvharidzira zviratidzo zveanthracnose yaisanganisira 74 cultivars, mitsara makumi matanhatu inowanikwa nekuyambuka kana kuberekesa, 5 mutants, uye makumi manomwe nematanhatu emusango kana epakutanga majeremusi.Mitsetse yakabva kunyika gumi nenomwe, kunyanya kubva kuPoland (58), Spain (47), Germany (27), Australia (26), Russia (19), Belarus (7), Italy (5) nemimwe mitsetse.kubva kunyika gumi.Iyo seti inosanganisirawo mitsara inopokana: 83A: 476, Tanjil, Wonga inotakura Lanr1 allele, uye Mandelup akatakura AnMan allele.Mitsetse yacho yakawanikwa kubva kuEuropean Lupine Genetic Resource Database inochengetwa nePoznań Plant Breeding Ltd., Wiatrowo, Poland (Supplementary Table S1).
Zvirimwa zvakarimwa pasi pemamiriro ekudzora (pikicha maawa gumi nematanhatu, tembiricha 25°C masikati uye 18°C ​​usiku).Zvinyorwa zviviri zvehupenyu zvakaongororwa.DNA yakaparadzaniswa nemavhiki matatu mashizha achishandisa iyo DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) maererano neprotocol.Hunhu uye kusungirirwa kweiyo yega DNA yakaongororwa nenzira dze spectrophotometric (NanoDrop 2000; Thermo Fisher Scientific, Waltham, MA, USA).Iyo AnManM1 inomaka iyo anthracnose resistance gene AnMan (yakatorwa mu cv. Mandelup) uye ma marker Anseq3 ne Anseq4 ari kumberi kwegene Lanr1 (yakabva mu cv. Tanjil) yakaongororwa 11,26,28.MaHomozygotes echidziviriro chinopikisa akaonekwa se "1", anotapukira - se "0", uye heterozygotes - se 0.5.
Zvichienderana nemhedzisiro yekuongororwa kwemakaki AnManM1, AnSeq3 neAnSeq4 uye kuwanikwa kwembeu dzekupedzisira yekutevera miedzo, mitsetse ye50 NLL yakasarudzwa yeanthracnose resistance phenotyping.Ongororo iyi yakaitwa muduplicate mucomputer inodzorwa greenhouse ine 14 hour photoperiod ine tembiricha ye22°C masikati uye 19°C usiku.Mbeu dzinokweshwa (kucheka bhachi rembeu kune rimwe divi re "embryo" nebadza rakapinza) isati yadyarwa kudzivirira kusarara kwembeu nekuda kwekuoma kwembeu uye kuti mbeu yamera.Zvirimwa zvakarimwa muhari (11 × 11 × 21 cm) nevhu risingabereki (TS-1 REC 085 Medium Basic, Klasmann-Deilmann Polska, Warsaw, Poland).Inoculation yakaitwa neColletotrichum lupini Col-08 strain, yakakura muna 1999 kubva pamadzinde emiti ine mashizha matete-mashizha akarimwa mumunda muVerzhenitsa, Greater Poland (52° 27′ 42″ N 17° 04′ 05″ E ).Wana nzvimbo.Iwo akaparadzana akagadzirwa muSNA svikiro pa20° C. pasi pechiedza chitema kwemazuva 21 kuti aite sporulation.Vhiki ina mushure mekudyara, apo zvinomera zvasvika pamashizha 4-6, inoculation yakaitwa nekupfapfaidza nekumiswa kwekonidia pamusangano we 0.5 x 106 conidia per ml.Mushure mekudzivirira, zvinomera zvakachengetwa murima kwemaawa makumi maviri nemana pane hunyoro hunenge 98% uye tembiricha ye25 ° C kuitira kuti kumera kwekonidia uye hutachiona hwehutachiona.Miti yacho yakabva yarimwa pasi pemaawa gumi nemana photoperiod pa 22 ° C zuva / 19 ° C usiku uye 70% humidity.Chirwere chechirwere chakaitwa mazuva makumi maviri nemaviri mushure mekudzivirira uye kubva ku0 (immune) kusvika ku9 (inonyanya kunetseka) zvichienderana nekuvapo kana kusavapo kwezvipembenene necrotic pamatanda nemashizha.Mukuwedzera, mushure mekupa zvibodzwa, uremu hwemiti hwakayerwa.Ukama pakati pemarker genotypes uye chirwere chephenotypes chakaverengerwa senge mbiri-yakatevedzana kuwirirana (kusavapo kweiyo heterozygous mamakisi mune yakagadzika mitsetse yekuongorora yeanthracnose resistance phenotype).