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Angustifolius lupine (NLL, Lupinus angustifolius L.) chirimwa chembeu chinoshandiswa kugadzira chikafu nekuvandudza ivhu. Kukura kweNLL pasi rose sechirimwa kwakakwezva fungus dzakawanda dzinokonzera hutachiona, kusanganisira lupine anthracnose, iyo inokonzera chirwere chinoparadza cheanthracnose. Maallele maviri, Lanr1 naAnMan, anopa simba rekudzivirira, akashandiswa mukubereka NLL, asi mashandiro emamorekuru ari pasi haazivikanwe. Muchidzidzo ichi, zviratidzo zveLanr1 naAnMan zvakashandiswa kuongorora sampuli dzeEuropean NLL. Kuedzwa kwemushonga munzvimbo yakadzorwa kwakasimbisa kushanda kwevanopa mushonga vanoramba. Kuongororwa kwemajini akasiyana kwakaitwa pamitsetse inomiririra inoramba uye inobatwa nehutachiona. Kuramba kweAnthracnose kwakabatana nekuwanda kwemashoko e "ontology" e "GO:0006952 Defense Response", "GO:0055114 Redox Process", uye "GO:0015979 Photosynthesis". Pamusoro pezvo, mutsetse weLanr1(83A:476) wakaratidza kudzokororwa kukuru kwetranscriptome nekukurumidza mushure mekubayiwa, nepo mimwe mitsara yakaratidza kunonoka kwemhinduro iyi nemaawa angangoita makumi mana nemaviri. Mhinduro dzekudzivirira dzakabatana nemajini eTIR-NBS, CC-NBS-LRR uye NBS-LRR, mapuroteni gumi anobatanidzwa mupathogenesis, mapuroteni ekutamisa lipid, endoglucan-1,3-β-glucosidase, mapuroteni emadziro emasero ane glycine yakawanda, uye majini kubva munzira yeoxygen inochinja. Mhinduro dzekutanga ku83A:476, kusanganisira kudzvinyirirwa kwemajini ane chekuita ne photosynthesis, dzakaenderana nekudzivirirwa kwakabudirira panguva yekukura kwezvirimwa zvefungi, zvichiratidza kuti effector inokonzera dziviriro. Maitiro eMandeloop anononoka, pamwe chete nekudhonzwa kwakatarwa.
Lupine ine mashizha matete (NLL, Lupinus angustifolius L.) imhando yezvirimwa zvine mapuroteni akawanda zvinobva kumadokero kweMediterranean. Parizvino inorimwa sechikafu chemhuka nevanhu. Inoonekwawo semupfudze wegirini muzvirongwa zvekutenderera zvirimwa nekuda kwenitrogen fixation ne symbiotic nitrogen fixing bacteria uye kuvandudzwa kwemamiriro evhu. NLL yave nenguva yekukurumidza kuchengetwa mudzimba muzana remakore rapfuura uye ichiri pasi pekumanikidzwa kukuru kwekubereka3,4,5,6,7,8,9,10,11,12. Nekurimwa kwakapararira kweNLL, kutevedzana kwefungus dzinokonzera hutachiona kwakagadzira nzvimbo itsva dzekurima uye kwakakonzera zvirwere zvitsva zvinoparadza zvirimwa. Chinhu chinonyanya kushamisa kuvarimi nevapfuyi vemiti yelupin kwaive kuonekwa kweanthracnose, yakakonzerwa nefungus inokonzera hutachiona, Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13. Chinhu chinonyanya kushamisa kuvarimi nevapfuyi vemiti yelupin kwaive kuonekwa kweanthracnose, yakakonzerwa nefungus inokonzera hutachiona, Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13. Наиболее примечательным для фермеров и селекционеров люпина было появление антракноза, вызванного патогенным грибком Colletotrichum (Feletotrichum) Hagedorn13. Chinhu chinonyanya kuzivikanwa nevarimi vemiti yelupine nekuberekesa maruva kwaive kubuda kweanthracnose yakakonzerwa nefungus inonzi Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13.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) Handler & Bondar引起的.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 патогененым гриtotricбком Colleire Hagedorn13. Chinonyanya kushamisa varimi nevapfuyi vemiti yelupine kubuda kweanthracnose inokonzerwa nefungus inonzi Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13.Mishumo yekutanga yechirwere ichi yakabva kuBrazil neUnited States, zviratidzo zvakajairika zvichionekwa muna 1912 na1929 zvichiteerana. Zvisinei, mushure memakore angangoita makumi matatu, utachiona hwakazonzi Colletotrichum gloeosporioides (Penz.) Penz. & Sacc., teleomorph Glomerella cingulata (Stoneman) Spauld. & Sacc., teleomorph Glomerella cingulata (Stoneman) Spauld. & Sacc., телеоморф Glomerella cingulata (Stoneman) Spauld. & Sacc., teleomorph yeGlomerella 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 muTargeted Morphology. & H. Schrenk,. & H. Schrenk,.naH. Schrenk. & H.施伦克，. & H.施伦克，.naH. Schlenk,.Kuongororwa kwechirwere kwekutanga kwakaitwa pakati pezana remakore rechi20 kwakaratidza kusasimba kwemiti muNLL uye yellow lupine (L. luteus L.), asi mishonga yese yewhite lupine (L. albus L.) yakaedzwa yaive nyore kubatwa nayo. Zvidzidzo zvakaratidza kuti kukura kweanthracnose kwakabatana nekuwedzera kwemvura (hunyoro hwemhepo) uye tembiricha (iri pakati pe12-28°C), zvichikonzera kusasimba kwekusasimba patembiricha yepamusoro. Kutaura zvazviri, nguva yaidiwa kuti conidia imere uye chirwere ichi chitange, yaive pfupi kane kana pa24°C (maawa mana) pane pa12°C (maawa gumi nematanhatu) pasi pemamiriro ehunyoro hwakanyanya. Saka, kudziya kwepasi rose kuri kuramba kuchienderera mberi kwakakonzera kupararira kweanthracnose. Zvisinei, chirwere ichi chakaonekwa muFrance (1982) neUkraine (1983) sechiratidzo chekutyisidzira kuri kuuya, asi zvinoita sekunge chakaregeredzwa neindasitiri yelupine panguva iyoyo. Makore mashoma gare gare, chirwere ichi chinoparadza chakapararira pasi rose uye chakabatawo nyika dzinogadzira lupine dzakadai seAustralia, Poland neGermany22,23,24. Mushure mekubuda kweanthracnose pakati pemakore ekuma1990, kuongororwa kwakawanda kwakaita kuti pave nevakawanda vanopa mishonga isingapindi mishonga muNLL19 samples. Kusapindirana kweNLL neanthracnose kunodzorwa nemarudzi maviri akasiyana ealleles anowanikwa mumanyuko akasiyana egermplasm: Lanr1 mumhando yeTanjil neWonga neAnMan mumhando yegerm. Mandalay 25, 26. Maalleles aya anozadzisa mamakisi emamorekuru anotsigira kusarudzwa kwegermplasm isingapindi mishonga muzvirongwa zvekuberekesa 25, 26, 27, 28, 29, 30. Mutsetse wekubereka unodzivirira 83A:476 une Lanr1 allele wakasanganiswa nemutsetse wemusango unodzivirirwa P27255 kuti uwane huwandu hweRIL hwakapatsanurwa kuti huve neanthracnose resistance, izvo zvakaita kuti zvikwanisike kupa Lanr1 locus kune chromosome NLL-1131, 32, 33. Kurongeka kwemamaki ekubatanidza kubva ku flanking resistance loci kuenda kuanthracnose ne genomic framework, NLL yakaratidza nzvimbo yema alleles ese matatu ari pa chromosome imwe chete (NLL-11), asi munzvimbo dzakasiyana29,34,35. Zvisinei, nekuda kwehuwandu hushoma hweRILs uye daro guru remajini pakati pemamaki nema alleles anoenderana, hapana mhedziso dzakavimbika dzinogona kutorwa nezvemajini avo ari pasi. Kune rumwe rutivi, kushandiswa kwemajini e reverse mu lupins kwakaoma nekuda kwesimba rawo rekugadzirisa, izvo zvinoita kuti genetic manipulation iome37.
Kugadzirwa kwegermplasm yakachengetwa inotakura allele inodiwa mumamiriro ehomozygous, akadai se83A:476 (Lanr1) neMandelup (AnMan), kwakavhura mukana wekudzidza anthracnose resistance pamberi pekuvapo kwemarudzi akasiyana ealleles mumapoka emusango. Mikana yemaitiro emamorekuru. Enzanisa mhinduro dzekudzivirira dzinogadzirwa nema genotypes chaiwo. Chidzidzo ichi chakaongorora mhinduro yekutanga yeNLL kuC. lupini vaccination. Kutanga, panel yeEuropean NLL germplasm ine mitsara 215 yakaongororwa uchishandisa mamorekuru markers anoratidza Lanr1 neAnMan alleles. Anthracnose phenotyping yakabva yaitwa pamitsara makumi mashanu yeNLL, yakasarudzwa kare yemamakuru emorekuru, pasi pemamiriro akadzorwa. Zvichibva pakuedza uku, mitsara mina yakasiyana muanthracnose resistance uye Lanr1/AnMan allelic composition yakasarudzwa kuti iratidze differential defense gene expression profile uchishandisa nzira mbiri dzinoenderana: high-throughput RNA sequencing uye real-time PCR quantification.
Kuongororwa kweseti yeNLL germplasm (N = 215) nezviratidzo Lanr1 (Anseq3 naAnseq4) naAnMan (Anseq4) naAnMan (AnManM1) kwakaratidza kuti mutsetse mumwe chete (95726, pedyo neSalamanca-b) unowedzera "resistance" allele yezviratidzo zvese, nepo "Kuvapo kwe'susceptible' alleles" kwakawana huwandu hwezviratidzo zvese mumitsara 158 (~73.5%). Mitsetse gumi nemitatu yakagadzira maallele maviri "resistance" echiratidzo cheLanr1, uye mitsara 8 yakagadzira maallele "resistance" echiratidzo cheLanr1. . "resistance" allele yechiratidzo cheAnMan (Supplementary Table S1). Mitsetse miviri yaive heterozygous yechiratidzo cheAnseq3 uye imwe heterozygous yechiratidzo cheAnManM1. Mitsetse makumi mana nemaviri (19.5%) yaive nezvikamu zvakasiyana zveAnseq3 neAnseq4 alleles, zvichiratidza kuwanda kwekusangana kwemarudzi maviri aya. Mafungiro eAnthracnose pasi pemamiriro akadzorwa (Supplementary Table S2) akaratidza kusiyana kwekusawirirana kwemajini akaedzwa, izvo zvakaratidzwa mukuoma kweanthracnose. Kusiyana kwepakati kwaive kubva pa1.8 (inodzivirira zvishoma) kusvika pa6.9 (inodzivirira) uye kusiyana kwehuremu hwezvirimwa kwaive kubva pa0.62 (inodzivirira) kusvika pa4.45 g (inodzivirira). Paiva nehukama hwakakosha pakati pezvinhu zvakaonekwa mukudzokorora kaviri kwekuyedza (0.51 yehuwandu hwechirwere, P = 0.00017 uye 0.61 yehuremu hwechirimwa, P < 0.0001) pamwe chete nepakati pezvikamu zviviri izvi (− 0.59 uye − 0.77, P < 0.0001). Paiva nehukama hwakakosha pakati pezvinhu zvakaonekwa mukudzokorora kaviri kwekuyedza (0.51 yezviratidzo zvekuoma kwechirwere, P = 0.00017 uye 0.61 yehuremu hwechirimwa, P < 0.0001) pamwe chete nepakati pezvikamu zviviri izvi (− 0.59 uye − 0.77, P < 0.0001). Выявлена ​​достоверная корреляция между значениями, наблюдаемыми в двух повторностях эксперимента (0,51 для баллов тяжез00, 1 = для массы растения, P < 0,0001), а также между этими двумя параметрами (-0,59 и -0,77, Р < 0,0001) 0,0001). Kuwirirana kwakakosha kwakawanikwa pakati pezvinhu zvakaonekwa mukudzokorora kaviri kwekuyedza (0.51 yezviratidzo zvekuoma kwechirwere, P = 0.00017 uye 0.61 yehuremu hwechirimwa, P < 0.0001), pamwe chete nepakati pezvikamu zviviri izvi (- 0.59 uye -0.77, P < 0.0001) 0.0001).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 = 0.00017，植物重量為0.61，P < 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 门 存在 相关性 （疾病 严重 程度 评分为 5 为分为 5 為 1 Φ. 0.00017 , 植物 為 為 0.61 , p <0.0001） 以及 两 个 参数 之间 （（（- 0.59 和– 0.50 和– 0.59 和– 0.59 和– 9 和– 9 .– 9 和– 9 .– 9 0.77, P < 0.0001). Наблюдалась значительная корреляция между значениями, наблюдаемыми в двух повторностях (оценка тяжести заболевания 0,51, P = 10,000 = 0,61, P <0,0001), и между этими двумя параметрами (-0,59 и -0,0001) 0,77, P <0,0001. Paiva nehukama hwakakosha pakati pezvinhu zvakaonekwa muzvikamu zviviri (chikero chehurwere 0.51, P = 0.00017 uye huremu hwechirimwa 0.61, P < 0.0001) uye pakati pezvikamu zviviri izvi (-0.59 uye -0 .0001) 0.77, P<0.0001. ).Zviratidzo zvinowanzoonekwa mumiti inobatwa nechirwere ichi zvinosanganisira kukwenya nekumonereka kwehunde kwakaita se "shepherd's bow", zvichiteverwa nekukuvara kweoval ne sporozoites yeorenji/pink (Mufananidzo 1). Majini eAustralia ane Lanr1 (83A:476 naTanjil) uye AnMan (Mandelup) anodzivirira zviri pakati nepakati, 0.0331 na 0.0036). Mimwe mitsetse inewo Lanr1 uye/kana AnMan alleles "inodzivirira" inoratidza zviratidzo zvechirwere ichi.
Zvinofadza kuti, mitsetse mishoma yeNLL isina allele ye "resistant" yakaratidza huwandu hwakanyanya hweanthracnose resistance (yakaenzana kana yakakwirira kupfuura yeLanr1 kana AnMan genotypes), yakadai seBoregine (P value < 0.0001 ye parameters dzese dziri mbiri), Bojar (P value < 0.0001 ye score uye 0.001 yehuremu hwemiti) uye Population B-549/79b (P value < 0.0001 ye score uye isingakoshi yehuremu). Zvinofadza kuti, mitsetse mishoma yeNLL isina allele ye "resistant" yakaratidza huwandu hwakanyanya hweanthracnose resistance (yakaenzana kana yakakwirira kupfuura yeLanr1 kana AnMan genotypes), yakadai seBoregine (P value < 0.0001 ye parameters dzese dziri mbiri), Bojar (P value < 0.0001 ye score uye 0.001 yehuremu hwemiti) uye Population B-549/79b (P value < 0.0001 ye score uye isingakoshi yehuremu). Интересно, что несколько линий NLL, лишенных какого-либо резистентного или более высокий, чем для генотипов Lanr1 или AnMan), таких как Boregine (значение P <0,0001 для обоих параметров), Bojar (значение P0 10,0001 P <0,0001 для обоих параметров), Bojar (значение P0 10,000 для массы растения) и популяции B-549/79b (значение P <0,0001 для оценки и незначимо для массы). Zvinofadza kuti, mitsara yakati wandei yeNLL isina allele yezviratidzo 'inodzivirira' yakaratidza mwero wepamusoro wekudzivirira anthracnose (yakaenzana kana yakakwirira kupfuura yeLanr1 kana AnMan genotypes), yakadai seBoregine (P kukosha < 0.0001 yematanho ese ari maviri ), Bojar (P kukosha < 0.0001 yekuongorora uye 0.001 yehuremu hwezvirimwa) uye huwandu hweB-549/79b (P kukosha < 0.0001 yekuongorora uye kwete kukosha kwehuremu).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（与LanAn1基因型相当或更高），例如Boregine（两个参数的P 值< 0.0001）、Bojar（P 值<得分為0.0001，植物重量為0.001）和种群B-549/79b（得分P 值< 0.0001，重量不显着）. Zvinonakidza kuti mamwe masisitimu eNLL asina zviratidzo zve "antigenic" anoratidza kuramba kwakadzikama (kwakaenzana nemajini eLanr1 kana AnMan kana kupfuura), akadai seBoregine (ma parameter ese ari maviri P < 0.0001), Bojar (P kukosha < 0.0001, huremu hwezvirimwa 0.001) uye strain B-549/79b (P kukosha < 0.0001, huremu hahuna kukosha). Интересно, что некоторые линии NLL, лишенные каких-либо маркерных аллелей «резистентности», показали высокие уровни устойчистинский выше, чем у генотипов Lanr1 или AnMan), такие как Boregine (значение P для обоих параметров <0,0001), Bojar (значение P <0,0001, маел001, мас001) B-549/79b (оценка P-значение <0,0001, масса незначительна). Zvinofadza kuti, mimwe mitsetse yeNLL isina zviratidzo zve'resistance' yakaratidza huwandu hwakanyanya hweanthracnose resistance (yakaenzana kana yakakwirira kupfuura Lanr1 kana AnMan genotypes), dzakadai seBoregine (P value yezvose zviri zviviri <0.0001), Bojar (P-value <0.0001, plant weight 0.001) uye population B-549/79b (P-value <0.0001, huremu hahuna kukosha).Chiitiko ichi chinoratidza mukana wekuti pave nechinhu chitsva chinorwisa majini, zvichitsanangura kusawirirana kwakaonekwa pakati pezviratidzo zvemajini uye phenotypes dzezvirwere (P values ​​​​kubva pa ~0.42 kusvika ~0.98). Saka, bvunzo yeKolmogorov-Smirnov yakaratidza kuti data pamusoro pekudzivirira anthracnose raiwanzo kugoverwa pama scores (P-values ​​​​0.25 na 0.11) uye plant mass (P-values ​​​​0.47 na 0.55), zvichiratidza kuti ndinofungidzira kuti ma alleles akawanda kupfuura Lanr1 naAnMan anobatanidzwa.
Zvichibva pamhedzisiro yekuongorora kwekudzivirira kweanthracnose, mitsara mina yakasarudzwa kuti iongororwe transcriptome: 83A:476, Boregine, Mandelup, uye Population 22660. Mitsetse iyi yakaongororwazve kuti ionekwe kuti haina simba reanthrax here mukuyedza kubaya neRNA sequencing, chero bedzi yakafanana neyemubvunzo wapfuura. Mapoinzi acho aive akadai: Boregin (1.71 ± 1.39), 83A: 476 (2.09 ± 1.38), Mandelup (3.82 ± 1.42) uye population 22660 (6.11 ± 1.29).
Nzira yeIllumina NovaSeq 6000 yakawana avhareji yemapeya e40.5 Mread pamuenzaniso wega wega (29.7 kusvika 54.4 Mreads) (Supplementary Table S3). Mapoinzi ekuenderana muchikamu chereferensi aibva pa75.5% kusvika 88.6%. Avhareji yehukama hwekuverenga data pakati pemhando dzekuedza pakati pezvakatorwa kubva muzvinhu zvipenyu yakabva pa0.812 kusvika 0.997 (avhareji 0.959). Pamajini 35,170 akaongororwa, 2917 haana kuratidza kuti pane zviratidzo, uye mamwe majini 4785 akataurwa padanho diki (base average <5). Pamajini 35,170 akaongororwa, 2917 haana kuratidza kuti pane zviratidzo, uye mamwe majini 4785 akataurwa padanho diki (base average <5). Из 35 170 проанализированных генов 2917 не проявляли экспрессии, а остальные 4785 генов экспрессировались на ненезнабрессии <5). Pamajini 35,170 akaongororwa, 2917 haana kuratidza kuratidzwa, uye mamwe majini 4785 akasara akataurwa padanho diki (base avhareji <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. Pamajini 35,170 akaongororwa, 2917 haana kuratidzwa uye mamwe majini 4785 akasara aive nekutaura kushoma (base avhareji <5).Saka, huwandu hwemajini akataurwa nezvawo (avhareji yepakati ≥ 5) panguva yekuedza kwacho hwaive 27,468 (78.1%) (Table Supplementary S4).
Kubva panguva yekutanga, mitsetse yese yeNLL yakapindura kubaiwa kweC. lupini (strain Col-08) nekugadzirazve transcriptome (Tafura 1), zvisinei, misiyano mikuru yakaonekwa pakati pemitsetse. Saka, resistance line 83A:476 (inotakura gene reLanr1) yakaratidza reprogramming yakakosha yetranscriptome panguva yekutanga (6 hpi) nekuwedzera kwakapetwa ka31-69 muhuwandu hwemajini ekukwira nekudzika akaparadzaniswa zvichienzaniswa nedzimwe nguva panguva iyi. Pamusoro pezvo, peak iyi yaive yenguva pfupi, sezvo kutaura kwemajini mashoma chete kwakaramba kuchinjwa zvakanyanya panguva yechipiri (12 hpi). Zvinofadza kuti, Boregine, iyo yakaratidzawo resistance yakanyanya mu graft test, haina kuitiswa reprogramming yakakura kudaro panguva yekuyedza. Zvisinei, huwandu hwemajini akaratidzwa zvakasiyana (DEG) hwaive hwakafanana neBoregine uye 83A:476 pa12 HPI. Mandelup pamwe nehuwandu hwevanhu 22660 zvakaratidza huwandu hwepamusoro hweDEG panguva yekupedzisira (48 l/s), zvichiratidza kunonoka kwemhinduro dzekudzivirira.
Nekuti 83A:476 yakagadziriswazve zvakanyanya mu transcriptome vachipindura C. lupini pa6 HPI zvichienzaniswa nemimwe mitsara yese, ~91% yemaDEG akaonekwa panguva iyi aive akatarisana nemutsara (Mufananidzo 1). Zvisinei, paive nekupindirana pakati pemhinduro dzekutanga pakati pemitsara yekudzidza, sezvo 68.5%, 50.9%, uye 52.6% DEG muBoregine, Mandelup, uye huwandu hwevanhu 22660, zvichiteerana, zvakapindirana neizvo zvakawanikwa mu83A:476 mune dzimwe nguva. Zvisinei, maDEG aya aive nechikamu chidiki chete (0.97–1.70%) chemaDEG ese akawanikwa pari zvino achishandisa 83A:476. Pamusoro pezvo, maDEG gumi nerimwe kubva mumitsara yese aive akabatana panguva iyi (Supplementary Tables S4-S6), kusanganisira zvikamu zvakajairika zvemhinduro dzekudzivirira zvirimwa: protein yekufambisa lipid (TanjilG_32225), endoglucan-1,3-β-glucoside enzyme (TanjilG_23384), mapuroteni maviri anokonzera kushushikana seSAM22 (TanjilG_31528 neTanjilG_31531), basic latex protein (TanjilG_32352), uye mapuroteni maviri emadziro emasero ane glycine yakawanda (TanjilG_19701 neTanjilG_19702). Paivawo nekupindirana kwakanyanya mumhinduro dzetranscriptome pakati pe83A:476 neBoregine pa24 HPI (16-38% DEG) uye pakati peMandelup nePopulation 22660 pa48 HPI (14-20% DEG).
Dhayagiramu yeVenn inoratidza huwandu hwemajini akabuda zvakasiyana (DEG) mumitsara yelupine ine mawere matete (NLL) yakabayiwa neColletotrichum lupini (rudzi rweCol-08 rwakawanikwa kubva kuminda yelupine muWierzhenice, Poland, 1999). Mitsetse yeNLL yakaongororwa yaive: 83A:476 (inodzivirira, ine Lanr1 allele), Boregine (inodzivirira, nhoroondo yemajini isingazivikanwe), Mandelup (inodzivirira zvishoma, ine AnMan allele) uye huwandu hwevanhu 22660 (inokurumidza kubatwa). Hpi inomirira maawa mushure mekubaiwa. Zero values ​​​​dzabviswa kuti girafu ive nyore.
Seti yemajini akawandisa pa6 hpi yakaongororwa kuti ionekwe kuti pane magene eR gene (Supplementary Table S7). Chidzidzo ichi chakaratidza kuti magene ekare ekudzivirira chirwere ane magene eNBS-LRR chete ari pa83A:476. Seti iyi yaive negene rimwe reTIR-NBS-LRR (tanjilg_05042), majini mashanu eCC-NBS-LRR (tanjilg_06165, tanjilg_06162, tanjilg_22773, tanjilg_22640, uye tanjilg_16162), uye Four NBS-LR, Tanjilg_16162), uye Four NBS-LRRE (tanjilg_16162) pamwe neFour NBS-Lrr (tanjilg_16162) uye Four NBS-LRR (TANJILG_16162). Majini ese aya ane magene ecanonical akarongwa muzvikamu zvakachengetedzwa. Pamusoro pemajini eNBS-LRR domain, maRLL kinases akati wandei akashanda pa6 hpi, kureva rimwe muBoregine (TanjilG_19877), maviri muMandelup (TanjilG_07141 naTanjilG_19877) uye muhuwandu hwevanhu 22660 (TanjilG_09014 naTanjilG_10361) uye maviri mu83A 27:476.
Majini ane chimiro chakachinja zvikuru mushure mekubayiwa neC. lupini (strain Col-08) akaongororwa Gene Ontology (GO) enrichment analysis (Supplementary Table S8). Izwi rekuti maitiro ezvebhayoloji ainyanya kutaurwa raive rekuti “GO:0006952 response yekudzivirira” iro rakaonekwa mumisanganiswa mitanhatu kubva pagumi nematanhatu (nguva × mutsetse) ine kukosha kukuru (P value <0.001) (Mufananidzo 2). Izwi rekuti maitiro ezvebhayoloji ainyanya kutaurwa raive rekuti “GO:0006952 response yekudzivirira” iro rakaonekwa mumisanganiswa mitanhatu kubva pagumi nematanhatu (nguva × mutsetse) ine kukosha kukuru (P value <0.001) (Mufananidzo 2). Наиболее часто чрезмерно представленным термином биологического процесса был «GO: 0006952 защитный ответ», который и вля 6 × линия) комбинаций с высокой значимостью (значение P <0,001) (рис. 2). Izwi rekuti maitiro ezvebhayoloji ainyanya kutaurwa raive 'GO:0006952 defense response', iro rakaonekwa muzvikamu zvitanhatu kubva pagumi nematanhatu (nguva × dzinza) zvine kukosha kukuru (P value <0.001) (Mufananidzo 2).最常被过度代表的生物过程术语是“GO:0006952 防御反应”，它出现在16 个（时间中×个中，具有高显着性（P 值< 0.001）（图2）。 Izwi rinonyanya kutaurwa nezvemaitiro ehupenyu ndi "GO:0006952 response defense", rinoonekwa muzvikamu zvitanhatu kubva pazvikamu gumi nematanhatu (时间×线), zvine kukosha kukuru (P value <0.001) (图2). Наиболее часто представленным термином биологического процесса был «GO: 0006952 Defense Response», который появлялся 1на × 6 линия) с высокой значимостью (значение P <0,001) (рис. 2). Izwi rekuti maitiro ezvebhayoloji ainyanya kutaurwa raive 'GO:0006952 Defense Response', iro rakaonekwa mumisanganiswa mitanhatu kubva pagumi nematanhatu (nguva × mutsetse) ine kukosha kukuru (P kukosha <0.001) (Mufananidzo 2).Izwi iri rakamiririrwa zvakanyanya panguva mbiri mu83A: 476 neBoregine (6 ne24 hpi) uye panguva imwe chete muMandelup nePopulation 22660 (12 ne6 hpi, zvichiteerana). Uyu mugumisiro unotarisirwa, uchiratidza mhinduro yehutachiona yemitsetse isingapindi. Pamusoro pezvo, 83A:476 yakapindura C. lupini nekukurumidza kukonzera majini ane chekuita nekuputika kweoxidative kunomiririrwa neshoko rekuti "GO:0055114 redox process", zvichiratidza mhinduro chaiyo yekudzivirira, nepo Boregine yakaratidza mhinduro chaiyo yekudzivirira, ine chekuita neshoko rekuti 'GO'. :0006950 Kupindura kwekushushikana”. Population 22660 yakamutsa mhinduro yekuramba yakatambanuka inosanganisira metabolites dzechipiri, zvichiratidza huwandu hwakawanda hwemashoko ekuti “GO:0016104 Maitiro ekugadzira triterpene biosynthesis” uye “GO:0006722 Maitiro ekugadzirisa triterpene” (mazwi ese ari maviri ndeemajini akafanana), tichifunga nezvemhedzisiro yekuongorora kweGO term enrichment, Mandelup reaction stability yaive pakati peBoregine nePopulation 22660. Pamusoro pezvo, reaction yekutanga 83A:476 (6 hpi) uye reaction yakanonoka Mandelup nePopulation 22660 zvinosanganisira izwi rekuti GO:0015979 'photosynthesis' nemamwe maitiro ane chekuita nehupenyu.
Mazwi e bioprocess gene ontology akasarudzwa mukutsanangurwa kwemajini akasiyana-siyana panguva yekupindura kwe transcriptome ye narrow-leaved lupine (NLL) yakabayiwa ne anthrax lupine (Col-08 strain yakawanikwa kubva kuminda ye lupine muWierzhenice, Poland, muna 1999) akawedzerwa zvakanyanya. Mitsetse yeNLL yakaongororwa yaive: 83A:476 (inodzivirira, ine homozygous Lanr1 allele), Boregine (inodzivirira, isingazivikanwe genetic background), Mandelup (inodzivirira zvishoma, ine homozygous AnMan allele) uye huwandu hwevanhu 22660 (inogona kubatwa).
Nekuti chidzidzo ichi chaive nechinangwa chekuona majini anobatsira mukusagadzikana kweanthracnose, majini akapihwa mazwi ekuti GO “GO: 0006952 Defensive responses” uye “GO: 0055114 Redox processes” akaongororwa ne cut-offs sezvo baseline means ≥ 30 ne mutsetse mumwe chete. × point in time ichibatanidza statistically significant values ​​​​​​ye log2 (fold change). Huwandu hwemajini anosangana nezvinodiwa izvi hwaive 65 yeGO:0006952 uye 524 yeGO:0055114.
83A:476 yakaratidza kuti maDEG peaks maviri akanyorwa neshoko rekuti GO:0006952, rekutanga pa6 majini pa inch (64 majini, up and down regulation) uye rechipiri pa24 majini pa inch (15 majini, up regulation chete). Boregine yakaratidzawo kuti GO:0006952 yakasvika pakakwirira panguva imwe chete, asi neDEG shoma (11 na8) uye preferential activation. Mandeloop yakaratidza maGO:0006952 pa12 na48 HPI, ese aine majini gumi nemaviri (ekutanga ane majini anobatsira, uye echipiri ane majini anodzvinyirira chete), nepo huwandu hwevanhu 22660 pa6 HPI (13 majini) hwaive nehuwandu hwakakura hwekuwedzera kwepeak. regulation. Zvinofanira kucherechedzwa kuti 96.4% yeGO:0006952 DEG muaya mapeaks yaive nerudzi rumwe chete rwemhinduro (kumusoro kana pasi), zvichiratidza kuwirirana kukuru mumhinduro dzekudzivirira pasinei nekusiyana kwehuwandu hwemajini anobatanidzwa. Boka guru remasequences ane chekuita neshoko rekuti GO:0006952 rinonyora Starvation Stress-Associated Message Protein 22 (SAM22-like), iyo iri muboka reprotein clade yeprotein 10 pathogenesis-associated (PR-10) uye protein protein inonzi latex. yakafanana (MLP-like) (Mufananidzo 3). Mapoka maviri aya akasiyana mumhando yekutaura uye gwara remhinduro. Majini anonyora mapuroteni akafanana neSAM22 akaratidza kupinza kwakasimba panguva dzekutanga (6 kana 12 hpi) uye kazhinji aisapindura pakupera kwekuyedza (48 hpi), nepo mapuroteni akafanana neMLP akaratidza kubatana pa6 hpi. hpi. 83A:476 uye Mandelup pa48 hp/in, anenge mamwe mapoinzi ese edata aisapindura. Pamusoro pezvo, kusiyana kwemaprofile ekutaura kwemajini akafanana neSAM22 kwakatevera kuona kusiyana kwekusagadzikana kweanthracnose, sezvo mitsetse isingapindiki yaive nenguva yakawanda ichikonzera majini aya kupfuura majini anogona kubatwa zvakanyanya. Imwe gene yakafanana neLlR18A/B yakaita sePR-10 yakaratidza maitiro akafanana negene reprotein rakafanana neSAM22.
Zvikamu zvikuru zvemaitiro ehupenyu hwemhuka zvinonzi "GO:0006952 Defense Response" uye maitiro ekutaura kwemajini akakodzera eLanr1 neAnMan alleles zvakaonekwa. Chikero cheLog2 chinomiririra log2 values ​​​​(fold change) pakati pemiti yakapinzwa (Colletotrichum lupini, strain Col-08, yakawanikwa kubva kuminda yelupine, Wizhenica, Poland, 1999) uye zvirimwa zvinodzora (sham-inoculated) panguva imwe chete. Mitsetse inotevera yelupine yakaongororwa: 83A:476 (inodzivirira, inotakura homozygous Lanr1 allele), Boregine (inodzivirira, genetic background haizivikanwe), Mandelup (inodzivirira zvishoma, inotakura homozygous AnMan allele), uye Population 22660 (inogona kubatwa).
Pamusoro pezvo, ma profiles ekuratidzwa kwemajini eRNA-seq candidate Lanr1 (TanjilG_05042) neAnMan (TanjilG_12861) akaongororwa (Mufananidzo 3). Jini reTanjilG_05042 rakaratidza mhinduro yakakosha (activation) pa83A:476 chete panguva yekutanga (6 hpi), nepo TanjilG_12861 yaive yakakosha muMandeloop panguva mbiri chete: 6 hpi (down regulation) uye 24 hpi (6 hpi). With.). adjustable) ).
Majini akanyanya kutaurwa mushoko rekuti GO:0055114 "redox process" aive majini anogadzira mapuroteni e cytochrome P450 uye peroxidase (Mufananidzo 4). Kune masampuro akaparadzaniswa kubva ku83A:476 pa6 HPI, huwandu hwepamusoro kana hushoma hwe log2 (kuchinja kwefold) (ye86.6% yemajini) aiwanzoonekwa pakati pezvirimwa zvakapinzwa nezvinodzora, zvichiratidza kupindura kwakanyanya kwerudzi urwu kune bonde rinopinzwa. 83A:476 yakaratidza GO yakakosha zvikuru: 0055114 DEG pa6 hpi (majini 503), nepo mamwe ma line ari pa48 hpi (Boregine, majini 31; Mandelup, majini 85; uye Population 22660, majini 78)). Mumajini mazhinji emhuri yeGO:0055114, mhando mbiri dzemhinduro pakubayiwa (activation uye inhibition) dzakaonekwa. Zvinofadza kuti, kusvika ku97.6% yemaDEG akawanikwa pashoko rekuti GO: 0055114 muMandelupe pa48 hp. Izvi zvinoratidza kuti, pasinei nechiyero chidiki zvikuru (kureva, huwandu hwemajini eredox akachinja, 85 zvichienderana ne503), maitiro ekunonoka kwemhinduro dzemandeloup kune anthracnose akafanana nemhinduro yekutanga ye83A:476. MuBoregine nePopulation 22660, kusangana uku kwakaderera pa51.6% uye 75.6%, zvichiteerana.
Maitiro ekuratidzwa kwezvikamu zvikuru zveshoko rekuti "GO:0055114 Redox process" akaziviswa. Chikero cheLog2 chinomiririra log2 values ​​​​(fold change) pakati pemiti yakapinzwa (Colletotrichum lupini, strain Col-08, yakawanikwa kubva kuminda yelupine, Wizhenica, Poland, 1999) uye zvirimwa zvinodzora (sham-inoculated) panguva imwe chete. Mitsetse inotevera yelupine yakaongororwa: 83A:476 (inodzivirira, inotakura homozygous Lanr1 allele), Boregine (inodzivirira, genetic background haizivikanwe), Mandelup (inodzivirira zvishoma, inotakura homozygous AnMan allele), uye Population 22660 (inogona kubatwa).
83A:476 Mhinduro dzeTranscriptomic pakubayiwa neC. lupini (strain Col-08) dzakabatanidzawo kunyaradzwa kwemajini kwakabatana neshoko rekuti GO:0015979 "photosynthesis" nedzimwe nzira dzehupenyu (Mufananidzo 5). Iyi seti yeGO:0015979 DEG yaive nemajini 105 akadzvinyirirwa zvakanyanya pa6 hpi pa83A:476. Muchikamu ichi, majini 37 akaderedzwawo muMandelup pa48 HPI uye 35 panguva imwe chete muhuwandu hwevanhu 22660, kusanganisira maDEG 19 akajairika kune ese ma genotypes. Hapana maDEG ane chekuita neshoko rekuti GO: 0015979 akashandiswa zvakanyanya musanganiswa chero upi zvawo (mutsetse x nguva).
Mapatani ekuratidzwa kwezvikamu zvikuru zveshoko rekuti "GO:0015979 Photosynthesis" akaburitswa pachena. Chikero cheLog2 chinomiririra log2 values ​​​​(fold change) pakati pemiti yakapinzwa (Colletotrichum lupini, strain Col-08, yakawanikwa kubva kuminda yelupine, Wizhenica, Poland, 1999) uye zvirimwa zvinodzora (sham-inoculated) panguva imwe chete. Mitsetse inotevera yelupine yakaongororwa: 83A:476 (inodzivirira, inotakura homozygous Lanr1 allele), Boregine (inodzivirira, genetic background haizivikanwe), Mandelup (inodzivirira zvishoma, inotakura homozygous AnMan allele), uye Population 22660 (inogona kubatwa).
Zvichibva pamhedzisiro yekuongorora kwakasiyana-siyana kwezviratidzo uye zvinofungidzirwa kuti zvine chekuita nekudzivirira fungus, seti iyi yemajini manomwe yakasarudzwa kuti iverenge ma profiles ezviratidzo nePCR chaiyo (Supplementary Table S9).
Jini reprotein rinofungidzirwa reTanjilG_10657 rakanyanya kukonzerwa mumitsara yese yakadzidzwa uye nguva dzakatarwa zvichienzaniswa nemiti yekudzora (mimic) (Supplementary Tables S10, S11). Pamusoro pezvo, chimiro cheTanjilG_10657 chakaratidza kuwedzera kwemaitiro mukati menguva yekuedza kwemitsara yese. Huwandu hwevanhu 22660 hwakaratidza kunzwisisika kwakanyanya kweTanjilG_10657 kubaiwa ne114-fold activation uye yepamusoro-soro relative expression level (4.4 ± 0.4) pa24 HPI (Mufananidzo 6a). Jini reprotein rePR10 LlR18A reTanjilG_27015 rakaratidzawo kushanda mumitsara yese nenguva, nekukosha kwehuwandu panhamba zhinji dzedata (Mufananidzo 6b). Kufanana neTanjilG_10657, huwandu hwepamusoro hweTanjilG_27015 hwakaonekwa muhuwandu hwevanhu 22660 vakabayiwa majekiseni pa24 HPI (19.5 ± 2.4). Jini reacid endochitinase reTanjilG_04706 rakakwira zvakanyanya mumitsara yese uye panguva dzese kunze kweBoregine 6 hpi (Mufananidzo 6c). Rakakonzereswa zvakanyanya panguva yekutanga (6 HPI) pa83A:476 (nenguva 10.5) uye rakawedzera zvine mwero mune dzimwe mitsara (nenguva 6.6-7.5). Munguva yekuyedza, kuratidzwa kweTanjilG_04706 kwakaramba kuri padanho rakafanana mu83A:476 neBoregine, nepo muMandelup nePopulation 22660 kwakawedzera zvakanyanya, kusvika pahuwandu hwakakwira (5.9 ± 1.5 uye 6.2 ± 1.5, zvichiteerana). Jini reTanjilG_23384 rakafanana ne endoglucan-1,3-β-glucosidase rakaratidza kushanda kwakanyanya panguva mbiri dzekutanga (6 ne12 hpi) mumitsara yese kunze kwevanhu 22660 (Mufananidzo 6d). Mazinga epamusoro ekutaura kweTanjilG_23384 akaonekwa panguva yechipiri (12 hpi) muMandelup (2.7 ± 0.3) uye 83A:476 (1.5 ± 0.1). Pa24 HPI, kutaura kweTanjilG_23384 kwaive kwakaderera mumitsara yese yakadzidzwa (kubva pa0.04 ± 0.009 kusvika pa0.44 ± 0.12).
Maprofile ezviratidzo zvemajini akasarudzwa (ag) anoratidzwa ne quantitative PCR. Nhamba 6, 12 na 24 dzinomiririra maawa mushure mekubayiwa. Majini eLanDExH7 naLanTUB6 akashandiswa kugadzirisa mamiriro emuviri uye LanTUB6 yakashandiswa kugadzirisa mamiriro emuviri. Mabhawa ezvikanganiso anomiririra kutsauka kwakajairwa zvichibva pamatanho matatu emuviri, rimwe nerimwe riri avhareji yemajini matatu ehunyanzvi. Kukosha kwehuwandu hwemusiyano muhuwandu hwekutaura pakati pemiti yakabayiwa (Colletotrichum lupini, strain Col-08, yakawanikwa muna 1999 kubva mumunda we lupin muWierzenica, Poland) nemiti yekudzora (mock-incocuted) kwakanyorwa pamusoro pe data points (*P value < 0.05, **P value ≤ 0.01, ***P value ≤ 0.001). Kukosha kwehuwandu hwemusiyano muhuwandu hwekutaura pakati pemiti yakabayiwa (Colletotrichum lupini, strain Col-08, yakawanikwa muna 1999 kubva mumunda we lupin muWierzenica, Poland) nemiti yekudzora (mock-incocuted) kwakanyorwa pamusoro pe data points (*P value < 0.05, **P value ≤ 0.01, ***P value ≤ 0.001). 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) Польша) и контрольными (ложно инокулированными) растениями отмечена над точками данных (*значение P < 0,05, **значение P ≤ 0,010, *** 0. Misiyano yakakosha muhuwandu hwezviratidzo pakati pemiti yakabayiwa (Colletotrichum lupini, strain Col-08, yakawanikwa muna 1999 kubva mumunda welupine muWierzhenice, Poland) uye zvirimwa zvekudzora (zvakabayiwa sham) zvakanyorwa pamusoro pemapoinzi edata (*P value < 0.05, **P-value ≤ 0.01, ***P-value ≤ 0.001).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）植物之间表达水平差异的统计学显着性标记在数据点者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 鼈接秗 接种 接种差异 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, штам Купин, в 1998 г. 0,001). Misiyano yakakosha muhuwandu hwezviratidzo pakati pemiti yakabayiwa (Colletotrichum lupini, strain Col-08, yakawanikwa kubva kuminda yelupine muVerzhenice, Poland, muna 1999) uye zvirimwa zvinodzora (zvakabayiwa) zvakanyorwa pamusoro pemapoinzi edata (*P value < 0.05 , **P-value ≤ 0.01, ***P-value ≤ 0.001).Mitsetse yeNLL yakaongororwa yaive: 83A:476 (inodzivirira, ine homozygous Lanr1 allele), Mandelup (inodzivirira zvishoma, ine homozygous AnMan allele), Boregine (inodzivirira, isingazivikanwe majini) uye huwandu hwevanhu 22660 (inogona kubatwa nechirwere ichi).
Jini reTanjilG_05042 panzvimbo yeLanr1 rakaratidza maitiro akasiyana ekutaura kubva kumaprofiles akawanikwa kubva kuongororo dzeRNA-seq (Mufananidzo 6e). Kushanda kwakakosha kwejini iri kwakaonekwa muMandelup uye muhuwandu hwevanhu 22660 (kusvika 39.7 uye 11.7 kamwechete, zvichiteerana), zvichikonzera mazinga ekutaura akakwira (kusvika 1.4 ± 0.14 uye 7.2 ± 1.3, zvichiteerana). 83A:476 yakaratidzawo kuwedzera kwehuwandu hwejini reTanjilG_05042 (kusvika 3.8-fold), zvisinei, mazinga ekutaura akawanikwa (0.044 ± 0.002) aive akaderera nekanopfuura 30 pane akaonekwa muMandelup uye huwandu hwevanhu 22660. yakaongororwa neqPCR yakaratidza mutsauko mukuru muhuwandu hwekutaura pakati pema genotypes mu mock-invaccined (control) variants, zvichisvika mutsauko we58-fold pakati pehuwandu hwevanhu 22660 ne83A:476, pamwe chete nepakati pehuwandu hwevanhu 22660 ne22660. Mutsauko wepakati wakawanikwa pakati peBoregine neMandalup.
Jini remhando yepamusoro paAnMan locus, TanjilG_12861, rakaitwa kuti rive nechirwere che 83A:476 uye Mandelup, rakanga risina kwarakarerekera muhuwandu hwevanhu 22660, uye rakaderedzwa muBoregine (Mufananidzo 6f). Kuonekwa kwejini reTanjilG_12861 kwaive kwakanyanya muhuwandu hwevanhu 83A yakabayiwa: 476 (0.14±0.01). Jini re 17.4 kDa class I heat shock protein gene TanjilG_05080 HSP17.4 rakaratidza mazinga ekuratidza akaderera mumhando dzese dzakadzidzwa uye nguva (Mufananidzo 6g). Kukosha kwakanyanya kwakaonekwa pa24 HPI muhuwandu hwevanhu 22660 (0.14±0.02, kuwedzera kasere mukupindura pakubaiwa).
Kuenzaniswa kwemajini ekutaura (Mufananidzo 7) kwakaratidza hukama hwakakura pakati peTanjilG_10657 nemamwe majini mana: TanjilG_27015 (r = 0.89), TanjilG_05080 (r = 0.85), TanjilG_05042 (r = 0.80), uye TanjilG_04706 (r = 0.79). Mhedzisiro yakadaro inogona kuratidza kubatanidzwa kwemajini aya panguva yekudzivirira. Majini eTanjilG_12861 neTanjilG_23384 akaratidza majini akasiyana ekutaura ane maPearson correlation coefficient values ​​​​akaderera (kubva pa0.08 kusvika 0.43 uye -0.19 kusvika 0.28, zvichiteerana) zvichienzaniswa nemamwe majini.
Kubatana pakati pemajini ekutaura kwakawanikwa uchishandisa quantitative PCR. Mitsetse inotevera yelupine yakaongororwa: 83A:476 (inodzivirira, ine homozygous Lanr1 allele), Mandelup (inodzivirira zvishoma, ine homozygous AnMan allele), Boregine (inodzivirira, genetic background haizivikanwe), uye Population 22660 (inogona kubatwa). Nguva nhatu dzakaverengerwa (maawa 6, 12 uye 24 mushure mekubatwa), kusanganisira yakabayiwa (Colletotrichum lupini, strain Col-08, yakawanikwa kubva kuminda yelupine muWierzhenice, Poland, muna 1999) uye zvirimwa zvinodzora (zvakabayiwa). Chikero chinoratidza kukosha kwePearson correlation coefficient.
Zvichibva padata rakawanikwa pa6 horsepower pa inch, WGCNA yakaitwa pa9981 DEG yakawanikwa nekuenzanisa zvirimwa zvakapinzwa nezvinodzora kuti zvitarise mhinduro dzekudzivirira kwekutanga (Supplementary Table S12). Mamodule makumi maviri nemaviri emagene (clusters) akawanikwa aine ma profiles ekutaura akabatana (akanaka kana asina kunaka) pakati pema genotypes uye akasiyana ekuyedza. Paavhareji, huwandu hwemajini aibuda hwakaderera zvichienderana ne83A:476 > Mandelup > Boregine > Population 22660 (mumhando mbiri idzi, zvisinei, maitiro aya aive akasimba muzvirimwa zvinodzora). Paavhareji, huwandu hwemajini aibuda hwakaderera zvichienderana ne83A:476 > Mandelup > Boregine > Population 22660 (mumhando mbiri idzi, zvisinei, maitiro aya aive akasimba muzvirimwa zvinodzora). В среднем уровни экспрессии генов снижались в порядке 83A:476 Mandelup > Boregine > Population 22660 (в обоих вариантах, однако, эта тенденый контрольных растений). Paavhareji, huwandu hwemajini hwakaderera muodha 83A:476 > Mandelup > Boregine > Population 22660 (mumhando mbiri idzi, zvisinei, maitiro aya aive akasimba muzvirimwa zvinodzora).平均而言，基因表达水平按83A:476 > Mandelup > Boregine > Population 22660的顺序下降（然而，在两种变体中，這种趋势在对照植物中更强）.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 ，更）........ В среднем уровни экспрессии генов снижались в ряду 83A:476 > Mandelup > Boregine > Population 22660 (однако в обоих вариантах эта тенденция син) контрольных растений). Paavhareji, huwandu hwemajini hwakaderera muchikamu che83A:476 > Mandelup > Boregine > Population 22660 (zvisinei, mumarudzi ese ari maviri, maitiro aya aive akasimba muzvirimwa zvinodzora).Kubaiwa majekiseni kwakaita kuti pave nekuwedzerwa kwehuwandu hwemajini, kunyanya mumamodule 18, 19, 14, 6 uye 1 (mukutevedzana kwemhedzisiro), kudzorwa kwemaitiro asina kunaka (semuenzaniso mamodule 9 uye 20) kana nemhedzisiro isina kwayakarerekera (semuenzaniso mamodule 11, 22, 8 uye 13). GO term enrichment analysis (Supplementary Table S13) yakaratidza "GO: 0006952 Protective responses" yemodule yakabayiwa majekiseni (18) ine activation yakanyanya, kusanganisira majini akaongororwa neqPCR (TanjilG_04706, TanjilG_23384, TanjilG_10657 uye TanjilG_27015), pamwe chete nemamodule akawanda eInoculate most depressed photosynthesis (9). Module 18 concentrator (Mufananidzo 8) yakaonekwa sejini reTanjilG_26536 rinonyora protein yePR-10-like LlR18B, uye module 9 concentrator yakaonekwa sejini reTanjilG_28955 rinonyora photosystem II PsbQ protein. Jini rekudzivirira anthracnose resistance Lanr1, TanjilG_05042, rakawanikwa mumodule 22 (Mufananidzo 9) uye rinobatanidzwa nemashoko ekuti “GO:0044260 Cellular macromolecular metabolic processes” uye “GO:0006355 Transcriptional regulation, DNA templating” yakatakura TanjilG_01212 hub. jini iri rinonyora heat stress transcription factor A-4a (HSFA4a).
Kuongororwa kwenetwork yakayerwa kwekushandiswa kwemajini pamwe chete kwemamodules ane mazwi ekuti "GO: 0006952 Defense responses". Kubatanidzwa kwemasero kwakarerutswa kuti kuratidze majini mana akaongororwa neqPCR (TanjilG_04706, TanjilG_23384, TanjilG_10657 naTanjilG_27015).
Kuongororwa kwe network ye weighted ye gene co-expression ye module ine izwi rekuti "GO: 0006355: Transcriptional regulation, DNA templating" uye kutakura candidate anthracnose resistance gene Lanr1 TanjilG_05042. Kusungana kwakarerutswa kuti kupatsanurwe TanjilG_05042 gene uye central TanjilG_01212 gene.
Kuongororwa kwekusagona kurwisa anthracnose kwakatorwa muAustralia kwakaratidza kuti mhando zhinji dzembeu dzakaburitswa pakutanga dzaive dzisinganyanyi kubatwa nechirwere; Kalya, Coromup neMandelup dzakatsanangurwa sedzisinganyanyi kubatwa nechirwere, nepo Wonga, Tanjil ne83A:476 dzakatsanangurwa sedzisinganyanyi kubatwa nechirwere zvakanyanya 26,27,31. dzaive neallele yakafanana yekusagona, dzakanzi Lanr1, uye Coromup neMandelup dzaive neallele yakasiyana, dzakanzi AnMan10, 26, 39, nepo Kalya akaparira allele yakasiyana. , Lanr2. Kuongororwa kwekusagona kurwisa anthracnose muGermany kwakaita kuti pave neBo7212 inosagona kubatwa nechirwere ichi ine allele imwe isiri Lanr1, inonzi LanrBo36.
Chidzidzo chedu chakaratidza kuwanda kwakaderera kwazvo (inenge 6%) kweLanr1 allele mugermplasm yakaedzwa. Kuongororwa uku kunoenderana nemhedzisiro yekuongorora germplasm yeEast Europe uchishandisa Anseq3 neAnseq4 markers, izvo zvakaratidza kuti Lanr1 allele iripo mumitsara miviri yeBelarusian chete. Izvi zvinoratidza kuti Lanr1 allele haisati yashandiswa zvakanyanya nezvirongwa zvekuberekesa zvemuno, kusiyana neAustralia, uko iri imwe yealleles dzakakosha dzekuberekesa dzinobatsirwa nemarker. Izvi zvinogona kunge zvichikonzerwa nedanho rakaderera rekuramba kupihwa neLanr1 allele mumamiriro eEurope zvichienzaniswa nemushumo weAustralia. Pamusoro pezvo, zvidzidzo zveanthracnose munzvimbo dzine mvura yakawanda muAustralia zvakaratidza kuti kuramba kunokonzerwa neLanr1 allele kunogona kusashanda mumamiriro ekunze anokurudzira kukura uye kukura nekukurumidza kwechirwere19,42. Kutaura zvazviri, muchidzidzo chino, zvimwe zviratidzo zveanthracnose zvakaonekwawo mu genotypes dzakatakura Lanr1 allele, zvichiratidza kuti kuramba kunogona kupera mumamiriro ezvinhu akanaka ekukura kweC. lupini. Pamusoro pezvo, kududzira kwenhema kwekuvapo kwezviratidzo zveAnseq3 neAnseq4, zviri pa1 cM kubva panzvimbo yeLanr1, zvinogoneka 28,30,43.
Chidzidzo chedu chakaratidza kuti 83A:476, yakatakura allele yeLanr1, yakapindura kubaiwa kweC. lupini nekugadzira transcriptome yakakura panguva yekutanga yakaongororwa (6 hpi), nepo muMandelup, yakatakura allele yeAnMan, mhinduro dzetranscriptomic dzakaonekwa gare gare. (kubva pa24 kusvika 48 hp). Kuchinja uku kwekudzivirira kwakabatana nekusiyana kwezviratidzo zvechirwere, zvichiratidza kukosha kwekuzivikanwa kwekutanga kwehutachiona kuti mhinduro ibudirire yekusagona. Kuti utapukire nyama yemiti, spores dzeanthrax dzinofanira kupfuura nematanho akati wandei ekukura pamusoro pemupi wemhuka, kusanganisira kumera, kupatsanurwa kwemasero, uye kuumbwa kweappressorium. Appendage chimiro chehutachiona chinonamatira pamusoro pemupi wemhuka uye chinoita kuti zvipinde munyama yemhuka. Saka, spores dzeC. gloeosporioides mupea extract dzakaratidza kupatsanurwa kwekutanga kwenucleus mushure memaminitsi 75-90 ekubatidzwa, kuumbwa kwechubhu yegerm mushure memaminitsi 90-120, uye kudzvinyirirwa mushure memaawa 4 45. Mango C. gloeosporioides yakaratidza kumera kweinopfuura 40% conidial mushure memaawa matatu ekuiswa muvhu uye kuumbwa kwe appressors dzinenge 20% mushure memaawa mana. Gene reCAP20 rine virulence-associated CAP20 reC. gloeosporioides rakaratidza transcriptional activity mu epiphyte-forming conidia mushure memaawa matatu nehafu ekuiswa mu avocado surface wax ine huwandu hwakawanda hweCAP20 protein mushure memaawa mana nemaminitsi makumi mana nematanhatu. Saizvozvowo, basa re melanin biosynthesis genes muC. trifolii rakakonzereswa panguva yekuiswa kwemaawa maviri zvichiteverwa nekuumbwa kwe appressorium mushure meawa imwe chete. Zvidzidzo zvemashizha zvakaratidza kuti mastrawberry akaiswa neC. acutatum ane kudzvinyirirwa kwekutanga pa8 hpi, nepo madomasi akaiswa neC. coccodes aine kudzvinyirirwa kwekutanga pa4 hpi48,49. Zvichienderana zvikuru nenguva yeColletotrichum spp. hutachiona. Mhinduro dzekudzivirira dzinokurumidza ku83A:476 dzinoratidza kubatanidzwa kwemajini ekudzivirira zvirimwa uye effector-triggered immunity (ETI) mumutsara uyu, nepo mhinduro dzaMandelup dzakanonoka dzichitsigira micro-associated molecular pattern-triggered immunity (MTI) hypothesis 50. Mhinduro dzekutanga ku83A: 476 naMandelup. Kubatana kwechikamu pakati pemajini akakwira kana akaderera mukunonoka kupindura kunotsigirawo pfungwa iyi, sezvo ETI inowanzoonekwa semhinduro yeMTI inokurumidza uye yakawedzerwa iyo inoguma nekufa kwemasero panzvimbo yehutachiona, inozivikanwa se anaphylactic shock 51,52.
Majini mazhinji anokonzerwa neshoko rekuti Gene Ontology GO:0006952 "Defense Response" ndiwo ma homologues gumi nerimwe eprotein ye stress-induced fasting message 22 (yakafanana neSAM22) uye mapuroteni manomwe makuru e latex protein-like (MLPs). Mapuroteni akafanana ne 31, 34, 43 uye 423 akaratidza kufanana kwe sequence. Majini akafanana neSAM22 akaratidza kushanda kwakasimba kwakagara kwenguva yakareba, zvichiratidza kuwedzera kwe anthracnose resistance (83A:476 uye Boregine). Zvisinei, majini akafanana neMLP akaderedzwa chete mumitsara ine candidate resistance allele (83A:476/Lanr1 pa 6 hpi uye Mandelup/AnMan pa 24 hpi). Zvinofanira kucherechedzwa kuti ma homologues ese akaonekwa eSAM22 anobva muboka remajini rinosvika 105 kb, nepo majini akafanana neMLP anobva munzvimbo dzakasiyana dze genome. Kushanda kwakarongeka kwemajini akadaro akafanana neSAM22 kwakawanikwawo muongororo yedu yapfuura yekuramba kweNLL kuDiaporthetoxica, zvichiratidza kuti ane chekuita nezvikamu zvakatwasuka zvemhinduro yekudzivirira. Mhedziso iyi inotsigirwawo nemishumo yemhinduro yakanaka yemajini akafanana neSAM22 pakukuvadzwa kana kurapwa nesalicylic acid, fungal inducers, kana hydrogen peroxide.
Majini akafanana neMLP akaratidzwa kuti anopindura kune zvakasiyana-siyana zvinonetsa zveabiotic uye biotic, kusanganisira hutachiona hwebhakitiriya, hutachiona uye hutachiona hwefungus mumarudzi mazhinji emiti55. Nzira dzekupindura kune kumwe kusangana pakati pezvirimwa nehutachiona dzakatangira pakuwedzera zvakanyanya (kureva, panguva yekupararira kwedonje neVerticillium dahliae) kusvika pakudzikira zvakanyanya (kureva, mushure mekupararira kwemuti weapuro neAlternaria spp.)56,57. Kudzikiswa kwakanyanya kwejini reMLP-like 423 kwakaonekwa panguva yekudzivirira kweavocado kuhutachiona hweF. niger uye panguva yekupararira kwemuti weapuro Botryosphaeria berengeriana f. cn. piricola uye Alternaria alternata imhando dzeapuro58,59. Pamusoro pezvo, apple calli yakawedzera kuburitsa jini reMLP-like 423 yaive nekushomeka kwemajini akabatana neresistance uye yaive nyore kubatwa nefungus59. Mushure meFusarium oxysporum f, jini reMLP-like 423 rakadzvinyirirwawo mu resistance common bean germplasm. cn. Bean infection60.
Dzimwe nhengo dzemhuri yePR-10 dzakaonekwa muchidzidzo chedu cheRNA-seq dzaive majini eLlR18A neLlR18B mukupindura kukwidziridzwa, pamwe chete nejini rekuwedzera (jini rimwe) kana risinganyanyi kudzorwa (majini matatu) reprotein yekufambisa mafuta DIR1. . Pamusoro pezvo, WGCNA inoratidza jini reLlR18B senzvimbo huru mumodule iyi, iyo inonyanya kubatwa nejekiseni uye ine majini akawanda ekudzivirira. Majini eLlR18A neLlR18B akakonzerwa nemashizha eyero elupine mukupindura mabhakitiriya ane pathogenic, pamwe nemuNLL stems mushure meD. toxica inoculation, nepo rice homologue yemajini aya, RSOsPR10, yakakurumidza kukonzerwa nehutachiona hwefungus hunofungidzirwa kuti hwakabatanidzwa mujasmonic acid signaling pathway53,61, 62. Jini reDIR1 rinoisa mapuroteni asina hunyanzvi ekutakura mafuta anodiwa kuti pave nesystemic acquired resistance (SAR). Nekukura kwemaitiro ekudzivirira, puroteni yeDIR1 inotakurwa kubva panzvimbo yehutachiona kuburikidza nephloem kuti iite kuti SAR ipinde munhengo dziri kure. Zvinofadza kuti, gene reTanjilG_02313 DIR1 rakakonzereswa zvakanyanya panguva yekutanga mumitsara 84A:476 nePopulation 22660, asi kuramba anthracnose kwakabudirira chete mumutsara 84A:476. Izvi zvinogona kuratidza kusashanda zvakanaka kwegene reDIR1 muNLL, sezvo mamwe matatu akafanana akagamuchira kubaiwa mumutsara 83A:476 chete pa6 hpi, uye mhinduro iyi yakatungamirirwa pasi.
Mukudzidza kwedu, zvikamu zvinonyanya kuenderana nemaitiro ehupenyu zvinonzi "GO:0055114 Redox process" zvaive cytochrome P450 protein, peroxidase, linoleic acid 9S-/13S-lipoxygenase, uye 1-aminocyclopropane-1-carboxylic acid oxidase. Pamusoro pezvo, WGCNA yedu inotsanangura homologue yeHSFA4a sema hub carrier modules akadai seLanr1 resistance gene candidate TanjilG_05042. HSFA4a chikamu che redox-dependent regulation ye nuclear transcription mumiti.
Mapuroteni eCytochrome P450 maoxidoreductases anokonzeresa NADPH uye/kana O2-dependent hydroxylation reactions mu primary and secondary metabolism, kusanganisira metabolism ye xenobiotics, pamwe nemahormone, fatty acids, sterols, cell wall components, biopolymers, uye biosynthesis ye protective compounds 69. Muchidzidzo chedu, kusiyana kwebasa re cytochrome P450 yezvirimwa kwakaderedzwa kubva pa -10.6 log2 (fold change) kusvika pa5.7 nekuda kwehuwandu hwakawanda hwe homologues dzakachinjwa (37) uye musiyano mu response patterns pakati pemajini chaiwo, zvichiratidza upward revision. . Kushandisa data reRNA-seq chete kujekesa fungidziro ye biological function yemajini eNLL muprotein superfamily yakakura kudaro kwaizova kufungidzira kwakanyanya. Zvisinei, zvakakosha kuziva kuti mamwe majini e cytochrome P450 anobatanidzwa nekuwedzera kwe resistance kune pathogenic fungi kana mabhakitiriya, kusanganisira mupiro kune allergic reactions69,70,71.
Maperoxidases eClass III maenzymes emiti anoshanda mumabasa akasiyana-siyana ekugadzirisa masero panguva yekukura nekukura kwezvirimwa, pamwe nekupindura kushushikana kwezvakatipoteredza zvakaita semunyu, kusanaya kwemvura, simba rechiedza, uye kurwiswa kwezvirwere72. Maperoxidases anobatanidzwa mukudyidzana kwemarudzi akati wandei emiti neAnthracis, anosanganisira Stylosanthes humilis neC. gloeosporioides, Lens culinaris neC. truncatum, Phaseolus vulgaris neC. lindemuthianum, Cucumis sativus neC. lagenarium73,74,75,76. Mhinduro yacho inokurumidza zvikuru, dzimwe nguva kunyangwe pa4 HPI, fungus isati yapinda munyama yemiti73. Jini reperoxidase rakapindurawo kuD. toxica NLL inoculation. Kuwedzera kune mabasa aro akajairika ekudzora kuputika kwe oxidative kana kubvisa oxidative stress, maperoxidases anogona kukanganisa kukura kwezvirwere nekugadzira zvipingamupinyi zvemuviri zvichibva pakusimbiswa kwemadziro esero panguva ye lignification, subunit kana cross-link yemakemikari chaiwo. Basa iri rinogona kuverengerwa mu silico kune gene reTanjilG_03329 rinonyora anion peroxidase inogadzira lignin iyo yakawedzera zvakanyanya muchidzidzo chedu mumutsara unodzivirira 83A:476 pa6 HPI, asi kwete mune mamwe marudzi uye nguva dzisina kupindura.
9S-/13S-lipoxygenase ye linoleic acid ndiyo nhanho yekutanga munzira ye oxidative ye lipid biosynthesis78. Zvigadzirwa zveiyi nzira zvine mabasa akawanda mukudzivirira zvirimwa, kusanganisira kusimbisa madziro emasero kuburikidza nekuumbwa kwe callose ne pectin deposits, uye kudzora oxidative stress kuburikidza nekugadzirwa kwe reactive oxygen species79,80,81,82,83. Muchidzidzo chino, kuratidzwa kwe linoleic acid 9S-/13S-lipoxygenase kwakachinjwa mumarudzi ese, asi muhuwandu hwevanhu vanobatwa nechirwere 22660, kuwedzera kwakapararira panguva dzakasiyana, nepo mumarudzi ane Lanr1 inodzivirira uye AnMan allele, inosimbisa kusiyana kwe oxylipin layer mukudzivirira anthrax reactions pakati pema genotypes aya.
Kubatana kwe1-aminocyclopropane-1-carboxylate oxidase (ACO) kwakawedzera zvakanyanya (majini mapfumbamwe) kana kuderedzwa (majini maviri) paakabayiwa lupine. Kunze kwezviviri, mhinduro dzese idzi dzakaitika pa6 hp. pa83A:476. Maitiro eenzyme anokonzerwa nemapuroteni eACO ndiyo danho rinoganhurira mukugadzirwa kwe ethylene uye nekudaro rinodzorwa zvakanyanya84. Ethylene ihormone yemiti inoita mabasa akasiyana-siyana mukudzora kukura kwemiti uye kupindura kumamiriro ekushushikana kweabiotic uye biotic. Kutangwa kweACO transcription uye kuvhurwa kwenzira ye ethylene signaling kunobatanidzwa mukuwedzera kuramba kwemupunga ku hemibiotrophic fungus oryzae oryzae nekudzora kugadzirwa kwe reactive oxygen species uye phytoalexins. Maitiro akafanana ehutachiona hwemashizha akawanikwa pakati peM. oryzae naC. lupini88,89, zvichitevera kuwedzera kukuru kweACO homologues mumutsara we83A:476 wakataurwa muchidzidzo ichi, anochinja mukana wekupa kuramba kuNLL anthracnose Ethylene sedanho guru rekuratidza nzira dzemamorekuru.
Muchidzidzo chazvino, kudzvinyirirwa kukuru kwemajini mazhinji ane chekuita ne photosynthesis kwakaonekwa pa6 hpi mu83A:476 uye pa48 hpi muMandeloop uye pa22660. Kukura uye kufambira mberi kweshanduko idzi zvinoenderana nedanho. Kuramba kweAnthracnose kwakaonekwa mukuyedza uku. Munguva pfupi yapfuura, kudzvinyirirwa kwakasimba uye kwekutanga kwezvinyorwa zvine chekuita ne photosynthesis kwakaratidzwa mumhando dzakasiyana dzekudyidzana kwezvirimwa nezvirwere, kusanganisira mabhakitiriya ane pathogenic nefungus. Kukurumidza (kubva pa2 HPI mune dzimwe nzira) uye kudzvinyirirwa kwepasi rose kwemajini ane chekuita ne photosynthesis mukupindura hutachiona kunogona kukonzera kusadzivirirwa kwezvirimwa zvichibva pakuiswa kwemhando dzeokisijeni dzinoshanda uye kusangana kwadzo ne salicylic acid pathway kuti igadzirise allergic reactions 90,94.
Mukupedzisa, nzira dzekudzivirira dzakakurudzirwa kune dzinowanzo dzivirira (83A:476) dzinosanganisira kukurumidza kuzivikanwa kwehutachiona nejini reR (zvichida TIR-NBS-LRR TanjilG_05042) uye salicylic acid uye ethylene signaling, zvichiteverwa nekugadzwa kweSAR refu. inotsigirwa neprotein yeDIR-1. Zvinofanira kucherechedzwa kuti nguva yebiotrophic yehutachiona hweC. lupini ipfupi kwazvo (inenge mazuva maviri), ichiteverwa nekukura kwemasero95. Kuchinja pakati pezvikamu izvi kunogona kusanganiswa ne necrosis uye kuratidzwa kwemapuroteni e-ethylene-inducible anoshanda sezvikonzero zve hypersensitivity reactions mumiti inogarwa. Nokudaro, nguva yekubata C. lupini zvakanaka padanho rebiotrophic yakamanikana zvikuru. Kugadziriswazve kwemajini ane chekuita ne redox uye photosynthesis kwakaonekwa mu83A:476 pa6 hpi kunoenderana nekufambira mberi kwefungus hyphae uye kunoratidza kukura kwemhinduro yekudzivirira inobudirira padanho rebiotrophic. Mhinduro dzeMandelup nevanhu 22660 dzinogona kunonoka kubata howa vasati vachinja kuita kukura kwechirwere, zvisinei, Mandelup inogona kushanda zvakanyanya kupfuura vanhu 22660 nekuti kudzorwa nekukurumidza kweprotein yePR-10 kunokurudzira kuramba kwakatwasuka.
ETI, inotungamirwa negene reR canonical, inoita senzira yakajairika yekudzivirira bhinzi kuanthracnose. Saka, mumuenzaniso wembeu Medicago truncatula, kudzivirira anthracnose kunopiwa negene reRCT1, nhengo yeTIR-NBS-LRR97 plant R gene class. Iri gene rinopawo kudzivirira anthracnose kwakafara mualfalfa kana ikaendeswa kumiti inobatwa nechirwere. Mubhinzi common (P. vulgaris), majini anopfuura makumi maviri ekudzivirira anthracnose akaonekwa kusvika pari zvino. Mamwe majini aya anowanikwa munzvimbo dzisina majini eR canonical, zvisinei mamwe akawanda ari kumucheto kwemachromosomes ane NBS-LRR gene cluster, kusanganisira TIR-NBS-LRRs99. Chidzidzo cheSSR che genome-wide chakasimbisawo hukama hwegene reNBS-LRR nekudzivirira anthracnose mubhinzi common. Geni reR canonical rakawanikwawo munzvimbo ye genomic yakatakura nzvimbo huru yekudzivirira anthracnose muwhite lupine 101.
Basa redu rinoratidza kuti maitiro ekudzivirira nekukasika, anotanga kushanda pakutanga kwehutachiona hwezvirimwa (zvichida kwete panosvika 12 hpi), anodzivirira lupine ine mashizha madiki kubva kuanthracnose inokonzerwa nefungus inonzi Collelotrichum lupini. Tichishandisa nzira yepamusoro yekutevedzana, takaratidza ma profiles akasiyana emaitiro ekudzivirira anthracnose mumiti yeNLL inotungamirwa nemajini ekudzivirira Lanr1 neAnMan. Kudzivirira kunobudirira kunosanganisira kugadzira majini emapuroteni anobatanidzwa mu redox, photosynthesis, uye pathogenesis mukati memaawa ekutanga emuti kusangana nehutachiona. Maitiro akafanana ekudzivirira, asi anononoka nenguva, haashande zvakanyanya mukudzivirira zvirimwa kubva kuzvirwere. Kudzivirira anthrax kunotungamirwa nejini reLanr1 kwakafanana nemhinduro yakajairika yejini reR (immune intensity inokonzereswa ne effector), nepo jini reAnMan ringangove richipa mhinduro yakatwasuka (immune intensity inokonzereswa ne microbe-associated molecular pattern), zvichipa mwero wepakati wekugara kwenguva refu.
Mitsetse yeNLL 215 yakashandiswa kuongorora zviratidzo zveanthracnose yaive nemarudzi makumi manomwe nemana emiti, mitsetse makumi matanhatu yakawanikwa nekupatsanura kana kubereka, mutants 5, uye germplasms dzemusango kana dzepakutanga 76. Mitsetse iyi yakabva kunyika gumi nenomwe, kunyanya kubva kuPoland (58), Spain (47), Germany (27), Australia (26), Russia (19), Belarus (7), Italy (5) nedzimwe mitsetse. kubva kunyika gumi. Seti iyi inosanganisirawo mitsetse isingatsungiriri: 83A:476, Tanjil, Wonga yakatakura Lanr1 allele, uye Mandelup yakatakura AnMan allele. Mitsetse iyi yakawanikwa kubva kuEuropean Lupine Genetic Resource Database inochengetwa nePoznań Plant Breeding Ltd., Wiatrowo, Poland (Supplementary Table S1).
Zvirimwa zvakarimwa mumamiriro ezvinhu akarongwa (photoperiod maawa gumi nematanhatu, tembiricha 25°C masikati uye 18°C ​​​​husiku). Zvakaongororwa zviviri zvezvipenyu. DNA yakabviswa pamashizha ane mavhiki matatu ekuberekwa uchishandisa DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) zvichienderana neprotocol. Hunhu uye huwandu hweDNA yakabviswa hwakaongororwa nenzira dze spectrophotometric (NanoDrop 2000; Thermo Fisher Scientific, Waltham, MA, USA). Chiratidzo cheAnManM1 chinoratidza gene resistance anthracnose AnMan (yakabva ku cv. Mandelup) uye zviratidzo zveAnseq3 neAnseq4 zvakakomberedza gene reLanr1 (yakabva ku cv. Tanjil) zvakaongororwa 11,26,28. Homozygotes yeallele isingapindi yakapihwa se "1″, inogona kubatwa - se "0″, uye heterozygotes - se 0.5.
Zvichibva pamhedzisiro yekuongororwa kwezviratidzo zveAnManM1, AnSeq3 neAnSeq4 uye kuwanikwa kwembeu dzekuedza kwekupedzisira, mitsara makumi mashanu yeNLL yakasarudzwa kuti ishandiswe kudzivirira anthracnose resistance phenotyping. Ongororo iyi yakaitwa kaviri mugreenhouse inodzorwa nekombuta ine nguva yekupisa kwemaawa gumi nemana ine tembiricha ye22°C masikati uye 19°C husiku. Mbeu dzinokwenywa (kucheka ganda rembeu riri kune rumwe rutivi rwe embryo nebanga rakapinza) vasati vadyara kudzivirira mbeu kurara nekuda kwekuti ganda rembeu rakaoma zvakanyanya uye kuti mbeu imere zvakaenzana. Zvirimwa zvakarimwa muhari (11 × 11 × 21 cm) dzine ivhu risina utachiona (TS-1 REC 085 Medium Basic, Klasmann-Deilmann Polska, Warsaw, Poland). Kubaya kwakaitwa nerudzi rweColletotrichum lupini Col-08, rwakarimwa muna 1999 kubva padzinde remiti yelupine ine mashizha matete yakadyarwa mumunda muVerzhenitsa, Greater Poland (52° 27′ 42″ N 17° 04′ 05″ E ). Wana nzvimbo. Miti yakatorwa yakadyarwa munzvimbo yeSNA pa20° C. pasi pechiedza chitema kwemazuva makumi maviri nerimwe kuti iite kuti mbeu ikure. Mavhiki mana mushure mekudyara, apo miti yasvika padanho remashizha mana kusvika matanhatu, kubaya kwakaitwa nekupfapfaidza ne conidia yakamiswa pahuwandu hwe 0.5 x 106 conidia pa ml. Mushure mekubaya, miti yakachengetwa murima kwemaawa makumi maviri nemana pahunyoro hweinenge 98% uye tembiricha ye25°C kuti zvive nyore kumera kwe conidia uye maitiro ehutachiona. Zvirimwa zvakazorimwa pasi penguva yekupisa kwemaawa gumi nemana pa22°C masikati/19°C husiku uye hunyoro hwe70%. Chiyero chechirwere chakaonekwa mazuva makumi maviri nemaviri mushure mekubayiwa uye chakatangira pa0 (chinodzivirira muviri) kusvika pa9 (chinonyanya kubatwa nechirwere) zvichienderana nekuvapo kana kusavapo kwezvironda zvinouraya padzinde nemashizha. Pamusoro pezvo, mushure mekubayiwa, huremu hwezvirimwa hwakayerwa. Hukama huripo pakati pema genotypes ezviratidzo uye phenotypes dzezvirwere hwakaverengerwa se point two-sequence correlations (kusavapo kwe heterozygous markers mu seti yemitsetse yekuongorora phenotype ye anthracnose resistance).