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Angustifolius lupine (NLL, Lupinus angustifolius L.) ni igihingwa cya leguminine gikoreshwa mu gutanga ibiribwa no kuzamura ubutaka.Kwiyongera kwisi yose NLL nkigihingwa cyakuruye ibihumyo byinshi bitera indwara, harimo na lupine anthracnose, itera indwara yangiza anthracnose.Alleles ebyiri, Lanr1 na AnMan, zitanga imbaraga zo kurwanya, zakoreshejwe mu bworozi bwa NLL, ariko uburyo bwa molekile bwibanze ntiburamenyekana.Muri ubu bushakashatsi, ibimenyetso bya Lanr1 na AnMan byakoreshejwe mugusuzuma ingero za NLL zi Burayi.Kwipimisha urukingo ahantu hagenzuwe byemeje imikorere yabaterankunga bombi barwanya.Imvugo itandukanye yerekana imiterere yakorwaga kumurongo uhagarariye kandi byoroshye.Kurwanya Anthracnose byari bifitanye isano no gukabya gukabije amagambo ya gene ontologiya “GO: 0006952 Defence Response”, “GO: 0055114 Redox Process”, na “GO: 0015979 Photosynthesis”.Byongeye kandi, umurongo wa Lanr1 (83A: 476) werekanye progaramu ya progaramu ya transcriptome yihuse nyuma yo guterwa, mugihe indi mirongo yerekanaga gutinda kwiki gisubizo amasaha agera kuri 42.Ibisubizo byokwirwanaho bifitanye isano na gen TIR-NBS, CC-NBS-LRR na NBS-LRR, poroteyine 10 zigira uruhare mu gutera indwara, poroteyine zoherejwe na lipide, endoglucan-1,3-β-glucosidase, poroteyine zikungahaye kuri glycine, hamwe na gen biva mu nzira ya ogisijeni.Ibisubizo hakiri kare kuri 83A: 476, harimo no guhagarika neza gen zigendana na fotosintezeza, byahuriranye no kurinda neza mugihe cyikura ryibimera cya biologiya yibihumyo, byerekana ko ingirakamaro itera ubudahangarwa.Imyitwarire ya Mandeloop itinda, nkuko bisanzwe bikurura.
Lupine ifite amababi magufi (NLL, Lupinus angustifolius L.) ni ibinyampeke byinshi bya poroteyine bikomoka mu burengerazuba bwa Mediterane1,2.Kuri ubu ihingwa nkigihingwa cyibiribwa ku nyamaswa n'abantu.Ifatwa kandi n’ifumbire mvaruganda muri sisitemu yo guhinduranya ibihingwa bitewe na azote ikosorwa na azbiyumu ya azote ikosora bagiteri no kuzamura muri rusange imiterere yubutaka.NLL yahuye n'inzira yihuse yo gutunga mu kinyejana gishize kandi iracyafite igitutu kinini cyo korora3,4,5,6,7,8,9,10,11,12.Hamwe n’ibihingwa byinshi bya NLL, urukurikirane rw’ibihumyo bitera indwara byateje imbere ubuhinzi bushya kandi bitera indwara nshya zangiza imyaka. Ikintu cyagaragaye cyane ku bahinzi n’aborozi ni isura ya anthracnose, yatewe na fungus itera indwara, Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13. Ikintu cyagaragaye cyane ku bahinzi n’aborozi ni isura ya anthracnose, yatewe na fungus itera indwara, Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13. Наиболее примечательным для кимеров и селекционеров люпина было появление антракноза, в »ванного патанным грибком Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13. Icyagaragaye cyane ku bahinzi n’aborozi ni ukugaragara kwa anthracnose yatewe na fungus Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13.Let 羽扇 豆 农民 和 饲养 者 来说 ， 最 的 真菌 let let 真菌 let let Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13 引起 的。对于 羽扇 豆 农民 和 饲养 者 来说 ， 引人注目 的 真菌 let let let let Colletotrichum lupini (Bondar) 嵵 Umusatsi。1 Наиболее поразительным для конмеров и селекционеров люпина является появление антракноза, в Igitangaje cyane ku bahinzi borozi n'aborozi ni ukuvuka kwa anthracnose iterwa na fungus yanduye Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13.Raporo ya mbere y’indwara yaturutse muri Berezile no muri Amerika, ibimenyetso bisanzwe bigaragara mu 1912 na 1929.Ariko, nyuma yimyaka igera kuri 30, virusi yise Colletotrichum gloeosporioides (Penz.) Penz. & Sacc., Teleomorph Glomerella cingulata (Stoneman) Spauld. & Sacc., Teleomorph Glomerella cingulata (Stoneman) Spauld. & Sacc., Омомомор Glomerella cingulata (Stoneman) Spauld. & Sacc., Teleomorph ya 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 muri Targeted Morphology. & H. Schrenk,. & H. Schrenk ,.na H. Schrenk. & H. 施伦克 ，。 & H. 施伦克 ，。na H. Schlenk ,.Indwara yibanze ya phenotyping yakozwe hagati yikinyejana cya 20 rwagaragaje ko hari ukurwanya muri NLL na lupine yumuhondo (L. luteus L.), ariko ibikoresho byose byera (L. albus L.) byapimwe byanduye cyane 15,16.Ubushakashatsi bwerekanye ko iterambere rya anthracnose rifitanye isano no kwiyongera kw'imvura (ubuhehere bwo mu kirere) n'ubushyuhe (mu ntera ya 12-28 ° C), biganisha ku kurenga ku barwanya ubushyuhe bwo hejuru17, 18. Mubyukuri, igihe cyasabwaga kugira ngo conidia imere kandi indwara itangire, yikubye inshuro enye kuri 24 ° C (amasaha 4) munsi ya 12 ° C (amasaha 16).Ubwo rero, ubushyuhe bukomeje kuba ku isi bwatumye antarakose ikwirakwira.Nyamara, iyi ndwara yagaragaye mu Bufaransa (1982) no muri Ukraine (1983) nk'intangiriro y’iterabwoba ryegereje, ariko bigaragara ko yirengagijwe n’inganda za lupine icyo gihe 20.21.Nyuma yimyaka mike, iyi ndwara yangiza ikwira isi yose ndetse inagira ingaruka mubihugu bikomeye bitanga lupine nka Ositaraliya, Polonye n'Ubudage 22,23,24.Nyuma y’icyorezo cya anthracnose rwagati mu myaka ya za 90 rwagati, isuzuma ryinshi ryatumye hamenyekana abaterankunga benshi barwanya ingero za NLL19.NLL irwanya anthracnose igenzurwa na alleles ebyiri zitandukanye ziganje ziboneka mumasoko atandukanye ya germplasm: Lanr1 mubihingwa Tanjil na Wonga na AnMan mubuhinzi.Mandalay 25, 26. Izi alleles zuzuza ibimenyetso bya molekuline bifasha guhitamo mikorobe idakira muri gahunda yo korora 25,26,27,28,29,30.Umurongo w’ubworozi udashobora kwihanganira 83A: 476 utwaye Lanr1 allele wambukijwe n'umurongo wo mu gasozi ushobora kwibasirwa na P27255 kugira ngo haboneke abaturage ba RIL batandukanya imiti igabanya ubukana bwa anthracnose, bituma bishoboka ko hashyirwaho inzige ya Lanr1 kuri chromosome NLL-1131, 32, 33. Guhuza ibimenyetso byerekana amakarita ahuza ibibanza bya LLL hamwe na antLomose. NLL-11), ariko mu myanya itandukanye 29,34,35.Nyamara, kubera umubare muto wa RIL hamwe nintera nini ya genetike hagati ya marikeri na alleles ihuye, nta mwanzuro wizewe ushobora gufata kubijyanye na gen.Ku rundi ruhande, gukoresha genetike ihindagurika muri lupine biragoye bitewe nubushobozi bwabo buke cyane bwo kuvuka, bigatuma manipulation genetique igorana37.
Iterambere rya germplasm yororerwa mu rugo itwara allele yifuzwa muri leta idahuje igitsina, nka 83A: 476 (Lanr1) na Mandelup (AnMan), yafunguye umuryango wo kwiga anthracnose irwanya guhangana na alleles mu baturage bo mu gasozi.Ibishoboka byimikorere ya molekile.Gereranya ibisubizo byo kwirwanaho byakozwe na genotypes yihariye.Ubu bushakashatsi bwasuzumye igisubizo cya mbere cya NLL ku rukingo rwa C. lupini.Ubwa mbere, akanama germplasm yuburayi NLL karimo imirongo 215 yerekanwe hifashishijwe ibimenyetso bya molekile biranga Lanr1 na AnMan alleles.Anthracnose phenotyping yakorwaga kumurongo wa NLL 50, mbere yatoranijwe kubimenyetso bya molekile, mugihe cyagenwe.Hashingiwe kuri ubwo bushakashatsi, imirongo ine itandukanye yo kurwanya anthracnose hamwe na Lanr1 / AnMan allelic yahimbwe yatoranijwe kugirango imvugo itandukanye yo kwirwanaho ikoreshwe hakoreshejwe uburyo bubiri bwuzuzanya: ikurikiranwa ryinshi rya RNA hamwe nigihe cyo kugereranya PCR.
Kugaragaza urutonde rwa NL DRMMLMSM (n = 215) hamwe na marindars Lanr1 (Anseq4) na Anman (Anman ", hafi ya 'Anman (Anman" .Imirongo 13 yabyaye alleles ebyiri "zihanganira" ikimenyetso cya Lanr1, naho imirongo 8 itanga alleles "irwanya" ya Lanr1.marikeri.“Kurwanya” allele yikimenyetso cya AnMan (Imbonerahamwe yinyongera S1).Imirongo ibiri yari itandukanye kuri marike ya Anseq3 hamwe na heterozygous kuri marike ya AnManM1.Imirongo 42 (19.5%) yatwaye ibice bitandukanye bya Anseq3 na Anseq4 alleles, byerekana inshuro nyinshi yo kwiyunga hagati yibi bibanza byombi.Anthracnose phenotypes mubihe byagenzuwe (Imbonerahamwe yinyongera S2) yerekanye itandukaniro mukurwanya genotypes zapimwe, byagaragaraga muburemere bwa antarakine.Itandukaniro riri hagati yamanota aringaniye kuva kuri 1.8 (irwanya iringaniye) kugeza kuri 6.9 (suseptible) naho itandukaniro ryibiro byibimera kuva kuri 0,62 (byoroshye) kugeza kuri 4.45 g (birwanya). Hariho isano rinini hagati yindangagaciro zagaragaye mubisubirwamo bibiri (0.51 kumanota yuburemere bwindwara, P = 0.00017 na 0.61 kuburemere bwibimera, P <0.0001) kimwe no hagati yibi bice byombi (- 0.59 na - 0.77, P <0.0001). Hariho isano rinini hagati yindangagaciro zagaragaye mubisubirwamo bibiri (0.51 kumanota yuburemere bwindwara, P = 0.00017 na 0.61 kuburemere bwibimera, P <0.0001) kimwe no hagati yibi bice byombi (- 0.59 na - 0.77, P <0.0001). Вызвлена ​​достоверная коррел мат между значениями, наблюдаемыми в двух повторностях мимимента (0,51 для адов т тестести болезни, P = 0,00017 и 0,61 для массы рас ретия, P <0,0001) 0,0001) 0,0001). Isano rikomeye ryabonetse hagati yagaciro kagaragaye mubisubiramo bibiri byubushakashatsi (0.51 kumanota yuburemere bwindwara, P = 0.00017 na 0.61 kuburemere bwibimera, P <0.0001), ndetse no hagati yibi bipimo byombi (- 0.59 na -0.77, P <0.0001) 0.0001).在 两次 重复 实验 中 观察 到 的 值 存在 显 着 相关 为 为 0.51 ， P = 0.00017 ， 植物 重量 为 0.61 ， P <0.0001） 以及在 两 次 重复 实验 中 观察 的 值 值 值 存在 相关性 (疾病 严重 程度 评 分为 - 0.59, 植物 - 0.59 和 - 0.77, p <0.0001). Наблюдалась значительная коррел ма между значениями, наблюдаемыми в двух повторностях (оценка т тестести заболевания 0,51, P = 0,00017 и масса рас приения 0,61, P <0,0001) 0,51 Hariho isano rinini hagati yindangagaciro zagaragaye muri duplicate (amanota yuburwayi bwindwara 0.51, P = 0.00017 nuburemere bwibimera 0.61, P <0.0001) no hagati yibi bipimo byombi (-0.59 na -0 .0001) 0.77, P <0.0001. ).Ibimenyetso bisanzwe bigaragara mu bimera byoroshye harimo gukubita no kugoreka uruti rusa n’imiterere y '“umuheto w’umwungeri”, hagakurikiraho ibikomere bya ova hamwe na orange / pink sporozoite (Ishusho yinyongera).Ibikoresho bya Australiya bitwara genr ya Lanr1 (83A: 476 na Tanjil) na AnMan (Mandelup) birwanya ubukana, 0.0331 na 0.0036).Imirongo imwe nimwe itwara "irwanya" Lanr1 na / cyangwa AnMan alleles yerekana ibimenyetso byindwara.
Igishimishije, imirongo mike ya NLL idafite ikimenyetso icyo ari cyo cyose "cyihanganira" allele yerekanye urwego rwo hejuru rwo kurwanya anthracnose (igereranywa cyangwa irenze iyo genotypes ya Lanr1 cyangwa AnMan), nka Boregine (P agaciro <0.0001 kubipimo byombi), Bojar (P agaciro <0.0001 kumanota na 0.001 kuburemere bwibiti) na B-549 / 79b (P). Igishimishije, imirongo mike ya NLL idafite ikimenyetso icyo ari cyo cyose "cyihanganira" allele yerekanye urwego rwo hejuru rwo kurwanya anthracnose (igereranywa cyangwa irenze iyo genotypes ya Lanr1 cyangwa AnMan), nka Boregine (P agaciro <0.0001 kubipimo byombi), Bojar (P agaciro <0.0001 kumanota na 0.001 kuburemere bwibiti) na B-549 / 79b (P). Интересно, что несколько matий NLL, лишенных какого-либо «Ристистентного» марерного аллеля, показали высокий оровень устойчивости к антракнозу (сопоставимый или блее в і ген ыс ыс ыс ыс . Igishimishije, imirongo myinshi ya NLL idafite ikimenyetso icyo aricyo cyose 'cyihanganira' allele yerekanaga urwego rwo hejuru rwo kurwanya anthracnose (ugereranije cyangwa irenga ugereranije na Lanr1 cyangwa AnMan genotypes), nka Boregine (P agaciro <0.0001 kubipimo byombi), Bojar (P agaciro <0.0001 kubisuzuma na 0.001 kuburemere bwibimera) nabaturage B-549/79000 (P).Lanr1 或 AnMan 基因型 相当 或 更高 ， ore ore ore ore 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. B-549 / 79b （得分 P 值 <0.0001 ， 重量 不 显 着）。 Birashimishije kubona sisitemu zimwe na zimwe za NLL zidafite ibimenyetso bya "antigenic" byerekana kwihanganira gutambitse cyane (bihwanye na genr ya Lanr1 cyangwa AnMan cyangwa irenga), nka Boregine (ibipimo byombi P <0.0001), Bojar (P agaciro <0.0001, uburemere bwibimera 0.001) hamwe na B-549 / 79b (P agaciro <0.0001, uburemere ntabwo ari ngombwa). Интересно, что некот мате киии NLL, лишенные каких-либо марерных аллена «Ристистентности», показали высокие оровни устойчивости к антракнозу (сравнимые или вще, кме у вое yin параметров <0,0001), Bojar (значение P <0,0001, масса растения 0,001) и поп под B-549 / 79b (оценка P-значение <0,0001, масса незначительна). Igishimishije, imirongo imwe ya NLL idafite 'resistance' marike alleles yerekanaga urwego rwo hejuru rwo kurwanya anthracnose (ugereranije cyangwa iruta genr1 ya Lanr1 cyangwa AnMan), nka Boregine (P agaciro k ibipimo byombi <0.0001), Bojar (P-agaciro <0.0001, uburemere bwibimera 0.001) nabaturage B-549 / 79b (P-agaciro <0.000).Iyi phenomenon yerekana ko hashobora kubaho inkomoko yubuvanganzo bushya yo kurwanya, isobanura ko nta sano ryagaragaye riri hagati ya genotypes ya marikeri na fenotipi yindwara (P agaciro kuva ~ 0.42 kugeza ~ 0.98).Rero, ikizamini cya Kolmogorov-Smirnov cyerekanye ko amakuru yerekeye kurwanya anthracnose yagereranijwe mubisanzwe yatanzwe kumanota (P-gaciro 0.25 na 0.11) hamwe nubwinshi bwibimera (P-gaciro 0.47 na 0.55), byerekana ko nkeka ko alleles nyinshi kurenza Lanr1 na AnMan zirimo.
Hashingiwe ku bisubizo byo gusuzuma anthracnose yo kurwanya, hatoranijwe imirongo 4 kugira ngo isesengurwe mu nyandiko: 83A: 476, Boregine, Mandelup, n’Abaturage 22660. Iyi mirongo yasubiwemo kugira ngo irwanye antarakera mu bushakashatsi bwakozwe na RNA ikurikirana, hashingiwe ko ari kimwe no mu kizamini cyabanjirije iki.Indangagaciro z'amanota zari zikurikira: Boregin (1.71 ± 1.39), 83A: 476 (2.09 ± 1.38), Mandelup (3.82 ± 1.42) n'abaturage 22660 (6.11 ± 1.29).
Porotokole ya Illumina NovaSeq 6000 yageze ku kigereranyo cya 40.5 Mugisoma byombi kuri sample (29.7 kugeza 54.4 Imisoma) (Imbonerahamwe yinyongera S3).Amanota yo guhuza urutonde rwerekanwe kuva kuri 75.5% kugeza kuri 88,6%.Impuzandengo ijyanye no kubara imibare yasomwe hagati yubushakashatsi bwakozwe hagati yimiterere yibinyabuzima kuva kuri 0.812 kugeza 0.997 (bivuze 0.959). Muri gen 35.170 zasesenguwe, 2917 zagaragaje ko nta mvugo, naho izindi 4785 zagaragajwe ku rwego ruto (ishingiro bivuze <5). Muri gen 35.170 zasesenguwe, 2917 zagaragaje ko nta mvugo, naho izindi 4785 zagaragajwe ku rwego ruto (ishingiro bivuze <5). Из 35 170 проанизированных баров 2917 не проявляли шәһәррессии, а остальные 4785 انانوۋ پرېسونوۋالись на незначительном оровне (басовое среднее <5). Muri gen 35.170 zasesenguwe, 2917 ntizigaragaza, naho 4785 zisigaye zagaragajwe ku rwego ruto (ishingiro bivuze <5).在 分析 的 35,170 个 基因 中 ， 2917 个 没有 表达 ， 其他 4785 个 基因 的 表达 可以 忽略 不 计 （基本 5 <5）。35.170 Из 35 170 проанизированных баров 2917 не нресрес гововались, а остальные 4785 କାନов имели незначительную өлкәрессию (басовое среднее значение <5). Muri gen 35,170 zasesenguwe, 2917 ntizagaragajwe kandi gen 4785 zisigaye zifite imvugo idahwitse (ishingiro risobanura <5).Niyo mpamvu, umubare w'ingirabuzima fatizo zafashwe zagaragajwe (ishingiro bivuze ≥ 5) mu gihe cy'igerageza ni 27.468 (78.1%) (Imbonerahamwe y'inyongera S4).
Kuva ku nshuro ya mbere, imirongo yose ya NLL yasubije urukingo rwa C. lupini (umurongo wa Col-08) usubiramo inyandiko-mvugo (Imbonerahamwe 1), ariko, itandukaniro rikomeye ryagaragaye hagati y'imirongo.Rero, umurongo wo kurwanya 83A: 476 (utwaye gene ya Lanr1) werekanye progaramu ya progaramu ya progaramu ya progaramu ya progaramu ya progaramu ya progaramu ya mbere (6 hpi) hiyongereyeho inshuro 31-69 umubare w’abatandukanijwe hejuru-na-genes ugereranije n’ibindi bihe byagenwe muri iki gihe.Byongeye kandi, iyi mpinga yabaye iyigihe gito, kuko imvugo ya genes nkeya yagumye ihindagurika cyane kumwanya wa kabiri (12 hpi).Igishimishije, Boregine, nayo yerekanaga urwego rwo hejuru rwo kurwanya ikizamini cya graft, ntabwo yigeze ikorerwa progaramu ya transcriptque nini mugihe cyo gukora ubushakashatsi.Nyamara, umubare wa genes zagaragajwe mu buryo butandukanye (DEG) wari umwe kuri Boregine na 83A: 476 kuri 12 HPI.Mandelup hamwe nabaturage 22660 berekanye impinga ya DEG mugihe cyanyuma (48 l / s), byerekana gutinda ugereranije nibisubizo byubwunganizi.
Kuberako 83A: 476 yakorewe progaramu ya transcriptque nini isubiza C. lupini kuri 6 HPI ugereranije nindi mirongo yose, ~ 91% bya DEGs byagaragaye muriki gihe byari byihariye umurongo (Ishusho 1).Nyamara, habayeho guhuzagurika mubisubizo hakiri kare hagati yumurongo w’inyigisho, kuko 68.5%, 50.9%, na 52,6% DEG muri Boregine, Mandelup, nabaturage 22660, byuzuzanya nababonetse muri 83A: 476 mugihe runaka mugihe runaka.Nyamara, aba DEG bagize igice gito gusa (0,97-1.70%) muri DEGs zose zagaragaye ubu ukoresheje 83A: 476.Byongeye kandi, DEGs 11 ziva kumirongo yose zahuzaga muriki gihe (Imbonerahamwe yinyongera S4-S6), harimo nibice bigize ibisubizo byokwirinda ibimera: protein yoherejwe na lipide (TanjilG_32225), endoglucan-1,3-β-glucoside enzyme (TanjilG_23384), proteine ​​ebyiri zidashobora guhangayika nka SAM22 (TanjilGanj_31528) ), hamwe na poroteyine ebyiri zikungahaye kuri glycine (TanjilG_19701 na TanjilG_19702).Habayeho kandi guhuzagurika cyane mubisubizo byanditse hagati ya 83A: 476 na Boregine kuri 24 HPI (yose hamwe 16-38% DEG) no hagati ya Mandelup nabaturage 22660 kuri 48 HPI (yose hamwe 14-20% DEG).
Igishushanyo cya Venn cyerekana umubare wa genes zitandukanye (DEG) mumirongo migufi ya lupine (NLL) yandujwe na Colletotrichum lupini (strain Col-08 yakuwe mumirima ya lupine i Wierzhenice, Polonye, ​​1999).Imirongo ya NLL yasesenguwe ni: 83A: 476 (irwanya, itwara Lanr1 allele), Boregine (irwanya, irondakoko ritazwi), Mandelup (irwanya iringaniye, itwara AnMan allele) n'abaturage 22660 (birashoboka cyane).Amagambo ahinnye hpi ahagarara kumasaha nyuma yo gukingirwa.Indangagaciro zeru zavanyweho kugirango byoroshye igishushanyo.
Igice cya genes zikabije kuri 6 hpi cyasesenguwe kugirango habeho domaine ya R ya Canonical R (Imbonerahamwe yinyongera S7).Ubu bushakashatsi bwerekanye inyandiko-mvugo y’ingirabuzima fatizo zirwanya indwara hamwe na NBS-LRR kuri 83A: 476.Iyi seti yari igizwe na gene imwe ya TIR-NBS-LRR (tanjilg_05042), genes eshanu CC-NBS-LRR (tanjilg_06165, tanjilg_06162, tanjilg_22773, tanjilg_22640, na tanjilg_16162), na bane NBS-LR16 ) kimwe na Bane NBS-Lrr (tanjilg_16162) na Bane NBS-LRR (TANJILG_16162).Izi genes zose zifite domaine zemewe zitondekanye muburyo bukurikiranye.Usibye ingirabuzimafatizo za NBS-LRR, kinase nyinshi za RLL zakorewe kuri 6 hpi, imwe muri Boregine (TanjilG_19877), ibiri i Mandelup (TanjilG_07141 na TanjilG_19877) no mu baturage 22660 (TanjilG_09014 na TanjilG_10361).
Imirasire ifite imvugo yahinduwe cyane mugusubiza urukingo rwa C. lupini (umurongo wa Col-08) bakorewe isesengura ryitunganyirizo rya Gene Ontology (GO) (Imbonerahamwe yinyongera S8). Ijambo ryibinyabuzima byakunze kugaragazwa cyane ni "GO: 0006952 igisubizo cyo kwirwanaho" cyagaragaye muri 6 kuri 16 (igihe × umurongo) hamwe bifite akamaro kanini (P agaciro <0.001) (Ishusho 2). Ijambo ryibinyabuzima byakunze kugaragazwa cyane ni "GO: 0006952 igisubizo cyo kwirwanaho" cyagaragaye muri 6 kuri 16 (igihe × umurongo) hamwe bifite akamaro kanini (P agaciro <0.001) (Ishusho 2). Наиболее часто чрезмерно предвенленным kомом биологического каса был «GO: 0006952 защитный ответ», который появлялся в 6 из 16 (время × дария) комбинаций с всокой значимостю (значение П <0,00). Ijambo ryibinyabuzima ryakunze kugaragara cyane ni 'GO: 0006952 igisubizo cyo kwirwanaho', cyagaragaye muri 6 kuri 16 (igihe × umurongo) hamwe bifite akamaro kanini (P agaciro <0.001) (Ishusho 2).GO 被 过度 代表 的 生物 过程 术语 “GO: 0006952 防御 反应” ， 它 出现 在 16 个 （时间 线） 组合 中 的 个 个 个 0.00 值 0.00 0.00 0.00 Ijambo ryibinyabuzima rihagarariwe cyane ni "GO: 0006952 igisubizo cyo kwirwanaho", kigaragara muri 6 kuri 16 (时间 × 线) hamwe, bifite akamaro kanini (P agaciro <0.001) (图 2). Наиболее часто чрезмерно предвенленным kомом биологического каса был «GO: 0006952 Defence Response», который появлялся в 6 из 16 комбинаций (время × дария) с высокой значимосту (значение П <0,001) (р. Ijambo ryibinyabuzima byakunze kugaragara cyane ni 'GO: 0006952 Defence Response', yagaragaye muri 6 kuri 16 ihuza (igihe × umurongo) ifite akamaro kanini (P agaciro <0.001) (Ishusho 2).Iri jambo ryagaragaye cyane ku manota abiri muri 83A: 476 na Boregine (6 na 24 hpi) naho icyarimwe muri Mandelup n'Abaturage 22660 (12 na 6 hpi).Iki nigisubizo giteganijwe, cyerekana antifungal igisubizo cyimirongo irwanya.Byongeye kandi, 83A: 476 yashubije C. lupini itera ingirabuzimafatizo zihuse zijyanye no guturika kwa okiside ihagarariwe nijambo "GO: 0055114 redox process", byerekana igisubizo cyihariye cyo kwirwanaho, mugihe Boregine yerekanye ibisubizo byihariye byo kwirwanaho, bijyanye nijambo 'GO'.: 0006950 Igisubizo cya Stress ”.Abaturage 22660 bashubije imbaraga zo guhangana na horizontal zirimo metabolite ya kabiri, bagaragaza umubare urenze urugero wamagambo "GO: 0016104 Inzira ya triterpene biosynthesis" na "GO: 0006722 Inzira ya triterpene metabolism" (ayo magambo yombi ni ay'ubwoko bumwe), urebye ibisubizo by'isesengura ryitunganyirizwa rya GO, Mandelup reaction yari iri hagati ya 226. Mandelup n'Abaturage 22660 harimo ijambo GO: 0015979 'photosynthesis' nibindi bijyanye na biologiya.
Amagambo ya bioprocess gene ontology yatoranijwe mu gutondekanya genes zagaragajwe mu buryo butandukanye mu gihe cyo gusubiza inyandiko za lupine zifite amababi magufi (NLL) yatewe na anthrax lupine (Col-08 ikomoka mu mirima ya lupine i Wierzhenice, muri Polonye, ​​mu 1999) irakabya cyane.Imirongo ya NLL yasesenguwe ni: 83A: 476 (irwanya, itwara abaryamana bahuje ibitsina Lanr1 allele), Boregine (irwanya, itazwi neza), Mandelup (irwanya iringaniye, itwara ababana bahuje ibitsina AnMan allele) hamwe nabaturage 22660 (birashoboka).
Kuberako ubu bushakashatsi bwari bugamije kumenya genes zigira uruhare mukurwanya anthracnose, genes zahawe ijambo GO "GO: 0006952 Ibisubizo birwanaho" na "GO: 0055114 Redox process" byasesenguwe no guca intege kuko ibyingenzi bivuze ≥ 30 byibuze umurongo umwe.× ingingo mugihe ihuza imibare ihambaye ya log2 (guhinduka inshuro).Umubare wa gen zujuje ibi bipimo ni 65 kuri GO: 0006952 na 524 kuri GO: 0055114.
83A: 476 yerekanye impinga ebyiri za DEG zasobanuwe nijambo GO: 0006952, iyambere kuri genes 6 kuri santimetero (genes 64, hejuru no kumanura) naho iya kabiri kuri gen 24 kuri santimetero (gen 15, hejuru gusa).Boregine yerekanye kandi ko GO: 0006952 yageze hejuru icyarimwe, ariko hamwe na DEG nkeya (11 na 8) no gukora cyane.Mandeloop yerekanye impinga ebyiri za GO: 0006952 kuri 12 na 48 HPI, zombi zitwara genes 12 (iyambere ifite genes ikora, naho iyakabiri ifite genes zikandamiza gusa), mugihe abaturage 22660 kuri 6 HPI (gen 13) bari bafite ubwiganze bwubwiyongere bukabije.amabwiriza.Twabibutsa ko 96.4% ya GO: 0006952 DEG muriyi mpinga yari ifite igisubizo kimwe (hejuru cyangwa hepfo), byerekana ihuzabikorwa rikomeye mubisubizo byokwirwanaho nubwo itandukaniro ryumubare wabyo.Itsinda rinini ryikurikiranya rijyanye nijambo GO: 0006952 rikubiyemo inzara ya Stress-Associated Message Protein 22 (SAM22-isa), ikaba iri mu cyiciro cya 10 cya poroteyine ziterwa na poroteyine (PR-10) hamwe na proteine ​​yibanze ya latex.poroteyine isa (MLP isa) na poroteyine (Ishusho 3).Amatsinda yombi yari atandukanye mumiterere yimvugo nicyerekezo cyigisubizo.Ingirabuzima fatizo zigizwe na poroteyine zisa na SAM22 zerekanaga ubwinjiriro buhoraho kandi bugaragara mu bihe bya mbere (6 cyangwa 12 hpi) kandi muri rusange ntibyitabiriwe nyuma y’ubushakashatsi (48 hpi), mu gihe poroteyine zisa na MLP zerekanaga guhuza kuri 6 hpi.hpi.83A: 476 na Mandelup kuri 48 hp / in, hafi yandi makuru yose yamakuru ntiyitabiriwe.Byongeye kandi, itandukaniro ryerekana imiterere ya SAM22 isa na poroteyine ya SAM22 yakurikiranye itandukaniro ryagaragaye mu kurwanya anthracnose, kubera ko imirongo irwanya imbaraga yari ifite umwanya munini utera izo genes kuruta genes zoroshye.Ubundi LlR18A / B isa na PR-10 yerekanye imiterere isa cyane na SAM22 isa na poroteyine.
Ibice byingenzi bigize ijambo ryibinyabuzima "GO: 0006952 Defence Response" hamwe nuburyo bwo kwerekana imiterere yabakandida ba Lanr1 na AnMan alleles bamenyekanye.Igipimo cya Log2 cyerekana indangagaciro za log2 (guhinduranya inshuro) hagati yatewe (Colletotrichum lupini, amoko ya Col-08, yakuwe mu murima wa lupine, Wizhenica, Polonye, ​​1999) no kugenzura (sham-yatewe) ibihingwa icyarimwe.Hasesenguwe imirongo migari ya lupine ikurikira: 83A: 476 (irwanya, itwara ababana bahuje ibitsina Lanr1 allele), Boregine (irwanya, irondakoko ritazwi), Mandelup (irwanya mu buryo bushyize mu gaciro, itwara AnMan allele bahuje ibitsina), hamwe n’abaturage 22660 (birashoboka).
Byongeye kandi, hasuzumwe imyirondoro yerekana imiterere y’abakandida ba RNA-seq Lanr1 (TanjilG_05042) na AnMan (TanjilG_12861) (Ishusho 3).Gene ya TanjilG_05042 yerekanye igisubizo gikomeye (activation) kuri 83A: 476 gusa kumwanya wambere (6 hpi), mugihe TanjilG_12861 yari ikomeye muri Mandeloop kumasaha abiri gusa: 6 hpi (amabwiriza yo hasi) na 24 hpi (6 hpi).Hamwe na.).guhinduka)).
Imirasire ikabije cyane mu ijambo GO: 0055114 “redox process” yari genes ikubiyemo poroteyine za cytochrome P450 na peroxidase (Ishusho 4).Kuburugero rwatandukanijwe na 83A: 476 kuri 6 HPI, agaciro ntarengwa cyangwa ntarengwa log2 (guhinduka inshuro) (kuri 86,6% ya gen) byagaragaye muri rusange hagati y ibihingwa byatewe no kugenzura, bikagaragaza igisubizo kinini cyiyi genotype yo gutera igitsina.83A: 476 yerekanye akamaro gakomeye GO: 0055114 DEG kuri 6 hpi (gen 503), mugihe imirongo isigaye kuri 48 hpi (Boregine, 31 gen; Mandelup, 85 gen; nabaturage 22660, 78 gen)).Muri genes nyinshi zumuryango wa GO: 0055114, habonetse ubwoko bubiri bwibisubizo byinkingo (activation no kubuza).Igishimishije, abagera kuri 97,6% ba DEGs bagaragaye ku ijambo GO: 0055114 i Mandelupe kuri 48 hp Izi nyigisho zerekana ko, nubwo igipimo gito cyane (ni ukuvuga umubare wa genoside ya redox yahinduwe, 85 na 503), uburyo bwo gutinda kwandikirwa inyandiko za mandeloup kuri antracnose bisa nkibisubizo byatanzwe na 83A: 476.Muri Boregine n'Abaturage 22660, uku guhuza ni hasi kuri 51,6% na 75,6%.
Uburyo bwo kwerekana ibice byingenzi bigize ijambo ryibinyabuzima “GO: 0055114 Redox process” byagaragaye.Igipimo cya Log2 cyerekana indangagaciro za log2 (guhinduranya inshuro) hagati yatewe (Colletotrichum lupini, amoko ya Col-08, yakuwe mu murima wa lupine, Wizhenica, Polonye, ​​1999) no kugenzura (sham-yatewe) ibihingwa icyarimwe.Hasesenguwe imirongo migari ya lupine ikurikira: 83A: 476 (irwanya, itwara ababana bahuje ibitsina Lanr1 allele), Boregine (irwanya, irondakoko ritazwi), Mandelup (irwanya mu buryo bushyize mu gaciro, itwara AnMan allele bahuje ibitsina), hamwe n’abaturage 22660 (birashoboka).
83A: 476 Ibisubizo byanditse ku guterwa hamwe na C. lupini (umurongo wa Col-08) byanashyizwemo gucecekesha guhuza genes bitirirwa ijambo GO: 0015979 “photosynthesis” nibindi bikorwa bijyanye na biologiya (FIG. 5).Iyi GO: 0015979 DEG yashyizwemo genes 105 zahagaritswe cyane kuri 6 hpi kuri 83A: 476.Muri iki gice, gen 37 nazo zagabanijwe muri Mandelup kuri 48 HPI na 35 icyarimwe icyarimwe mubaturage 22660, harimo 19 DEGs ihuriweho na genotypes zombi.Nta DEG ijyanye nijambo GO: 0015979 yakoreshejwe cyane muburyo ubwo aribwo bwose (umurongo x igihe).
Uburyo bwo kwerekana ibice byingenzi bigize ijambo ryibinyabuzima “GO: 0015979 Photosynthesis” byagaragaye.Igipimo cya Log2 cyerekana indangagaciro za log2 (guhinduranya inshuro) hagati yatewe (Colletotrichum lupini, amoko ya Col-08, yakuwe mu murima wa lupine, Wizhenica, Polonye, ​​1999) no kugenzura (sham-yatewe) ibihingwa icyarimwe.Hasesenguwe imirongo migari ya lupine ikurikira: 83A: 476 (irwanya, itwara ababana bahuje ibitsina Lanr1 allele), Boregine (irwanya, irondakoko ritazwi), Mandelup (irwanya mu buryo bushyize mu gaciro, itwara AnMan allele bahuje ibitsina), hamwe n’abaturage 22660 (birashoboka).
Hashingiwe ku bisubizo by’isesengura ry’imvugo itandukanye kandi birashoboka ko byagize uruhare mu gusubiza ibibazo birwanya ibihumyo bitera, iyi gen ya karindwi yatoranijwe kugirango igereranye imyirondoro y’imvugo na PCR nyayo (Imbonerahamwe S9).
Intungamubiri za poroteyine gene TanjilG_10657 yatewe cyane mumirongo yose yize hamwe nigihe cyagenwe ugereranije no kugenzura (mimic) ibihingwa (Imbonerahamwe yinyongera S10, S11).Mubyongeyeho, imvugo yerekana imvugo ya TanjilG_10657 yerekanye icyerekezo cyiyongera mugihe cyibigeragezo kumirongo yose.Abaturage 22660 berekanye ibyiyumvo byinshi bya TanjilG_10657 yo gukingirwa hamwe inshuro 114 hamwe nurwego rwo hejuru rwo kugereranya (4.4 ± 0.4) kuri 24 HPI (Ishusho 6a).PR10 LlR18A ya poroteyine gene TanjilG_27015 nayo yerekanye gukora cyane mumirongo yose hamwe nigihe, hamwe nibisobanuro bifatika mubisobanuro byinshi (Ishusho 6b).Kimwe na TanjilG_10657, urwego rwo hejuru rwerekana imvugo ya TanjilG_27015 rwagaragaye mu baturage 22660 batewe na 24 HPI (19.5 ± 2.4).Acide endochitinase gene TanjilG_04706 yagenzuwe cyane mumirongo yose kandi mugihe cyose usibye Boregine 6 hpi (Ishusho 6c).Byatewe cyane kumwanya wambere (6 HPI) kuri 83A: 476 (inshuro 10.5) kandi byiyongera kuburyo bugaragara mumirongo yindi (inshuro 6.6-7.5).Mu bushakashatsi, imvugo ya TanjilG_04706 yagumye ku rwego rumwe muri 83A: 476 na Boregine, mu gihe muri Mandelup n’Abaturage 22660 yariyongereye cyane, igera ku gaciro kari hejuru (5.9 ± 1.5 na 6.2 ± 1.5).Endoglucan-1,3-β-glucosidase imeze nka gene TanjilG_23384 yerekanye imbaraga nyinshi kumanota abiri yambere (6 na 12 hpi) mumirongo yose usibye abaturage 22660 (Ishusho 6d).Urwego rwo hejuru rwerekana imvugo ya TanjilG_23384 rwagaragaye kumwanya wa kabiri (12 hpi) i Mandelup (2.7 ± 0.3) na 83A: 476 (1.5 ± 0.1).Kuri 24 HPI, imvugo ya TanjilG_23384 yari mike ugereranije mumirongo yose yize (kuva 0.04 ± 0.009 kugeza 0.44 ± 0.12).
Imyirondoro yerekana genes zatoranijwe (ag) zagaragajwe na PCR yuzuye.Imibare 6, 12 na 24 yerekana amasaha nyuma yo gukingirwa.Imirasire ya LanDExH7 na LanTUB6 yakoreshejwe mubisanzwe kandi LanTUB6 yakoreshejwe muguhuza ibice.Utubari twibeshya twerekana gutandukana gushingiye kubinyabuzima bitatu byigana, buri kimwe nikigereranyo cya tekinike eshatu zisubiramo. Ubusobanuro bwibarurishamibare butandukanye murwego rwo kwerekana imvugo hagati yatewe (Colletotrichum lupini, strain Col-08, yabonetse mu 1999 mu murima wa lupine i Wierzenica, muri Polonye) no kugenzura (gushinyagurira) ibihingwa byashyizwe hejuru yamakuru (* P agaciro <0.05, ** P agaciro ≤ 0.01, *** P agaciro ≤ 0.001). Ubusobanuro bwibarurishamibare butandukanye murwego rwo kwerekana imvugo hagati yatewe (Colletotrichum lupini, strain Col-08, yabonetse mu 1999 mu murima wa lupine i Wierzenica, muri Polonye) no kugenzura (gushinyagurira) ibihingwa byashyizwe hejuru yamakuru (* P agaciro <0.05, ** P agaciro ≤ 0.01, *** P agaciro ≤ 0.001). Статистическая значимость различий в оровнях кресрессии между интокулированными (Colletotrichum lupini, штамм Col-08, получен в 1999 г. с поля люпина в Верженице, Польша) контроль ми чение P <0,05, ** значение P ≤ 0,01, *** значение P ≤ 0,001). Itandukaniro rishingiye ku mibare mu rwego rwo kwerekana imvugo hagati yatewe (Colletotrichum lupini, strain Col-08, yabonetse mu 1999 mu murima wa lupine i Wierzhenice, muri Polonye) no kugenzura (sham-yatewe) ibihingwa bigaragara hejuru yamakuru (* P agaciro <0.05, ** P-agaciro ≤ 0.01, *** P-agaciro ≤ 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 （表达 水平 差异 的 P 0.00 0.05 P P P P P P P P P P接种 （colletotrichum lupini ， ibara-08 株 ， 1999 年 波兰 波兰 wierzenica 的 羽扇 获得） 对照 （接种 植物 水平 p 性 标记 0.05 p * p 值 <0.05, ** P ≤ 0.01, *** P Статистически значимые различия в оровнях кресрессии между интокулированными (Colletotrichum lupini, штамм Col-08, полученный с поа люпина в Верженице, Польша, в 1999 г.) контроль начение P <0,05, ** P-значение ≤ 0,01, *** P-значение ≤ 0,001). Itandukaniro rinini cyane muburyo bwo kwerekana imvugo hagati yatewe (Colletotrichum lupini, strain Col-08, yakuwe mu mirima ya lupine i Verzhenice, muri Polonye, ​​mu 1999) hamwe no kugenzura (sham-yatewe) ibihingwa bigaragara hejuru yamakuru (* P agaciro <0.05, ** P-agaciro ≤ 0.01, *** P-agaciro ≤ 0.001).Imirongo ya NLL yasesenguwe ni: 83A: 476 (irwanya, itwara abaryamana bahuje ibitsina Lanr1 allele), Mandelup (irwanya mu buryo bushyize mu gaciro, itwara abahuje ibitsina AnMan allele), Boregine (irwanya, itazwi neza) kandi abaturage 22660 (birashoboka).
Umukandida gene TanjilG_05042 ku gace ka Lanr1 yerekanye uburyo butandukanye bwo kwerekana imvugo itandukanye n'imyirondoro yakuwe mu bushakashatsi bwa RNA-seq (Ishusho 6e).Gukora cyane kwa gene byagaragaye muri Mandelup no mubaturage 22660 (kugeza kuri 39.7 na 11.7 inshuro), bigatuma habaho imvugo yo hejuru (kugeza kuri 1.4 ± 0.14 na 7.2 ± 1.3).83A: 476 yanagaragaje ko hari igenzura rya gene ya TanjilG_05042 (kugeza ku nshuro 3,8), nyamara, urwego rwo kugereranya imvugo rwagezweho (0.044 ± 0.002) rwikubye inshuro zirenga 30 ugereranije n’ubwa Mandelup n’abaturage 22660.wasesenguwe na qPCR yerekanye itandukaniro rigaragara murwego rwimvugo hagati ya genotypes muburyo bwo gukingirwa urw'agashinyaguro (kugenzura), igera ku ntera inshuro 58 hagati yabaturage 22660 na 83A: 476, ndetse no hagati yabaturage 22660 na 22660. Itandukaniro ryibice bibiri ryagezweho hagati ya Boregine na Mandalup.
Gene y'abakandida ku kibanza cya AnMan, TanjilG_12861, yatangijwe mu rwego rwo gusubiza inkingo muri 83A: 476 na Mandelup, ntaho yari abogamiye mu baturage 22660, kandi ntiyagengwaga na Boregine (Ishusho 6f).Imvugo igereranije ya gen ya TanjilG_12861 niyo yari hejuru cyane muri 83A: 476 (0.14 ± 0.01).Icyiciro cya 17.4 kDa I ubushyuhe bwa poroteyine gene TanjilG_05080 HSP17.4 yerekanaga urwego rwo hasi rwerekana imvugo murwego rwose rwize hamwe nigihe cyagenwe (Ishusho 6g).Agaciro gakomeye kagaragaye kuri 24 HPI mu baturage 22660 (0.14 ± 0.02, kwiyongera inshuro umunani mu gusubiza inkingo).
Kugereranya imyirondoro ya gene (Isanamu 7) yerekanye isano iri hagati ya TanjilG_10657 nizindi genes enye: TanjilG_27015 (r = 0.89), TanjilG_05080 (r = 0.85), TanjilG_05042 (r = 0.80), na TanjilG_04706 (r = 0.79).Ibisubizo nkibi birashobora kwerekana guhuza genes mugihe cyo gusubiza.Ingirabuzimafatizo za TanjilG_12861 na TanjilG_23384 zerekanye imiterere itandukanye yerekana imvugo ifite agaciro ka coefficient ya Pearson yo hasi (kuva 0.08 kugeza 0.43 na -0.19 kugeza 0.28) ugereranije nizindi gen.
Isano riri hagati yerekana imvugo ya gene ryagaragaye hakoreshejwe PCR yuzuye.Hasesenguwe imirongo migufi ya lupine ikurikira: 83A: 476 (irwanya, itwara abaryamana bahuje ibitsina Lanr1 allele), Mandelup (irwanya mu buryo bushyize mu gaciro, itwara AnMan allele bahuje ibitsina), Boregine (irwanya, imiterere y’imiterere itazwi), n’abaturage 22660 (birashoboka).Ibihe bitatu byabazwe (nyuma yamasaha 6, 12 na 24 nyuma yo guterwa), harimo gutera (Colletotrichum lupini, amoko ya Col-08, yakuwe mumirima ya lupine i Wierzhenice, muri Polonye, ​​mumwaka wa 1999) no kugenzura (sham-yatewe).Igipimo cyerekana agaciro ka coefficient ya Pearson.
Hashingiwe ku makuru yabonetse kuri 6 mbaraga za santimetero imwe, WGCNA yakorewe kuri 9981 DEG yagaragajwe no kugereranya ibihingwa byatewe kandi bigenzura kugirango byibande kubisubizo hakiri kare (Imbonerahamwe yinyongera S12).22 ya modules ya gene (cluster) yabonetse hamwe na profili (positif cyangwa negative) imvugo yerekana imvugo hagati ya genotypes nuburyo bwo kugerageza. Ugereranije, imiterere ya gene yagabanutse uko bikurikirana 83A: 476> Mandelup> Boregine> Abaturage 22660 (muri ubwo buryo bwombi, ariko, iyi nzira yari ikomeye mu bimera bigenzura). Ugereranije, imiterere ya gene yagabanutse uko bikurikirana 83A: 476> Mandelup> Boregine> Abaturage 22660 (muri ubwo buryo bwombi, ariko, iyi nzira yari ikomeye mu bimera bigenzura). В. Ugereranije, imiterere ya gene yagabanutse kuri gahunda 83A: 476> Mandelup> Boregine> Abaturage 22660 (muri variants zombi, ariko, iyi nzira yari ikomeye cyane mubihingwa bigenzura).A 而言 ， 基因 表达 水平 按 83A: 476> Mandelup> Boregine> Abaturage 22660 的 顺序 下降 （， 在 在 变 体 中 ， 这种 趋势 在 对照。。。。。平均 而 言 ， 基因 水平 按 83a: 476> mandelup> boregine> abaturage 22660 的 顺序 下降 （， 种 中 ， 这 种 在 在 植物 中 更） В. Ugereranije, imiterere ya gene yagabanutse murukurikirane 83A: 476> Mandelup> Boregine> Abaturage 22660 (nyamara, muburyo bwombi, iyi nzira yari ikomeye mubimera bigenzura).Urukingo rwavuyemo kugenzura imvugo ya gene, cyane cyane mu isomo rya 18, 19, 14, 6 na 1 (mu buryo bugabanuka), amabwiriza mabi (urugero ni 9 na 20) cyangwa n'ingaruka zidafite aho zibogamiye (urugero: 11, 22, 8 na 13)GO isesengura ryitunganyirizwa rya GO (Imbonerahamwe yinyongera S13) ryerekanye "GO: 0006952 Ibisubizo birinda" kuri module yatewe (18) hamwe nibikorwa byinshi, harimo genes zasesenguwe na qPCR (TanjilG_04706, TanjilG_23384, TanjilG_10657 na TanjilG_27015), hamwe na modul nyinshi zahagaritswe.Icyiciro cya 18 cyibanze (Igicapo 8) cyamenyekanye nka gene ya TanjilG_26536 ikubiyemo poroteyine ya PR-10 isa na LlR18B, naho module 9 yibanze nka gene ya TanjilG_28955 ikubiyemo poroteyine ya II PsbQ.Umukandida anthracnose irwanya gene Lanr1, TanjilG_05042, yabonetse mu cyiciro cya 22 (Igicapo 9) kandi ifitanye isano n’amagambo "GO: 0044260 Cellular macromolecular metabolic process" na "GO: 0006355 Amabwiriza yo kwandukura, ADN yerekana" itwara hub ya TanjilG_01212.gene ikubiyemo ubushyuhe bwo kwanduza ibintu A-4a (HSFA4a).
Isesengura riremereye ryisesengura rya gene gufatanya module hamwe namagambo yerekana ibinyabuzima byerekanwe "GO: 0006952 Ibisubizo byingabo".Imanza zoroshe kwerekana genes enye zasesenguwe na qPCR (TanjilG_04706, TanjilG_23384, TanjilG_10657 na TanjilG_27015).
Isesengura riremereye ryisesengura rya gene ryerekana module hamwe nijambo ryibinyabuzima byerekanwe cyane "GO: 0006355: Amabwiriza yo kwandukura, ADN yerekana" no gutwara umukandida anthracnose irwanya gene Lanr1 TanjilG_05042.Imanza zoroshe gutandukanya gene ya TanjilG_05042 hamwe na gene yo hagati ya TanjilG_01212.
Isuzumabumenyi rya Anthracnose ryakusanyirijwe muri Ositaraliya ryerekanye ko ibihingwa byinshi byasohotse hakiri kare byoroshye;Kalya, Coromup na Mandelup bavuzwe ko barwanya mu rugero, mu gihe Wonga, Tanjil na 83A: 476 bavuzweho kwihanganira cyane 26,27,31.yari ifite allele imwe yo guhangana, yagenwe Lanr1, na Coromup na Mandelup bari bafite allele itandukanye, yagenwe AnMan10, 26, 39, mugihe Kalya yanyuze kuri allele itandukanye., Lanr2.Kwipimisha anthracnose mu Budage byatumye hamenyekana umurongo urwanya Bo7212 hamwe n'umukandida allele uretse Lanr1, wagenwe LanrBo36.
Ubushakashatsi bwacu bwerekanye inshuro nke cyane (hafi 6%) ya Lanr1 allele muri germplasm yapimwe.Iri genzura rihuye n’ibisubizo byo gusuzuma germplasm y’iburayi y’iburasirazuba ukoresheje ibimenyetso bya Anseq3 na Anseq4, byerekanaga ko Lanr1 allele iboneka mu mirongo ibiri gusa ya Biyelorusiya.Ibi birerekana ko Lanr1 allele itarakoreshwa cyane na gahunda y’ubworozi bwaho, bitandukanye na Ositaraliya, aho ari imwe muri alleles zingenzi mu bworozi bwafashijwe.Ibi birashobora guterwa nurwego rwo hasi rwo guhangana na Lanr1 allele mubihe byuburayi ugereranije na raporo ya Australiya.Byongeye kandi, ubushakashatsi bwakozwe na anthracnose mu turere tw’imvura nyinshi muri Ositaraliya bwerekanye ko ibisubizo by’imyigaragambyo byahujwe na Lanr1 allele bidashobora kuba ingirakamaro mu bihe by’ikirere bifasha gukura n’iterambere ryihuse ry’indwara 19.42.Mubyukuri, muri ubu bushakashatsi, ibimenyetso bimwe na bimwe bya anthracnose byagaragaye no muri genotypes zitwara Lanr1 allele, byerekana ko kurwanya bishobora gucika mugihe cyiza kugirango iterambere rya C. lupini.Mubyongeyeho, gusobanura ibinyoma byerekana ko hariho ibimenyetso bya Anseq3 na Anseq4, hafi ya cM 1 uvuye kuri Lanr1, birashoboka 28,30,43.
Ubushakashatsi bwacu bwerekanye ko 83A: 476, yitwaje Lanr1 allele, yashubije urukingo rwa C. lupini hamwe na progaramu nini ya transcriptome ya progaramu ya progaramu ya mbere yasesenguwe (6 hpi), mugihe i Mandelup, yitwaje AnMan allele, ibisubizo byanditse byagaragaye nyuma.(kuva 24 kugeza 48 hp).Ihinduka ryigihe gito mubisubizo byokwirwanaho rifitanye isano no gutandukanya ibimenyetso byindwara, byerekana akamaro ko kumenyekanisha hakiri kare indwara kugirango habeho igisubizo cyiza cyo guhangana.Kugira ngo yanduze ibimera, spora ya anthrax igomba kunyura mubyiciro byinshi byiterambere hejuru yabakiriye, harimo kumera, kugabana ingirabuzimafatizo, no gukora appressorium.Umugereka ni imiterere yanduye ifata hejuru yuwakiriye kandi ikorohereza kwinjira mubice byakira.Rero, spores ya C. gloeosporioide mumashanyarazi yamashanyarazi yerekanaga igabana ryambere rya nucleus nyuma yiminota 75-90 ya incububasi, gushiraho umuyoboro wa mikorobe nyuma yiminota 90-120, no guhagarika nyuma yamasaha 4 45.Umwembe C. gloeosporioides yerekanye hejuru ya 40% kumera nyuma yamasaha 3 yubushakashatsi hamwe no gushiraho 20% bya appressors nyuma yamasaha 4.Indwara ya virusi ya CAP20 ya C. gloeosporioide yerekanye ibikorwa byo kwanduza muri conidia ikora epiphyte nyuma ya 3.5 h incubation mu gishashara cya avoka hamwe na proteine ​​nyinshi za CAP20 nyuma ya 4 h 46 min.Mu buryo nk'ubwo, ibikorwa bya melanin biosynthesis genes muri C. trifolii byatewe mugihe cyubushakashatsi bwamasaha 2 hanyuma hakurikiraho gushiraho appressorium nyuma yisaha 1.Ubushakashatsi bwakozwe ku nyama z’amababi bwerekanye ko strawberry yatewe na C. acutatum yabanje guhagarika 8 hpi, mu gihe inyanya zatewe na C. coccode zabanje guhagarika kuri 4 hpi48,49.ahanini bihuye nigihe igipimo cya Colletotrichum spp.inzira yandura.Ibisubizo byihuse kuri 83A: 476 byerekana uruhare rwokurwanya ibimera hamwe ningirabuzimafatizo ziterwa na immunite (ETI) muri uyu murongo, mugihe ibisubizo bitinze bya Mandelup bishyigikira mikorobe ifitanye isano na mikorobe iterwa n'ubudahangarwa (MTI) 50. Ibisubizo hakiri kare kuri 83A: 476 na Mandelup.Guhuza igice hagati ya gen-hejuru cyangwa munsi-bigengwa nigisubizo cyatinze nacyo gishyigikira iki gitekerezo, kuko ETI ikunze gufatwa nkigisubizo cyihuse kandi cyongerewe imbaraga cya MTI kirangirira ku rupfu rwa selile aho rwanduye, ruzwi nka anaphylactique 51,52.
Ubwinshi muri gen bwitirirwa ijambo ryerekanwe cyane Gene Ontology GO: 0006952 "Defence Response" ni 11 homologues yubutumwa bwatewe no kwiyiriza ubusa buterwa na poroteyine 22 (bisa na SAM22) hamwe na karindwi nini ya proteine ​​ikunda (MLPs).-bisa na poroteyine 31, 34, 43 na 423 zerekanye uko zikurikirana.SAM22 isa na gen yerekanaga ibikorwa byingenzi bimara igihe kirekire, byerekana urwego rwinshi rwo kurwanya anthracnose (83A: 476 na Boregine).Nyamara, gen zimeze nka MLP zagabanijwe gusa kumurongo utwara abakandida barwanya allele (83A: 476 / Lanr1 kuri 6 hpi na Mandelup / AnMan kuri 24 hpi).Twabibutsa ko abantu bose bamenyekanye nka SAM22 bameze nka homologues bakomoka kuri cluster ya gene ingana na kb 105, mugihe gen-imeze nka MLP ikomoka mubice bitandukanye bya genome.Guhuza ibikorwa bya gen bisa na SAM22 twabisanze no mubushakashatsi bwibanze bwerekeranye no kurwanya NLL kurwanya Diaporthetoxica, byerekana ko bafite uruhare muri horizontal yibisubizo byokwirwanaho.Uyu mwanzuro ushyigikiwe kandi na raporo zerekana igisubizo cyiza cya SAM22 imeze nkimvune cyangwa kuvurwa na acide salicylic, inducers fungal, cyangwa hydrogen peroxide.
Imirasire ya MLP yerekanwe ko isubiza ibibazo bitandukanye bya abiotic na biotic, harimo indwara ziterwa na bagiteri, virusi na virusi zandurira mu moko menshi y'ibimera55.Icyerekezo cyo gusubiza imikoranire imwe n'imwe hagati y'ibimera na virusi byatangiraga kwiyongera cyane (ni ukuvuga mugihe cyo gutera ipamba hamwe na Verticillium dahliae) kugeza kugabanuka cyane (ni ukuvuga, nyuma yo kwanduza igiti cya pome na spari ya Alternariya.) 56,57.Kugabanuka gukabije kwa MLP isa na 423 byagaragaye mugihe cyo kwirinda avoka kwandura F. niger no mugihe cyo kwanduza igiti cya pome Botryosphaeria berengeriana f.cn.piricola na Alternariya alternata ni pome ya pome 58,59.Byongeye kandi, pome ya pome ya pome ikabije ya MLP isa na 423 gene yari ifite imvugo yo hasi ya genes zifitanye isano no kurwanya indwara kandi byandura cyane kwandura fungal 59.Nyuma ya Fusarium oxysporum f, gene ya MLP isa na 423 nayo yahagaritswe muri germplasme isanzwe yibishyimbo.cn.Indwara y'ibishyimbo 60.
Abandi bagize umuryango wa PR-10 bagaragaye mu bushakashatsi bwacu bwa RNA-seq ni gen ya LlR18A na LlR18B mu rwego rwo gusubiza ibibazo, hamwe na gen (1 gen) cyangwa igengwa na gen (3 genes) ya poroteyine yohereza lipide DIR1..Byongeye kandi, WGCNA yerekana gene ya LlR18B nk'ihuriro muri iyi module, ishobora gukingirwa cyane kandi ikagira genes nyinshi zo gukingira.Genos ya LLR18A na LLR18B yatewe mu mababi y'umuhondo asubiza acteri y'umuhondo asubiza ibiti bya StHegenic, ndetse no mu biti by'umuceri byatanzwe na Acide, mu gihe Rasip PID Preteins Protents isabwa kugirango utange sisitemu yabonetse (sar).Hamwe niterambere ryibisubizo birinda, poroteyine ya DIR1 itwarwa kuva yibandwaho binyuze muri floem kugirango itere SAR mu ngingo za kure.Igishimishije, gene ya TanjilG_02313 DIR1 yatewe cyane mugihe cyambere kumurongo wa 84A: 476 nabaturage 22660, ariko kurwanya antarakose byateye imbere gusa kumurongo 84A: 476.Ibi birashobora kwerekana imikorere idahwitse ya DIR1 muri NLL, kubera ko homologues eshatu zisigaye zashubije urukingo gusa kumurongo wa 83A: 476 kuri 6 hpi, kandi igisubizo cyerekejwe hepfo.
Mu bushakashatsi bwacu, ibice bikunze kugaragara bijyanye na biologiya yiswe “GO: 0055114 Redox process” ni protein ya cytochrome P450, peroxidase, aside linoleque 9S- / 13S-lipoxygenase, na 1-aminocyclopropane-1-karubasi ya aside aside.Byongeye kandi, WGCNA yacu isobanura homologue ya HSFA4a nk'ihuriro ritwara modul nk'umukandida wa Lanr1 wo kurwanya gene TanjilG_05042.HSFA4a ni kimwe mu bigize redox-iterwa no kugenzura iyandikwa rya kirimbuzi mu bimera.
Intungamubiri za Cytochrome P450 ni oxydeoreductase itera hydroxylation ya NADPH na / cyangwa O2 iterwa na hydroxylation reaction ya metabolisme yambere na kabiri, harimo metabolisme ya xenobiotics, hamwe na hormone, acide fatty, sterol, ibice byurukuta rwa selile, biopolymers, hamwe na biosynthesis yibintu bikingira 6 byaragabanutse kuri 5. yahinduye homologues (37) nibitandukaniro muburyo bwo gusubiza hagati ya gen runaka, byerekana isubiramo hejuru..Gukoresha gusa amakuru ya RNA-seq kugirango usobanure imikorere yibinyabuzima ya gen ya NLL muri proteine ​​nini cyane birenze urugero byaba ari impuguke.Ariko, birakwiye ko tumenya ko ingirabuzimafatizo zimwe na zimwe za cytochrome P450 zifitanye isano no kongera imbaraga zo kurwanya ibihumyo cyangwa bagiteri ziterwa na virusi, harimo n’umusanzu mu myitwarire ya allergique 69,70,71.
Icyiciro cya III peroxidase ni imisemburo myinshi yibimera bigira uruhare muburyo butandukanye bwo guhindagurika mugihe cyo gukura no gutera imbere, ndetse no gukemura ibibazo by’ibidukikije nkumunyu, amapfa, ubukana bwumucyo mwinshi, nigitero cya patogene72.Peroxidase igira uruhare mu mikoranire y’ibinyabuzima byinshi na Anthracis, harimo Stylosanthes humilis na C. gloeosporioides, Lens culinaris na C. truncatum, Phaseolus vulgaris na C. lindemuthianum, Cucumis sativus na C. lagenarium73,74,75,76.Igisubizo kirihuta cyane, rimwe na rimwe ndetse no kuri 4 HPI, mbere yuko igihumyo cyinjira mubice by'ibihingwa73.Gene ya peroxidase nayo yasubije D. toxica NLL.Usibye imirimo yabo isanzwe yo kugenzura okiside iturika cyangwa gukuraho stress ya okiside, peroxidase irashobora kubangamira imikurire ya patogene itera inzitizi zumubiri zishingiye ku gushimangira urukuta rw'utugingo mugihe cyo guhuza, subunit cyangwa guhuza ibice byihariye.Iyi mikorere irashobora kwitirirwa muri silico na gene ya TanjilG_03329 irimo kodegisi ya lignin ikora anion peroxidase yagenzuwe cyane mubushakashatsi bwacu mumurongo wa 83A: 476 wihanganira kuri 6 HPI, ariko ntabwo biri mubindi bice hamwe nigihe cyigihe kitashubije.
9S- / 13S-lipoxygenase ya acide linoleike nintambwe yambere mumihanda ya okiside ya lipide biosynthesis78.Ibicuruzwa byiyi nzira bifite ibikorwa byinshi mukurinda ibimera, harimo urukuta rw'ingirabuzimafatizo binyuze mu gushiraho ububiko bwa callose na pectine, no kugenzura imihangayiko ya okiside binyuze mu gukora amoko ya ogisijeni ikora neza 79,80,81,82,83.Muri ubu bushakashatsi, imvugo ya acide linoleque 9S- / 13S-lipoxygenase yahinduwe mu bice byose, ariko mu baturage bakunze kwibasirwa 22660, igenzura ryiganje mu bihe bitandukanye, mu gihe mu miterere itwara Lanr1 irwanya na AnMan allele, ishimangira itandukaniro ry’imiterere ya oxylipine ikingira anthrax irinda izo genotypes.
1-aminocyclopropane-1-carboxylate oxydease (ACO) homologue yagenzuwe cyane (genes 9) cyangwa yagabanijwe (genes 2) mugihe yatewe na lupine.Usibye bibiri, ibisubizo byose byabaye kuri 6 hp.kuri 83A: 476.Enzymatique reaction yahujwe na proteyine za ACO nintambwe igabanya igipimo mubikorwa bya Ethylene bityo ikagengwa cyane84.Ethylene ni imisemburo yibimera igira uruhare runini muguhuza iterambere ryibimera no guhangana nibibazo bya abiotic na biotic.Kwinjiza inyandiko-mvugo ya ACO no gukora inzira yerekana ibimenyetso bya Ethylene bigira uruhare mukongera imbaraga zumuceri kuri hemibiotrophic fungus oryzae oryzae muguhuza umusaruro wubwoko bwa ogisijeni ikora na phytoalexine.Uburyo bwo kwanduza amababi busa cyane buboneka hagati ya M. oryzae na C. lupini88,89, bitewe n’uko hagaragaye igenzura rikomeye ry’abahuje ibitsina ba ACO ku murongo wa 83A: 476 wavuzwe muri ubu bushakashatsi, bihindura uburyo bwo gutanga imbaraga zo kurwanya NLL anthracnose Ethylene yerekana intambwe nyamukuru mu nzira ya molekile.
Muri ubu bushakashatsi, guhagarika cyane genes nyinshi zijyanye na fotosintezeza byagaragaye kuri 6 hpi muri 83A: 476 no kuri 48 hpi muri Mandeloop nabaturage 22660.Ingano niterambere ryizo mpinduka biragereranijwe nurwego.Kurwanya Anthracnose byagaragaye muri ubu bushakashatsi.Vuba aha, gukandamizwa gukomeye kandi hakiri kare inyandiko-mvugo ifitanye isano na fotosintezeza byavuzwe mu buryo butandukanye bwo guhuza ibimera-bitera indwara, harimo na bagiteri ziterwa na virusi.Kwihuta (kuva 2 HPI mubikorwa bimwe na bimwe) no guhagarika isi yose ifitanye isano na fotosintezeza mugusubiza kwandura irashobora gukingira ubudahangarwa bw’ibimera hashingiwe ku kohereza amoko ya ogisijeni yangiza ndetse n’imikoranire yabo n'inzira ya acide salicylique yo guhuza allergique 90,94.
Mu gusoza, uburyo bwo gusubiza bwirwanaho bwasabwe kumirongo irwanya cyane (83A: 476) burimo kumenyekanisha byihuse indwara ya gene ya R (birashoboka ko TIR-NBS-LRR TanjilG_05042) hamwe na allergic reaction-yifashishije aside salicylic acide na signal ya Ethylene, hanyuma hakurikiraho SAR ndende.ibikorwa bishyigikiwe na poroteyine ya DIR-1.Twabibutsa ko igihe cya biotropique yanduye C. lupini ari kigufi cyane (hafi iminsi 2), hagakurikiraho gukura kwa nekrotike95.Inzibacyuho hagati yibi byiciro irashobora kuba ifitanye isano na necrosis no kwerekana poroteyine za Ethylene-zidashobora gukora zitera imbaraga zo gukabya gukabije mu bimera byakira.Kubwibyo, igihe cyamadirishya yo gufata neza C. lupini kurwego rwa biotrophique ni nto cyane.Gusubiramo porogaramu za gen zifitanye isano na redox na fotosintezeza byagaragaye muri 83A: 476 kuri 6 hpi bihuye niterambere rya hyphae ya fungal kandi itangaza ko habaho igisubizo cyiza cyo gukingira kurwego rwa biotropique.Ibisubizo byanditse kuri Mandelup hamwe nabaturage 22660 birashobora gutinda gufata ibihumyo mbere yo guhindukira gukura kwa nerotique, nyamara, Mandelup irashobora gukora neza kurusha abaturage 22660 kuko amategeko yihuse ya poroteyine PR-10 atera guhangana na horizontal.
ETI, itwarwa na gene ya Canonical R, bigaragara ko ari uburyo rusange bwo kurwanya ibishyimbo birwanya anthracnose.Rero, mubinyamisogwe by'icyitegererezo Medicago truncatula, kurwanya anthracnose bitangwa na gene RCT1, umwe mubagize uruganda rwa TIR-NBS-LRR97.Iyi gene kandi itanga imiti myinshi ya anthracnose irwanya alfalfa iyo yimuriwe mubihingwa byoroshye.Mu bishyimbo bisanzwe (P. vulgaris), kugeza ubu hamenyekanye ingirabuzimafatizo zirenga ebyiri za anthracnose.Amwe murayo gen aboneka mu turere tudafite ingirabuzimafatizo zose za R, nyamara izindi nyinshi ziri ku nkombe za chromosomes zitwara cluster ya NBS-LRR, harimo TIR-NBS-LRRs99.Ubushakashatsi bwakozwe na SSR bugizwe na genoside yemeje kandi isano ya gene NBS-LRR hamwe na anthracnose irwanya ibishyimbo bisanzwe.Gene ya Canonical R yabonetse kandi mukarere ka genomic itwara inzige zikomeye zo kurwanya anthracnose muri lupine yera 101.
Igikorwa cacu kirerekana ko guhita twirwanya, bigakorwa mugihe cyambere cyo kwandura ibimera (byaba byiza bitarenze 12 hpi), birinda neza lupine ifite amababi mato ya anthracnose yatewe na fungus Collelotrichum lupini.Twifashishije uburyo bwinshi bwo kwinjiza ibintu, twerekanye imvugo itandukanye yerekana imiterere ya anthracnose irwanya ibimera bya NLL byahujwe na genr1 yo kurwanya Lanr1 na AnMan.Kwirinda neza bikubiyemo gutegura neza gen zigizwe na poroteyine zigira uruhare muri redox, fotosintezeza, na patogenezi mu masaha make nyuma y’ikimera kibonanye na virusi.Ibintu nkibi birinda, ariko bitinze mugihe, ntabwo bigira ingaruka nziza mukurinda ibimera indwara.Kurwanya Anthrax byahujwe na gene ya Lanr1 bisa nigisubizo gisanzwe cyihuta cya R (ubudahangarwa bw'umubiri), mugihe gene ya AnMan ishobora gutanga igisubizo gitambitse (ubudahangarwa buterwa na mikorobe ifitanye isano na mikorobe), butanga urwego ruciriritse rwo kuramba.
Imirongo 215 NLL yakoreshwaga mu kwerekana ibimenyetso bya anthracnose yari igizwe n’ibihingwa 74, imirongo 60 yabonetse mu kwambuka cyangwa korora, mutant 5, na mikorobe 76 zo mu gasozi cyangwa umwimerere.Imirongo yaturutse mu bihugu 17, cyane cyane yaturutse muri Polonye (58), Espagne (47), Ubudage (27), Ositaraliya (26), Uburusiya (19), Biyelorusiya (7), Ubutaliyani (5) n'indi mirongo.baturutse mu bihugu 10.Iyi seti ikubiyemo kandi imirongo irwanya: 83A: 476, Tanjil, Wonga itwaye Lanr1 allele, na Mandelup itwaye AnMan allele.Imirongo yabonetse mububiko bwiburayi bwa Lupine Genetic Resource Data base yabitswe na Poznań Plant Breeding Ltd, Wiatrowo, Polonye (Imbonerahamwe yinyongera S1).
Ibimera byakuze mubihe byagenzuwe (Photoperiod amasaha 16, ubushyuhe 25 ° C kumanywa na 18 ° C nijoro).Hasesenguwe ibinyabuzima bibiri.ADN yatandukanijwe n'amababi y'ibyumweru bitatu ikoresheje DNeasy Plant Mini Kit (Qiagen, Hilden, Ubudage) nk'uko protocole ibivuga.Ubwiza nubunini bwa ADN yitaruye byasuzumwe nuburyo bwa spekitifotometometrike (NanoDrop 2000; Thermo Fisher Scientific, Waltham, MA, USA).Ikimenyetso cya AnManM1 kiranga antancnose irwanya gene AnMan (ikomoka kuri cv.Homozygote kuri allele irwanya amanota yatsinzwe nka "1 ″, byoroshye - nka" 0 ″, na heterozygote - nka 0.5.
Hashingiwe ku bisubizo byo gusuzuma ku bimenyetso AnManM1, AnSeq3 na AnSeq4 no kuboneka kw'imbuto zo gukora ubushakashatsi bwa nyuma, imirongo 50 ya NLL yatoranijwe kuri fenotipi ya anthracnose.Isesengura ryakozwe mu buryo bubiri muri pariki igenzurwa na mudasobwa hamwe na Photoperiod yamasaha 14 ifite ubushyuhe bwa 22 ° C ku manywa na 19 ° C nijoro.Imbuto zishushanyije (gukata ikote ry'imbuto ku rundi ruhande rw'isoro hamwe n'icyuma gikarishye) mbere yo kubiba kugira ngo imbuto zidasinzira bitewe n'ikoti ry'imbuto rikomeye kandi bikamera neza.Ibimera byahinzwe mu nkono (11 × 11 × 21 cm) hamwe nubutaka butemewe (TS-1 REC 085 Hagati Yibanze, Klasmann-Deilmann Polska, Warsaw, Polonye).Gutera inshinge byakozwe hamwe na Colletotrichum lupini Col-08, byahinzwe mu 1999 bivuye ku giti cy’ibiti bya lupine bifite amababi magufi ahingwa mu murima i Verzhenitsa, muri Polonye Nkuru (52 ° 27 ′ 42 ″ N 17 ° 04 ′ 05 ″ E).Shaka akarere.Akato kari karahawe umuco muri SNA kuri 20 ° C. munsi yumucyo wumukara iminsi 21 kugirango utere sporulation.Ibyumweru bine nyuma yo kubiba, igihe ibihingwa byari bigeze ku kibabi cya 4-6, gutera inshinge byakozwe mu gutera hamwe no guhagarika conidia ku gipimo cya 0.5 x 106 conidia kuri ml.Nyuma yo gukingirwa, ibimera byabitswe mu mwijima amasaha 24 mu butumburuke bwa 98% nubushyuhe bwa 25 ° C kugirango byoroherezwe kwandura no kwandura.Ibimera byahise bikura munsi ya fotoperiyo yamasaha 14 kumunsi wa 22 ° C / 19 ° C nijoro nubushuhe bwa 70%.Amanota yindwara yakozwe nyuma yiminsi 22 nyuma yo gukingirwa kandi kuva kuri 0 (immunite) kugeza kuri 9 (birashoboka cyane) bitewe nuko hari ibisebe bya nekrotike bihari cyangwa bitabaho.Byongeye, nyuma yo gutanga amanota, hapimwe uburemere bwibimera.Isano iri hagati ya genotypes ya marikeri na fenotipi yindwara yabazwe nkingingo ebyiri zifitanye isano (kutagira ibimenyetso bya heterozygous mumurongo wumurongo wo gusesengura fenotipike ya anthracnose).