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I-Angustifolius lupine (NLL, Lupinus angustifolius L.) iyisitshalo esinamaqabunga aluhlaza esisetshenziselwa ukukhiqizwa kokudla kanye nokuthuthukiswa kwenhlabathi. Ukwanda komhlaba wonke kwe-NLL njengesitshalo kudonsele isikhunta esiningi esibangela izifo, okuhlanganisa i-lupine anthracnose, ebangela isifo esibhubhisayo se-anthracnose. Ama-allele amabili, i-Lanr1 kanye ne-AnMan, anikeza ukumelana okukhulu, asetshenziswe ekuzaleni kwe-NLL, kodwa izindlela eziyisisekelo zama-molecule azikaziwa. Kulolu cwaningo, izimpawu ze-Lanr1 kanye ne-AnMan zasetshenziswa ukuhlola amasampula e-NLL aseYurophu. Ukuhlolwa komjovo endaweni elawulwayo kuqinisekisile ukusebenza kahle kwabahlinzeki abamelana nawo. Ukuhlolwa kokubonakaliswa kwezakhi zofuzo okuhlukile kwenziwa emigqeni emelene neyasengozini. Ukumelana ne-anthracnose kwakuhlotshaniswa nokubonakaliswa ngokweqile kwamagama e-gene ontology athi “GO:0006952 Defense Response”, “GO:0055114 Redox Process”, kanye nethi “GO:0015979 Photosynthesis”. Ngaphezu kwalokho, umugqa we-Lanr1(83A:476) ubonise ukuhlelwa kabusha kwe-transcriptome okuphawulekayo ngokushesha ngemva kokugonywa, kuyilapho eminye imigqa ibonise ukubambezeleka kwale mpendulo cishe ngamahora angu-42. Izimpendulo zokuzivikela zihlotshaniswa nezakhi zofuzo ze-TIR-NBS, i-CC-NBS-LRR kanye ne-NBS-LRR, amaprotheni ayi-10 ahilelekile ekuguleni, amaprotheni okudlulisa amafutha, i-endoglucan-1,3-β-glucosidase, amaprotheni odonga lweseli acebile nge-glycine, kanye nezakhi zofuzo ezivela endleleni yokusabela yomoya-mpilo. Izimpendulo zakuqala ku-83A:476, kufaka phakathi ukucindezelwa ngokucophelela kwezakhi zofuzo ezihlotshaniswa ne-photosynthesis, kuhambisane nokuvikelwa okuphumelelayo ngesikhathi sesigaba sokukhula kwezitshalo sebhayoloji yefungal, okuphakamisa ukuthi i-effector ibangela ukuzivikela. Ukusabela kwe-Mandeloop kuyehliswa, njengoba kunjalo nokuhudula okuvundlile.
I-lupine enamahlamvu amancane (i-NLL, i-Lupinus angustifolius L.) iyisitshalo esinamaprotheni amaningi esivela esifundeni saseMedithera esentshonalanga. Njengamanje ikhuliswa njengesitshalo sokudla sezilwane nabantu. Iphinde ibhekwe njengomanyolo oluhlaza ezinhlelweni zokujikeleza kwezitshalo ngenxa yokufakwa kwe-nitrogen ngamagciwane aqinisa i-nitrogen kanye nokuthuthukiswa okuphelele kwesakhiwo somhlabathi. I-NLL iye yadlula enkambisweni esheshayo yokufuywa kwezilwane kuleli khulu leminyaka elidlule futhi isengaphansi kwengcindezi ephezulu yokuzalanisa3,4,5,6,7,8,9,10,11,12. Ngokutshalwa kabanzi kwe-NLL, ukulandelana kwesikhunta esibangela izifo kwakha izindawo ezintsha zezolimo futhi kwabangela izifo ezintsha ezibhubhisa izitshalo. Okuphawuleka kakhulu kubalimi be-lupin kanye nabafuyi bezitshalo kwaba ukuvela kwe-anthracnose, okubangelwa yisikhunta esibangela izifo, i-Colletotrichum lupini (Bondar) Nirenberg, Feiler kanye ne-Hagedorn13. Okuphawuleka kakhulu kubalimi be-lupin kanye nabafuyi bezitshalo kwaba ukuvela kwe-anthracnose, okubangelwa yisikhunta esibangela izifo, i-Colletotrichum lupini (Bondar) Nirenberg, Feiler kanye ne-Hagedorn13. Наиболее примечательным для фермеров и селекционеров люпина было появление антракноза, вызванного патогенным грибком Colletotrichum (Feiler) Hagedorn13. Okuphawuleka kakhulu kubalimi be-lupine kanye nabafuyi bezitshalo kwaba ukuvela kwe-anthracnose okubangelwa yisikhunta esibangela izifo i-Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13.对于羽扇豆农民和饲养者來说，最引人注目的是炭疽病的出现，它是由病原真菌Colletotrichum lupini) Handler &3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 гриtricбком Colle Hagedorn13. Okuhlaba umxhwele kakhulu kubalimi be-lupine kanye nabafuyi ukuvela kwe-anthracnose okubangelwa yi-fungus ebangela izifo i-Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13.Imibiko yokuqala yalesi sifo ivela eBrazil nase-United States, kanti izimpawu ezivamile zavela ngo-1912 nango-1929 ngokulandelana. Kodwa-ke, ngemva kweminyaka engaba ngu-30, leli gciwane labizwa ngokuthi i-Colletotrichum gloeosporioides (Penz.) Penz. & Sacc., teleomorph Glomerella cingulata (Stoneman) Spauld. & Sacc., teleomorph Glomerella cingulata (Stoneman) Spauld. & Sacc., телеоморф Glomerella cingulata (Stoneman) Spauld. & Sacc., i-teleomorph ye-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 ku-Targeted Morphology. kanye no-H. Schrenk,. kanye no-H. Schrenk,.kanye no-H. Schrenk. & H.施伦克，. & H.施伦克，.kanye no-H. Schlenk,.Ukuhlolwa kwezifo kwasekuqaleni okwenziwe maphakathi nekhulu lama-20 kwabonisa ukumelana okuthile kuma-NLL kanye nama-lupine aphuzi (L. luteus L.), kodwa wonke ama-lupine amhlophe (L. albus L.) ahlolwe ayesengozini enkulu.15,16. Izifundo zibonise ukuthi ukuthuthukiswa kwe-anthracnose kuhlotshaniswa nokwanda kwemvula (umswakama womoya) kanye nokushisa (ebangeni lika-12-28°C), okuholela ekwephulweni kokumelana emazingeni okushisa aphezulu.17, 18. Eqinisweni, isikhathi esidingekayo ukuze i-conidia ikhule futhi lesi sifo siqale, sasifushane ngokuphindwe kane ku-24°C (amahora ama-4) kunaku-12°C (amahora ayi-16) ngaphansi kwezimo zomswakama ophezulu.19 Ngakho-ke, ukufudumala komhlaba okuqhubekayo kuholele ekusabalaleni kwe-anthracnose. Kodwa-ke, lesi sifo sabonwa eFrance (1982) nase-Ukraine (1983) njengesibonakaliso sosongo oluzayo, kodwa kubonakala sengathi imboni ye-lupine ayizange inakwe ngaleso sikhathi. Eminyakeni embalwa kamuva, lesi sifo esibhubhisayo sasakazeka emhlabeni wonke futhi sathinta namazwe amakhulu akhiqiza i-lupine njenge-Australia, iPoland neJalimane. Ngemuva kokuqubuka kwe-anthracnose maphakathi nawo-1990, ukuhlolwa okubanzi kwaholela ekutholakaleni kwabanikeli abaningana abangazweli kumasampula e-NLL19. Ukumelana ne-NLL ku-anthracnose kulawulwa yi-alleles ezimbili ezihlukene ezitholakala emithonjeni ehlukene ye-germplasm: i-Lanr1 ku-cultivar Tanjil kanye ne-Wonga kanye ne-AnMan ku-cultivar. I-Mandalay 25, 26. Lawa ma-allele agcwalisa izimpawu zama-molecule ezisekela ukukhethwa kwe-germplasm engazweli ezinhlelweni zokuzalanisa. Umugqa wokuzala omelana nokumelana 83A:476 ophethe i-allele ye-Lanr1 wahlanganiswa nomugqa wasendle osengozini P27255 ukuze kutholakale inani le-RIL elihlukanisa ukumelana ne-anthracnose, okwenza kwaba nokwenzeka ukwabela indawo ye-Lanr1 ku-chromosome NLL-1131, 32, 33. Ukuqondanisa izimpawu zemephu yokuxhumanisa kusukela endaweni yokumelana ehlangothini kuya ku-anthracnose ngohlaka lwe-genomic, i-NLL yembule indawo yazo zonke izinhlobo ezintathu ze-alleles ku-chromosome efanayo (NLL-11), kodwa ezindaweni ezahlukene 29,34,35. Kodwa-ke, ngenxa yenani elincane lama-RIL kanye nebanga elikhulu lezakhi zofuzo phakathi kwezimpawu nama-allele ahambisanayo, azikho iziphetho ezithembekile ezingathathwa mayelana nezakhi zofuzo zazo ezingaphansi. Ngakolunye uhlangothi, ukusetshenziswa kwezakhi zofuzo ezibuyela emuva kuma-lupin kunzima ngenxa yamandla azo okuvuselela aphansi kakhulu, okwenza ukuphathwa kwezakhi zofuzo kube nzima 37.
Ukuthuthukiswa kwe-germplasm yasekhaya ephethe i-allele efiselekayo esimweni se-homozygous, njenge-83A:476 (Lanr1) kanye ne-Mandelup (AnMan), kuvule ithuba lokufunda ukumelana ne-anthracnose lapho kukhona inhlanganisela ephikisanayo yama-allele ezilwaneni zasendle. Amathuba ezindlela zama-molecule. Qhathanisa izimpendulo zokuzivikela ezikhiqizwe yizinhlobo ezithile ze-genotype. Lolu cwaningo luhlole impendulo yokuqala ye-transcriptome ye-NLL ekugonyweni kwe-C. lupini. Okokuqala, iphaneli ye-germplasm yaseYurophu ye-NLL equkethe imigqa engu-215 yahlolwa kusetshenziswa izimpawu zama-molecule eziphawula ama-allele e-Lanr1 kanye ne-AnMan. I-phenotyping ye-anthracnose yabe isikwenziwa emigqeni engu-50 ye-NLL, eyayikhethwe ngaphambilini kuma-marker ama-molecule, ngaphansi kwezimo ezilawulwayo. Ngokusekelwe kulezi zivivinyo, imigqa emine ehlukene ekumelaneni ne-anthracnose kanye nokwakheka kwe-allelic kwe-Lanr1/AnMan kwakhethwa ukuze kuchazwe ngokuhlukile ukubonakaliswa kwezakhi zofuzo zokuzivikela kusetshenziswa izindlela ezimbili ezihambisanayo: ukulandelana kwe-RNA ephezulu kanye nokulinganisa kwe-PCR ngesikhathi sangempela.
Ukuhlolwa kwesethi ye-NLL germplasm (N = 215) ngamamaki i-Lanr1 (Anseq3 kanye ne-Anseq4) kanye ne-AnMan (Anseq4) kanye ne-AnMan (AnManM1) kubonise ukuthi umugqa owodwa kuphela (95726, eduze neSalamanca-b) owandisa i-allele "yokumelana" yazo zonke izimpawu, kuyilapho "Ukuba khona kwama-allele 'athintekayo'" kuthole isilinganiso sawo wonke amamaki emigqeni engu-158 (~73.5%). Imigqa eyishumi nantathu ikhiqize ama-allele amabili "amelana" nomaki we-Lanr1, kanye nemigqa engu-8 ikhiqize ama-allele "amelana" nomaki we-Lanr1. I-allele "yokumelana" nomaki we-AnMan (Ithebula Elingeziwe S1). Imigqa emibili yayiyi-heterozygous komaki we-Anseq3 kanye ne-heterozygous eyodwa komaki we-AnManM1. Imigqa engu-42 (19.5%) yayinezigaba eziphambene zama-allele e-Anseq3 kanye ne-Anseq4, okubonisa imvamisa ephezulu yokuhlangana kabusha phakathi kwalezi zindawo ezimbili. Ama-phenotype e-Anthracnose ngaphansi kwezimo ezilawulwayo (Ithebula Elingeziwe S2) aveze ukuguquguquka kokumelana kwezinhlobo ze-genotype ezivivinyiwe, okwabonakala ebunzimeni be-anthracnose. Umehluko kumaphuzu aphakathi wawusukela ku-1.8 (omelana nokulingana) kuya ku-6.9 (omelana nokulingana) kanti umehluko wesisindo sesitshalo wawusukela ku-0.62 (omelana nokulingana) kuya ku-4.45 g (omelana nokulingana). Kube nokuhlobana okuphawulekayo phakathi kwamanani abonwe ekuphindaphindweni okubili kokuhlolwa (0.51 ngamaphuzu obunzima besifo, P = 0.00017 kanye no-0.61 ngesisindo sesitshalo, P < 0.0001) kanye naphakathi kwalezi zinhlaka ezimbili (− 0.59 kanye no-− 0.77, P < 0.0001). Kube nokuhlobana okuphawulekayo phakathi kwamanani abonwe ekuphindulweni okubili kokuhlolwa (0.51 ngamaphuzu obunzima besifo, P = 0.00017 kanye no-0.61 ngesisindo sesitshalo, P < 0.0001) kanye naphakathi kwala mapharamitha amabili (− 0.59 kanye no-− 0.77, P < 0.0001). Выявлена ​​достоверная корреляция между значениями, наблюдаемыми в двух повторностях эксперимента (0,51 для баллов тяжез00, 1 б, 1 = для массы растения, P < 0,0001), а также между этими двумя параметрами (-0,59 futhi -0,77, Р < 0,0001) 0,0001). Kutholakale ubudlelwano obubalulekile phakathi kwamanani abonwe ekuphindaphindweni okubili kokuhlolwa (0.51 ngamanani obunzima besifo, P = 0.00017 kanye no-0.61 ngesisindo sesitshalo, P < 0.0001), kanye naphakathi kwala mapharamitha amabili (- 0.59 kanye no--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 之间 存在 相关性 （疾病 严重 程度 评 分为 5 為 1 為0.00017 , 植物 为 為 0.61 , p <0.0001） 以及 两 个 参数 之间 (((((- 0.59 和– 0.50 和– 0.59 和– 9 和– 9) 0.77, p < 0.0001). Наблюдалась значительная корреляция между значениями, наблюдаемыми в двух повторностях (оценка тяжести заболевания 0,51, P = 10 0,61, P <0,0001), и между этими двумя параметрами (-0,59 futhi -0,0001) 0,77, P <0,0001. Kube nokuhlobana okuphawulekayo phakathi kwamanani abonwe ngokuphindwe kabili (isilinganiso sobunzima besifo 0.51, P = 0.00017 kanye nesisindo sesitshalo 0.61, P < 0.0001) kanye naphakathi kwalezi zinhlaka ezimbili (-0.59 kanye -0 .0001) 0.77, P<0.0001. ).Izimpawu ezivamile ezibonakala ezitshalweni ezisengozini zihlanganisa ukugoba nokusonta kwesiqu okufana nesakhiwo “somnsalo womalusi”, kulandelwe izilonda ezi-oval ezine-sporozoites ezi-orenji/ezipinki (Isithombe Esingeziwe 1). Izakhi zofuzo zase-Australia ezithwala izakhi zofuzo ze-Lanr1 (83A:476 kanye ne-Tanjil) kanye ne-AnMan (Mandelup) zimelana kahle, 0.0331 kanye no-0.0036). Eminye imigqa ephethe ne-Lanr1 kanye/noma i-AnMan alleles “emelana” ikhombisa izimpawu zesifo.
Ngokuthakazelisayo, imigqa embalwa ye-NLL engenayo i-allele yophawu "olumelana" iveze izinga eliphezulu lokumelana ne-anthracnose (okufanayo noma okuphakeme kune-Lanr1 noma i-AnMan genotypes), njenge-Boregine (inani le-P < 0.0001 kuzo zombili izinhlaka), i-Bojar (inani le-P < 0.0001 le-score kanye ne-0.001 lesisindo sesitshalo) kanye ne-Population B-549/79b (inani le-P < 0.0001 le-score kanye nelingabalulekanga ngesisindo). Ngokuthakazelisayo, imigqa embalwa ye-NLL engenayo i-allele yophawu "olumelana" iveze izinga eliphezulu lokumelana ne-anthracnose (okufanayo noma okuphakeme kune-Lanr1 noma i-AnMan genotypes), njenge-Boregine (inani le-P < 0.0001 kuzo zombili izinhlaka), i-Bojar (inani le-P < 0.0001 le-score kanye ne-0.001 lesisindo sesitshalo) kanye ne-Population B-549/79b (inani le-P < 0.0001 le-score kanye nelingabalulekanga ngesisindo). Интересно, что несколько линий NLL, лишенных какого-либо «резистентного» маркерного аллеля, показали высокий уровень устойчивонсти или более высокий, чем для генотипов Lanr1 noma AnMan), таких как Boregine (значение P <0,0001 для обоих параметров), Bojar (значение P0 10,0,0 для массы растения) и популяции B-549/79b (значение P <0,0001 для оценки и незначимо для массы). Ngokuthakazelisayo, imigqa eminingana ye-NLL engenayo i-allele yophawu 'olumelana' ibonise izinga eliphezulu lokumelana ne-anthracnose (eqhathaniswa noma ephakeme kune-Lanr1 noma i-AnMan genotypes), njenge-Boregine (inani le-P < 0.0001 kuzo zombili izinhlaka ), i-Bojar (inani le-P < 0.0001 lokuhlola kanye ne-0.001 yesisindo sesitshalo) kanye nenani labantu i-B-549/79b (inani le-P < 0.0001 lokuhlola futhi alibalulekile ngesisindo).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<得分为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）。 Kuyathakazelisa ukuthi ezinye izinhlelo ze-NLL ezingenazo izimpawu "zokubulala i-genic" zibonisa ukumelana okuphezulu okuvundlile (okulingana nezakhi zofuzo ze-Lanr1 noma ze-AnMan noma ngaphezulu), njenge-Boregine (zombili izinhlaka P < 0.0001), i-Bojar (inani le-P < 0.0001, isisindo sesitshalo 0.001) kanye nohlobo lwe-B-549/79b (inani le-P < 0.0001, isisindo asibalulekile). Интересно, что некоторые линии NLL, лишенные каких-либо маркерных аллелей «резистентности», показали высокие уровни устойчанизинский выше, чем у генотипов I-Lanr1 noma i-AnMan), такие как Boregine (значение P для обоих параметров <0,0001), Bojar (значение P <0,0001, мас001, мас001) B-549/79b (оценка P-значение <0,0001, масса незначительна). Ngokuthakazelisayo, eminye imigqa ye-NLL engenazo izimpawu 'zokumelana' ibonise amazinga aphezulu okumelana ne-anthracnose (okuqhathaniswa noma okungaphezu kwe-Lanr1 noma i-AnMan genotypes), njenge-Boregine (inani le-P lazo zombili izinhlaka <0.0001), i-Bojar (inani le-P <0.0001, isisindo sesitshalo 0.001) kanye nenani labantu B-549/79b (inani le-P <0.0001, isisindo asibalulekile).Lesi simo sibonisa ukuthi kungenzeka ukuthi kube nomthombo omusha wokumelana nezakhi zofuzo, okuchaza ukuntuleka okubonakalayo kokuhlobana phakathi kwezinhlobo ze-marker genotypes kanye ne-phenotypes yesifo (amanani e-P ​​kusukela ku-~0.42 kuya ku-~0.98). Ngakho-ke, ukuhlolwa kwe-Kolmogorov-Smirnov kubonise ukuthi idatha yokumelana ne-anthracnose yayisakazwa cishe ngokujwayelekile kumaphuzu (amanani e-P ​​0.25 no-0.11) kanye nesisindo sesitshalo (amanani e-P ​​0.47 no-0.55), okuphakamisa ukuthi ngicabanga ukuthi kuhileleke ama-allele amaningi kune-Lanr1 ne-AnMan.
Ngokusekelwe emiphumeleni yokuhlolwa kokumelana ne-anthracnose, imigqa emi-4 yakhethwa ukuze kuhlaziywe i-transcriptome: 83A:476, Boregine, Mandelup, kanye ne-Population 22660. Le migqa yahlolwa kabusha ukuze kutholakale ukumelana ne-anthrax ekuhlolweni kokugonywa ngokulandelelana kwe-RNA, uma nje ifana nasovivinyweni lwangaphambilini. Amanani amaphuzu ayelandelayo: Boregin (1.71 ± 1.39), 83A: 476 (2.09 ± 1.38), Mandelup (3.82 ± 1.42) kanye ne-population 22660 (6.11 ± 1.29).
Iphrothokholi ye-Illumina NovaSeq 6000 ifinyelele isilinganiso sama-pair angu-40.5 Mread ngesampula ngayinye (29.7 kuya ku-54.4 Mreads) (Ithebula Elingeziwe S3). Amaphuzu okuhambisana kuchungechunge lokubhekisela asukela ku-75.5% kuya ku-88.6%. Ukuxhumana okumaphakathi kwedatha yokubalwa kokufundwa phakathi kwezinhlobo zokuhlola phakathi kokuphindaphindwa kwezinto eziphilayo kusukela ku-0.812 kuya ku-0.997 (isilinganiso esingu-0.959). Kuma-gene angu-35,170 ahlaziywe, angu-2917 awembulanga ukuvezwa, kanti amanye ama-gene angu-4785 abonakaliswe ezingeni eliphansi (isilinganiso esiyisisekelo < 5). Kuma-gene angu-35,170 ahlaziywe, angu-2917 awembulanga ukuvezwa, kanti amanye ama-gene angu-4785 abonakaliswe ezingeni eliphansi (isilinganiso esiyisisekelo < 5). Из 35 170 проанализированных генов 2917 не проявляли экспрессии, а остальные 4785 генов экспрессировались на ненезнабрессии <5). Kuma-gene angu-35,170 ahlaziywe, angu-2917 awazange abonise ukuvezwa, kanti ama-gene angu-4785 asele abonakaliswe ezingeni elincane (isilinganiso esiyisisekelo <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 (базение 5). Kuma-gene angu-35,170 ahlaziywe, angu-2917 awazange avele kanti ama-gene angu-4785 asele ayenokubonakaliswa okuncane (isilinganiso esiyisisekelo <5).Ngakho-ke, inani lezakhi zofuzo ezicatshangelwe eziveziwe (isilinganiso esiyisisekelo ≥ 5) ngesikhathi sokuhlola lalingu-27,468 (78.1%) (Ithebula Elingeziwe S4).
Kusukela ephuzwini lokuqala, yonke imigqa ye-NLL yasabela ekugonyelweni kwe-C. lupini (uhlobo lwe-Col-08) ngokuhlela kabusha i-transcriptome (Ithebula 1), nokho, umehluko omkhulu wabonwa phakathi kwemigqa. Ngakho-ke, umugqa wokumelana 83A:476 (ophethe i-gene ye-Lanr1) ubonise ukuhlelwa kabusha okukhulu kwe-transcriptome endaweni yokuqala (6 hpi) ngokukhuphuka okuphindwe ka-31-69 enanini lama-gene ahlukanisiwe aphezulu naphansi uma kuqhathaniswa namanye amaphuzu esikhathi kulesi sikhathi. Ngaphezu kwalokho, leli nani eliphakeme lahlala isikhathi esifushane, njengoba ukubonakaliswa kwama-gene ambalwa kuphela kwahlala kushintshiwe kakhulu endaweni yesibili (12 hpi). Ngokuthakazelisayo, i-Boregine, nayo ebonise izinga eliphezulu lokumelana ekuhlolweni kwe-graft, ayizange ibhekane nokuhlelwa kabusha okukhulu kangaka kwe-transcriptal ngesikhathi sokuhlolwa. Kodwa-ke, inani lama-gene achazwe ngokuhlukile (DEG) lalifana ne-Boregine kanye ne-83A:476 ku-12 HPI. Kokubili uMandelup kanye nenani labantu abangu-22660 kubonise ukuphakama kwe-DEG ngesikhathi sokugcina (48 l/s), okubonisa ukubambezeleka okulinganiselwe ezimpendulweni zokuzivikela.
Ngenxa yokuthi i-83A:476 yahlelwa kabusha kakhulu nge-transcriptome ngenxa ye-C. lupini ku-6 HPI uma kuqhathaniswa nazo zonke ezinye imigqa, ama-~91% ama-DEG abonwe ngalesi sikhathi ayeqondene nohlu lozalo (Isithombe 1). Kodwa-ke, kube nokufana okuthile ezimpendulweni zokuqala phakathi kwemigqa yocwaningo, njengoba ama-68.5%, ama-50.9%, kanye nama-52.6% DEG eBoregine, eMandelup, kanye nenani labantu abangu-22660, ngokulandelana, ahambisana nalawo atholakale ku-83A:476 emaphuzwini athile ngesikhathi. Kodwa-ke, la ma-DEG abalelwa engxenyeni encane kuphela (0.97–1.70%) yawo wonke ama-DEG atholakale kusetshenziswa i-83A:476. Ngaphezu kwalokho, ama-DEG angu-11 avela kuyo yonke imigqa ayehambisana ngalesi sikhathi (Amathebula Angeziwe S4-S6), kufaka phakathi izingxenye ezivamile zezimpendulo zokuzivikela kwezitshalo: iphrotheni yokudlulisa amafutha (iTanjilG_32225), i-enzyme ye-endoglucan-1,3-β-glucoside (iTanjilG_23384), amaprotheni amabili angena ekucindezelekeni njenge-SAM22 (iTanjilG_31528 neTanjilG_31531), iphrotheni ye-latex eyisisekelo (iTanjilG_32352), kanye namaprotheni amabili odonga lweseli olucebile nge-glycine (iTanjilG_19701 neTanjilG_19702). Kwakukhona futhi ukufana okuphezulu kwezimpendulo ze-transcriptome phakathi kuka-83A:476 neBoregine ku-24 HPI (isamba esingu-16-38% DEG) kanye naphakathi kweMandelup kanye nePopulation 22660 ku-48 HPI (isamba esingu-14-20% DEG).
Umdwebo we-Venn okhombisa inani lezakhi zofuzo ezivezwe ngokuhlukile (DEG) emigqeni ye-lupine enamahlamvu amancane (NLL) efakwe i-Colletotrichum lupini (uhlobo lwe-Col-08 olutholwe emasimini e-lupine e-Wierzhenice, ePoland, 1999). Imigqa ye-NLL ehlaziyiwe yile: 83A:476 (emelana nomzimba, ephethe i-allele ye-Lanr1), i-Boregine (emelana nomzimba, isizinda sofuzo esingaziwa), i-Mandelup (emelana nomzimba ngokulinganiselwe, ephethe i-allele ye-AnMan) kanye nenani labantu abangu-22660 (abasengozini kakhulu). Isifinyezo se-hpi simele amahora ngemva kokugonywa. Amanani awekho asusiwe ukuze kube lula igrafu.
Isethi yezakhi zofuzo ezivezwe ngokweqile ku-6 hpi ihlaziywe ukuze kutholakale ukuthi kukhona izizinda zezakhi zofuzo ze-R ezingokomfanekiso (Ithebula Elingeziwe S7). Lolu cwaningo lwembule ukungeniswa kwe-transcriptome kwezakhi zofuzo zokumelana nezifo zakudala ezinezizinda ze-NBS-LRR kuphela ku-83A:476. Leli sethi laliqukethe i-gene eyodwa ye-TIR-NBS-LRR (tanjilg_05042), ama-gene amahlanu e-CC-NBS-LRR (tanjilg_06165, tanjilg_06162, tanjilg_22773, tanjilg_22640, kanye ne-tanjilg_16162), kanye ne-Four NBS-LR, Tanjilg_16162), kanye ne-Four NBS-LRRE (tanjilg_16162) kanye ne-Four NBS-Lrr (tanjilg_16162) kanye ne-Four NBS-LRR (TANJILG_16162). Zonke lezi zakhi zofuzo zinezizinda ze-canonical ezihlelwe ngokulandelana okugciniwe. Ngaphezu kwezakhi zofuzo zesizinda se-NBS-LRR, ama-kinase amaningana e-RLL asebenze ku-6 hpi, okungukuthi elilodwa ku-Boregine (TanjilG_19877), amabili ku-Mandelup (TanjilG_07141 kanye no-TanjilG_19877) kanye nenani labantu abangu-22660 (TanjilG_09014 kanye no-TanjilG_10361) kanye namabili ku-83A 27:476.
Izakhi zofuzo ezishintshe kakhulu ekuphenduleni ekugonyweni nge-C. lupini (uhlobo lwe-Col-08) zahlolwa ngokuhlaziywa kokunongwa kwe-Gene Ontology (GO) (Ithebula Elingeziwe S8). Igama lenqubo yezinto eziphilayo elivame ukumelwa ngokweqile kwakuyi-“GO:0006952 impendulo yokuzivikela” evele ezinhlanganisweni ezingu-6 kwezingu-16 (isikhathi × umugqa) ezinokubaluleka okuphezulu (inani le-P <0.001) (Isithombe 2). Igama lenqubo yezinto eziphilayo elivame ukumelwa ngokweqile kwakuyi-“GO:0006952 impendulo yokuzivikela” evele ezinhlanganisweni ezingu-6 kwezingu-16 (isikhathi × umugqa) ezinokubaluleka okuphezulu (inani le-P <0.001) (Isithombe 2). Наиболее часто чрезмерно представленным термином биологического процесса был «GO: 0006952 защитный ответ», которлый повля 6 × 6 линия) комбинаций с высокой значимостью (значение P <0,001) (рис. 2). Igama lenqubo yezinto eziphilayo elivame ukumelwa ngokweqile kwakuyi-'GO:0006952 impendulo yokuzivikela', evele ezinhlanganisweni ezingu-6 kwezingu-16 (isikhathi × uhlu) ezinokubaluleka okuphezulu (inani le-P <0.001) (Isithombe 2).最常被过度代表的生物过程术语是“GO:0006952 防御反应”个中，具有高显着性(P 值< 0.001）(图2). Igama lenqubo yezinto eziphilayo elimele kakhulu yi-“GO:0006952 defense response”, elivela ezinhlanganisweni ezingu-6 kwezingu-16 (时间×线), ezinokubaluleka okuphezulu (inani le-P < 0.001) (图2). Наиболее часто чрезмерно представленным термином биологического процесса был «GO: 0006952 Defence Response», который появлялся 1на × 6 им линия) с высокой значимостью (значение P <0,001) (рис. 2). Igama lenqubo yezinto eziphilayo elivame ukumelwa ngokweqile kwakuyi-'GO:0006952 Defense Response', elivele ezinhlanganisweni ezingu-6 kwezingu-16 (isikhathi × umugqa) ngokubaluleka okuphezulu (inani le-P < 0.001) (Isithombe 2).Leli gama lamelelwa ngokweqile ezindaweni ezimbili zesikhathi ku-83A: 476 kanye ne-Boregine (6 kanye ne-24 hpi) kanye nephuzu elilodwa eMandelup kanye ne-Population 22660 (12 kanye ne-6 hpi, ngokulandelana). Lokhu kuwumphumela olindelekile, oqokomisa impendulo ye-antifungal yemigqa ephikisanayo. Ngaphezu kwalokho, i-83A:476 iphendule ku-C. lupini ngokusheshisa ukusheshisa izakhi zofuzo ezihlobene nokuqhuma kwe-oxidative okumelelwe yigama elithi “GO:0055114 redox process”, okubonisa impendulo ethile yokuzivikela, kanti i-Boregine iveze izimpendulo ezithile zokuzivikela, ezihlobene negama elithi 'GO'. :0006950 Impendulo yokucindezeleka”. Isibalo sabantu abangu-22660 sisebenze impendulo yokumelana okuvundlile ehilela ama-metabolite esibili, siqokomisa inani elikhulu lamagama athi “GO:0016104 Inqubo ye-triterpene biosynthesis” kanye nelithi “GO:0006722 Inqubo ye-triterpene metabolism” (womabili la magama angowesethi efanayo yezakhi zofuzo), uma kucatshangelwa imiphumela yokuhlaziywa kokunotha kwegama le-GO, ukuzinza kokusabela kwe-Mandelup kwakuphakathi kwe-Boregine kanye ne-Population 22660. Ngaphezu kwalokho, ukusabela kwasekuqaleni okungu-83A:476 (6 hpi) kanye nokusabela okulibazisekile i-Mandelup kanye ne-Population 22660 kuhlanganisa igama elithi GO:0015979 elithi 'photosynthesis' kanye nezinye izinqubo zebhayoloji ezihlobene.
Amagama e-bioprocess gene ontology akhethwe ekuchazeni izakhi zofuzo ezivezwe ngokuhlukile ngesikhathi sempendulo ye-transcriptome ye-lupine enamahlamvu amancane (i-NLL) efakwe i-anthrax lupine (uhlobo lwe-Col-08 olutholwe emasimini e-lupine eWierzhenice, ePoland, ngo-1999) aqisiwe kakhulu. Imigqa ye-NLL ehlaziyiwe yile: 83A:476 (emelana nokumelana, ethwele i-allele ye-Lanr1 efana ne-homozygous), i-Boregine (emelana nokumelana, isizinda sezakhi zofuzo esingaziwa), i-Mandelup (emelana nokumelana ngokulinganiselwe, ethwele i-allele ye-AnMan efana ne-homozygous) kanye nenani labantu abangu-22660 (elithintekayo).
Ngenxa yokuthi lolu cwaningo luhlose ukuhlonza izakhi zofuzo ezifaka isandla ekuphikisweni kwe-anthracnose, izakhi zofuzo ezinikezwe amagama athi GO “GO: 0006952 Izimpendulo Zokuzivikela” kanye nelithi “GO: 0055114 Izinqubo ze-Redox” zahlaziywa ngezikhawulo njengoba kusho isisekelo ≥ 30 okungenani ngomugqa owodwa. × iphuzu ngesikhathi elihlanganisa amanani abalulekile ngokwezibalo e-log2 (ushintsho olugoqekayo). Inani lamazakhi zofuzo ahlangabezana nalezi zindlela lalingu-65 ku-GO:0006952 kanye no-524 ku-GO:0055114.
I-83A:476 yembule iziqongo ezimbili ze-DEG ezichazwe ngegama elithi GO:0006952, elokuqala kuma-genes angu-6 nge-intshi (ama-genes angu-64, umthethonqubo wokukhuphuka nokuwa) kanye nelesibili kuma-genes angu-24 nge-intshi (ama-genes angu-15, umthethonqubo wokukhuphuka kuphela). I-Boregine iphinde yabonisa ukuthi i-GO:0006952 ifinyelele esicongweni ngesikhathi esifanayo, kodwa nge-DEG encane (11 no-8) kanye nokusebenza okuthandwayo. I-Mandeloop ibonise iziqongo ezimbili ze-GO:0006952 ku-12 no-48 HPI, zombili ziphethe ama-genes angu-12 (elokuqala elinama-genes asebenzayo, kanye nelesibili elinama-genes acindezelayo kuphela), kuyilapho inani labantu abangu-22660 ku-6 HPI (ama-genes angu-13) lalinobukhulu obukhulu bomthethonqubo wokukhuphuka kwe-peak. Kufanele kuqashelwe ukuthi ama-96.4% e-GO:0006952 DEG kulezi ziqongo ayenohlobo olufanayo lokuphendula (phezulu noma phansi), okubonisa ukufana okukhulu ezimpendulweni zokuzivikela naphezu kokungafani kwenani lama-genes ahilelekile. Iqembu elikhulu kunawo wonke lama-sequence ahlobene negama elithi GO:0006952 lifaka ikhodi ye-Starvation Stress-Associated Message Protein 22 (SAM22-like), engaphansi kwe-class 10 pathogenesis-associated protein (PR-10) protein clade kanye ne-core protein latex. efanayo (MLP-like) protein) (Isithombe 3). La maqembu amabili ahluke ngendlela yokuvezwa kanye nendlela yokusabela. Ama-gene afaka amaprotheni afana ne-SAM22 abonise ukungeniswa okuhambisanayo nokubalulekile ezikhathini zokuqala (6 noma 12 hpi) futhi ngokuvamile ayengaphenduli ekupheleni kokuhlolwa (48 hpi), kuyilapho amaprotheni afana ne-MLP abonise ukuhambisana ku-6 hpi. hpi. 83A:476 kanye ne-Mandelup ku-48 hp/in, cishe wonke amanye amaphuzu edatha ayengaphenduli. Ngaphezu kwalokho, umehluko kumaphrofayili okubonakaliswa kwama-gene amaprotheni afana ne-SAM22 alandele ukuhlukahluka kokumelana ne-anthracnose, njengoba imigqa ephikisana kakhulu yayinezikhathi eziningi ezibangela kakhulu la ma-gene kunezakhi zofuzo ezisengozini enkulu. Esinye isakhi sofuzo se-PR-10 esifana ne-LlR18A/B sibonise iphethini yokubonakaliswa efana kakhulu nesakhi sofuzo se-protein esifana ne-SAM22.
Izingxenye eziyinhloko zenqubo yezinto eziphilayo ethi “GO:0006952 Defense Response” kanye namaphethini okubonakaliswa kwezakhi zofuzo ezifanelekayo ze-Lanr1 kanye ne-AnMan alleles kwatholakala. Isikali se-Log2 simelela amanani e-log2 (ushintsho olugoqekayo) phakathi kwezitshalo ezifakwe i-incubator (Colletotrichum lupini, uhlobo lwe-Col-08, olutholwe emasimini e-lupine, eWizhenica, ePoland, 1999) kanye nezitshalo zokulawula (ezifakwe i-incubator) ngesikhathi esifanayo. Imigqa elandelayo ye-lupine enamahlamvu amancane yahlaziywa: 83A:476 (emelana nezifo, ethwele i-allele ye-Lanr1 efana ne-homozygous), i-Boregine (emelana nezifo, isizinda sofuzo esingaziwa), i-Mandelup (emelana nezifo ngokulinganisela, ethwele i-allele ye-AnMan efana ne-homozygous), kanye ne-Population 22660 (etholakala kalula).
Ngaphezu kwalokho, amaphrofayili okubonakaliswa kwezakhi zofuzo ze-RNA-seq i-Lanr1 (TanjilG_05042) kanye ne-AnMan (TanjilG_12861) ahlolwe (Isithombe 3). Izakhi zofuzo ze-TanjilG_05042 zibonise impendulo ebalulekile (ukusebenza) ku-83A:476 kuphela ngesikhathi sokuqala (6 hpi), kuyilapho i-TanjilG_12861 yayibalulekile eMandeloop kuphela ngesikhathi samaphoyinti amabili: i-6 hpi (ukulawulwa kwe-down) kanye ne-24 hpi (6 hpi). With.). adjustable) ).
Izakhi zofuzo ezivezwe kakhulu egameni elithi GO:0055114 “inqubo ye-redox” kwakuyizakhi zofuzo ezifaka amaprotheni e-cytochrome P450 kanye ne-peroxidase (Isithombe 4). Kumasampula ahlukaniswe ku-83A:476 ku-6 HPI, amanani aphezulu noma aphansi kakhulu e-log2 (ushintsho olugoqekayo) ​​(ku-86.6% wezakhi zofuzo) ngokuvamile abonwa phakathi kwezitshalo ezifakwe i-incubator kanye nezitshalo ezilawulwayo, okugqamisa impendulo ephezulu yalolu hlobo lwe-genotype ebulilini obufaka i-incubator. I-83A:476 ibonise i-GO ebaluleke kakhulu: 0055114 DEG ku-6 hpi (amazakhi zofuzo angu-503), kuyilapho eminye imigqa ku-48 hpi (i-Boregine, amazakhi zofuzo angu-31; i-Mandelup, amazakhi zofuzo angu-85; kanye ne-Population 22660, amazakhi zofuzo angu-78)). Kuzakhi zofuzo eziningi zomndeni we-GO:0055114, kwabonwa izinhlobo ezimbili zezimpendulo ekugonyweni (ukusebenza kanye nokuvinjelwa). Ngokuthakazelisayo, kufika ku-97.6% wama-DEG ahlonziwe ngegama elithi GO: 0055114 eMandelupe ku-48 hp Lokhu okubonwe kusikisela ukuthi, naphezu kwesilinganiso esincane kakhulu (okungukuthi, inani lama-gene e-redox aguquliwe, angu-85 uma kuqhathaniswa no-503), iphethini yezimpendulo ze-transcriptome ezibambezelekile ze-mandeloup ku-anthracnose ifana nempendulo yokuqala ka-83A:476. Ku-Boregine kanye ne-Population 22660, lokhu kuhlangana kuphansi ku-51.6% kanye no-75.6%, ngokulandelana.
Amaphethini okubonakaliswa kwezingxenye eziyinhloko zegama lenqubo yezinto eziphilayo elithi “GO:0055114 Redox process” embulwe. Isikali se-Log2 simelela amanani e-log2 (ushintsho olugoqekayo) phakathi kwezitshalo ezifakwe i-incubator (Colletotrichum lupini, uhlobo lwe-Col-08, olutholwe emasimini e-lupine, eWizhenica, ePoland, 1999) kanye nezitshalo zokulawula (ezifakwe i-incubator) ngesikhathi esifanayo. Imigqa elandelayo ye-lupine enamahlamvu amancane yahlaziywa: 83A:476 (emelana nemithi, ethwala i-allele ye-Lanr1 efana ne-homozygous), i-Boregine (emelana nemithi, isizinda sofuzo esingaziwa), i-Mandelup (emelana nemithi, ethwala i-allele ye-AnMan efana ne-homozygous), kanye ne-Population 22660 (etholakala kalula).
83A:476 Izimpendulo ze-Transcriptomic ekufakweni kwe-C. lupini (uhlobo lwe-Col-08) nazo zazihlanganisa ukuthuliswa okuhlanganisiwe kwezakhi zofuzo ezibangelwa igama elithi GO:0015979 “photosynthesis” kanye nezinye izinqubo zebhayoloji ezihlobene (UMfanekiso 5). Leli sethi le-GO:0015979 DEG laliqukethe izakhi zofuzo eziyi-105 ezacindezelwa kakhulu ku-6 hpi ku-83A:476. Kulesi sigaba esincane, izakhi zofuzo ezingama-37 nazo zehlisiwe eMandelup ku-48 HPI kanye no-35 ngesikhathi esifanayo kubantu abangu-22660, kufaka phakathi ama-DEG ayi-19 ajwayelekile kuzo zombili izinhlobo zezakhi zofuzo. Awekho ama-DEG ahlobene negama elithi GO: 0015979 asebenze kakhulu kunoma iyiphi inhlanganisela (umugqa x isikhathi).
Amaphethini okubonakaliswa kwezingxenye eziyinhloko zegama lenqubo yezinto eziphilayo ethi “GO:0015979 Photosynthesis” embulwe. Isikali se-Log2 simelela amanani e-log2 (ushintsho olugoqekayo) phakathi kwezitshalo ezifakwe i-incubator (Colletotrichum lupini, uhlobo lwe-Col-08, olutholwe emasimini e-lupine, eWizhenica, ePoland, 1999) kanye nezitshalo zokulawula (ezifakwe i-incubator) ngesikhathi esifanayo. Imigqa elandelayo ye-lupine enamahlamvu amancane yahlaziywa: 83A:476 (emelana nemithi, ethwala i-allele ye-Lanr1 efana ne-homozygous), i-Boregine (emelana nemithi, isizinda sofuzo esingaziwa), i-Mandelup (emelana nemithi, ethwala i-allele ye-AnMan efana ne-homozygous), kanye ne-Population 22660 (etholakala kalula).
Ngokusekelwe emiphumeleni yokuhlaziywa kokubonakaliswa okuhlukile kanye nokucatshangelwa ukuthi kuhilelekile ekuphenduleni kokuzivikela ngokumelene nesikhunta esibangela izifo, leli sethi lama-gene ayisikhombisa lakhethwa ukuze kulinganiswe amaphrofayili okubonakaliswa yi-PCR yesikhathi sangempela (Ithebula Elingeziwe S9).
I-gene yephrotheni ecatshangelwayo i-TanjilG_10657 ibangelwe kakhulu kuyo yonke imigqa efundwe kanye namaphuzu esikhathi uma kuqhathaniswa nezitshalo zokulawula (ezilingisayo) (Amathebula Angeziwe S10, S11). Ngaphezu kwalokho, iphrofayili yokubonakaliswa kwe-TanjilG_10657 ibonise ukuthambekela okwandayo phakathi nokuhlolwa kwayo yonke imigqa. Inani labantu abangu-22660 libonise ukuzwela okuphezulu kakhulu kwe-TanjilG_10657 ekufakweni ngokusebenza okungu-114-fold kanye nezinga eliphezulu kakhulu lokubonakaliswa (4.4 ± 0.4) ku-24 HPI (Isithombe 6a). I-gene yephrotheni ye-PR10 LlR18A i-TanjilG_27015 nayo ibonise ukusebenza kuyo yonke imigqa namaphuzu esikhathi, ngokubaluleka kwezibalo kumaphuzu amaningi edatha (Isithombe 6b). Ngokufanayo ne-TanjilG_10657, izinga eliphezulu kakhulu lokubonakaliswa kwe-TanjilG_27015 labonwa kubantu abangu-22660 abafakwe imijovo ku-24 HPI (19.5 ± 2.4). I-acid endochitinase gene i-TanjilG_04706 yanda kakhulu kuyo yonke imigqa nakuzo zonke izikhathi ngaphandle kwe-Boregine 6 hpi (Isithombe 6c). Yabangelwa kakhulu endaweni yokuqala (6 HPI) ku-83A:476 (ngezikhathi eziyi-10.5) futhi yanda ngokulinganisela kweminye imigqa (ngezikhathi eziyi-6.6-7.5). Ngesikhathi sokuhlola, ukubonakaliswa kwe-TanjilG_04706 kwahlala kumazinga afanayo ku-83A:476 kanye ne-Boregine, kuyilapho e-Mandelup kanye ne-Population 22660 kwanda kakhulu, kwafinyelela amanani aphezulu kakhulu (5.9 ± 1.5 kanye no-6.2 ± 1.5, ngokulandelana). I-gene efana ne-endoglucan-1,3-β-glucosidase-like TanjilG_23384 ibonise ukusebenza okuphezulu ezindaweni ezimbili zokuqala zesikhathi (6 kanye ne-12 hpi) kuyo yonke imigqa ngaphandle kwenani labantu abangu-22660 (Isithombe 6d). Amazinga aphezulu kakhulu okubonakaliswa kwe-TanjilG_23384 abonwe endaweni yesibili yesikhathi (12 hpi) eMandelup (2.7 ± 0.3) kanye no-83A:476 (1.5 ± 0.1). Ku-24 HPI, ukubonakaliswa kwe-TanjilG_23384 kwakuphansi kakhulu kuyo yonke imigqa efundwe (kusukela ku-0.04 ± 0.009 kuya ku-0.44 ± 0.12).
Amaphrofayili okubonakaliswa kwezakhi zofuzo ezikhethiwe (ag) avezwe yi-quantitative PCR. Izinombolo 6, 12 kanye no-24 zimele amahora ngemva kokugonywa. Izakhi zofuzo ze-LanDExH7 kanye ne-LanTUB6 zasetshenziswa ukuze kulungiswe futhi i-LanTUB6 yasetshenziswa ekulinganisweni phakathi kochungechunge. Amabha amaphutha amelela ukuphambuka okujwayelekile okusekelwe ekuphindaphindweni kwezinto ezintathu eziphilayo, ngayinye eyisilinganiso sokuphindaphindwa kwezobuchwepheshe ezintathu. Ukubaluleka kwezibalo komehluko emazingeni okubonakaliswa phakathi kwezitshalo ezifakwe umjovo (i-Colletotrichum lupini, uhlobo lwe-Col-08, olutholwe ngo-1999 ensimini ye-lupin eWierzenica, ePoland) kanye nezitshalo zokulawula (ezifakwe umjovo) kumakwe ngaphezulu kwamaphuzu edatha (*Inani le-P < 0.05, **Inani le-P ≤ 0.01, ***Inani le-P ≤ 0.001). Ukubaluleka kwezibalo komehluko emazingeni okubonakaliswa phakathi kwezitshalo ezifakwe umjovo (i-Colletotrichum lupini, uhlobo lwe-Col-08, olutholwe ngo-1999 ensimini ye-lupin eWierzenica, ePoland) kanye nezitshalo zokulawula (ezifakwe umjovo) kumakwe ngaphezulu kwamaphuzu edatha (*Inani le-P < 0.05, **Inani le-P ≤ 0.01, ***Inani le-P ≤ 0.001). Статистическая значимость различий в уровнях экспрессии между инокулированными (Colletotrichum lupini, штам Col-08, получен ngo-1999 lniv, получен в 1999 г. Польша) и контрольными (ложно инокулированными) растениями отмечена над точками данных (*значение P < 0,05, **значение P ≤ 0,010, ***≤ 0,010, *** 0, *** Umehluko obalulekile ngokwezibalo emazingeni okuvezwa phakathi kwezitshalo ezifakwe umjovo (i-Colletotrichum lupini, uhlobo lwe-Col-08, olutholwe ngo-1999 ensimini ye-lupine eWierzhenice, ePoland) kanye nezitshalo zokulawula (ezifakwe umjovo) kuphawulwe ngaphezu kwamaphuzu edatha (*Inani le-P < 0.05, **Inani le-P ≤ 0.01, ***Inani le-P ≤ 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 в 1999 г.) и контрольными (ложно инокулированными) растениями отмечены над точками данных (* значение P < 0,05, ** P-значение ≤ 0-P, 0,001). Umehluko obalulekile ngokwezibalo emazingeni okuvezwa phakathi kwezitshalo ezifakwe umjovo (i-Colletotrichum lupini, uhlobo lwe-Col-08, olutholwe emasimini e-lupine eVerzhenice, ePoland, ngo-1999) kanye nezitshalo zokulawula (ezifakwe umjovo) kuphawulwe ngaphezu kwamaphuzu edatha (*Inani le-P < 0.05 , **Inani le-P ≤ 0.01, ***Inani le-P ≤ 0.001).Imigqa ye-NLL ehlaziyiwe yile: 83A:476 (emelana nomzimba, ethwele i-allele ye-Lanr1 ene-homozygous), i-Mandelup (emelana nomzimba ngokumaphakathi, ethwele i-allele ye-AnMan ene-homozygous), i-Boregine (emelana nomzimba, isizinda sofuzo esingaziwa) kanye nenani labantu abangu-22660 (elithintekayo).
I-gene ekhethiwe i-TanjilG_05042 endaweni ye-Lanr1 ibonise iphethini yokubonakaliswa ehluke kakhulu kumaphrofayili atholwe ezifundweni ze-RNA-seq (Isithombe 6e). Ukusebenza okuphawulekayo kwalesi sigine kubonwe eMandelup kanye nenani labantu abangu-22660 (kufika ku-39.7 kanye nezikhathi ezingu-11.7, ngokulandelana), okuholele emazingeni aphezulu okuvezwa (kufika ku-1.4 ± 0.14 kanye no-7.2 ± 1.3, ngokulandelana). I-83A:476 iphinde yembula ukwanda okuthile kwe-gene ye-TanjilG_05042 (kufika ku-3.8-fold), noma kunjalo, amazinga okuvezwa ahlobene afinyelelwe (0.044 ± 0.002) ayengaphansi ngokuphindwe ka-30 kunalawo abonwe eMandelup kanye nenani labantu abangu-22660. okuhlaziywe yi-qPCR kubonise umehluko omkhulu emazingeni okubonakaliswa phakathi kwezinhlobo ze-genotypes ezinhlotsheni ezigonywe (zokulawula) ezigonywe nge-mock, kwafinyelela umehluko ophindwe ka-58 phakathi kwezinhlobo ezingu-22660 no-83A:476, kanye naphakathi kwezinhlobo ezingu-22660 no-22660. Umehluko ophindwe kabili watholakala phakathi kweBoregine neMandalup.
I-gene efanelekile endaweni ye-AnMan, i-TanjilG_12861, yavuselelwa ngenxa yokugonywa ku-83A:476 kanye ne-Mandelup, yayingathathi hlangothi kubantu abangu-22660, futhi yehliswa ku-Boregine (Isithombe 6f). Ukuvezwa okuhlobene kwe-gene ye-TanjilG_12861 kwakuphakeme kakhulu ku-83A egonyiwe: 476 (0.14±0.01). I-gene ye-17.4 kDa class I heat shock protein gene i-TanjilG_05080 HSP17.4 ibonise amazinga aphansi okubonakaliswa okuhlobene kuzo zonke izinhlobo ezifundwe kanye namaphuzu esikhathi (Isithombe 6g). Inani eliphakeme kakhulu labonwa ku-24 HPI kubantu abangu-22660 (0.14 ± 0.02, ukwanda okuphindwe kasishiyagalombili ekuphenduleni ekugonyweni).
Ukuqhathaniswa kwamaphrofayili okubonakaliswa kwezakhi zofuzo (Isithombe 7) kwembule ubudlelwano obuphezulu phakathi kweTanjilG_10657 nezinye izakhi zofuzo ezine: TanjilG_27015 (r = 0.89), TanjilG_05080 (r = 0.85), TanjilG_05042 (r = 0.80), kanye neTanjilG_04706 (r = 0.79). Imiphumela enjalo ingabonisa ukulawulwa ngokubambisana kwalezi zakhi zofuzo ngesikhathi sezimpendulo zokuzivikela. Izakhi zofuzo zeTanjilG_12861 kanye neTanjilG_23384 zibonise amaphrofayili okubonakaliswa ahlukene anamanani aphansi e-Pearson correlation coefficient ​​(kusukela ku-0.08 kuya ku-0.43 kanye no--0.19 kuya ku-0.28, ngokulandelana) uma kuqhathaniswa nezinye izakhi zofuzo.
Ubudlelwano phakathi kwamaphrofayili okubonakaliswa kwezakhi zofuzo butholakale kusetshenziswa i-PCR yokulinganisa. Imigqa elandelayo ye-lupine enama-narrowleaf ihlaziywe: 83A:476 (emelana nokumelana, ethwele i-allele ye-Lanr1 ene-homozygous), i-Mandelup (emelana nokumelana nokulingana, ethwele i-allele ye-AnMan ene-homozygous), i-Boregine (emelana nokumelana, isizinda sezakhi zofuzo esingaziwa), kanye ne-Population 22660 (emelana nokumelana ...
Ngokusekelwe kudatha etholwe ku-6 horsepower per inch, i-WGCNA yenziwe ku-9981 DEG ehlonzwe ngokuqhathanisa izitshalo ezifakwe umjovo kanye nezilawulayo ukuze kugxilwe ezimpendulweni zokuzivikela zakuqala (Ithebula Elingeziwe S12). Amamojula ezizakhi zofuzo angamashumi amabili nambili (amaqoqo) atholakale enamaphrofayili okubonakaliswa ahlobene (amahle noma amabi) phakathi kwezinhlobo zezakhi zofuzo kanye nezinhlobo zokuhlola. Ngokwesilinganiso, amazinga okubonakaliswa kwezakhi zofuzo ayehla ngokulandelana okungu-83A:476 > Mandelup > Boregine > Inani labantu abangu-22660 (kuzo zombili izinhlobo, nokho, lo mkhuba wawunamandla ezitshalweni ezilawulayo). Ngokwesilinganiso, amazinga okubonakaliswa kwezakhi zofuzo ayehla ngokulandelana okungu-83A:476 > Mandelup > Boregine > Inani labantu abangu-22660 (kuzo zombili izinhlobo, nokho, lo mkhuba wawunamandla ezitshalweni ezilawulayo). В среднем уровни экспрессии генов снижались в порядке 83A:476 > Mandelup > Boregine > Population 22660 (в обоих вариантах, однако, эта тенден) контрольных растений). Ngokwesilinganiso, amazinga okubonakaliswa kwezakhi zofuzo ehle ngokulandelana okungu-83A:476 > Mandelup > Boregine > Inani labantu abangu-22660 (kuzo zombili izinhlobo, nokho, lo mkhuba wawunamandla ezitshalweni ezilawulayo).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> inani labantu 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). Ngokwesilinganiso, amazinga okubonakaliswa kwezakhi zofuzo ehlile ochungechungeni lwe-83A:476 > Mandelup > Boregine > Inani labantu abangu-22660 (noma kunjalo, kuzo zombili izinhlobo, lo mkhuba wawunamandla ezitshalweni ezilawulayo).Ukugoma kuholele ekukhulisweni kokubonakaliswa kwezakhi zofuzo, ikakhulukazi kumamojula 18, 19, 14, 6 kanye no-1 (ngokulandelana komphumela okwehla), ukulawulwa okungekuhle (isb. amamojula 9 kanye no-20) noma ngemiphumela engathathi hlangothi (isb. amamojula 11, 22, 8 kanye no-13). Ukuhlaziywa kokunongwa kwe-GO term (Ithebula Elingeziwe S13) kwembule “Izimpendulo Zokuvikela ze-GO: 0006952” zemojula efakwe i-incubator (18) esebenza kahle kakhulu, kufaka phakathi izakhi zofuzo ezihlaziywe yi-qPCR (TanjilG_04706, TanjilG_23384, TanjilG_10657 kanye no-TanjilG_27015), kanye namamojula amaningi e-Inoculate most suppressed photosynthesis (9). I-module 18 concentrator (Isithombe 8) ikhonjwe njenge-gene ye-TanjilG_26536 ebhala iphrotheni ye-PR-10-like LlR18B, kanti i-module 9 concentrator ikhonjwe njenge-gene ye-TanjilG_28955 ebhala iphrotheni ye-photosystem II PsbQ. I-gene yokumelana ne-anthracnose efanelekile i-Lanr1, i-TanjilG_05042, itholakale ku-module 22 (Isithombe 9) futhi ihlotshaniswa namagama athi “GO:0044260 Izinqubo ze-metabolic ze-macromolecular zeselula” kanye ne-“GO:0006355 Ukulawulwa kokubhalwa phansi, i-DNA templating” ephethe i-TanjilG_01212 hub. I-gene ibhala i-heat stress transcription factor A-4a (HSFA4a).
Ukuhlaziywa kwenethiwekhi enesisindo sokubonakaliswa kwezakhi zofuzo kwamamojula anamagama enqubo yezinto eziphilayo amelwe ngokweqile athi “GO: 0006952 Izimpendulo Zokuzivikela”. Ukuhlanganiswa kwenziwe lula ukuze kuqokonyiswe izakhi zofuzo ezine ezihlaziywe yi-qPCR (TanjilG_04706, TanjilG_23384, TanjilG_10657 kanye neTanjilG_27015).
Ukuhlaziywa kwenethiwekhi enesisindo sokubonakaliswa kwezakhi zofuzo kwemojuli enegama lenqubo yezinto eziphilayo elimelelwe ngokweqile elithi “GO: 0006355: Ukulawulwa kokubhalwa phansi, ukulinganisa i-DNA” kanye nokuthwala isakhi sokumelana ne-anthracnose esifanelekile i-Lanr1 TanjilG_05042. Ukuhlanganiswa kwenziwe lula ukuze kuhlukaniswe isakhi sofuzo i-TanjilG_05042 kanye nesakhi sofuzo esiphakathi i-TanjilG_01212.
Ukuhlolwa kokumelana ne-anthracnose okuqoqwe e-Australia kubonise ukuthi iningi lezinhlobo zokuqala ezikhishwe zazisengozini; i-Kalya, i-Coromup kanye ne-Mandelup zichazwe njengezimelana nokumelana okulinganiselwe, kuyilapho i-Wonga, i-Tanjil kanye ne-83A:476 zichazwe njengezimelana nokumelana kakhulu ne-26,27,31. zazine-allele efanayo yokumelana, i-Lanr1 eqokiwe, kanti i-Coromup kanye ne-Mandelup zazine-allele ehlukile, i-AnMan10, 26, 39, kuyilapho i-Kalya idlulele ku-allele ehlukile. , Lanr2. Ukuhlolwa kokumelana ne-anthracnose eJalimane kwaholela ekutholakaleni komugqa omelana no-Bo7212 one-allele engaba khona ngaphandle kwe-Lanr1, i-LanrBo36.
Ucwaningo lwethu luveze imvamisa ephansi kakhulu (cishe u-6%) we-allele ye-Lanr1 ku-germplasm evivinyiwe. Lokhu kuqaphela kuhambisana nemiphumela yokuhlola i-germplasm yase-East Europe kusetshenziswa izimpawu ze-Anseq3 kanye ne-Anseq4, okubonise ukuthi i-allele ye-Lanr1 ikhona emigqeni emibili yaseBelarus kuphela. Lokhu kusikisela ukuthi i-allele ye-Lanr1 ayikasetshenziswa kabanzi yizinhlelo zokuzalanisa zasendaweni, ngokungafani nase-Australia, lapho ingenye yama-allele ayisihluthulelo okuzalanisa okusizayo. Lokhu kungase kube ngenxa yezinga eliphansi lokumelana elinikezwa yi-allele ye-Lanr1 ezimweni zasensimini yaseYurophu uma kuqhathaniswa nombiko wase-Australia. Ngaphezu kwalokho, izifundo ze-anthracnose ezindaweni ezinemvula eningi e-Australia zibonise ukuthi izimpendulo zokumelana ezibangelwa yi-allele ye-Lanr1 kungenzeka zingasebenzi ezimweni zezulu ezikhuthaza ukukhula kanye nokuthuthukiswa okusheshayo kwe-pathogen19,42. Eqinisweni, kulesi sifundo samanje, ezinye izimpawu ze-anthracnose nazo zabonwa kuma-genotypes aphethe i-allele ye-Lanr1, okuphakamisa ukuthi ukumelana kungase kuphele ngaphansi kwezimo ezifanele zokuthuthukiswa kwe-C. lupini. Ngaphezu kwalokho, ukuhumusha okungamanga okuhle kokuba khona kwezimpawu ze-Anseq3 kanye ne-Anseq4, ezingaba yi-1 cM ukusuka endaweni ye-Lanr1, kungenzeka kube yi-28,30,43.
Ucwaningo lwethu lukhombisile ukuthi i-83A:476, ephethe i-allele ye-Lanr1, yasabela ekufakweni kwe-C. lupini ngokuhlelwa kabusha kwe-transcriptome enkulu ngesikhathi sokuqala sokuhlaziywa (6 hpi), kuyilapho eMandelup, ephethe i-allele ye-AnMan, izimpendulo ze-transcriptomic zabonwa kamuva kakhulu. (kusukela ku-24 kuya ku-48 hp). Lokhu kuhlukahluka kwesikhashana kwezimpendulo zokuzivikela kuhlotshaniswa nokwehluka kwezimpawu zesifo, okugqamisa ukubaluleka kokuqashelwa kwe-pathogen kusenesikhathi ukuze kuphendulwe ngempumelelo ekumelaneni. Ukuze kutheleleke izicubu zezitshalo, ama-spores e-anthrax kumele adlule ezigabeni eziningana zokukhula ebusweni bombusi, okuhlanganisa ukuhluma, ukuhlukaniswa kwamaseli, kanye nokwakheka kwe-appressorium. I-appendage iyisakhiwo esithelelanayo esinamathela ebusweni bombusi futhi sisiza ukungena ezicutshini zombusi. Ngakho-ke, ama-spores e-C. gloeosporioides ku-pea extract abonise ukuhlukaniswa kokuqala kwe-nucleus ngemuva kwemizuzu engu-75-90 yokufukamela, ukwakheka kwe-germ tube ngemuva kwemizuzu engu-90-120, kanye nokucindezelwa ngemuva kwamahora angu-4 45. I-Mango C. gloeosporioides ibonise ukuhluma okungaphezu kuka-40% kwe-conidial ngemva kwamahora ama-3 okufukamela kanye nokwakheka kwama-appressor angaba ngu-20% ngemva kwamahora ama-4. I-gene ye-CAP20 ehlobene ne-virulence ye-C. gloeosporioides ibonise umsebenzi wokubhala ku-conidia eyakha i-epiphyte ngemva kwamahora ama-3.5 okufukamela ku-wax engaphezulu kwe-avocado enokugxila okuphezulu kweprotheyini ye-CAP20 ngemva kwamahora ama-4 nemizuzu engama-46. Ngokufanayo, umsebenzi wezakhi zofuzo ze-melanin biosynthesis ku-C. trifolii wabangelwa ngesikhathi sokufukamela kwamahora ama-2 kulandelwa ukwakheka kwe-appressorium ngemva kwehora eli-1. Izifundo zezicubu zamaqabunga zibonise ukuthi ama-strawberry afakwe i-C. acutatum anokucindezelwa kokuqala ku-8 hpi, kanti utamatisi afakwe i-C. coccodes anokucindezelwa kokuqala ku-4 hpi48,49. Ngokuhambisana kakhulu nesilinganiso sesikhathi senqubo yokutheleleka ye-Colletotrichum spp. Izimpendulo zokuzivikela ezisheshayo ku-83A:476 ziphakamisa ukubandakanyeka kwezakhi zofuzo zokumelana nezitshalo kanye ne-effector-triggered immunity (ETI) kulo mugqa, kuyilapho izimpendulo ezibambezelekile zikaMandelup zisekela i-micro-associated molecular pattern-triggered immunity (MTI) hypothesis 50. Izimpendulo zakuqala ku-83A: 476 kanye neMandelup. Ukunqwabelana okuyingxenye phakathi kwezakhi zofuzo ezilawulwa phezulu noma phansi ekuphenduleni okubambezelekile nakho kusekela lo mqondo, njengoba i-ETI ivame ukubhekwa njengempendulo ye-MTI esheshisiwe futhi ethuthukisiwe ephetha ekufeni kweseli okuhleliwe endaweni yokutheleleka, okwaziwa ngokuthi i-anaphylactic shock 51,52.
Iningi lezakhi zofuzo ezibangelwa igama elithi Gene Ontology GO:0006952 elithi “Defense Response” yizakhi zofuzo ezingu-11 zeprotheni yokuzila ukudla ebangelwa ukucindezeleka engu-22 (efana ne-SAM22) kanye namaprotheni ayisikhombisa amakhulu e-latex protein-like (MLPs). Amaprotheni afana ne-31, 34, 43 kanye ne-423 abonise ukufana kokulandelana. Izakhi zofuzo ezifana ne-SAM22 zibonise ukusebenza okuphawulekayo okuhlale isikhathi eside, okubonisa amazinga akhuphukile okumelana ne-anthracnose (83A:476 kanye ne-Boregine). Kodwa-ke, izakhi zofuzo ezifana ne-MLP zehlisiwe kuphela emigqeni ephethe i-allele yokumelana (83A:476/Lanr1 ku-6 hpi kanye ne-Mandelup/AnMan ku-24 hpi). Kufanele kuqashelwe ukuthi zonke izakhi zofuzo ezifana ne-SAM22 ezikhonjwe zivela eqenjini lezakhi zofuzo elihlanganisa cishe i-105 kb, kuyilapho izakhi zofuzo ezifana ne-MLP zivela ezindaweni ezihlukene ze-genome. Ukusebenza okuhlelekile kwezakhi zofuzo ezifana ne-SAM22 kutholakale futhi ocwaningweni lwethu lwangaphambilini lokumelana ne-NLL ekugonyelweni kwe-Diaporthetoxica, okuphakamisa ukuthi zihilelekile ezingxenyeni ezivundlile zempendulo yokuzivikela. Lesi siphetho sisekelwa futhi yimibiko yempendulo enhle yezakhi zofuzo ezifana ne-SAM22 ekulimaleni noma ekwelashweni nge-salicylic acid, i-fungus inducers, noma i-hydrogen peroxide.
Izakhi zofuzo ezifana ne-MLP zikhonjiswe ukuthi zisabela ekucindezelekeni okuhlukahlukene okungenabungozi kanye ne-biotic, okuhlanganisa ukutheleleka kwesikhunta kwamagciwane, amagciwane kanye ne-pathogenic ezinhlotsheni eziningi zezitshalo55. Iziqondiso zokuphendula ekusebenzisaneni okuthile phakathi kwezitshalo namagciwane zazisukela ekukhuleni okukhulu (okungukuthi, ngesikhathi sokutheleleka kwekotini nge-Verticillium dahliae) kuya ekuncipheni okukhulu (okungukuthi, ngemva kokutheleleka kwesihlahla sama-apula nge-Alternaria spp.)56,57. Ukwehla okukhulu kwezakhi zofuzo ezifana ne-MLP-like 423 kuye kwabonwa ngesikhathi sokuzivikela kwe-avocado ekuthelelekeni kwe-F. niger kanye nangesikhathi sokutheleleka kwesihlahla sama-apula i-Botryosphaeria berengeriana f. cn. piricola kanye ne-Alternaria alternata kuyizinhlobo ze-apula58,59. Ngaphezu kwalokho, i-apple calli eveza ngokweqile i-gene efana ne-MLP-like 423 yayinezimpawu eziphansi zezakhi zofuzo ezihlobene nokumelana futhi yayisengozini enkulu yokutheleleka nge-fungus59. Ngemva kwe-Fusarium oxysporum f, i-gene efana ne-MLP-like 423 nayo yacindezelwa ku-common bean germplasm engamelana nayo. cn. Ukutheleleka kwebhontshisi 60.
Amanye amalungu omndeni we-PR-10 ahlonzwe ocwaningweni lwethu lwe-RNA-seq kwakuyizakhi zofuzo ze-LlR18A kanye ne-LlR18B ekuphenduleni ukwanda kokuguquguquka, kanye nezakhi zofuzo eziphakanyisiwe (i-gene eli-1) noma eziphansi (izakhi zofuzo ezi-3) zeprotheyini yokudlulisa amafutha i-DIR1. . Ngaphezu kwalokho, i-WGCNA iqokomisa i-gene ye-LlR18B njengesizinda kule module, esengozini enkulu yokugonywa futhi ephethe izakhi zofuzo eziningana zokuzivikela. Izakhi zofuzo ze-LlR18A kanye ne-LlR18B zabangelwa amaqabunga e-lupine aphuzi ekuphenduleni amagciwane abangela izifo, kanye naseziqwini ze-NLL ngemuva kokugonywa kwe-D. toxica, kuyilapho i-homologue yelayisi yalezi zakhi zofuzo, i-RSOsPR10, yabangelwa ngokushesha ukutheleleka kwesikhunta okungenzeka ukuthi kuhilelekile endleleni yokubonisana ye-jasmonic acid53,61, 62. Izakhi zofuzo ze-DIR1 ziqukethe amaprotheni okuthutha amafutha angenawo akhethekile adingekayo ekuqaleni kokumelana okutholiwe kwesistimu (SAR). Ngokuthuthuka kokusabela kokuzivikela, iphrotheni ye-DIR1 ithuthwa isuka endaweni yokutheleleka nge-phloem ukuze ibangele i-SAR ezithweni ezikude. Ngokuthakazelisayo, i-gene ye-TanjilG_02313 DIR1 yabangelwa kakhulu endaweni yokuqala emigqeni engu-84A:476 kanye ne-Population 22660, kodwa ukumelana ne-anthracnose kwathuthuka ngempumelelo kuphela emgqeni ongu-84A:476. Lokhu kungase kubonise ukusebenza okuncane kwe-gene ye-DIR1 ku-NLL, njengoba amanye ama-homologue amathathu asele asabela ekugonyelweni kuphela emgqeni ongu-83A:476 ku-6 hpi, futhi le mpendulo yaqondiswa phansi.
Esifundweni sethu, izingxenye ezivame kakhulu ezihambisana nenqubo yezinto eziphilayo ebizwa ngokuthi “GO:0055114 Redox process” kwakuyi-cytochrome P450 protein, i-peroxidase, i-linoleic acid 9S-/13S-lipoxygenase, kanye ne-1-aminocyclopropane-1-carboxylic acid oxidase. Ngaphezu kwalokho, i-WGCNA yethu ichaza i-HSFA4a homologue njengamamojula athwala ihabhu njenge-Lanr1 resistance gene candidate TanjilG_05042. I-HSFA4a iyisici sokulawulwa kwe-redox kokuqoshwa kwe-nuclear ezitshalweni.
Amaprotheni e-Cytochrome P450 angama-oxidoreductase avuselela ukusabela kwe-NADPH kanye/noma i-O2-dependent hydroxylation ku-metabolism eyinhloko neyesibili, okuhlanganisa i-metabolism yama-xenobiotics, kanye nama-hormone, ama-fatty acids, ama-sterols, izingxenye zodonga lweseli, ama-biopolymers, kanye ne-biosynthesis yama-protective compounds 69. Esifundweni sethu, ukuguquguquka komsebenzi we-plant cytochrome P450 kwehlisiwe kusuka ku--10.6 log2 (ushintsho oluphindaphindayo) kuya ku-5.7 ngenxa yenani elikhulu lama-homologues ashintshiwe (37) kanye nomehluko kumaphethini okuphendula phakathi kwezakhi zofuzo ezithile, okubonisa ukubuyekezwa okuphezulu. . Ukusebenzisa idatha ye-RNA-seq kuphela ukuchaza umsebenzi webhayoloji obekayo wezakhi zofuzo ze-NLL kumndeni omkhulu kangaka weprotheni kungaba ukuqagela okukhulu. Kodwa-ke, kubalulekile ukuqaphela ukuthi ezinye izakhi zofuzo ze-cytochrome P450 zihlotshaniswa nokumelana okukhulu nesikhunta noma amabhaktheriya e-pathogenic, okuhlanganisa negalelo ekuphenduleni kokungezwani komzimba69,70,71.
Ama-peroxidase ekilasi lesi-3 angama-enzyme ezitshalo asebenza ngezindlela eziningi ahilelekile ezinqubweni eziningi ze-metabolic ngesikhathi sokukhula kwezitshalo nokukhula kwazo, kanye nokuphendula ekucindezelekeni kwemvelo njengosawoti, isomiso, ukukhanya okukhulu, kanye nokuhlaselwa yi-pathogen72. Ama-peroxidase ahilelekile ekusebenzisaneni kwezinhlobo eziningana zezitshalo ne-Anthracis, okuhlanganisa i-Stylosanthes humilis kanye ne-C. gloeosporioides, i-Lens culinaris kanye ne-C. truncatum, i-Phaseolus vulgaris kanye ne-C. lindemuthianum, i-Cucumis sativus kanye ne-C. lagenarium73,74,75,76. Impendulo ishesha kakhulu, ngezinye izikhathi ngisho naku-4 HPI, ngaphambi kokuba isikhunta singene ezicutshini zezitshalo73. I-gene ye-peroxidase nayo yasabela ekufakweni kwe-D. toxica NLL. Ngaphezu kwemisebenzi yayo evamile yokulawula ukuqhuma kwe-oxidative noma ukuqeda ukucindezeleka kwe-oxidative, ama-peroxidase angaphazamisa ukukhula kwe-pathogen ngokudala izithiyo zomzimba ezisekelwe ekuqinisweni kodonga lweseli ngesikhathi sokufakwa kwe-lignification, i-subunit noma ukuxhumanisa kwamakhemikhali athile. Lo msebenzi ungahlotshaniswa ku-silico ne-gene ye-TanjilG_03329 ebhala i-anion peroxidase eyakha i-lignin eyakhushulwa kakhulu ocwaningweni lwethu kumugqa omelana ne-83A:476 ku-6 HPI, kodwa hhayi kwezinye izinhlobo kanye namaphoyinti esikhathi angaphendulanga.
I-9S-/13S-lipoxygenase ye-linoleic acid iyisinyathelo sokuqala endleleni ye-oxidative ye-lipid biosynthesis78. Imikhiqizo yale ndlela inemisebenzi eminingi ekuvikeleni izitshalo, okuhlanganisa ukuqinisa udonga lwamaseli ngokwakhiwa kwama-callose nama-pectin deposits, kanye nokulawula ukucindezeleka kwe-oxidative ngokukhiqizwa kwezinhlobo ze-oxygen ezisebenzayo79,80,81,82,83. Kulesi sifundo samanje, ukuvezwa kwe-linoleic acid I-9S-/13S-lipoxygenase yashintshwa kuzo zonke izinhlobo, kodwa kubantu abasengozini 22660, ukwanda kwanda ngezikhathi ezahlukene, kuyilapho ezinhlotsheni ezithwala i-Lanr1 engamelana ne-allele ye-AnMan, kugcizelela ukuhlukahluka kwengqimba ye-oxylipin ekuphenduleni kwe-anthrax evikelayo phakathi kwalezi zinhlobo ze-genotype.
I-homologue ye-1-aminocyclopropane-1-carboxylate oxidase (ACO) yakhushulwa kakhulu (ama-gene ayi-9) noma yehliswa (ama-gene ama-2) lapho ifakwa i-lupine. Ngaphandle kwezimo ezimbili, zonke lezi zimpendulo zenzeke ku-6 hp. ku-83A:476. Ukusabela kwe-enzyme okuqondiswa amaprotheni e-ACO kuyisinyathelo esinciphisa izinga ekukhiqizweni kwe-ethylene futhi ngenxa yalokho kulawulwa kakhulu84. I-Ethylene iyi-hormone yesitshalo edlala indima ehlukahlukene ekulawuleni ukuthuthukiswa kwezitshalo kanye nokusabela ezimweni zokucindezeleka kwe-abiotic kanye ne-biotic. Ukuqaliswa kokubhalwa kwe-ACO kanye nokusebenza kwendlela yokubonisa i-ethylene kuhilelekile ekwandiseni ukumelana kwelayisi ne-hemibiotrophic fungus oryzae oryzae ngokulawula ukukhiqizwa kwezinhlobo ze-oxygen ezisabelayo kanye nama-phytoalexins. Inqubo yokutheleleka kwamaqabunga efanayo kakhulu etholakale phakathi kwe-M. oryzae ne-C. lupini88,89, ngokumelene nokwanda okukhulu kwezinhlobo ezifanayo ze-ACO emgqeni we-83A:476 obikwe kulolu cwaningo, ishintsha amathuba okunikeza ukumelana ne-NLL anthracnose I-Ethylene isinyathelo esiyinhloko esikhombayo ezindleleni zama-molecule.
Esifundweni samanje, ukucindezelwa okukhulu kwezakhi zofuzo eziningi ezihlobene ne-photosynthesis kwabonwa ku-6 hpi ku-83A:476 kanye naku-48 hpi eMandeloop kanye nabantu abangu-22660. Ububanzi kanye nokuqhubeka kwalezi zinguquko kuhambisana nezinga. Ukumelana ne-anthracnose kwabonwa kulolu cwaningo. Muva nje, ukucindezelwa okunamandla nokusesekuqaleni kwemibhalo ehlobene ne-photosynthesis kubikwe kumamodeli amaningana okusebenzisana kwezitshalo nezifo, kufaka phakathi amabhaktheriya abangela izifo kanye nesikhunta. Ukushesha (kusukela ku-2 HPI kwezinye izindlela zokuxhumana) kanye nokucindezelwa komhlaba wonke kwezakhi zofuzo ezihlobene ne-photosynthesis ekuphenduleni ukutheleleka kungabangela ukuzivikela kwezitshalo ngokusekelwe ekufakweni kwezinhlobo ze-oxygen ezisebenzayo kanye nokusebenzisana kwazo nendlela ye-salicylic acid ukuze kuxazululwe ukusabela kokungezwani komzimba 90,94.
Ekuphetheni, izindlela zokuphendula ukuzivikela eziphakanyisiwe zohlu olumelana kakhulu (83A:476) zifaka phakathi ukuqashelwa okusheshayo kwe-pathogen yi-R gene (mhlawumbe i-TIR-NBS-LRR TanjilG_05042) kanye ne-salicylic acid kanye ne-ethylene signaling ebangelwa ukuphendula kwe-allergy, okulandelwa ukusungulwa kwesenzo se-SAR eside. kusekelwa yi-DIR-1 protein. Kufanele kuqashelwe ukuthi isikhathi se-biotrophic sokutheleleka kwe-C. lupini sifushane kakhulu (cishe izinsuku ezi-2), kulandelwe ukukhula kwe-necrotic95. Ukuguquka phakathi kwalezi zigaba kungahlotshaniswa ne-necrosis kanye nokubonakaliswa kwamaprotheni angena e-ethylene asebenza njengezimbangela zokusabela kwe-hypersensitivity ezitshalweni ezisingatha. Ngakho-ke, isikhathi sokubamba ngempumelelo i-C. lupini esigabeni se-biotrophic sincane kakhulu. Ukuhlelwa kabusha kwezakhi zofuzo ezihlotshaniswa ne-redox kanye ne-photosynthesis okubonwe ku-83A:476 ku-6 hpi kuhambisana nokuqhubeka kwe-fungal hyphae futhi kumemezela ukuthuthukiswa kwempendulo ephumelelayo yokuvikela esigabeni se-biotrophic. Izimpendulo ze-transcriptomic zikaMandelup kanye nenani labantu abangu-22660 zingase zibambezeleke kakhulu ukubamba isikhunta ngaphambi kokushintshela ekukhuleni kwe-necrotic, noma kunjalo, iMandelup ingasebenza kangcono kunenani labantu abangu-22660 ngoba ukulawulwa okusheshayo kweprotheyini ye-PR-10 kukhuthaza ukumelana okuvundlile.
I-ETI, eqhutshwa yi-gene ye-canonical R, ibonakala iyindlela evamile yokumelana nobhontshisi ne-anthracnose. Ngakho-ke, kumodeli we-legume Medicago truncatula, ukumelana ne-anthracnose kunikezwa yi-gene ye-RCT1, ilungu lesigaba se-gene yesitshalo i-TIR-NBS-LRR97. Lesi gene siphinde sinikeze ukumelana ne-anthracnose ebanzi ku-alfalfa uma idluliselwa ezitshalweni ezisengozini. Kubhontshisi ovamile (i-P. vulgaris), sekutholakale izakhi zofuzo ezingaphezu kwamashumi amabili nambili zokumelana ne-anthracnose kuze kube manje. Ezinye zalezi zakhi zofuzo zitholakala ezindaweni ezingenazo izakhi zofuzo ze-canonical R, nokho ezinye eziningi zitholakala emaphethelweni ama-chromosome aphethe iqoqo lezakhi zofuzo ze-NBS-LRR, kufaka phakathi i-TIR-NBS-LRRs99. Ucwaningo lwe-SSR oluphelele lwe-genome luphinde lwaqinisekisa ukuhlangana kwezakhi zofuzo ze-NBS-LRR nokumelana ne-anthracnose kubhontshisi ovamile. Izakhi zofuzo ze-canonical R nazo zitholakale esifundeni se-genomic esithwele indawo enkulu yokumelana ne-anthracnose ku-lupine emhlophe 101.
Umsebenzi wethu ukhombisa ukuthi ukusabela okusheshayo kokumelana, okusebenza esigabeni sokuqala sokutheleleka kwezitshalo (kungcono kungakapheli ama-hpi angu-12), kuvikela ngempumelelo i-lupine enamahlamvu amancane ku-anthracnose ebangelwa yi-fungus ebangela izifo i-Collelotrichum lupini. Sisebenzisa ukulandelana okuphezulu kokuphuma, sibonise amaphrofayili okubonakaliswa okuhlukile kwezakhi zofuzo zokumelana ne-anthracnose ezitshalweni ze-NLL eziqondiswa yizakhi zofuzo zokumelana ne-Lanr1 ne-AnMan. Ukuzivikela okuphumelelayo kuhilela ukuklama ngokucophelela izakhi zofuzo zamaprotheni ahilelekile ku-redox, i-photosynthesis, kanye ne-pathogenesis emahoreni ambalwa kusukela ekuthinteni kokuqala kwesitshalo ne-pathogen. Ukusabela okufanayo kokuvikela, kodwa okubambezelekile ngesikhathi, akuphumelelanga kangako ekuvikeleni izitshalo ezifweni. Ukumelana ne-anthrax okuqondiswa yizakhi zofuzo i-Lanr1 kufana nokusabela okusheshayo okuvamile kwezakhi zofuzo i-R (ukuvikeleka okubangelwa yi-effector), kuyilapho izakhi zofuzo i-AnMan cishe zinikeza impendulo evundlile (ukuvikeleka okubangelwa iphethini yamangqamuzana ehlobene ne-microbe), okunikeza izinga eliphakathi lokuzinza.
Imigqa engu-215 ye-NLL esetshenziselwa ukuhlola izimpawu ze-anthracnose yayihlanganisa izinhlobo ezingu-74 zezitshalo, imigqa engu-60 etholwe ngokuwela noma ukuzalanisa, ama-mutant angu-5, kanye nama-germplasm angu-76 asendle noma okuqala. Imigqa yayivela emazweni angu-17, ikakhulukazi evela ePoland (58), eSpain (47), eJalimane (27), e-Australia (26), eRussia (19), eBelarus (7), e-Italy (5) nakwamanye amazwe. Isethi ihlanganisa nemigqa engabhekisi: 83A:476, eTanjil, eWonga ephethe i-allele yeLanr1, kanye naseMandelup ephethe i-allele ye-AnMan. Imigqa itholwe ku-European Lupine Genetic Resource Database egcinwe yiPoznań Plant Breeding Ltd., eWiatrowo, ePoland (Supplementary Table S1).
Izitshalo zakhuliswa ngaphansi kwezimo ezilawulwayo (isikhathi sokukhanya amahora angu-16, izinga lokushisa lingu-25°C emini kanye no-18°C ​​ebusuku). Kwahlaziywa amakhophi amabili ezinto eziphilayo. I-DNA yahlukaniswa emaqabungeni amasonto amathathu kusetshenziswa i-DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) ngokwephrothokholi. Ikhwalithi kanye nokuhlushwa kwe-DNA ehlukanisiwe kwahlolwa ngezindlela ze-spectrophotometric (NanoDrop 2000; Thermo Fisher Scientific, Waltham, MA, USA). Umaka we-AnManM1 omaka i-gene yokumelana ne-anthracnose i-AnMan (ethathwe ku-cv. Mandelup) kanye namamaka i-Anseq3 kanye ne-Anseq4 eceleni kwe-gene i-Lanr1 (ethathwe ku-cv. Tanjil) ahlaziywe angu-11,26,28. Ama-Homozygotes e-allele yokumelana abekwe njengo-“1″, asengozini – njengo-“0″, kanye nama-heterozygotes – njengo-0.5.
Ngokusekelwe emiphumeleni yokuhlolwa kwezimpawu ze-AnManM1, i-AnSeq3 kanye ne-AnSeq4 kanye nokutholakala kwembewu yokuhlolwa kokugcina okulandelayo, kukhethwe imigqa engu-50 ye-NLL yokumelana ne-anthracnose phenotyping. Ukuhlaziywa kwenziwe kabili endaweni yokushisa elawulwa yikhompyutha enesikhathi sokukhanya samahora angu-14 enobubanzi bokushisa obungu-22°C emini kanye no-19°C ebusuku. Imbewu iyaklwejwa (inqunywe ungqimba lwembewu ohlangothini oluphambene nombungu ngensingo ebukhali) ngaphambi kokuhlwanyela ukuvimbela ukungalali kwembewu ngenxa yokuthi ungqimba lwembewu luqinile kakhulu nokuqinisekisa ukuhluma okufanayo. Izitshalo zakhuliswa ezitsheni (11 × 11 × 21 cm) ezinenhlabathi engcolile (TS-1 REC 085 Medium Basic, Klasmann-Deilmann Polska, Warsaw, Poland). Ukugonywa kwenziwa ngohlobo lwe-Colletotrichum lupini Col-08, olwakhuliswa ngo-1999 kusuka eziqwini zezitshalo ze-lupine ezinamahlamvu amancane ezikhuliswe ensimini eVerzhenitsa, eGreater Poland (52° 27′ 42″ N 17° 04′ 05″ E). Thola indawo. Ama-isolates atshalwa endaweni yokushisa ye-SNA ku-20° C. ngaphansi kokukhanya okumnyama izinsuku ezingu-21 ukuze kubangele ukubola. Emasontweni amane ngemva kokuhlwanyela, lapho izitshalo sezifinyelele esigabeni samaqabunga angu-4-6, ukugonywa kwenziwa ngokufafaza ngokumiswa kwe-conidia ekugxilweni okungu-0.5 x 106 conidia nge-ml. Ngemva kokugonywa, izitshalo zagcinwa ebumnyameni amahora angu-24 kumswakama ongaba ngu-98% kanye nokushisa okungu-25°C ukuze kube lula ukuhluma kwe-conidia kanye nenqubo yokutheleleka. Izitshalo zabe sezikhuliswa ngaphansi kwesikhathi sokukhanya samahora ayi-14 ku-22°C ngosuku/19°C ebusuku kanye nomswakama ongu-70%. Isilinganiso sesifo senziwe ezinsukwini ezingama-22 ngemuva kokugonywa futhi sasukela ku-0 (esivikela amasosha omzimba) saya ku-9 (esisengozini enkulu) kuye ngokuthi kukhona noma akukho yini izilonda ezifile eziqwini nasemaqabungeni. Ngaphezu kwalokho, ngemva kokuthola amaphuzu, isisindo sezitshalo salinganiswa. Ubudlelwano phakathi kwezinhlobo ze-genotype ezimaka kanye nezinhlobo zezifo babalwe njengezixhumanisi ezimbili (ukungabikho kwezimpawu ze-heterozygous kusethi yemigqa yokuhlaziya i-phenotype yokumelana ne-anthracnose).