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I-Angustifolius lupine (NLL, Lupinus angustifolius L.) sisityalo esinemidumba esisetyenziselwa ukuvelisa ukutya kunye nokuphucula umhlaba. Ukwanda kwe-NLL kwihlabathi liphela njengesityalo kuye kwatsala iifungi ezininzi ezibangela izifo, kuquka i-lupine anthracnose, ebangela isifo esibi se-anthracnose. Ii-alleles ezimbini, i-Lanr1 kunye ne-AnMan, ezinika ukumelana okuphezulu, zisetyenzisiwe ekuzaleni i-NLL, kodwa iindlela ezisisiseko zeemolekyuli azikaziwa. Kolu phononongo, iimpawu ze-Lanr1 kunye ne-AnMan zisetyenzisiwe ukuhlola iisampulu ze-NLL zaseYurophu. Uvavanyo lwe-vaccine kwindawo elawulwayo luqinisekisile ukusebenza kakuhle kwabanikeli bobabini abachaseneyo. Uhlalutyo lokubonakaliswa kwezakhi zofuzo ezahlukeneyo lwenziwe kwimigca emeleyo echaseneyo nesesichengeni. Ukumelana ne-Anthracnose kunxulunyaniswe nokubonakaliswa kakhulu kwamagama e-gene ontology athi "GO:0006952 Defense Response", "GO:0055114 Redox Process", kunye ne "GO:0015979 Photosynthesis". Ukongeza, umgca weLanr1(83A:476) ubonise ukuphinda-phinda okuphawulekayo kwe-transcriptome ngokukhawuleza emva kokugonywa, ngelixa eminye imigca ibonise ukulibaziseka kule mpendulo malunga neeyure ezingama-42. Iimpendulo zokhuselo zinxulunyaniswa ne-TIR-NBS, i-CC-NBS-LRR kunye ne-NBS-LRR genes, iiproteni ezili-10 ezibandakanyeka kwi-pathogenesis, iiproteni zokudlulisa i-lipid, i-endoglucan-1,3-β-glucosidase, iiproteni zodonga lweseli ezityebileyo nge-glycine, kunye nee-genes ezivela kwindlela ye-oxygen esebenzayo. Iimpendulo zokuqala kwi-83A:476, kubandakanya ukucinezelwa ngononophelo kwe-genes ezinxulumene ne-photosynthesis, zihambelana nokukhuselwa ngempumelelo ngexesha lesigaba sokukhula kwezityalo kwibhayoloji yefungal, nto leyo ebonisa ukuba i-effector ibangela ukuzikhusela. Impendulo yeMandeloop iyacothiswa, njengoko kunjalo nokurhuqa okuthe tye ngokubanzi.
I-lupine enamagqabi amafutshane (i-NLL, i-Lupinus angustifolius L.) yinkozo eneproteni eninzi evela kummandla osentshona weMeditera1,2. Okwangoku ikhuliswa njengesityalo sokutya sezilwanyana nabantu. Ikwathathwa njengomgquba oluhlaza kwiinkqubo zokujikeleza izityalo ngenxa yokufakwa kwe-nitrogen yibhaktiriya elungisa i-nitrogen kunye nokuphuculwa ngokubanzi kwesakhiwo somhlaba. I-NLL iye yadlula kwinkqubo ekhawulezayo yokugcinwa ekhaya kwinkulungwane edlulileyo kwaye isephantsi koxinzelelo oluphezulu lokuzalisa3,4,5,6,7,8,9,10,11,12. Ngokulinywa ngokubanzi kwe-NLL, ukulandelana kweefungi ezibangela izifo kwavelisa iindawo ezintsha zezolimo kwaye kwabangela izifo ezintsha ezitshabalalisa izityalo. Eyona nto iphawulekayo kubalimi be-lupin kunye nabafuyi yayikukuvela kwe-anthracnose, ebangelwa yi-fungus ebangela izifo, iColletotrichum lupini (Bondar) Nirenberg, Feiler kunye neHagedorn13. Eyona nto iphawulekayo kubalimi be-lupin kunye nabafuyi yayikukuvela kwe-anthracnose, ebangelwa yi-fungus ebangela izifo, iColletotrichum lupini (Bondar) Nirenberg, Feiler kunye neHagedorn13. Наиболее примечательным для фермеров и селекционеров люпина было появление антракноза, вызванного патогенным грибком Nitrotrichum Hagedorn13. Eyona nto iphawulekayo kubalimi be-lupine kunye nabafuyi yayikukuvela kwe-anthracnose ebangelwa yi-fungus ebangela izifo i-Colletotrichum lupini (Bondar) Nirenberg, Feiler kunye ne-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 &3 Niler &3 Fedor (Bondar Lupini)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 патогеным гриtricбком Colle Hagedorn13. Eyona nto ibangela umdla kubalimi be-lupine kunye nabafuyi kukuvela kwe-anthracnose ebangelwa yi-fungus ebangela izifo i-Colletotrichum lupini (Bondar) Nirenberg, Feiler kunye ne-Hagedorn13.Iingxelo zokuqala zesi sifo zivela eBrazil nase-United States, kwaye iimpawu eziqhelekileyo zavela ngo-1912 nango-1929 ngokulandelelana. Nangona kunjalo, emva kweminyaka engama-30, i-pathogen yabizwa ngokuba yiColletotrichum gloeosporioides (Penz.) Penz. kunye noSacc., i-teleomorph Glomerella cingulata (Stoneman) Spauld. kunye noSacc., i-teleomorph Glomerella cingulata (Stoneman) Spauld. & Sacc., телеоморф Glomerella cingulata (Stoneman) Spauld. kunye noSacc., i-teleomorph yeGlomerella cingulata (Stoneman) Spauld. & Sacc., 有目的形态的Glomerella cingulata (Stoneman) Spauld. & Sacc., 有目的形态的Glomerella cingulata (Stoneman) Spauld. & Sacc., Glomerella cingulata (Stoneman) Spauld в Целенаправленной морфологии. kunye noSacc., Glomerella cingulata (Stoneman) Spauld kwiMofology eJongiweyo. kunye no-H. Schrenk,. kunye no-H. Schrenk,.kunye no-H. Schrenk. & H.施伦克,. & H.施伦克,.kunye no-H. Schlenk,.Uvavanyo lokuqala lwesifo olwenziwe phakathi kwinkulungwane yama-20 lubonise ukumelana okuthile kwi-NLL kunye ne-yellow lupine (L. luteus L.), kodwa zonke i-white lupine (L. albus L.) accessions ezivavanyiweyo zazisengozini enkulu15,16. Izifundo zibonise ukuba uphuhliso lwe-anthracnose lunxulunyaniswa nokwanda kwemvula (umswakama womoya) kunye nobushushu (kwi-12-28°C), nto leyo ekhokelela ekwaphulweni kokumelana kumaqondo obushushu aphezulu17, 18. Enyanisweni, ixesha elifunekayo ukuze i-conidia ikhule kwaye isifo siqale, lalifutshane ngokuphindwe kane kwi-24°C (iiyure ezi-4) kune-12°C (iiyure ezili-16) phantsi kweemeko zomswakama ophezulu19. Ke ngoko, ukufudumala kwehlabathi okuqhubekayo kukhokelele ekusasazekeni kwe-anthracnose. Nangona kunjalo, esi sifo sabonwa eFransi (1982) nase-Ukraine (1983) njengesibonakaliso sesongelo esizayo, kodwa kubonakala ngathi asizange sihoywe lishishini le-lupine ngelo xesha20,21. Kwiminyaka embalwa kamva, esi sifo sibulalayo sasasazeka kwihlabathi liphela saza sachaphazela amazwe amakhulu avelisa i-lupine afana ne-Australia, iPoland neJamani22,23,24. Emva kokuqhambuka kwe-anthracnose phakathi kwiminyaka yoo-1990, ukuvavanywa okubanzi kwakhokelela ekuchongweni kwabanikeli abaninzi abachasene nentsholongwane kwiisampulu ze-NLL19. Ukumelana ne-NLL kwi-anthracnose kulawulwa zii-alleles ezimbini ezahlukeneyo ezifumaneka kwimithombo eyahlukeneyo ye-germplasm: i-Lanr1 kwi-cultivar i-Tanjil kunye ne-Wonga kunye ne-AnMan kwi-cultivar. I-Mandalay 25, 26. Ezi alleles zizalisekisa iimpawu ze-molecular ezixhasa ukukhethwa kwe-germplasm echasene nentsholongwane kwiinkqubo zokuzalisa25, 26, 27, 28, 29, 30. Umgca wokuzalela oxhathisayo 83A:476 ophethe i-allele ye-Lanr1 wanqunyulwa nomgca wasendle osengozini P27255 ukuze kufunyanwe inani le-RIL elahlulahlulayo ukumelana ne-anthracnose, nto leyo eyenza ukuba kube nokwenzeka ukwabela indawo ye-Lanr1 kwi-chromosome NLL-1131, 32, 33. Ukulungelelaniswa kweempawu zemephu yonxibelelwano ukusuka kwindawo yokumelana ejikelezileyo ukuya kwi-anthracnose ngesakhelo se-genomic, i-NLL ityhile indawo yazo zonke ii-alleles ezintathu kwi-chromosome efanayo (NLL-11), kodwa kwiindawo ezahlukeneyo 29,34,35. Nangona kunjalo, ngenxa yenani elincinci lee-RIL kunye nomgama omkhulu we-genetic phakathi kweempawu kunye nee-alleles ezihambelanayo, akukho zigqibo zithembekileyo ezinokuthathwa malunga nee-genes zazo ezingaphantsi. Kwelinye icala, ukusetyenziswa kwe-genetics ebuyela umva kwi-lupins kunzima ngenxa yamandla azo okuhlaziya aphantsi kakhulu, okwenza ukuguqulwa kwe-genetic kube nzima 37.
Uphuhliso lwe-germplasm yasekhaya ephethe i-allele efunekayo kwimeko ye-homozygous, efana ne-83A:476 (Lanr1) kunye neMandelup (AnMan), ivule ithuba lokufunda ukumelana ne-anthracnose xa kukho indibaniselwano echaseneyo yee-alleles kwiindawo zasendle. Amathuba eendlela ze-molecular. Thelekisa iimpendulo zokuzikhusela eziveliswa zii-genotypes ezithile. Olu phononongo luvavanye impendulo yokuqala ye-transcriptome ye-NLL kwi-C. lupini vaccination. Okokuqala, iphaneli ye-germplasm ye-NLL yaseYurophu equlethe imigca engama-215 yahlolwa kusetyenziswa iimpawu ze-molecular eziphawula ii-alleles ze-Lanr1 kunye ne-AnMan. I-Anthracnose phenotyping yenziwa kwimigca engama-50 ye-NLL, eyayikhethwe ngaphambili kwiimpawu ze-molecular, phantsi kweemeko ezilawulwayo. Ngokusekelwe kolu vavanyo, imigca emine eyahlukileyo kukumelana ne-anthracnose kunye nokwakheka kwe-allelic ye-Lanr1/AnMan yakhethwa ukuze kuchazwe i-gene defense expression profiling kusetyenziswa iindlela ezimbini ezihambelanayo: ukulandelelana kwe-RNA ephezulu kunye nokulinganisa kwe-PCR ngexesha langempela.
Ukuhlolwa kweseti ye-NLL germplasm (N = 215) ngee-markers Lanr1 (Anseq3 kunye ne-Anseq4) kunye ne-AnMan (Anseq4) kunye ne-AnMan (AnManM1) kubonise ukuba umgca omnye kuphela (95726, kufutshane neSalamanca-b) wandisa i-allele "yokumelana" kuzo zonke ii-markers , ngelixa "Ubukho bee-allele 'ezinokuchaphazeleka'" bufumene umlinganiselo wazo zonke ii-markers kwimigca eli-158 (~73.5%). Imigca elishumi elinesithathu ivelise ii-allele ezimbini "ezinokuxhathisa" ze-marker Lanr1, kwaye imigca esi-8 ivelise ii-allele "ezinokuxhathisa" ze-marker Lanr1. I-allele "yokuxhathisa" ye-marker AnMan (Itheyibhile eyongezelelweyo S1). Imigca emibini yayiyi-heterozygous ye-marker Anseq3 kunye ne-heterozygous ye-marker AnManM1. Imigca engama-42 (19.5%) yayinezigaba ezichaseneyo ze-alleles ze-Anseq3 kunye ne-Anseq4, nto leyo ebonisa ukuba kukho ukuphindaphindeka okuphezulu phakathi kwezi loci zimbini. Ii-phenotypes ze-Anthracnose phantsi kweemeko ezilawulwayo (Itheyibhile eyongezelelekileyo S2) zityhile umahluko ekuchaseni kwe-genotypes ezivavanyiweyo, nto leyo ebonakaliswe kubukhali be-anthracnose. Umahluko kumanqaku aphakathi wawususela kwi-1.8 (emelana nomlinganiselo) ukuya kwi-6.9 (emelana nomlinganiselo) kwaye umahluko kubunzima bezityalo wawususela kwi-0.62 (emelana nomlinganiselo) ukuya kwi-4.45 g (emelana nomlinganiselo). Bekukho ulwalamano olubalulekileyo phakathi kwamaxabiso abonwe kwiimpendulo ezimbini zovavanyo (0.51 yamanqaku obunzima besifo, P = 0.00017 kunye no-0.61 yobunzima besityalo, P < 0.0001) kunye naphakathi kwezi parameter zimbini (− 0.59 kunye no-− 0.77, P < 0.0001). Bekukho ulwalamano olubalulekileyo phakathi kwamaxabiso abonwe kwiimpendulo ezimbini zovavanyo (0.51 yamanqaku obunzima besifo, P = 0.00017 kunye no-0.61 yobunzima besityalo, P < 0.0001) kunye naphakathi kwezi parameter zimbini (− 0.59 kunye no-− 0.77, P < 0.0001). Выявлена ​​достоверная корреляция между значениями, наблюдаемыми в двух повторностях эксперимента (0,51 для баллов тяжез00, 10 для массы растения, P < 0,0001), а также между этими двумя параметрами (-0,59 и -0,77, Р < 0,0001) 0,0001). Kufunyenwe ulwalamano olubalulekileyo phakathi kwamaxabiso abonwe kuphindaphindo oluphindwe kabini lovavanyo (0.51 yamanqaku obunzima besifo, P = 0.00017 kunye no-0.61 yobunzima besityalo, P < 0.0001), kunye naphakathi kwezi parameter zimbini (- 0.59 kunye 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（疾病严重程度评分为0.51，P = 0.00017，植物重量為0.61，P <0.0001）以及這两个参数之间（- 0.59 和- 0.77，P <0.0001).在 两 次 重复 实验 中 观察 的 值 门间 存在 相关性 （疾病 严重 程度 程度 评分為 1 ithole = 10 intengiso = 10 intengiso. 0.00017 , 植物 為為 0.61 , p <0.0001） 以及 两 个 参数 之间 （（（- 0.59 和– 0.50 和– 0.59 和–9 和–9 0.77, P < 0.0001). Наблюдалась значительная корреляция между значениями, наблюдаемыми в двух повторностях (оценка тяжести заболевания 0,51, 10, P = 0,61, P <0,0001), и между этими двумя параметрами (-0,59 и -0,0001) 0,77, P <0,0001. Bekukho ulwalamano olubalulekileyo phakathi kwamaxabiso abonwe kabini (amanqaku obunzima besifo 0.51, P = 0.00017 kunye nobunzima besityalo 0.61, P < 0.0001) naphakathi kwezi parameter zimbini (-0.59 kunye -0 .0001) 0.77, P<0.0001. ).Iimpawu eziqhelekileyo ezibonwa kwizityalo ezisengozini ziquka ukukrwitshwa nokujijwa kwesiqu okufana nesakhiwo "somphetho womalusi", kulandele izilonda ezimbhoxo ezinee-sporozoites ezi-orenji/ezipinki (Umfanekiso Ongezelelweyo 1). Izakhi zofuzo zase-Australia ezithwala i-Lanr1 (83A:476 kunye neTanjil) kunye ne-AnMan (Mandelup) ziyakwazi ukumelana nobunzima, 0.0331 kunye ne-0.0036). Eminye imigca ekwaphethe ii-alleles ze-Lanr1 kunye/okanye ze-AnMan "ezichaseneyo" ibonisa iimpawu zesifo.
Okunomdla kukuba, imigca embalwa ye-NLL engenazo naziphi na ii-allele ze-marker "ezimelana" ityhile inqanaba eliphezulu lokumelana ne-anthracnose (elinokuthelekiseka okanye elingaphezulu kune-Lanr1 okanye i-AnMan genotypes), njengeBoregine (ixabiso le-P < 0.0001 kuzo zombini iiparameter), iBojar (ixabiso le-P < 0.0001 lamanqaku kunye ne-0.001 lamanqaku obunzima besityalo) kunye nePopulation B-549/79b (ixabiso le-P < 0.0001 lamanqaku kwaye alibalulekanga lamanqaku obunzima). Okunomdla kukuba, imigca embalwa ye-NLL engenazo naziphi na ii-allele ze-marker "ezimelana" ityhile inqanaba eliphezulu lokumelana ne-anthracnose (elinokuthelekiseka okanye elingaphezulu kune-Lanr1 okanye i-AnMan genotypes), njengeBoregine (ixabiso le-P < 0.0001 kuzo zombini iiparameter), iBojar (ixabiso le-P < 0.0001 lamanqaku kunye ne-0.001 lamanqaku obunzima besityalo) kunye nePopulation B-549/79b (ixabiso le-P < 0.0001 lamanqaku kwaye alibalulekanga lamanqaku obunzima). Интересно, что несколько линий NLL, лишенных какого-либо «резистентного» маркерного аллеля, показали высокий уровень или более высокий, чем для генотипов Lanr1 okanye AnMan), таких как Boregine (значение P <0,0001 для обоих параметров), Bojar (значение P0 <10,0001 для массы растения) и популяции B-549/79b (значение P <0,0001 для оценки и незначимо для массы). Okubangel’ umdla kukuba, imigca eliqela ye-NLL engenazo naziphi na ii-allele ze-'resistant' ibonise inqanaba eliphezulu lokuxhathisa i-anthracnose (ethelekiswa okanye engaphezulu kune-Lanr1 okanye i-AnMan genotypes), efana ne-Boregine (ixabiso le-P < 0.0001 kuzo zombini iiparameter ), i-Bojar (ixabiso le-P < 0.0001 lovavanyo kunye ne-0.001 yobunzima besityalo) kunye nenani labantu i-B-549/79b (ixabiso le-P < 0.0001 lovavanyo kwaye alibalulekanga kubunzima).有趣的是，一些缺乏任何“抗性”标记等位基因的NLL 系显示出高水平的炭疽病抗性（与LanAn1基因型相当或更高），例如Boregine（两个参数的P 值< 0.0001）、Bojar（P 值<得分為0.0001，植物重量為0.001）和种群B-549/79b（得分P 值< 0.0001，重量不显着）。 Kuyathakazelisa ukuba ezinye iinkqubo ze-NLL ezingenazo iimpawu "ezichasene ne-genic" zibonisa ukumelana okuphezulu okuthe tye (okufana ne-Lanr1 okanye i-AnMan genes okanye ngaphezulu), njenge-Boregine (zombini iiparameters P < 0.0001), i-Bojar (ixabiso le-P < 0.0001, ubunzima besityalo 0.001) kunye nohlobo lwe-B-549/79b (ixabiso le-P < 0.0001, ubunzima abubalulekanga). Интересно, что некоторые линии NLL, лишенные каких-либо маркерных аллелей «резистентности», показали высокие уровни устойчанизсти 30 B-549/79b (оценка P-значение <0,0001, масса незначительна). Okunomdla kukuba, eminye imigca ye-NLL engenazo naziphi na ii-alleles ze-'resistance' ibonise amanqanaba aphezulu okumelana ne-anthracnose (ethelekiswa okanye engaphezulu kune-Lanr1 okanye i-AnMan genotypes), njenge-Boregine (ixabiso le-P kuzo zombini iiparameter <0.0001), i-Bojar (ixabiso le-P <0.0001, ubunzima besityalo 0.001) kunye nenani labantu i-B-549/79b (ixabiso le-P <0.0001, ubunzima abubalulekanga).Le nto ibonisa ukuba kungenzeka ukuba kubekho umthombo omtsha wemfuza wokuxhathisa, ichaza ukungabikho konxibelelwano olubonakalayo phakathi kweempawu ze-genotypes kunye ne-phenotypes yesifo (amaxabiso e-P ukusuka kwi-~0.42 ukuya kwi-~0.98). Ngoko ke, uvavanyo lweKolmogorov-Smirnov lubonise ukuba idatha malunga nokuxhathisa i-anthracnose yayisasazwa phantse ngokuqhelekileyo kumanqaku (amaxabiso e-P 0.25 kunye ne-0.11) kunye nobunzima bezityalo (amaxabiso e-P 0.47 kunye ne-0.55), nto leyo ebonisa ukuba ndicinga ukuba kukho ii-alleles ezingaphezulu kuneLanr1 kunye ne-AnMan.
Ngokusekelwe kwiziphumo zovavanyo lokumelana ne-anthracnose, imigca emi-4 ikhethwe kuhlalutyo lwe-transcriptome: 83A:476, Boregine, Mandelup, kunye nePopulation 22660. Le migca iphinde yavavanywa ukuxhathisa i-anthrax kwiimvavanyo zokugonya nge-RNA sequencing, ukuba nje ifana neyovavanyo lwangaphambili. Amaxabiso amanqaku ebengolu hlobo lulandelayo: Boregin (1.71 ± 1.39), 83A: 476 (2.09 ± 1.38), Mandelup (3.82 ± 1.42) kunye nepopulation 22660 (6.11 ± 1.29).
Iprotokholi ye-Illumina NovaSeq 6000 ifikelele kumndilili wee-40.5 Mread pairs ngesampulu nganye (29.7 ukuya kwi-54.4 Mreads) (Itheyibhile eyongezelelekileyo S3). Amanqaku olungelelwaniso kulandelelwano lwesalathiso asusela kwi-75.5% ukuya kwi-88.6%. Ulwalamano oluphakathi kwedatha yokubalwa kokufundwa phakathi kwezahluko zovavanyo phakathi kweekopi zebhayoloji lususela kwi-0.812 ukuya kwi-0.997 (umyinge we-0.959). Kwizakhi zofuzo ezingama-35,170 ezihlalutyiweyo, ezingama-2917 azizange zibonakalise zimpawu, kwaye ezinye izakhi zofuzo ezingama-4785 zavezwa kwinqanaba elingaphantsi (i-base average <5). Kwizakhi zofuzo ezingama-35,170 ezihlalutyiweyo, ezingama-2917 azizange zibonakalise zimpawu, kwaye ezinye izakhi zofuzo ezingama-4785 zavezwa kwinqanaba elingaphantsi (i-base average <5). Из 35 170 проанализированных генов 2917 не проявляли экспрессии, а остальные 4785 генов экспрессировались на ненезнабрессии <5). Kwizakhi zofuzo ezingama-35,170 ezihlalutyiweyo, ezingama-2917 azizange zibonakalise ukubonakaliswa, kwaye ezinye izakhi zofuzo ezingama-4785 zichazwe kwinqanaba elingaphantsi (i-base mean <5).在分析的35,170 个基因中，2917 个沒有表达，其他4785 个基因的表达可以忽略不计（基本平均倉。35,170 Из 35 170 проанализированных генов 2917 не экспрессировались, а остальные 4785 генов имели незначительную экспрессировались, а остальные 4785 генов имели незначительную экспрессировались. Kwizakhi zofuzo ezingama-35,170 ezihlalutyiweyo, ezingama-2917 azizange zibonakaliswe kwaye ezinye izakhi zofuzo ezingama-4785 zazinembonakalo engaphantsi (i-base mean <5).Ngoko ke, inani lee-genes ezijongiweyo eziveziweyo (i-base average ≥ 5) ngexesha lovavanyo yayiyi-27,468 (78.1%) (Itheyibhile eyongezelelweyo S4).
Ukususela kwinqanaba lokuqala, yonke imigca ye-NLL yasabela ekufakweni kwe-C. lupini (uhlobo lwe-Col-08) ngokuyiphinda-phinda i-transcriptome (Itheyibhile 1), nangona kunjalo, umahluko omkhulu wabonwa phakathi kwemigca. Ngoko ke, umgca wokuxhathisa 83A:476 (ophethe i-gene ye-Lanr1) ubonise ukuphinda-phinda kwe-transcriptome kwindawo yokuqala (6 hpi) kunye nokwanda okuphindwe ka-31-69 kwinani le-genes ezizimeleyo eziphezulu nezisezantsi xa kuthelekiswa nezinye iindawo zexesha ngeli xesha. Ukongeza, le ncopho yayifutshane, njengoko ukubonakaliswa kwe-genes ezimbalwa kuphela kwahlala kutshintshile kakhulu kwinqanaba lesibini lexesha (12 hpi). Okunomdla kukuba, i-Boregine, nayo ebonise izinga eliphezulu lokuxhathisa kuvavanyo lwe-graft, ayizange idlule kukuphinda-phinda kwe-transcriptional okukhulu kangaka ngexesha lovavanyo. Nangona kunjalo, inani le-genes ezichazwe ngokwahlukileyo (DEG) lalifana kwi-Boregine kunye ne-83A:476 kwi-12 HPI. Bobabini uMandelup kunye nabemi abangama-22660 babonise iincopho ze-DEG kwixesha lokugqibela (48 l/s), nto leyo ebonisa ukulibaziseka okulinganiselweyo kwiimpendulo zokhuselo.
Ngenxa yokuba i-83A:476 ifumene uhlengahlengiso olukhulu lwe-transcriptome ukuphendula i-C. lupini kwi-6 HPI xa kuthelekiswa nayo yonke eminye imigca, malunga ne-91% yee-DEG ezibonwe ngeli xesha zazixhomekeke kumgca othile (Umzobo 1). Nangona kunjalo, bekukho ukudibana okuthile kwiimpendulo zokuqala phakathi kwemigca yophando, njengoko i-68.5%, i-50.9%, kunye ne-52.6% DEG eBoregine, eMandelup, kunye nabemi abangama-22660, ngokwahlukeneyo, zidibene nezo zifunyenwe kwi-83A:476 kwiindawo ezithile ngexesha. Nangona kunjalo, ezi DEG zibalwa kuphela yingxenyana encinci (0.97–1.70%) yazo zonke ii-DEG ezifunyenweyo ngoku kusetyenziswa i-83A:476. Ukongeza, ii-DEG ezili-11 ezivela kuzo zonke iimigca zazihambelana ngeli xesha (IiTheyibhile ezongezelelweyo S4-S6), kuquka nezinto eziqhelekileyo zeempendulo zokhuselo lwezityalo: iproteni yokudlulisa amafutha (iTanjilG_32225), i-enzyme ye-endoglucan-1,3-β-glucoside (iTanjilG_23384), iiproteni ezimbini ezibangela uxinzelelo ezifana ne-SAM22 (iTanjilG_31528 kunye neTanjilG_31531), iproteni ye-latex esisiseko (iTanjilG_32352), kunye neeproteni ezimbini zodonga lweeseli ezityebileyo nge-glycine (iTanjilG_19701 kunye neTanjilG_19702). Kwakukho nokudibana okuphezulu kwiimpendulo ze-transcriptome phakathi kwe-83A:476 kunye neBoregine kwi-24 HPI (iyonke yi-16-38% DEG) kunye naphakathi kweMandelup kunye nePopulation 22660 kwi-48 HPI (iyonke yi-14-20% DEG).
Umzobo weVenn obonisa inani leejini ezivezwe ngokwahlukileyo (DEG) kwimigca ye-lupine enamagqabi amancinci (NLL) efakwe i-Colletotrichum lupini (uhlobo lwe-Col-08 olufunyenwe kwiintsimi ze-lupine eWierzhenice, ePoland, ngo-1999). Imigca ye-NLL ehlalutyiweyo yayiyi: 83A:476 (enganyangekiyo, ephethe i-allele ye-Lanr1), i-Boregine (enganyangekiyo, imvelaphi ye-genetic ayaziwa), i-Mandelup (enganyangekiyo, ephethe i-allele ye-AnMan) kunye nenani labantu abangama-22660 (abasengozini enkulu). Isifinyezo se-hpi simele iiyure emva kokugonywa. Amaxabiso angama-zero asusiwe ukuze kube lula ukwenza igrafu ibe lula.
Iseti yezakhi zofuzo ezivezwe kakhulu kwi-6 hpi ihlalutywe ukuze kubonwe ukuba kukho ii-canonical R gene domains (Itheyibhile Eyongezelelekileyo S7). Olu phononongo lutyhile ukungeniswa kwe-transcriptome yezakhi zofuzo zokuchasana nezifo zakudala ezinee-NBS-LRR domains kuphela kwi-83A:476. Le seti yayine-TIR-NBS-LRR gene enye (tanjilg_05042), ii-CC-NBS-LRR genes ezintlanu (tanjilg_06165, tanjilg_06162, tanjilg_22773, tanjilg_22640, kunye ne-tanjilg_16162), kunye ne-Four NBS-LR, Tanjilg_16162), kunye ne-Four NBS-LRRE (tanjilg_16162) kunye ne-Four NBS-Lrr (tanjilg_16162) kunye ne-Four NBS-LRR (TANJILG_16162). Zonke ezi zakhi zofuzo zine-canonical domains ezicwangciswe ngokwee-sequences ezigciniweyo. Ukongeza kwi-NBS-LRR domain genes, ii-RLL kinases ezininzi zenziwe zasebenza kwi-6 hpi, enye kwiBoregine (TanjilG_19877), ezimbini kwiMandelup (TanjilG_07141 kunye neTanjilG_19877) kunye nenani labantu abangama-22660 (TanjilG_09014 kunye neTanjilG_10361) kunye nezimbini kwi-83A 27:476.
Iijini ezinembonakalo etshintshileyo kakhulu xa ziphendulwe yi-C. lupini (uhlobo lwe-Col-08) ziye zavavanywa kuhlalutyo lokunongwa kwe-Gene Ontology (GO) (Itheyibhile eyongezelelekileyo S8). Igama lenkqubo yebhayoloji elidla ngokumelwa kakhulu yayili-“GO:0006952 impendulo yokuzikhusela” evele kwiindidi ezi-6 kwezili-16 (ixesha × umgca) ezinokubaluleka okuphezulu (ixabiso le-P <0.001) (Umzobo 2). Igama lenkqubo yebhayoloji elidla ngokumelwa kakhulu yayili-“GO:0006952 impendulo yokuzikhusela” evele kwiindidi ezi-6 kwezili-16 (ixesha × umgca) ezinokubaluleka okuphezulu (ixabiso le-P <0.001) (Umzobo 2). Наиболее часто чрезмерно представленным термином биологического процесса был GO: 0006952 защитный ответ», который изявля 6 × 6 линия) комбинаций с высокой значимостью (значение P <0,001) (рис. 2). Igama lenkqubo yebhayoloji elidla ngokumelwa kakhulu yayiyi-'GO:0006952 defense response', evele kwiindidi ezi-6 kwezili-16 (ixesha × umgca) ezindibaniselwano ezinokubaluleka okuphezulu (ixabiso le-P <0.001) (Umzobo 2).最常被过度代表的生物过程术语是“GO:0006952 防御反应”，它出现在16 个（时间中×个中，具有高显着性(P 值< 0.001）(图2）). Igama lenkqubo yebhayoloji elimele kakhulu yi-“GO:0006952 defense response”, elivela kwiindidi ezi-6 kwezili-16 (时间×线), ezinokubaluleka okuphezulu (ixabiso le-P <0.001) (图2). Наиболее часто чрезмерно представленным термином биологического процесса был GO: 0006952 Impendulo yoKhuselo», который появлялся 1на × 6 линия) с высокой значимостью (значение P <0,001) (рис. 2). Igama lenkqubo yebhayoloji elidla ngokumelwa kakhulu yayiyi-'GO:0006952 Defense Response', evele kwiindidi ezi-6 kwezili-16 (ixesha × umgca) ngokubaluleka okuphezulu (ixabiso le-P <0.001) (Umzobo 2).Eli gama limelwe kakhulu kwiindawo ezimbini zexesha kwi-83A: i-476 kunye ne-Boregine (6 kunye ne-24 hpi) kunye nexesha elinye kwiMandelup kunye ne-Population 22660 (12 kunye ne-6 hpi, ngokulandelelana). Esi sisiphumo esilindelekileyo, sigxininisa impendulo yokubola kwemigca enganyangekiyo. Ukongeza, i-83A:476 iphendule kwi-C. lupini ngokubangela ngokukhawuleza iijini ezinxulumene nokuqhuma kwe-oxidative okumelwe ligama elithi "GO:0055114 redox process", ebonisa impendulo ethile yokuzikhusela, ngelixa i-Boregine ityhile iimpendulo ezithile zokuzikhusela, ezinxulumene negama elithi 'GO'. :0006950 Impendulo yoxinzelelo”. I-Population 22660 isebenze impendulo yokumelana ngokuthe tye equka ii-metabolites zesibini, igxininisa inani eligqithisileyo lamagama athi “GO:0016104 Inkqubo ye-triterpene biosynthesis” kunye nethi “GO:0006722 Inkqubo ye-triterpene metabolism” (zombini ezi magama zezeseti enye ye-genes), kuthathelwa ingqalelo iziphumo zohlalutyo lwe-GO term enrichment, uzinzo lwe-reaction ye-Mandelup lwaluphakathi kweBoregine kunye ne-Population 22660. Ukongeza, i-reaction yokuqala 83A:476 (6 hpi) kunye ne-delayed reaction Mandelup kunye ne-Population 22660 ziquka igama elithi GO:0015979 'photosynthesis' kunye nezinye iinkqubo zebhayoloji ezinxulumeneyo.
Amagama e-bioprocess gene ontology akhethwe kwinkcazo ye-differentially expressed genes ngexesha le-transcriptome responses ye-narrow-leaved lupine (NLL) efakwe i-anthrax lupine (uhlobo lwe-Col-08 olufunyenwe kwiintsimi ze-lupine eWierzhenice, ePoland, ngo-1999) agqithisiwe kakhulu. Imigca ye-NLL ehlalutyiweyo yayiyi: 83A:476 (enganyangekiyo, ephethe i-homozygous Lanr1 allele), i-Boregine (enganyangekiyo, imvelaphi ye-genetic engaziwayo), i-Mandelup (enganyangekiyo, ephethe i-homozygous AnMan allele) kunye nenani labantu abangama-22660 (abanokuchaphazeleka).
Ngenxa yokuba olu phononongo lujolise ekuchongeni ii-genes ezinegalelo ekuchaseni i-anthracnose, ii-genes ezinikwe amagama athi GO “GO: 0006952 Defensive responses” kunye nelithi “GO: 0055114 Redox processes” zihlalutywe nge-cut-offs kuba i-baseline means ≥ 30 kunye nomgca omnye ubuncinane. × i-point in time edibanisa amaxabiso abalulekileyo ngokwezibalo ze-log2 (utshintsho olugobileyo). Inani lee-genes ezihlangabezana nale migangatho yayiyi-65 kwi-GO:0006952 kunye ne-524 kwi-GO:0055114.
I-83A:476 ityhile iincochoyi ezimbini ze-DEG ezichazwe ngegama elithi GO:0006952, eyokuqala kwiijini ezi-6 nge-intshi (iijini ezingama-64, ummiselo wokunyuka nokuhla) kwaye eyesibini kwiijini ezingama-24 nge-intshi (iijini ezili-15, ummiselo wokunyuka kuphela). I-Boregine ikwabonise ukuba i-GO:0006952 ifikelele kwinqanaba elifanayo, kodwa nge-DEG encinci (11 kunye no-8) kunye nokusebenza okukhethekileyo. I-Mandeloop ibonise iincochoyi ezimbini ze-GO:0006952 kwi-12 kunye ne-48 HPI, zombini zithwele iijini ezili-12 (eyokuqala ene-genes esebenzayo, kwaye eyesibini ene-genes ezicinezelayo kuphela), ngelixa inani labantu abangama-22660 kwi-6 HPI (iijini ezili-13) lalinobuninzi obukhulu bomgaqo wokunyuka kwe-peak. Kufuneka kuqatshelwe ukuba i-96.4% ye-GO:0006952 DEG kwezi ncochoyi yayinehlobo olufanayo lokuphendula (phezulu okanye ezantsi), nto leyo ebonisa ukufana okukhulu kwiimpendulo zokuzikhusela nangona kukho umahluko kwinani leejini ezibandakanyekayo. Iqela elikhulu leenkqubo ezinxulumene negama elithi GO:0006952 libhala iProtein yoMyalezo weNdlala iStanger Stress-Associated Message 22 (SAM22-like), ekwi-class 10 pathogenesis-associated protein (PR-10) protein clade kunye neprotein yeprotein ye-core latex. efanayo (MLP-like) (Umzobo 3). La maqela mabini ahluke ngohlobo lokubonakaliswa kunye nendlela yokuphendula. Iijini ezibhala iiprotein ezifana ne-SAM22 zibonise ukungeniswa okuzinzileyo nokubalulekileyo kwiindawo zokuqala (6 okanye 12 hpi) kwaye ngokubanzi azizange ziphendule ekupheleni kovavanyo (48 hpi), ngelixa iiprotein ezifana ne-MLP zibonise ukulungelelana kwi-6 hpi. hpi. 83A:476 kunye neMandelup kwi-48 hp/in, phantse zonke ezinye iindawo zedatha azizange ziphendule. Ukongeza, umahluko kwiiprofayili zokubonakaliswa kweejini zeprotein ezifana ne-SAM22 ulandele ukubonwa kokuguquguquka kokuxhathisa i-anthracnose, njengoko imigca enganyangekiyo ngakumbi yayinexesha elininzi elibangela kakhulu ezi jini kuneejini ezisengozini enkulu. Esinye i-gene efana ne-LlR18A/B efana ne-PR-10 sibonise ipatheni yokubonakalisa efana kakhulu ne-gene efana ne-SAM22 efana ne-protein.
Izinto eziphambili zenkqubo yebhayoloji ethi “GO:0006952 Defense Response” kunye neepateni zokubonakaliswa kwezakhi zofuzo ezikhethiweyo ze-Lanr1 kunye ne-AnMan alleles zichongiwe. Isikali se-Log2 simele ixabiso le-log2 (utshintsho olugobileyo) phakathi kwezityalo ezifakwe i-incocuted (Colletotrichum lupini, uhlobo lwe-Col-08, olufunyenwe kwiintsimi ze-lupine, eWizhenica, ePoland, 1999) kunye nezityalo zokulawula (ezifakwe i-incocuted) ngexesha elinye. Le migca ye-lupine ilandelayo yahlalutywa: 83A:476 (enganyangekiyo, ethwele i-allele ye-Lanr1 efana ne-homozygous), i-Boregine (enganyangekiyo, imvelaphi yezakhi zofuzo ayaziwa), i-Mandelup (enganyangekiyo, ethwele i-allele ye-AnMan efana ne-homozygous), kunye ne-Population 22660 (enganyangekiyo).
Ukongeza, iiprofayili zokubonakaliswa kwe-RNA-seq candidate genes Lanr1 (TanjilG_05042) kunye ne-AnMan (TanjilG_12861) zivavanyiwe (Umzobo 3). I-gene yeTanjilG_05042 ibonise impendulo ebalulekileyo (ukusebenza) kwi-83A:476 kuphela kwindawo yokuqala (6 hpi), ngelixa iTanjilG_12861 yayibalulekile kwiMandeloop kuphela kwiindawo ezimbini zexesha: i-6 hpi (ulawulo oluphantsi) kunye ne-24 hpi (6 hpi). With.). adjustable) ).
Ezona genes zigqithisileyo kwigama elithi GO:0055114 “inkqubo ye-redox” yayizigenes ezifaka iiproteni ze-cytochrome P450 kunye ne-peroxidase (Umzobo 4). Kwiisampuli ezithathwe kwi-83A:476 kwi-6 HPI, amaxabiso aphezulu okanye aphantsi e-log2 (utshintsho olugobileyo) (kwi-86.6% yegenes) ajongwa ngokubanzi phakathi kwezityalo ezifakwe i-incubated kunye nezolawulo, nto leyo egxininisa impendulo ephezulu yale genotype kwisini esifake i-incubating. I-83A:476 ibonise i-GO ebaluleke kakhulu: 0055114 DEG kwi-6 hpi (iigenes ezingama-503), ngelixa ezinye iigenes kwi-48 hpi (iBoregine, iigenes ezingama-31; iMandelup, iigenes ezingama-85; kunye nePopulation 22660, iigenes ezingama-78)). Kwiigenes ezininzi zosapho lwe-GO:0055114, iintlobo ezimbini zeempendulo zokugonywa (ukusebenza kunye nokuthintela) zibonwe. Okunomdla kukuba, ukuya kuthi ga kwi-97.6% yee-DEG ezichongiweyo kwigama elithi GO: 0055114 eMandelupe nge-48 hp Ezi ngqwalasela zibonisa ukuba, nangona isikali sincinci kakhulu (oko kukuthi, inani lee-genes ze-redox eziguquliweyo, ezingama-85 xa kuthelekiswa nama-503), ipateni yeempendulo ze-transcriptome ezilibazisekileyo ze-mandeloup kwi-anthracnose ifana nempendulo yokuqala ye-83A:476. Kwi-Boregine kunye ne-Population 22660, oku kuhlangana kuphantsi kwi-51.6% kunye ne-75.6%, ngokwahlukeneyo.
Iipateni zokubonakaliswa kwezinto eziphambili zegama lenkqubo yebhayoloji ethi “GO:0055114 Redox process” zityhilwe. Isikali seLog2 simele amaxabiso e-log2 (utshintsho olugobileyo) phakathi kwezityalo ezifakwe i-incocuted (Colletotrichum lupini, uhlobo lwe-Col-08, olufunyenwe kwiintsimi ze-lupine, eWizhenica, ePoland, 1999) kunye nezityalo ezilawulwayo (ezifakwe i-incocuted) ngexesha elinye. Le migca ye-lupine ilandelayo yahlalutywa: 83A:476 (enganyangekiyo, ethwele i-allele ye-Lanr1 e-homozygous), i-Boregine (enganyangekiyo, imvelaphi yemfuza ayaziwa), i-Mandelup (enganyangekiyo, ethwele i-allele ye-AnMan e-homozygous), kunye ne-Population 22660 (enganyangekiyo).
83A:476 Iimpendulo zeTranscriptomic kwi-inculation nge-C. lupini (uhlobo lwe-Col-08) nazo zaziquka ukuthuliswa kweejini okunxulunyaniswa negama elithi GO:0015979 elithi “photosynthesis” kunye nezinye iinkqubo zebhayoloji ezinxulumeneyo (UMfanekiso 5). Le seti ye-GO:0015979 DEG yayiqulethe iijini ezili-105 ezazicinezelwe kakhulu kwi-6 hpi kwi-83A:476. Kule nxalenye incinci, iijini ezingama-37 nazo zancitshiswa kwiMandelup kwi-48 HPI kunye ne-35 ngexesha elinye kubemi abangama-22660, kuquka ii-DEG ezili-19 eziqhelekileyo kuzo zombini iijini. Akukho DEG ezinxulumene negama elithi GO: 0015979 ezasebenza kakhulu kuyo nayiphi na indibaniselwano (umgca x ixesha).
Iipateni zokubonakaliswa kwezinto eziphambili zegama lenkqubo yebhayoloji ethi “GO:0015979 Photosynthesis” zatyhilwa. Isikali seLog2 simele amaxabiso e-log2 (utshintsho olugobileyo) phakathi kwezityalo ezifakwe i-incocuted (Colletotrichum lupini, uhlobo lweCol-08, olufunyenwe kwiintsimi ze-lupine, eWizhenica, ePoland, 1999) kunye nezityalo zolawulo (ezifakwe i-incocuted) ngexesha elinye. Le migca ye-lupine ilandelayo yahlalutywa: 83A:476 (enganyangekiyo, ethwele i-allele ye-Lanr1 ene-homozygous), iBoregine (enganyangekiyo, imvelaphi yemfuza ayaziwa), iMandelup (enganyangekiyo, ethwele i-allele ye-AnMan ene-homozygous), kunye nePopulation 22660 (enganyangekiyo).
Ngokusekelwe kwiziphumo zohlalutyo lokubonakaliswa okwahlukileyo kunye nokuba kubandakanyeke kwiimpendulo zokuzikhusela kwifungi ezibangela izifo, olu seti lweejini ezisixhenxe lukhethwe ukuze kulinganiswe iiprofayili zokubonakaliswa nge-PCR yexesha langempela (Itheyibhile eyongezelelekileyo S9).
I-gene yeproteni eqikelelwayo iTanjilG_10657 ibangelwe kakhulu kuzo zonke imigca efundweyo kunye namanqaku exesha xa kuthelekiswa nezityalo zolawulo (ezifanayo) (Iitheyibhile ezongezelelweyo S10, S11). Ukongeza, iprofayili yokubonakaliswa kweTanjilG_10657 ibonise umkhwa okhulayo ngexesha lovavanyo lwazo zonke imigca. Inani labantu abangama-22660 libonise uvakalelo oluphezulu lweTanjilG_10657 ekufakweni kwe-inculation nge-activation ye-114-fold kunye nenqanaba eliphezulu lokubonakaliswa (4.4 ± 0.4) kwi-24 HPI (Umzobo 6a). I-gene yeproteni ye-PR10 LlR18A iTanjilG_27015 ikwabonise ukusebenza kuyo yonke imigca kunye namanqaku exesha, ngokubaluleka kwezibalo kwiindawo ezininzi zedatha (Umzobo 6b). Ngokufanayo neTanjilG_10657, inqanaba eliphezulu lokubonakaliswa kweTanjilG_27015 labonwa kubemi abangama-22660 abafakwe i-incubator kwi-24 HPI (19.5 ± 2.4). I-acid endochitinase gene iTanjilG_04706 yanda kakhulu kuyo yonke imigca nakuzo zonke iindawo zexesha ngaphandle kweBoregine 6 hpi (Umzobo 6c). Yabangelwa ngamandla kwindawo yokuqala (6 HPI) kwi-83A:476 (ngamaxesha ali-10.5) kwaye yanda ngokuphakathi kwezinye imigca (ngamaxesha ali-6.6-7.5). Ngexesha lovavanyo, ukubonakaliswa kweTanjilG_04706 kwahlala kumanqanaba afanayo kwi-83A:476 nakwiBoregine, ngelixa kwiMandelup nakwiPopulation 22660 kwanda kakhulu, kwafikelela kumaxabiso aphezulu (5.9 ± 1.5 kunye ne-6.2 ± 1.5, ngokulandelanayo). I-endoglucan-1,3-β-glucosidase-like gene TanjilG_23384 ibonise ukusebenza okuphezulu kwiindawo zokuqala ezimbini (6 kunye ne-12 hpi) kuyo yonke imigca ngaphandle kwenani labantu abangama-22660 (Umzobo 6d). Amanqanaba aphezulu okubonakaliswa kweTanjilG_23384 abonwe kwindawo yesibini (12 hpi) eMandelup (2.7 ± 0.3) kunye ne-83A:476 (1.5 ± 0.1). Kwi-24 HPI, ukubonakaliswa kweTanjilG_23384 bekuphantsi kakhulu kuyo yonke imigca efundwayo (ukusuka kwi-0.04 ± 0.009 ukuya kwi-0.44 ± 0.12).
Iiprofayili zokubonakaliswa kwezakhi zofuzo ezikhethiweyo (ag) ezityhilwe yi-quantitative PCR. Amanani 6, 12 kunye no-24 amele iiyure emva kokugonywa. Izakhi zofuzo zeLanDExH7 kunye neLanTUB6 zisetyenziselwe ukulungelelanisa kwaye iLanTUB6 yasetyenziswa ukulinganisa phakathi kothotho. Iibha zempazamo zimele ukuphambuka okuqhelekileyo okusekelwe kwiikopi ezintathu zebhayoloji, nganye kuzo ingumyinge weekopi ezintathu zobugcisa. Ukubaluleka kwezibalo komahluko kumanqanaba okubonakaliswa phakathi kwezityalo ezifakwe i-incubator (iColletotrichum lupini, uhlobo lweCol-08, olufunyenwe ngo-1999 kwintsimi ye-lupin eWierzenica, ePoland) kunye nezityalo zolawulo (ezifakwe i-mock-incubator) ziphawulwe ngaphezulu kwamanqaku edatha (*Ixabiso le-P < 0.05, **Ixabiso le-P ≤ 0.01, ***Ixabiso le-P ≤ 0.001). Ukubaluleka kwezibalo komahluko kumanqanaba okubonakaliswa phakathi kwezityalo ezifakwe i-incubator (iColletotrichum lupini, uhlobo lweCol-08, olufunyenwe ngo-1999 kwintsimi ye-lupin eWierzenica, ePoland) kunye nezityalo zolawulo (ezifakwe i-mock-incubator) ziphawulwe ngaphezulu kwamanqaku edatha (*Ixabiso le-P < 0.05, **Ixabiso le-P ≤ 0.01, ***Ixabiso le-P ≤ 0.001). Статистическая значимость различий в уровнях экспрессии между инокулированными (Colletotrichum lupini, штамм Col-08, получен в 1999 г. Польша) и контрольными (ложно инокулированными) растениями отмечена над точками данных (*значение P < 0,05, **значение P ≤ 0,010, *** 0,01, *** Umahluko obalulekileyo ngokwezibalo kumanqanaba okubonakaliswa phakathi kwezityalo ezifakwe i-incubator (iColletotrichum lupini, uhlobo lweCol-08, olufunyenwe ngo-1999 kwintsimi ye-lupine eWierzhenice, ePoland) kunye nezityalo zolawulo (ezifakwe i-incubator) ziphawulwe ngaphezulu kwamanqaku edatha (*Ixabiso le-P < 0.05, **Ixabiso le-P ≤ 0.01, ***Ixabiso le-P ≤ 0.001).接种（Colletotrichum lupini，Col-08株，1999年从波兰Wierzenica的羽扇豆田获得2 0.05, **P 值≤ 0.01, ***P 值≤ 0.001).接种 （colletorichum lupini , color-08 株 ， 1999 年 波兰 波兰 wierzenica的 羽扇 获得） 和对照 鼈接秗种 波兰差异 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 люпин, в 1999 г.) и контрольными (ложно инокулированными) растениями отмечены над точками данных (* значение P < 0,05, *** 0,001). Umahluko obalulekileyo ngokwezibalo kumanqanaba okubonakaliswa phakathi kwezityalo ezifakwe i-incubator (iColletotrichum lupini, uhlobo lweCol-08, olufunyenwe kwiintsimi ze-lupine eVerzhenice, ePoland, ngo-1999) kunye nezityalo zolawulo (ezifakwe i-incubator) ziphawulwe ngaphezulu kwamanqaku edatha (*Ixabiso le-P < 0.05 , **Ixabiso le-P ≤ 0.01, ***Ixabiso le-P ≤ 0.001).Imigca ye-NLL ehlalutyiweyo yayiyi: 83A:476 (enganyangekiyo, ethwele i-allele ye-Lanr1 ene-homozygous), iMandelup (enganyangekiyo ngokuphakathi, ethwele i-allele ye-AnMan ene-homozygous), iBoregine (enganyangekiyo, imvelaphi yemfuza engaziwayo) kunye nenani labantu abangama-22660 (abanokuchaphazeleka).
I-gene ekhethiweyo iTanjilG_05042 kwindawo yeLanr1 ibonise ipatheni yokubonakalisa eyahlukileyo kakhulu kwiiprofayili ezifunyenwe kwizifundo ze-RNA-seq (Umzobo 6e). Ukusebenza okubalulekileyo kwale gene kubonwe eMandelup nakwinani labantu abangama-22660 (ukuya kuthi ga kwi-39.7 kunye ne-11.7 amaxesha, ngokulandelelana), nto leyo ebangele amanqanaba aphezulu okubonakalisa (ukuya kuthi ga kwi-1.4 ± 0.14 kunye ne-7.2 ± 1.3, ngokulandelelana). I-83A:476 ikwatyhile ukunyuka okuthile kwe-gene yeTanjilG_05042 (ukuya kuthi ga kwi-3.8-fold), nangona kunjalo, amanqanaba okubonakalisa afunyenweyo (0.044 ± 0.002) ayengaphantsi ngokuphindwe kathathu kunalawo abonwe eMandelup nakwinani labantu abangama-22660. Ihlalutywe yi-qPCR ibonise umahluko omkhulu kumanqanaba okubonakaliswa phakathi kwee-genotypes kwiindidi ezahlukeneyo ezigonywe (ulawulo), ifikelela kumahluko ophindwe ka-58 phakathi kwabemi abangama-22660 kunye nama-83A:476, kunye naphakathi kwabemi abangama-22660 kunye nama-22660. Umahluko ophindwe kabini ufezekisiwe phakathi kweBoregine kunye neMandalup.
I-gene ekhethiweyo kwi-AnMan locus, iTanjilG_12861, yavuselelwa ngenxa yokugonywa kwi-83A:476 kunye neMandelup, yayingathathi cala kubemi abangama-22660, kwaye yancitshiswa kwiBoregine (Umzobo 6f). Ukubonakaliswa kwe-gene yeTanjilG_12861 yayiyeyona iphezulu kwi-83A egonyiweyo: 476 (0.14±0.01). I-gene ye-17.4 kDa class I heat shock protein gene yeTanjilG_05080 HSP17.4 ibonise amanqanaba aphantsi okubonakaliswa kuzo zonke iintlobo ezifundweyo kunye namanqaku exesha (Umzobo 6g). Ixabiso eliphezulu labonwa kwi-24 HPI kubemi abangama-22660 (0.14±0.02, ukwanda okuphindwe kasibhozo kwimpendulo yokugonywa).
Ukuthelekiswa kweeprofayili zokubonakaliswa kwezakhi zofuzo (Umzobo 7) kubonise ulwalamano oluphezulu phakathi kweTanjilG_10657 kunye nezinye izakhi zofuzo ezine: TanjilG_27015 (r = 0.89), TanjilG_05080 (r = 0.85), TanjilG_05042 (r = 0.80), kunye neTanjilG_04706 (r = 0.79). Ezo ziphumo zinokubonisa ukulawulwa ngokubambisana kwezi zakhi zofuzo ngexesha lokuphendula kokuzikhusela. Izakhi zofuzo zeTanjilG_12861 kunye neTanjilG_23384 zibonise iiprofayili zokubonakaliswa ezahlukeneyo ezinexabiso eliphantsi lePearson correlation coefficient ​​(ukusuka kwi-0.08 ukuya kwi-0.43 kunye ne--0.19 ukuya kwi-0.28, ngokulandelelana) xa kuthelekiswa nezinye izakhi zofuzo.
Ulwalamano phakathi kweeprofayili zokubonakaliswa kwezakhi zofuzo lufunyenwe kusetyenziswa i-quantitative PCR. Le migca ye-lupine ilandelayo enamaqhezu amancinci ihlalutywe: 83A:476 (enganyangekiyo, ethwele i-allele ye-homozygous Lanr1), iMandelup (enganyangekiyo ngokuphakathi, ethwele i-allele ye-homozygous AnMan), iBoregine (enganyangekiyo, imvelaphi yezakhi zofuzo ayaziwa), kunye ne-Population 22660 (enganyangekiyo). Amanqaku amathathu exesha abalwe (iiyure ezi-6, ezili-12 kunye nezingama-24 emva kokugonywa), kuquka ukugonywa (iColletotrichum lupini, uhlobo lweCol-08, olufunyenwe kwiintsimi ze-lupine eWierzhenice, ePoland, ngo-1999) kunye nezityalo zolawulo (ezingenwe yi-sham). Isikali sibonisa ixabiso le-coefficient yokudibanisa iPearson.
Ngokusekelwe kwidatha efunyenwe kwi-6 horsepower nge-intshi nganye, i-WGCNA yenziwe kwi-9981 DEG echongiweyo ngokuthelekisa izityalo ezifakwe i-incubator kunye nezolawulo ukuze kugxilwe kwiimpendulo zokuzikhusela kwangethuba (Itheyibhile Eyongezelelekileyo S12). Iimodyuli ze-gene ezingamashumi amabini anesibini (ii-clusters) zifunyenwe zineprofayili zokubonakalisa ezinxulumeneyo (ezilungileyo okanye ezimbi) phakathi kwe-genotypes kunye neendidi zovavanyo. Ngokomndilili, amanqanaba okubonakaliswa kwezakhi zofuzo ayehla ngokolandelelwano lwe-83A:476 > Mandelup > Boregine > Abemi 22660 (nangona kunjalo, kuzo zombini ezi ntlobo, olu hlobo lotshintsho lwalunamandla kwizityalo ezilawulayo). Ngokomndilili, amanqanaba okubonakaliswa kwezakhi zofuzo ayehla ngokolandelelwano lwe-83A:476 > Mandelup > Boregine > Abemi 22660 (nangona kunjalo, kuzo zombini ezi ntlobo, olu hlobo lotshintsho lwalunamandla kwizityalo ezilawulayo). В среднем уровни экспрессии генов снижались в порядке 83A:476 > Mandelup > Boregine > Population 22660 контрольных растений). Ngokomndilili, amanqanaba okubonakaliswa kwezakhi zofuzo ehle ngolandelelwano lwe-83A:476 > Mandelup > Boregine > Abemi abangama-22660 (nangona kunjalo, kuzo zombini ezi ntlobo, olu hlobo lotshintsho lwalunamandla kwizityalo 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 按 83a: 476> mandelup> boregine> labemi 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). Ngokomndilili, amanqanaba okubonakaliswa kwezakhi zofuzo ehlile kuthotho lwe-83A:476 > Mandelup > Boregine > Abemi abangama-22660 (nangona kunjalo, kuzo zombini iinguqulelo, olu hlobo lotshintsho lwalunamandla kwizityalo ezilawulayo).Ukugonywa kubangele ukwanda kokubonakaliswa kwezakhi zofuzo, ingakumbi kwiimodyuli 18, 19, 14, 6 kunye no-1 (ngokwendlela ehla ngayo isiphumo), ulawulo olubi (umz. iimodyuli 9 kunye no-20) okanye ngeziphumo ezingathathi cala (umz. iimodyuli 11, 22, 8 kunye no-13). Uhlalutyo lwe-GO term enrichment (Itheyibhile eyongezelelekileyo S13) lutyhile "GO: 0006952 Iimpendulo zoKhuseleko" kwimodyuli efakwe i-incubated (18) ene-activation ephezulu, kubandakanya izakhi zofuzo ezihlalutywe yi-qPCR (TanjilG_04706, TanjilG_23384, TanjilG_10657 kunye no-TanjilG_27015), kunye nezinye iimodyuli ezininzi ze-Inoculate most depressed photosynthesis (9). I-module 18 concentrator (Umzobo 8) ichongiwe njenge-gene yeTanjilG_26536 ebhala iproteni ye-PR-10 efana ne-LlR18B, kwaye i-module 9 concentrator ichongiwe njenge-gene yeTanjilG_28955 ebhala iproteni ye-photosystem II PsbQ. I-gene yokumelana ne-anthracnose ekhethiweyo i-Lanr1, iTanjilG_05042, ifunyenwe kwi-module 22 (Umzobo 9) kwaye inxulunyaniswa namagama athi “GO:0044260 Iinkqubo ze-metabolic ze-macromolecular zeselula” kunye nethi “GO:0006355 Ulawulo lwe-transcriptional, i-DNA templating” ephethe i-TanjilG_01212 hub. I-gene ibhala i-heat stress transcription factor A-4a (HSFA4a).
Uhlalutyo lwenethiwekhi olunesisindo sokubonakaliswa kwezakhi zofuzo kwiimodyuli ezinamagama enkqubo yebhayoloji amelwe kakhulu athi “GO: 0006952 Iimpendulo zoKhuselo”. Ukuhlanganiswa kwenziwe lula ukuze kugqanyiswe izakhi zofuzo ezine ezihlalutywe yi-qPCR (TanjilG_04706, TanjilG_23384, TanjilG_10657 kunye neTanjilG_27015).
Uhlalutyo lwenethiwekhi olunesisindo sokubonakaliswa kwezakhi zofuzo kwimodyuli enegama eligqithisileyo lenkqubo yebhayoloji elithi “GO: 0006355: Ulawulo lokubhalwa kwezakhi zofuzo, i-DNA templating” kunye nokuthwala i-gene yokumelana ne-anthracnose ekhethiweyo i-Lanr1 TanjilG_05042. Ukuhlanganiswa kwenziwe lula ukuze kwahlulwe i-gene ye-TanjilG_05042 kunye ne-gene ephakathi ye-TanjilG_01212.
Ukuhlolwa kokuxhathisa i-anthracnose okuqokelelwe e-Australia kubonise ukuba uninzi lweentlobo zezityalo ezikhutshwe kwangoko zazisengozini; iKalya, iCoromup kunye neMandelup zichazwe njengezingamelaniyo nezifo, ngelixa iWonga, iTanjil kunye ne-83A:476 zichazwe njengezingamelani nezifo kakhulu26,27,31. zazine-allele efanayo yokuxhathisa, ezibizwa ngokuba yiLanr1, kwaye iCoromup kunye neMandelup zazine-allele eyahlukileyo, ezibizwa ngokuba yi-AnMan10, 26, 39, ngelixa iKalya idlule kwi-allele eyahlukileyo. , Lanr2. Ukuhlolwa kokuxhathisa i-anthracnose eJamani kuphumele ekuchongweni komgca onganyamezeliyo weBo7212 one-allele ekhethiweyo ngaphandle kweLanr1, ebizwa ngokuba yiLanrBo36.
Uphononongo lwethu lutyhile ukuba i-allele yeLanr1 iphantsi kakhulu (malunga ne-6%) kwi-germplasm evavanyiweyo. Olu phononongo luhambelana neziphumo zokuhlola i-germplasm yaseMpuma Yurophu kusetyenziswa iimpawu ze-Anseq3 kunye ne-Anseq4, ezibonise ukuba i-allele yeLanr1 ikhona kwimigca emibini kuphela yaseBelarus. Oku kubonisa ukuba i-allele yeLanr1 ayikasetyenziswa kakhulu ziinkqubo zokuzalisa zasekuhlaleni, ngokungafaniyo nase-Australia, apho yenye yee-allele eziphambili zokuzala ezincediswa zii-marker. Oku kusenokubangelwa linqanaba eliphantsi lokuxhathisa elibonelelwa yi-allele yeLanr1 kwiimeko zasentsimini zaseYurophu xa kuthelekiswa nengxelo yase-Australia. Ukongeza, izifundo ze-anthracnose kwiindawo ezinemvula eninzi e-Australia zibonise ukuba iimpendulo zokuxhathisa ezibangelwa yi-allele yeLanr1 zisenokungasebenzi kwiimeko zemozulu ezikhuthaza ukukhula kunye nophuhliso olukhawulezileyo lwe-pathogen19,42. Enyanisweni, kolu phononongo, ezinye iimpawu ze-anthracnose nazo zabonwa kwii-genotypes ezithwala i-allele yeLanr1, nto leyo ebonisa ukuba ukuxhathisa kunokuphela phantsi kweemeko ezifanelekileyo zophuhliso lwe-C. lupini. Ukongeza, utoliko olungelulo oluchanekileyo lokubakho kweempawu ze-Anseq3 kunye ne-Anseq4, ezimalunga ne-1 cM ukusuka kwindawo ye-Lanr1, zinokwenzeka 28,30,43.
Uphononongo lwethu lubonise ukuba i-83A:476, ephethe i-allele ye-Lanr1, yaphendula kwi-C. lupini inoculation ngokuphinda-phinda i-transcriptome enkulu ngexesha lokuqala lokuhlalutywa (6 hpi), ngelixa eMandelup, ephethe i-allele ye-AnMan, iimpendulo ze-transcriptomic zabonwa kamva kakhulu. (ukusuka kwi-24 ukuya kwi-48 hp). Olu tshintsho lwexesha kwiimpendulo zokuzikhusela lunxulunyaniswa nomahluko kwiimpawu zesifo, nto leyo egxininisa ukubaluleka kokuqatshelwa kwangoko kwe-pathogen ukuze kuphendulwe ngempumelelo kwi-resistance. Ukuze kosulelwe izicubu zezityalo, ii-anthrax spores kufuneka zidlule kumanqanaba aliqela ophuhliso kumphezulu we-host, kubandakanya ukuhluma, ukwahlulwa kweeseli, kunye nokwakheka kwe-appressorium. I-appendage sisakhiwo sosulelo esinamathela kumphezulu we-host kwaye senza kube lula ukungena kwizicubu ze-host. Ngoko ke, ii-spores ze-C. gloeosporioides kwi-pea extract zibonise ukwahlulwa kokuqala kwe-nucleus emva kwemizuzu engama-75-90 yokufunxwa, ukwakheka kwetyhubhu yentsholongwane emva kwemizuzu engama-90-120, kunye nokucinezelwa emva kweeyure ezi-4 45. IMango C. gloeosporioides ibonakalise ukuhluma okungaphezulu kwe-40% emva kweeyure ezi-3 zokufukama kunye nokwakheka kwe-appressors malunga ne-20% emva kweeyure ezi-4. I-gene ye-CAP20 enxulumene ne-virulence ye-C. gloeosporioides ibonise umsebenzi wokubhala kwi-conidia eyenza i-epiphyte emva kweeyure ezi-3.5 zokufukama kwi-wax yomphezulu we-avocado kunye noxinzelelo oluphezulu lwe-protein ye-CAP20 emva kweeyure ezi-4 nemizuzu engama-46. Ngokufanayo, umsebenzi we-melanin biosynthesis genes kwi-C. trifolii wenziwa ngexesha lokufukama kweeyure ezi-2 kulandele ukwenziwa kwe-appressorium emva kweyure e-1. Izifundo zezicubu zamagqabi zibonise ukuba ama-strawberries afakwe i-C. acutatum anokucinywa kokuqala kwi-8 hpi, ngelixa iitumato ezifakwe i-C. coccodes zinokucinywa kokuqala kwi-4 hpi48,49. Ngokuhambelana kakhulu nesikali sexesha senkqubo yosulelo ye-Colletotrichum spp. Iimpendulo zokuzikhusela ezikhawulezayo kwi-83A:476 zibonisa ukubandakanyeka kwezakhi zofuzo zokuxhathisa izityalo kunye ne-effector-triggered immunity (ETI) kulo mgca, ngelixa iimpendulo zikaMandelup ezilibazisekileyo zixhasa i-micro-associated molecular pattern-triggered immunity (MTI) hypothesis 50. Iimpendulo zokuqala kwi-83A: 476 kunye neMandelup. Ukudibana okuncinci phakathi kwezakhi zofuzo ezilawulwa phezulu okanye eziphantsi kwimpendulo elibazisekileyo kukwaxhasa le ngcamango, njengoko i-ETI idla ngokuthathwa njengempendulo ye-MTI ekhawulezileyo nephuculweyo efikelela ekugqibeleni ekufeni kweeseli okucwangcisiweyo kwindawo yosulelo, eyaziwa ngokuba yi-anaphylactic shock 51,52.
Uninzi lwezakhi zofuzo ezibangelwa ligama elithi Gene Ontology GO:0006952 elithi “Defense Response” zii-homologues ezili-11 zeprotheyini yokuzila ukutya ebangelwa luxinzelelo (ezifana ne-SAM22) kunye neeprotheyini ezisixhenxe eziphambili ze-latex protein-like (MLPs). Iiprotheyini ezifana ne-31, 34, 43 kunye ne-423 zibonise ukufana kolandelelwano. Izakhi zofuzo ezifana ne-SAM22 zibonise ukusebenza okubonakalayo okuthathe ixesha elide, zibonisa amanqanaba aphezulu okumelana ne-anthracnose (83A:476 kunye ne-Boregine). Nangona kunjalo, izakhi zofuzo ezifana ne-MLP zancitshiswa kuphela kwimigca ephethe i-allele yokumelana (83A:476/Lanr1 kwi-6 hpi kunye ne-Mandelup/AnMan kwi-24 hpi). Kufuneka kuqatshelwe ukuba zonke izakhi zofuzo ezifana ne-SAM22 ezichongiweyo zivela kwiqoqo lezakhi zofuzo elimalunga ne-105 kb, ngelixa izakhi zofuzo ezifana ne-MLP zivela kwiindawo ezahlukeneyo ze-genome. Ukusebenza okudibeneyo kwee-gene ezifana ne-SAM22 kufunyenwe nakwisifundo sethu sangaphambili sokuchasana ne-NLL kwi-Diaporthetoxica inoculation, nto leyo ebonisa ukuba zibandakanyeka kwiindawo ezithe tye zempendulo yokuzikhusela. Esi sigqibo sikwaxhaswa ziingxelo zempendulo entle yee-gene ezifana ne-SAM22 ekwenzakaleni okanye kunyango nge-salicylic acid, i-fungus inducers, okanye i-hydrogen peroxide.
Izakhi zofuzo ezifana ne-MLP zibonakalisiwe ukuba zisabela kwiingxaki ezahlukeneyo ze-abiotic kunye ne-biotic, kubandakanya usulelo lwebhaktiriya, intsholongwane kunye ne-pathogenic fungal kwiintlobo ezininzi zezityalo55. Imiyalelo yokusabela kwiintsebenziswano ezithile phakathi kwezityalo kunye ne-pathogens yayiqala ekwandeni kakhulu (oko kukuthi, ngexesha lokuhlasela komqhaphu ngeVerticillium dahliae) ukuya ekwehleni okukhulu (oko kukuthi, emva kokosulelwa ngumthi weapile nge-Alternaria spp.)56,57. Ukuncipha okukhulu kwezakhi zofuzo ezifana ne-MLP-like 423 kuye kwabonwa ngexesha lokukhuselwa kwe-avocado kwi-F. niger usulelo kwaye ngexesha lokosulelwa komthi weapile iBotryosphaeria berengeriana f. cn. piricola kunye ne-Alternaria alternata zii-apple pathotypes58,59. Ukongeza, i-apple calli eveza kakhulu i-MLP-like 423 gene yayine-expression ephantsi yezakhi zofuzo ezinxulumene nokuchasana kwaye yayisengozini enkulu yokosulelwa ngumngundo59. Emva kweFusarium oxysporum f, i-MLP-like 423 gene nayo yacinezelwa kwi-common bean germplasm enganyangekiyo. cn. ukosulelwa ngumthi weapile 60.
Amanye amalungu osapho lwe-PR-10 achazwe kwisifundo sethu se-RNA-seq yayizizakhi zofuzo ze-LlR18A kunye ne-LlR18B ekuphenduleni ukunyuka kwe-regulation, kunye nezakhi zofuzo eziphakanyisiweyo (1 gene) okanye eziphantsi kwe-regulated (3 gene) zeprotheyini yokudlulisa i-lipid DIR1. . Ukongeza, i-WGCNA igxininisa i-LlR18B gene njengesixhobo kule module, esichaphazeleka kakhulu kukugonywa kwaye sithwala iizakhi zofuzo ezininzi zokukhusela. Iizakhi zofuzo ze-LlR18A kunye ne-LlR18B zenziwe ngamagqabi e-lupine atyheli ekuphenduleni iibhaktheriya ezibangela izifo, kunye nakwiziqu ze-NLL emva kokugonywa kwe-D. toxica, ngelixa i-homologue yerayisi yezi zakhi zofuzo, i-RSOsPR10, yabangelwa ngokukhawuleza lusulelo lwefungus olucingelwayo olubandakanyeka kwindlela yokubonisa i-jasmonic acid53,61, 62. Izakhi zofuzo ze-DIR1 ziqulathe iiproteni zokuthutha i-lipid ezingezizo ezithile ezifunekayo ekuqaleni kokumelana ne-systemic acquired resistance (SAR). Ngophuhliso lweempendulo zokukhusela, iproteni ye-DIR1 ithuthwa ukusuka kwindawo yosulelo nge-phloem ukuze ibangele i-SAR kwizitho ezikude. Okubangela umdla kukuba, i-gene ye-TanjilG_02313 DIR1 yaveliswa kakhulu kwindawo yokuqala kwimigca 84A:476 kunye ne-Population 22660, kodwa ukumelana ne-anthracnose kwaphuhliswa ngempumelelo kuphela kumgca 84A:476. Oku kunokubonisa ukusebenza okungaphantsi kwe-gene ye-DIR1 kwi-NLL, kuba ezinye ii-homologues ezintathu zasabela ekufakweni kwe-inculation kuphela kumgca we-83A:476 kwi-6 hpi, kwaye le mpendulo yajoliswa ezantsi.
Kwisifundo sethu, ezona zinto zixhaphakileyo ezihambelana nenkqubo yebhayoloji ebizwa ngokuba yi-“GO:0055114 Redox process” yayiyi-cytochrome P450 protein, i-peroxidase, i-linoleic acid 9S-/13S-lipoxygenase, kunye ne-1-aminocyclopropane-1-carboxylic acid oxidase. Ukongeza, i-WGCNA yethu ichaza i-HSFA4a homologue njengeemodyuli ezithwala i-hub ezifana ne-Lanr1 resistance gene candidate TanjilG_05042. I-HSFA4a yinxalenye yokulawulwa kwe-redox yokubhalwa kwenyukliya kwizityalo.
Iiproteni zeCytochrome P450 zii-oxidoreductases ezivuselela i-NADPH kunye/okanye ii-hydroxylation reactions ezixhomekeke kwi-O2 kwi-metabolism yokuqala neyesibini, kubandakanya i-metabolism ye-xenobiotics, kunye neehomoni, ii-fatty acids, ii-sterols, ii-cell wall components, ii-biopolymers, kunye ne-biosynthesis yeekhompawundi ezikhuselayo 69. Kwisifundo sethu, umahluko kumsebenzi we-cytochrome P450 yesityalo wehlisiwe ukusuka kwi--10.6 log2 (utshintsho oluphindwe kabini) ukuya kwi-5.7 ngenxa yenani elikhulu lee-homologues ezitshintshileyo (37) kunye nomahluko kwiipatheni zempendulo phakathi kweejini ezithile, okubonisa uhlaziyo oluphezulu. . Ukusebenzisa idatha ye-RNA-seq kuphela ukucacisa umsebenzi webhayoloji ocingayo weejini ze-NLL kwi-superfamily enkulu yeproteni kuya kuba yinto eqikelelweyo kakhulu. Nangona kunjalo, kubalulekile ukuqaphela ukuba ezinye iijini ze-cytochrome P450 zinxulunyaniswa nokumelana okwandileyo kwi-fungi ye-pathogenic okanye iibhaktheriya, kubandakanya negalelo kwiimpendulo ze-allergy69,70,71.
Iiperoxidases zeKlasi lesiThathu ziienzymes zezityalo ezisebenza ngeendlela ezininzi ezibandakanyeka kwiinkqubo ezahlukeneyo ze-metabolic ngexesha lokukhula nophuhliso lwezityalo, kunye nokuphendula kwiingcinezelo zokusingqongileyo ezifana netyuwa, imbalela, ukukhanya okuphezulu, kunye nokuhlaselwa yi-pathogen72. Iiperoxidases zibandakanyeka ekusebenzisaneni kweentlobo ezahlukeneyo zezityalo ne-Anthracis, kubandakanya iStylosanthes humilis kunye neC. gloeosporioides, iLens culinaris kunye neC. truncatum, iPhaseolus vulgaris kunye neC. lindemuthianum, iCucumis sativus kunye neC. lagenarium73,74,75,76. Impendulo ikhawuleza kakhulu, ngamanye amaxesha ide ibe kwi-4 HPI, ngaphambi kokuba ifungus ingene kwizicubu zezityalo73. I-peroxidase gene ikwaphendule kwi-D. toxica NLL inoculation. Ukongeza kwimisebenzi yayo eqhelekileyo yokulawula ukuqhuma kwe-oxidative okanye ukuphelisa uxinzelelo lwe-oxidative, ii-peroxidases zinokuphazamisa ukukhula kwe-pathogen ngokudala imiqobo ebonakalayo esekelwe ekuqiniseni udonga lweseli ngexesha lokuqina, i-subunit okanye ukudibanisa iikhompawundi ezithile. Lo msebenzi unokunxulunyaniswa kwi-silico ne-gene yeTanjilG_03329 ebhala i-anion peroxidase eyenza i-lignin eyanyuswa kakhulu kuphononongo lwethu kumgca oxhathisayo we-83A:476 kwi-6 HPI, kodwa kungekhona kwezinye iintlobo kunye namaxesha angaphendulanga.
I-9S-/13S-lipoxygenase ye-linoleic acid linyathelo lokuqala kwindlela ye-oxidative ye-lipid biosynthesis78. Iimveliso zale ndlela zinemisebenzi emininzi ekukhuseleni izityalo, kubandakanya ukuqinisa udonga lweseli ngokwenziwa kwee-callose kunye nee-pectin deposits, kunye nokulawula uxinzelelo lwe-oxidative ngokuvelisa iintlobo ze-oxygen ezisebenzayo79,80,81,82,83. Kolu phononongo lwangoku, ukubonakaliswa kwe-linoleic acid I-9S-/13S-lipoxygenase yatshintshwa kuzo zonke iintlobo, kodwa kubemi abasengozini 22660, ukunyuka kwanda kwabakho ngamaxesha ahlukeneyo, ngelixa kwiintlobo ezithwala i-Lanr1 enganyangekiyo kunye ne-AnMan allele, igxininisa ukwahlukahlukana komaleko we-oxylipin kwiimpendulo ze-anthrax ezikhuselayo phakathi kwezi genotypes.
I-1-aminocyclopropane-1-carboxylate oxidase (ACO) homologue yanda kakhulu (ii-genes ezili-9) okanye yancitshiswa (ii-genes ezi-2) xa yafakwa i-lupine. Ngaphandle kweemeko ezimbini, zonke ezi mpendulo zenzeke kwi-6 hp. kwi-83A:476. I-enzyme reaction elawulwa ziiproteni ze-ACO linyathelo elithintela izinga lokuveliswa kwe-ethylene kwaye ke ngoko ilawulwa kakhulu84. I-Ethylene yi-hormone yesityalo edlala indima eyahlukeneyo ekulawuleni uphuhliso lwezityalo kunye nokuphendula kwiimeko zoxinzelelo lwe-abiotic kunye ne-biotic. Ukuqaliswa kokubhalwa kwe-ACO kunye nokusebenza kwendlela yokubonisa i-ethylene kubandakanyeka ekwandiseni ukumelana kwerayisi kwi-hemibiotrophic fungus oryzae oryzae ngokulawula ukuveliswa kweentlobo ze-oxygen ezisebenzayo kunye ne-phytoalexins. Inkqubo efanayo kakhulu yosulelo lwamagqabi efunyenwe phakathi kwe-M. oryzae kunye ne-C. lupini88,89, ngokuchasene nokunyuka okukhulu kwee-homologues ze-ACO kumgca we-83A:476 oxelwe kolu phononongo, itshintsha amathuba okunika ukumelana ne-NLL anthracnose I-Ethylene inyathelo eliphambili lokubonisa kwiindlela zeemolekyuli.
Kolu phononongo lwangoku, ukuncitshiswa okukhulu kwezakhi zofuzo ezininzi ezinxulumene ne-photosynthesis kubonwe kwi-6 hpi kwi-83A:476 nakwi-48 hpi eMandeloop nakwinani labantu abangama-22660. Ubungakanani kunye nokuqhubela phambili kwezi nguqu kuhambelana nenqanaba. Ukumelana ne-Anthracnose kubonwe kolu vavanyo. Kutshanje, ukuncitshiswa okunamandla nokukhawulezileyo kwemibhalo enxulumene ne-photosynthesis kuye kwaxelwa kwiimodeli ezininzi zokunxibelelana kwezityalo nezifo, kubandakanya iibhaktheriya ezibangela izifo kunye nefungi. Ukukhawulezisa (ukusuka kwi-2 HPI kwezinye iindlela zokunxibelelana) kunye nokuncitshiswa kwezakhi zofuzo zehlabathi ezinxulumene ne-photosynthesis ekuphenduleni usulelo kunokubangela ukhuseleko lwezityalo ngokusekelwe ekusetyenzisweni kweentlobo zeoksijini ezisebenzayo kunye nokusebenzisana kwazo nendlela ye-salicylic acid ukuze kulawulwe ukusabela kwe-allergy 90,94.
Ukuqukumbela, iindlela zokuphendula ukhuselo ezicetywayo kwi-lineage exhathisayo kakhulu (83A:476) ziquka ukuqatshelwa ngokukhawuleza kwe-pathogen yi-R gene (mhlawumbi i-TIR-NBS-LRR TanjilG_05042) kunye ne-salicylic acid kunye ne-ethylene signaling ehambelana ne-allergy response-mediated reaction, elandelwa kukusekwa kwe-long-range SAR action. ixhaswa yi-DIR-1 protein. Kufuneka kuqatshelwe ukuba ixesha le-biotrophic losulelo lwe-C. lupini lifutshane kakhulu (malunga neentsuku ezi-2), lilandelwe kukukhula kwe-necrotic95. Utshintsho phakathi kwezi zigaba lunokunxulunyaniswa ne-necrosis kunye nokubonakaliswa kweeproteni ezingasebenzi njengezinto ezibangela ukusabela kwe-hypersensitivity kwizityalo ezisingethe. Ke ngoko, ixesha lokubamba ngempumelelo i-C. lupini kwinqanaba le-biotrophic lincinci kakhulu. Ukuhlelwa kwakhona kwe-genes ezinxulumene ne-redox kunye ne-photosynthesis okubonwe kwi-83A:476 kwi-6 hpi kuhambelana nokuqhubela phambili kwe-fungus hyphae kwaye kubonisa uphuhliso lwempendulo yokukhusela ephumelelayo kwinqanaba le-biotrophic. Iimpendulo ze-transcriptomic zikaMandelup kunye nabantu abangama-22660 zinokubambezeleka kakhulu ukuba zingabamba i-fungus ngaphambi kokuba zitshintshele ekukhuleni kwe-necrotic, nangona kunjalo, iMandelup inokuba luncedo ngakumbi kunabantu abangama-22660 kuba ulawulo olukhawulezayo lweprotheyini ye-PR-10 lukhuthaza ukumelana ngokuthe tye.
I-ETI, eqhutywa yi-gene ye-canonical R, ibonakala iyindlela eqhelekileyo yokuxhathisa iimbotyi kwi-anthracnose. Ngoko ke, kwimodeli ye-legume Medicago truncatula, ukuxhathisa i-anthracnose kunikwa yi-gene ye-RCT1, ilungu le-gene ye-plant R ye-TIR-NBS-LRR97. Le gene ikwanika ukuxhathisa i-anthracnose kwi-alfalfa xa idluliselwa kwizityalo ezisengozini. Kwi-common bean (P. vulgaris), ii-genes ezingaphezu kwamashumi amabini anesibini zokuxhathisa i-anthracnose zichongiwe ukuza kuthi ga ngoku. Ezinye zezi genes zifumaneka kwiindawo ezingenazo naziphi na ii-genes ze-canonical R, nangona kunjalo ezinye ezininzi zifumaneka emaphethelweni ee-chromosomes ezithwele i-NBS-LRR gene cluster, kubandakanya i-TIR-NBS-LRRs99. Uphononongo lwe-SSR olupheleleyo lwe-genome luqinisekisile ukudibana kwe-gene ye-NBS-LRR nokuxhathisa i-anthracnose kwi-common bean. I-gene ye-canonical R ikwafunyenwe kwindawo ye-genomic ethwele i-major anthracnose resistance locus kwi-white lupine 101.
Umsebenzi wethu ubonisa ukuba impendulo yokumelana kwangoko, esebenze kwinqanaba lokuqala losulelo lwezityalo (kungcono ukuba kungadluli i-12 hpi), ikhusela ngokufanelekileyo i-lupine enamagqabi amancinci kwi-anthracnose ebangelwa yi-fungus ebangela izifo i-Collelotrichum lupini. Sisebenzisa ulandelelwano oluphezulu lwe-throughput, sibonise iiprofayili zokubonakaliswa okwahlukileyo kwe-anthracnose resistance genes kwizityalo ze-NLL ezilawulwa yi-Lanr1 kunye ne-AnMan resistance genes. Ukuzikhusela okuphumelelayo kubandakanya ukuyila ngononophelo ii-genes zeeproteni ezibandakanyeka kwi-redox, i-photosynthesis, kunye ne-pathogenesis kwiiyure emva kokudibana kokuqala kwesityalo ne-pathogen. Iimpendulo ezifanayo zokukhusela, kodwa ezilibazisekayo ngexesha, azisebenzi kakuhle ekukhuseleni izityalo kwizifo. Ukumelana ne-anthrax okulawulwa yi-Lanr1 gene kufana nempendulo eqhelekileyo ekhawulezayo ye-R gene (immunity ebangelwa yi-effector), ngelixa i-AnMan gene inokuthi inike impendulo ethe tyaba (immunity ebangelwa yi-microbe-associated molecular pattern), ebonelela ngenqanaba eliphakathi lokuzinza.
Imigca ye-NLL engama-215 esetyenziselwa ukuvavanya iimpawu ze-anthracnose yayiquka ii-cultivar ezingama-74, imigca engama-60 efunyenwe ngokunqumla okanye ukuzala, ii-mutants ezi-5, kunye nee-germplasms zasendle okanye zokuqala ezingama-76. Imigca yayivela kumazwe ali-17, ikakhulu evela ePoland (58), eSpain (47), eJamani (27), eOstreliya (26), eRashiya (19), eBelarus (7), e-Itali (5) nakwezinye imigca. evela kumazwe ali-10. Iseti ikwabandakanya imigca enganyangekiyo: 83A:476, eTanjil, eWonga ephethe i-allele yeLanr1, kunye neMandelup ephethe i-allele yeAnMan. Imigca ifunyenwe kwi-European Lupine Genetic Resource Database egcinwe yiPoznań Plant Breeding Ltd., eWiatrowo, ePoland (Supplementary Table S1).
Izityalo zikhuliswe phantsi kweemeko ezilawulwayo (ixesha le-photoperiod iiyure ezili-16, ubushushu obungama-25°C emini kunye ne-18°C ​​ebusuku). Kuhlalutywe iikopi ezimbini zebhayoloji. I-DNA yahlulwa kumagqabi aneeveki ezintathu ubudala kusetyenziswa i-DNeasy Plant Mini Kit (Qiagen, Hilden, eJamani) ngokwemigaqo. Umgangatho kunye noxinzelelo lwe-DNA ehlukanisiweyo kwavavanywa ngeendlela ze-spectrophotometric (NanoDrop 2000; Thermo Fisher Scientific, Waltham, MA, e-USA). Uphawu lwe-AnManM1 oluphawula i-anthracnose resistance gene i-AnMan (ethathwe kwi-cv. Mandelup) kunye neempawu ze-Anseq3 kunye ne-Anseq4 ecaleni kwe-gene i-Lanr1 (ethathwe kwi-cv. Tanjil) zahlalutywa nge-11,26,28. Ii-Homozygotes ze-allele enganyangekiyo zanikwa amanqaku njenge-"1″, enokuchaphazeleka - njenge-"0″, kunye nee-heterozygotes - njenge-0.5.
Ngokusekelwe kwiziphumo zokuhlolwa kweempawu ze-AnManM1, i-AnSeq3 kunye ne-AnSeq4 kunye nokufumaneka kwembewu kwiimvavanyo zokugqibela zokulandelela, kukhethwe imigca engama-50 ye-NLL yokuthintela i-anthracnose. Uhlalutyo lwenziwe ngokuphindwe kabini kwi-greenhouse elawulwa yikhompyutha ene-photoperiod yeeyure ezili-14 enomgangatho wobushushu obuyi-22°C emini kunye ne-19°C ebusuku. Imbewu iyakrwelwa (isike i-seed coat kwicala elichaseneyo le-embryo nge-blade ebukhali) ngaphambi kokuhlwayela ukuthintela ukungakwazi kwembewu ukulala ngenxa yokuba i-seed coat inzima kakhulu kwaye kuqinisekiswe ukuhluma okufanayo. Izityalo zikhuliswe kwiimbiza (11 × 11 × 21 cm) ezinomhlaba ongengomhlwa (TS-1 REC 085 Medium Basic, Klasmann-Deilmann Polska, Warsaw, Poland). Ukugonywa kwenziwa ngohlobo lweColletotrichum lupini Col-08, olwakhuliswa ngo-1999 kwiziqu zezityalo zelupine ezinamagqabi amancinane ezikhuliswe kwintsimi eVerzhenitsa, eGreater Poland (52° 27′ 42″ N 17° 04′ 05″ E). Fumana indawo. Ezi zahlulwe zatyalwa kwindawo yobushushu ye-SNA kuma-20° C. phantsi kokukhanya okumnyama kangangeentsuku ezingama-21 ukuze kubangele ukubola. Kwiiveki ezine emva kokutyala, xa izityalo zifikelele kwinqanaba lamagqabi ama-4-6, ukugonywa kwenziwa ngokutshiza nge-suspension ye-conidia kuxinzelelo lwe-0.5 x 106 conidia nge ml nganye. Emva kokugonywa, izityalo zigcinwe ebumnyameni iiyure ezingama-24 kumswakama omalunga nama-98% kunye nobushushu obungama-25°C ukuze kube lula ukuhluma kwe-conidia kunye nenkqubo yosulelo. Emva koko izityalo zakhuliswa phantsi kwexesha leeyure ezili-14 kwi-photoperiod yama-22°C emini/19°C ebusuku kunye nomswakama ongama-70%. Amanqaku esifo enziwa emva kweentsuku ezingama-22 emva kokugonywa kwaye aqala kwi-0 (ayikhuselekanga) ukuya kwi-9 (isengozini enkulu) kuxhomekeke ekubeni ikhona okanye ayikho izilonda ezifileyo kwiziqu nakumagqabi. Ukongeza, emva kokufumana amanqaku, ubunzima bezityalo balinganiswa. Ubudlelwane phakathi kweempawu ze-genotypes kunye neempawu zesifo babalwe njenge-point two-sequence correlations (ukungabikho kweempawu ze-heterozygous kwisethi yemigca yokuhlalutya i-phenotype yokumelana ne-anthracnose).