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IAngustifolius lupine (NLL, Lupinus angustifolius L.) sisityalo esiyimidumba esisetyenziselwa imveliso yokutya kunye nokuphucula umhlaba.Ukwandiswa kwehlabathi jikelele kwe-NLL njengesityalo kutsale umngundo omninzi we-pathogenic, kubandakanya i-lupine anthracnose, ebangela isifo esonakalisayo se-anthracnose.I-alleles ezimbini, i-Lanr1 kunye ne-AnMan, ezenza ukwanda kokuchasa, zisetyenzisiwe ekuzaleni kwe-NLL, kodwa iindlela ezisisiseko zeemolekyuli azikaziwa.Kolu phononongo, abamakishi be-Lanr1 kunye ne-AnMan basetyenziselwe ukuhluza iisampuli ze-NLL yaseYurophu.Uvavanyo lwesitofu sokugonya kwindawo elawulwayo luqinisekise ukusebenza kakuhle kwabo bobabini abaxhasi abaxhathisayo.Iprofayile yembonakalo yemfuza eyahlukileyo yenziwa kummeli onganyangekiyo kunye nemigca esesichengeni.Ukuxhathisa kwe-anthracnose kwakunxulunyaniswa nokuchazwa ngokugqithisileyo kwegama le-ontology yemfuza "GO: 0006952 Impendulo yoKhuselo", "GO: 0055114 Inkqubo yeRedox", kunye ne "GO: 0015979 Photosynthesis".Ukongezelela, umgca we-Lanr1 (83A: 476) ubonise i-reprogramming ephawulekayo ye-transcriptome ngokukhawuleza emva kwe-inoculation, ngelixa enye imigca ibonise ukulibaziseka kule mpendulo malunga neeyure ze-42.Iimpendulo zokukhusela zidibene ne-TIR-NBS, i-CC-NBS-LRR kunye ne-NBS-LRR izakhi zofuzo, iiprotheyini ze-10 ezibandakanyekayo kwi-pathogenesis, iiprotheni zokudlulisa i-lipid, i-endoglucan-1,3-β-glucosidase, i-glycine-rich-rich cell wall proteins, kunye ne-genes ukusuka kwindlela esebenzayo ye-oxygen.Iimpendulo zakwangoko kwi-83A: 476, kubandakanywa ukunyanzeliswa ngononophelo kwezakhi zofuzo ezinxulumene ne-photosynthesis, zihambelana nokukhuselwa okuyimpumelelo ngexesha lesigaba sokukhula kwezityalo ze-fungal biology, ebonisa ukuba i-effector ibangela ukugonywa.I-reaction ye-Mandeloop iyacotha, njengokuba iyonke idonsa ethe tye.
Ilupine enamagqabi amxinwa (NLL, Lupinus angustifolius L.) yicereal ephezulu yeprotheni evela kwingingqi yentshona yeMeditera1,2.Ngoku ikhuliswa njengesityalo sokutya kwezilwanyana kunye nabantu.Ikwagqalwa njengomgquba oluhlaza kwiinkqubo zokujikeleziswa kwezityalo ngenxa yokulungiswa kwenitrogen nge-symbiotic nitrogen yokulungisa ibhaktiriya kunye nokuphuculwa ngokubanzi kobume bomhlaba.I-NLL iye yanenkqubo ekhawulezileyo yokwenziwa kwasekhaya kwinkulungwane edlulileyo kwaye isephantsi koxinzelelo oluphezulu lokuzala3,4,5,6,7,8,9,10,11,12.Ngokulinywa ngokubanzi kwe-NLL, ukulandelelana kwefungi ye-pathogenic yavelisa iindawo ezintsha zezolimo kwaye yabangela izifo ezitsha ezitshabalalisa izityalo. Eyona nto yayiphawuleka kumafama elupine nakubalimi yaba yinkangeleko ye anthracnose, ebangelwa yintsholongwane yokungunda, Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13. Eyona nto yayiphawuleka kumafama elupine nakubalimi yaba yinkangeleko ye anthracnose, ebangelwa yintsholongwane yokungunda, Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13. Наиболее примечательным для фермеров и селекционеров люпина было появление антракноза, вызванного патогенным грибком Nitrogendorn (3) Okona kuphawuleka kumafama welupine nakubalimi yaba kukuvela kwe anthracnose ebangelwa yintsholongwane yokungunda iColletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13.对于羽扇豆农民和饲养者來说，最引人注目的是炭疽病的起现，它是由病原真菌Colletotrichum lupini).对于羽扇豆农民和饲养者來說，最引人注目的是炭疽病的出现，它是由病原真菌Colletotrichum lupini). Наиболее поразительным для фермеров и селекционеров люпина является появление антракноза, вызываемого патогенгеным гриtricбком Colle . Okona kutsala umdla kumafama welupine nakubalimi kukuvela kwe anthracnose ebangelwa yintsholongwane yokungunda iColletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13.Iingxelo zokuqala zesi sifo zavela eBrazil nase-United States, zineempawu eziqhelekileyo ezibonakala ngo-1912 nango-1929 ngokulandelelanayo.Noko ke, emva kweminyaka emalunga nama-30, le pathogen yabizwa ngokuba yiColletotrichum gloeosporioides (Penz.) Penz. & Sacc., teleomorph Glomerella cingulata (Stoneman) Spauld. & Sacc., teleomorph Glomerella cingulata (Stoneman) Spauld. & Sacc., телеоморф Glomerella cingulata (Stoneman) Spauld. & Sacc., teleomorph of Glomerella cingulata (Stoneman) Spauld. & Sacc., 有目的形态的Glomerella cingulata (Stoneman) Spauld. & Sacc., 有目的形态的Glomerella cingulata (Stoneman) Spauld. & Sacc., Glomerella cingulata (Stoneman) Spauld в Целенаправленной морфологии. & Sacc., Glomerella cingulata (Stoneman) Spauld kwiMorphology ekuJoliswe kuyo. & H. Schrenk,. & H. Schrenk,.kunye noH. Schrenk. & H.施伦克,. & H.施伦克,.kunye noH. Schlenk,.I-phenotyping yesifo sokuqala esenziwe phakathi kwekhulu le-20 ibonise ukuxhathisa kwi-NLL kunye ne-yellow lupine (L. luteus L.) accessions, kodwa yonke i-lupine emhlophe (L. albus L.) i-accessions evavanyiweyo yayichaphazeleka kakhulu15,16.Uphononongo lubonise ukuba ukuphuhliswa kwe-anthracnose kuhambelana nokunyuka kwemvula (umswakama womoya) kunye nobushushu (kuluhlu lwe-12-28 ° C), okukhokelela ekwaphuleni ukuchasana kumaqondo okushisa aphezulu17, 18. Enyanisweni, ixesha elifunekayo ukuze i-conidia intshule kwaye isifo siqale , yayiphindwe kane mfutshane kwi-24 ° C (kwi-12 ° C (iiyure ze-12) ngaphantsi kweeyure ze-12 ze-12 C (iiyure ze-12 eziphezulu).Ngaloo ndlela, ukufudumala kwehlabathi okuqhubekayo kuye kwabangela ukusasazeka kwe-anthracnose.Nangona kunjalo, esi sifo sabonwa eFransi (1982) nase-Ukraine (1983) njengesandulela sesoyikiso esizayo, kodwa ngokucacileyo sasingahoywa lishishini le-lupine ngelo xesha20,21.Kwiminyaka embalwa kamva, esi sifo sitshabalalisayo sanwenwa kwihlabathi liphela kwaye sachaphazela namazwe avelisa ilupine anjengeOstreliya, iPoland neJamani22,23,24.Ukulandela ukuqhambuka kwe-anthracnose phakathi kwiminyaka yoo-1990, ukuhlolwa okubanzi kubangele ukuchongwa kwabanikeli abaninzi abaxhathisayo kwiisampulu ze-NLL19.Ukuxhathisa kwe-NLL kwi-anthracnose kulawulwa ngama-alleles amabini ahlukeneyo ahlukeneyo afumaneka kwimithombo eyahlukeneyo ye-germplasm: iLanr1 kwicultivar Tanjil kunye neWonga kunye ne-AnMan kwicultivar.Mandalay 25, 26. Ezi alleles zincedisana namanqaku emolekyuli axhasa ukukhethwa kwe-germplasm enganyangekiyo kwiiprogram zokuzalanisa25,26,27,28,29,30.Umgca wokuzala oxhathisayo 83A: 476 othwele i-Lanr1 allele wawelwa kunye nomgca wasendle othintekayo we-P27255 ukufumana i-RIL eyahlulahlula ukuxhatshazwa kwe-anthracnose, okwenza kube lula ukwabela i-Lanr1 locus kwi-chromosome NLL-1131, 32, 33, ukusuka kwi-map resistance marker ukuya kwi-maphu ehambelana ne-flanking-marketing. isakhelo, i-NLL iveze indawo yazo zontathu iialleles kwichromosome enye (NLL-11), kodwa kwizikhundla ezahlukeneyo29,34,35.Nangona kunjalo, ngenxa yenani elincinci le-RIL kunye nomgama omkhulu wemfuza phakathi kwabamakishi kunye ne-alleles ehambelanayo, akukho zigqibo ezinokuthenjwa ezinokuthathwa malunga neegene zabo eziphantsi.Kwelinye icala, ukusetyenziswa kwe-reverse genetics kwi-lupins kunzima ngenxa yesakhono sazo sohlaziyo oluphantsi kakhulu, nto leyo eyenza ukuguqulwa kofuzo kube nzima37.
Ukuphuhliswa kwe-germplasm yasekhaya ethwele i-allele efunwayo kwimeko ye-homozygous, efana ne-83A: 476 (Lanr1) kunye ne-Mandelup (AnMan), ivule umnyango wokufunda ukuxhathisa i-anthracnose ebusweni bobukho beendibaniselwano ezichasayo ze-alleles kuluntu lwasendle.Amathuba eendlela zemolekyuli.Thelekisa iimpendulo zokukhusela eziveliswa yi-genotypes ethile.Olu phononongo luvavanye impendulo yokuqala ye-transcriptome ye-NLL kugonyo lwe-C. lupini.Okokuqala, iphaneli ye-germplasm ye-NLL yaseYurophu equlethe imigca ye-215 ihlolwe kusetyenziswa iimpawu ze-molecular eziphawula i-Lanr1 kunye ne-AnMan alleles.I-Anthracnose phenotyping emva koko yenziwa kwimigca ye-50 ye-NLL, ekhethiweyo ngaphambili kwiimpawu ze-molecular, phantsi kweemeko ezilawulwayo.Ngokusekwe kolu vavanyo, imigca emine eyahlukileyo kuxhathiso lwe-anthracnose kunye ne-Lanr1/AnMan allelic composition yakhethelwa ukuchazwa kohlobo lokhuselo olwahlukileyo kusetyenziswa iindlela ezimbini ezincedisanayo: ukulandelelana kwe-RNA ephezulu kunye ne-PCR yexesha lokwenyani.
Ukuhlolwa kweseti ye-NLL germplasm (N = 215) eneziphawuli i-Lanr1 (Anseq3 kunye ne-Anseq4) kunye ne-AnMan (Anseq4) kunye ne-AnMan (AnManM1) ibonise ukuba ngumgca omnye kuphela (95726, kufuphi ne-Salamanca-b) owandisa "ukumelana" ne-allele kuzo zonke iimpawu ze-'susceptible's zonke (~ 73.5%) imigca.Imigca elishumi elinesithathu ivelise iiallele ezimbini "ezixhathisayo" ze-marker ye-Lanr1, kwaye imigca esi-8 yavelisa "resistant" alleles of Lanr1.isiphawuli."Ukuchasa" ialle ye-AnMan marker (iTheyibhile eyoNgezelelweyo S1).Imigca emibini ibiyi-heterozygous ye-marker ye-Anseq3 kunye ne-heterozygous enye ye-AnManM1 marker.Imigca ye-42 (19.5%) iphethe izigaba ezichaseneyo ze-Anseq3 kunye ne-Anseq4 alleles, ebonisa ukuphindaphinda okuphindaphindiweyo phakathi kwezi loci zimbini.I-Anthracnose phenotypes phantsi kweemeko ezilawulwayo (i-Supplementary Table S2) ibonakalise ukuhluka kokumelana ne-genotypes evavanyiweyo, ebonakaliswe kubunzima be-anthracnose.Umahluko kumanqaku aphakathi ukusuka kwi-1.8 (enganyangekiyo ngokuphakathi) ukuya kwi-6.9 (inokuchaphazeleka) kunye nokwahlukana kobunzima bezityalo ukusuka kwi-0.62 (enokuchaphazeleka) ukuya kwi-4.45 g (i-resistant). Kwakukho ukulungelelaniswa okubalulekileyo phakathi kwamaxabiso abonwe kwiimpendulo ezimbini zovavanyo (i-0.51 yamanqaku obunzima besifo, i-P = 0.00017 kunye ne-0.61 yobunzima besityalo, i-P <0.0001) kunye naphakathi kwezi zimbini iiparameters (- 0.59 kunye - 0.77, P0100). Kwakukho ulungelelwaniso olubalulekileyo phakathi kwamaxabiso ajongwe kwiimpendulo ezimbini zovavanyo (i-0.51 yamanqaku obunzima besifo, P = 0.00017 kunye ne-0.61 yobunzima besityalo, P <0.0001) kunye naphakathi kwezi parameters zimbini (- 0.59 kunye - 0.0.0. Выявлена ​​достоверная корреляция между значениями, наблюдаемыми в двух повторностях эксперимента (0,51 для баллов тяжезд00, 11 массы растения, P < 0,0001), а также между этими двумя параметрами (-0,59 и -0,77, Р < 0,0001) 0,0001). Ulungelelwaniso olubalulekileyo lufunyenwe phakathi kwamaxabiso ajongwe ngokuphindaphindiweyo kovavanyo (0.51 kumanqaku obunzima besifo, P = 0.00017 kunye ne-0.61 yobunzima besityalo, P <0.0001), kunye naphakathi kwezi parameters zimbini (- 0.59 kunye -0.77, P0 <0.0) 0.0).在两次重复实验中观察到的值之间存在显着相关性（疾病严重程度评分為0.51,P = 70.000. 0.0001）以及這两个参数之间（- 0.59 kunye- 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 分为 分为 0.51 ， p = 0.00017 ， 植物 为 为 0.61 ， p <0.0001） 以及 两 个 参数 之间 （（（（- 0.59 和– 0.59 和– 0.59 和– 0.59 和- 0.77，P < 0.0001)。 Наблюдалась значительная корреляция между значениями, наблюдаемыми в двух повторностях (оценка тяжести заболевания 0,51, 60, 10,60 P <0,0001), и между этими двумя параметрами (-0,59 и -0,0001) 0,77, P <0,0001. Kwakukho ulungelelwaniso olubalulekileyo phakathi kwamaxabiso ajongwe ngokuphindiweyo (ubunzima besifo amanqaku 0.51, P = 0.00017 kunye nobunzima besityalo 0.61, P <0.0001) naphakathi kwezi parameters zimbini (-0.59 kunye -0 .0001) 0.77, P <0.000 ).Iimpawu eziqhelekileyo ezibonwa kwizityalo ezithintekayo ziquka i-kinking kunye nokujika kwesiqu esifana nesakhiwo "somalusi," kulandelwa izilonda ezi-oval ezine-orenji / pink sporozoites (Fig. 1).Ukufikelela kwe-Australian ephethe i-Lanr1 (83A: 476 kunye ne-Tanjil) kunye ne-AnMan (i-Mandelup) i-general resistant, i-0.0331 kunye ne-0.0036).Eminye imigca ekwaphethe “resistant” Lanr1 kunye/okanye iAnMan alleles ibonisa iimpawu zesi sifo.
Kuyathakazelisa ukuba imigca embalwa ye-NLL ayinayo nayiphi na i-marker "resistant" ye-allele ibonise izinga eliphezulu lokumelana ne-anthracnose (enokuthelekiswa okanye ephezulu kune-Lanr1 okanye i-AnMan genotypes), njenge-Boregine (ixabiso le-P <0.0001 kuzo zombini iiparameters), i-Bojar (ixabiso le-P <0.0001 kunye ne-5 yexabiso le-Population ye-7 ye-P9. I-0.0001 yamanqaku kwaye ingabalulekanga kubunzima). Kuyathakazelisa ukuba imigca embalwa ye-NLL ayinayo nayiphi na i-marker "resistant" ye-allele ibonise izinga eliphezulu lokumelana ne-anthracnose (enokuthelekiswa okanye ephezulu kune-Lanr1 okanye i-AnMan genotypes), njenge-Boregine (ixabiso le-P <0.0001 kuzo zombini iiparameters), i-Bojar (ixabiso le-P <0.0001 kunye ne-5 yexabiso le-Population ye-7 ye-P9. I-0.0001 yamanqaku kwaye ingabalulekanga kubunzima). Интересно, что несколько линий NLL, лишенных какого-либо «резистентного» маркерного аллеля, показали высокий уровень е высокий, чем для генотипов Lanr1 или AnMan), таких как Boregine (значение P <0,0001 для обоих параметров), Bojar (значение P < 0,00001,0001 стения) и популяции B-549/79b (значение P <0,0001 для оценки и незначимо для массы). Kuyathakazelisa ukuba imigca emininzi ye-NLL ayinayo nayiphi na i-marker 'resistant' allele ibonise izinga eliphezulu lokuchasana ne-anthracnose (enokuthelekiswa okanye ephezulu kune-Lanr1 okanye i-AnMan genotypes), njenge-Boregine (ixabiso le-P <0.0001 kuzo zombini iiparameters), i-Bojar (ixabiso le-P <0.0000 kunye ne-7 yabantu kunye ne-9b yesityalo se-9b) kunye ne-9 yesityalo se-9b) Ixabiso le-P <0.0001 yokuvavanya kwaye ayibalulekanga 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（帎LanAn1更高），例如Boregine（两个参数的P值< 0.0001）、Bojar（P 值<得分為0.0001，植物重和量為0/B-9得（9得（9得（9)值< 0.0001，重量不显着）。 Kuyathakazelisa ukuba ezinye iinkqubo ze-NLL ezingenazo iimpawu "ze-antigenic" zibonisa ukuchasana okuphezulu okuthe tye (okulingana ne-Lanr1 okanye i-AnMan genes okanye ngaphezulu), njenge-Boregine (zombini iiparamitha P <0.0001), i-Bojar (P ixabiso <0.0001, ubunzima besityalo 0.001) kunye ne-490 yexabiso le-7b ebaluleke kakhulu, i-0 000 0.001 (0.001) kunye ne-490 ephawulekayo ye-1b, ubunzima be-0. Интересно, что некоторые линии NLL, лишенные каких-либо маркерных аллелей «резистентности», показали высокие уровни устойчанизы, чем у генотипов Lanr1 или AnMan), такие как Boregine (значение P для обоих параметров <0,0001), Bojar (значение P <0,0001, масса раст00лцои 5/9) енка P-значение <0,0001, масса незначительна). Into ebangela umdla kukuba, imigca ethile ye-NLL iswelekile 'I-Arrekence' ye-Alleles ibonise amanqanaba aphezulu okuxhathisa i-anthracnose (i-0.000), i-Biner ye-Witter (i-0.000) kunye ne-B-0.0001, ubunzima obungabalulekanga).Lo mcimbi ucebisa ukuba kunokwenzeka ukuba kubekho inoveli yomthombo wofuzo wokuchasa, ucacisa ukungabikho kolungelelwaniso phakathi kwe-marker genotypes kunye ne-phenotypes yesifo (amaxabiso e-P ukusuka ~ 0.42 ukuya ~ 0.98).Ngaloo ndlela, uvavanyo lwe-Kolmogorov-Smirnov lubonise ukuba idatha malunga nokuxhathisa i-anthracnose yayiye yasasazwa ngokuqhelekileyo kumanqaku (i-P-values ​​​​0.25 kunye ne-0.11) kunye nobunzima bezityalo (i-P-values ​​0.47 kunye ne-0.55), iphakamisa ukuba ndiqikelele ukuba i-alleles ezininzi kune-Lanr1 kunye ne-AnMan zibandakanyeka.
Ngokusekelwe kwiziphumo zokuhlola ukuchasana kwe-anthracnose, imigca ye-4 ikhethwe ukuhlalutya i-transcriptome: 83A: 476, i-Boregine, i-Mandelup, kunye ne-Population 22660. Le migca iphinde ihlolwe ukuxhathisa i-anthrax kwiimvavanyo ze-inoculation ngokulandelelana kwe-RNA, ngaphandle kokuba zifana novavanyo lwangaphambili.Amaxabiso amanqaku aye ngolu hlobo lulandelayo: Boregin (1.71 ± 1.39), 83A: 476 (2.09 ± 1.38), Mandelup (3.82 ± 1.42) kunye nabemi 22660 (6.11 ± 1.29).
I-Illumina NovaSeq 6000 protocol izuze umyinge we-40.5 Mread pairs kwisampuli nganye (29.7 ukuya ku-54.4 Mreads) (Itheyibhile eyoNgezelelweyo ye-S3).Amanqaku olungelelwaniso kulandelelwano lwereferensi asuka kuma-75.5% ukuya kuma-88.6%.Umndilili wolungelelwaniso lwedatha yokubala efundiweyo phakathi kokwahluka kovavanyo phakathi kokuphindaphindwa kwezinto eziphilayo ukusuka kwi-0.812 ukuya kwi-0.997 (ithetha i-0.959). Ngaphandle kwee-genes ze-35,170 ezihlalutyiweyo, i-2917 ingabonakali inkcazo, kwaye ezinye ii-genes ze-4785 zibonakaliswe kwinqanaba elingenakulinganiswa (isiseko sithetha <5). Ngaphandle kwee-genes ze-35,170 ezihlalutyiweyo, i-2917 ingabonakali inkcazo, kwaye ezinye ii-genes ze-4785 zibonakaliswe kwinqanaba elingenakulinganiswa (isiseko sithetha <5). Из 35 170 проанализированных генов 2917 не проявляли экспрессии, а остальные 4785 генов экспрессировались на ненезнабрессии. Kwiintlobo ze-35,170 ezihlalutyiweyo, i-2917 ayizange ibonise inkcazo, kwaye i-4785 i-gene esele ibonakaliswe kwinqanaba elingenakulinganiswa (isiseko sithetha <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. Kwiintlobo ze-35,170 ezihlalutyiweyo, i-2917 ayizange ibonakaliswe kwaye i-4785 eseleyo ye-gene yayinentetho engafanelekanga (isiseko sithetha <5).Ngaloo ndlela, inani leezakhi zofuzo ezicatshangelwayo zichazwe (isiseko sithetha ≥ 5) ngexesha lovavanyo lwaluyi-27,468 (78.1%) (i-Supplementary Table S4).
Ukususela kwixesha lokuqala, yonke imigca ye-NLL iphendule kwi-inoculation ye-C. lupini (uxinzelelo lwe-Col-08) ngokucwangcisa kwakhona i-transcriptome (Itheyibhile 1), nangona kunjalo, ukungafani okuphawulekayo kwabonwa phakathi kwemigca.Ngaloo ndlela, umgca wokuchasa i-83A: i-476 (ephethe i-Lanr1 gene) ibonise i-reprogramming ephawulekayo ye-transcriptome kwindawo yokuqala (i-6 hpi) kunye nokunyuka kwe-31-69-fold of the single up and down-genes xa kuthelekiswa namanye amanqaku exesha ngeli xesha.Ukongezelela, le ncopho yayihlala ixesha elifutshane, njengoko ukubonakaliswa kweegenes ezimbalwa kuphela kwahlala kutshintshile kakhulu kwixesha lesibini (12 hpi).Okubangel 'umdla kukuba, i-Boregine, ekwabonise inqanaba eliphezulu lokuchasa kuvavanyo loxhulumaniso, ayizange iphinde iphinde iphinde iphinde ibhalwe ngexesha lovavanyo.Nangona kunjalo, inani leejeni ezichazwe ngokwahlukileyo (DEG) lalifana ne-Boregine kunye ne-83A: 476 kwi-12 HPI.Bobabini i-Mandelup kunye nabemi be-22660 babonisa i-DEG iphakamileyo kwixesha lokugqibela (48 l / s), ebonisa ukulibaziseka okuhambelanayo kwiimpendulo zokukhusela.
Ngenxa yokuba i-83A: i-476 yafumana i-reprogramming enkulu ye-transcriptome ekuphenduleni i-C. lupini kwi-6 HPI xa kuthelekiswa nayo yonke eminye imigca, ~ 91% yee-DEGs eziqatshelwe ngeli xesha zithe ngqo kumgca (umzobo 1).Nangona kunjalo, bekukho ukugqithelana kwiimpendulo zangaphambili phakathi kwemigca yokufunda, njenge-68.5%, 50.9%, kunye ne-52.6% ye-DEG e-Boregine, Mandelup, kunye nabemi be-22660, ngokulandelanayo, zidibene nezo zifunyenwe kwi-83A: 476 kumanqaku athile ngexesha.Nangona kunjalo, ezi DEGs zibalwa kuphela iqhezu elincinci (0.97-1.70%) yazo zonke ii-DEGs ngoku zichongiwe zisebenzisa i-83A: 476.Ukongezelela, ii-DEG ze-11 ezivela kuyo yonke imigca zihambelana ngeli xesha (i-Supplementary Tables S4-S6), kubandakanywa amacandelo aqhelekileyo kwiimpendulo zokukhusela izityalo: iprotheni yokudlulisa i-lipid (i-TanjilG_32225), i-endoglucan-1,3-β-glucoside enzyme (TanjilG_23384), i-protein ezimbini ze-stress-inducible-inducible i-3G35 i-Tanjil-3 i-Tanjil-3 i-Tanjil-3 (i-TanjilG3384) 31), iprotheni ye-latex esisiseko (i-TanjilG_32352), kunye ne-glycine-rich-rich structural cell wall proteins (TanjilG_19701 kunye ne-TanjilG_19702).Kwakhona kwakukho ukugqithisa okuphezulu kwiimpendulo ze-transcriptome phakathi kwe-83A: i-476 kunye ne-Boregine kwi-24 HPI (i-16-38% ye-DEG iyonke) kunye naphakathi kwe-Mandelup kunye ne-Population 22660 kwi-48 HPI (i-14-20% ye-DEG iyonke).
Umzobo weVenn obonisa inani lemizila yemfuza ebonakaliswe ngokwahlukileyo (DEG) kwimigca ye-lupine enamagqabi (NLL) etofwe nge-Colletotrichum lupini (uxinzelelo lwe-Col-08 olufunyenwe kwiindawo zelupine e-Wierzhenice, ePoland, ngo-1999).Imigca ye-NLL ehlalutyiweyo yayiyi: 83A: 476 (i-resistant, ephethe i-Lanr1 allele), i-Boregine (i-resistant, imvelaphi yofuzo engaziwa), i-Mandelup (i-resistant resistant, ithwele i-AnMan allele) kunye nabemi be-22660 (ixhaphake kakhulu).Isishunqulelo esithi hpi simela iiyure emva kogonyo.Amaxabiso angu-Zero asusiwe ukwenza lula igrafu.
Isethi yeejene ezigqithisiweyo kwi-6 hpi yahlalutywa ubukho be-canonical R gene domains (Itheyibhile eyoNgezelelweyo ye-S7).Olu pho nonongo lubonakalise ukufakwa kwe-transcriptome ye-classical disease resistance genes kunye ne-NBS-LRR domains kuphela kwi-83A: 476.Le seti yayine-TIR-NBS-LRR yofuzo enye (tanjilg_05042), iijini ezintlanu zeCC-NBS-LRR (tanjilg_06165, tanjilg_06162, tanjilg_22773, tanjilg_22640, kunye ne-tanjilg_16162) kunye ne-Four NBS-L1 I-RRE (tanjilg_16162) kunye ne-NBS-Lrr eZine (tanjilg_16162) kunye ne-NBS-LRR eZine (TANJILG_16162).Yonke le mizila yemfuza ineendawo eziphefumlelweyo ezilungelelaniswe ngokolandelelwano olugciniweyo.Ukongeza kwiijene zesizinda se-NBS-LRR, ezininzi ze-RLL kinases zenziwe zasebenza kwi-6 hpi, ezizezi enye e-Boregine (TanjilG_19877), ezimbini e-Mandelup (TanjilG_07141 kunye ne-TanjilG_19877) kunye nabemi be-22660 (TanjilG_068G3) kunye ne-2 e-TanjilG_09014 kunye ne-2 Tanjil 19014_1 kunye ne-2 Tanjil 19014 6.
I-Gene enentetho eguquguqukayo kakhulu ekuphenduleni i-inoculation kunye ne-C. lupini (i-strain Col-08) yayiphantsi kohlalutyo lwe-Gene Ontology (GO) (i-Supplementary Table S8). Ixesha eligqithisiweyo kakhulu lenkqubo yezinto eziphilayo "GO: 0006952 impendulo yokukhusela" ebonakala kwi-6 ngaphandle kwe-16 (ixesha × umgca) ukudibanisa nokubaluleka okuphezulu (ixabiso le-P <0.001) (Umfanekiso 2). Ixesha eligqithisiweyo kakhulu lenkqubo yezinto eziphilayo "GO: 0006952 impendulo yokukhusela" ebonakala kwi-6 ngaphandle kwe-16 (ixesha × umgca) ukudibanisa nokubaluleka okuphezulu (ixabiso le-P <0.001) (Umfanekiso 2). Наиболее часто чрезмерно представленным термином биологического процесса был GO: 0006952 защитный отвенным термином биологического процесса был GO: 0006952 комбинаций с высокой значимостью (значение P <0,001) (рис. 2). Elona gama lenkqubo yebhayoloji elixhaphakileyo laliyi-'GO: 0006952 impendulo yokhuselo', evele kwi-6 ye-16 (ixesha × umnombo) indibaniselwano nokubaluleka okuphezulu (ixabiso le-P <0.001) (Umfanekiso 2).最常被过度代表的生物过程术语是“GO:0006952 防御反应”着性 (P <0.001) (图2) Ixesha elimele kakhulu lenkqubo yezinto eziphilayo "GO: 0006952 impendulo yokukhusela", ebonakala kwi-6 ye-16 (时间× 线) indibaniselwano, ngokubaluleka okuphezulu (ixabiso le-P <0.001) (图2) . Наиболее часто чрезмерно представленным термином биологического процесса был GO: 0006952 Impendulo yoKhuselo», который появлялся 10 × 6 с высокой значимостью (значение P <0,001) (рис. 2). Elona gama lenkqubo yebhayoloji elixhaphakileyo laliyi-'GO: 0006952 Impendulo yoKhuselo', evele kwi-6 kwi-16 indibaniselwano (ixesha × umgca) ngokubaluleka okuphezulu (ixabiso le-P <0.001) (Umfanekiso 2).Eli gama laligqithisekile kumanqaku amabini exesha kwi-83A: 476 kunye ne-Boregine (6 kunye ne-24 hpi) kunye nexesha elinye kwi-Mandelup kunye ne-Population 22660 (12 kunye ne-6 hpi, ngokulandelanayo).Esi sisiphumo esilindelekileyo, sigxininisa impendulo ye-antifungal yemigca echasayo.Ukongezelela, i-83A: i-476 iphendule kwi-C. lupini ngokukhupha ngokukhawuleza izakhi zofuzo ezinxulumene nokuqhuma kwe-oxidative emele igama elithi "GO: 0055114 inkqubo ye-redox", ebonisa impendulo ethile yokukhusela, ngelixa i-Boregine ibonakalise iimpendulo ezithile zokukhusela, ezinxulumene negama elithi 'GO'.: 0006950 impendulo yoxinzelelo ".I-Population 22660 isebenze impendulo ethe tye yokuchasana ebandakanya i-metabolites yesibini, igxininisa inani eligqithisileyo lamagama "GO: 0016104 Inkqubo ye-triterpene biosynthesis" kunye ne "GO: 0006722 Inkqubo ye-triterpene metabolism" (zombini amagama akwiseti efanayo yofuzo), kuthathelwa ingqalelo iziphumo ze-Mandell kunye nokuhlaziywa kwe-Popure 2660. Ukongeza, ukusabela kwangoko 83A: 476 (6 hpi) kunye nokulibaziseka ukusabela kweMandelup kunye nabemi be-22660 kubandakanya igama elithi GO: 0015979 'photosynthesis' kunye nezinye iinkqubo zebhayoloji ezinxulumene nazo.
Amagama e-bioprocess gene ontology akhethwe kwinkcazo yeegenes ezivezwa ngokwahlukileyo ngexesha leempendulo ze-transcriptome ze-lupine enamagqabi amancinci (NLL) efakwe i-anthrax lupine (uhlobo lwe-Col-08 olufunyenwe kumasimi e-lupine e-Wierzhenice, e-Poland, ngo-1999) zibaxiwe kakhulu.Imigca ye-NLL ehlalutyiweyo yayiyi: 83A: 476 (i-resistant, ethwele i-homozygous Lanr1 allele), i-Boregine (i-resistant, imvelaphi engaziwayo yofuzo), i-Mandelup (i-resistant resistant, ithwele i-homozygous AnMan allele) kunye ne-population 22660 (i-susceptible).
Ngenxa yokuba olu pho nonongo lujolise ekuchongeni izakhi zofuzo ezifaka isandla ekuxhathiseni i-anthracnose, izakhi zofuzo ezinikezelwe kwimigaqo ethi GO "GO: 0006952 iimpendulo ezikhuselayo" kunye ne "GO: 0055114 iinkqubo ze-Redox" zahlaziywa nge-cut-offs ukususela ekubeni isiseko sithetha ≥ 30 ubuncinane kunye nomgca omnye.× inqaku ngexesha elidibanisa amaxabiso abalulekileyo elog2 (utshintsho olusongekileyo).Inani lemizila yemfuza edibana nale migaqo yayingama-65 kwi-GO:0006952 kunye ne-524 ye-GO:0055114.
I-83A: I-476 ibonise iincopho ezimbini ze-DEG ezichazwe ngegama elithi GO: 0006952, eyokuqala kwi-6 yemfuza nge-intshi nganye (i-64 genes, up and down regulation) kunye neyesibini kwi-24 genes nge-intshi nganye (i-15 genes, up regulation only).U-Boregine uphinde wabonisa ukuba i-GO: i-0006952 iphakame ngexesha elifanayo, kodwa nge-DEG engaphantsi (11 kunye ne-8) kunye nokusebenza okukhethiweyo.I-Mandeloop ibonise iincopho ezimbini ze-GO: i-0006952 kwi-12 kunye ne-48 HPI, zombini ithwele i-gene ye-12 (eyokuqala i-genenes esebenzayo, kunye neyesibini i-genenes ecinezelayo kuphela), ngelixa i-22660 yabantu kwi-6 HPI (i-13 genes) yayinobukhulu obukhulu bokwanda kwe-peak.ummiselo.Kufuneka kuqatshelwe ukuba i-96.4% ye-GO: i-0006952 DEG kwezi zincopho zazinohlobo olufanayo lokuphendula (phezulu okanye phantsi), ebonisa ukugqithiswa okubalulekileyo kwiimpendulo zokukhusela nangona kukho ukungafani kwinani leegene ezibandakanyekayo.Elona qela likhulu lolandelelwano olunxulumene negama elithi GO:0006952 lifakela iProtein ye-Stress-Associated Message Protein 22 (SAM22-like), eyeklasi ye-10 ye-pathogenesis-associated protein (PR-10) protein clade kunye ne-core protein latex.iprotheni efanayo (i-MLP-like)) iprotheni (Umfanekiso 3).La maqela mabini ahluka ngohlobo lwentetho kunye nesalathiso sempendulo.I-gene encoding iiprotheyini ezinjenge-SAM22-ezifana ne-SAM22 zibonise ukunyanzeliswa okuhambelanayo kunye nokubalulekileyo kwixesha elidlulileyo (6 okanye i-12 hpi) kwaye ngokuqhelekileyo ayiphenduli ekupheleni kovavanyo (48 hpi), ngelixa iiprotheni ezinjenge-MLP zibonise ukulungelelaniswa kwi-6 hpi.hpi.I-83A: 476 kunye ne-Mandelup kwi-48 hp / in, phantse zonke ezinye iindawo zedatha azizange ziphendule.Ukongeza, iiyantlukwano kwiiprofayili zenkcazo ye-SAM22-efana neprotheyini yejini ilandele ukuguquguquka okubonwayo ekuchaseni i-anthracnose, njengoko imigca exhathisayo yayinamanqaku amaninzi exesha elibangela kakhulu ezi genes kuneendlela ezinokuthi zichaphazeleke.Enye i-LlR18A / B-efana ne-PR-10 i-gene ibonise ipateni yokubonakalisa efana kakhulu ne-SAM22-like protein gene.
Amacandelo aphambili enkqubo yebhayoloji igama elithi "GO:0006952 Impendulo yoKhuselo" kunye neepatheni zokubonisa iijene zabaviwa beLanr1 kunye ne-AnMan alleles ichongiwe.Isikali se-Log2 simele amaxabiso e-log2 (utshintsho olusongekileyo) phakathi kokutofa (i-Colletotrichum lupini, i-strain Col-08, efunyenwe kumasimi e-lupine, e-Wizhenica, e-Poland, ngo-1999) kunye nokulawula (i-sham-inoculated) izityalo ngexesha elifanayo.Le migca ilandelayo ye-lupine yamagqabi ihlalutyiweyo: 83A: 476 (i-resistant, ethwele i-homozygous Lanr1 allele), i-Boregine (i-resistant, imvelaphi yofuzo engaziwayo), i-Mandelup (ixhathise ngokuphakathi, ithwele i-homozygous AnMan allele), kunye ne-Population 22660 (inokuchaphazeleka).
Ukongeza, iiprofayili zenkcazo yomgqatswa we-RNA-seq kuhlobo lwe-Lanr1 (TanjilG_05042) kunye ne-AnMan (TanjilG_12861) zavavanywa (umzobo 3).I-gene ye-TanjilG_05042 ibonise impendulo ebalulekileyo (ukusebenza) kwi-83A: i-476 kuphela ngexesha lokuqala (6 hpi), ngelixa i-TanjilG_12861 yayibalulekile kwi-Mandeloop kuphela ngamaxesha amabini: i-6 hpi (umgaqo ophantsi) kunye ne-24 hpi (6 hpi).kunye.).iyahlengahlengiswa) ).
Iigenes ezigqithisiweyo kakhulu kwixesha elithi GO: 0055114 "inkqubo ye-redox" yayiyi-gene encoding cytochrome P450 proteins kunye ne-peroxidase (Fig. 4).Kwiisampuli ezihlukanisiweyo kwi-83A: i-476 kwi-6 HPI, ubuninzi okanye ubuncinci be-log2 (utshintsho olutshintshileyo) amaxabiso (kwi-86.6% yezakhi zofuzo) zibonwa ngokubanzi phakathi kwezityalo ezigonyelwe kunye nokulawula, zigxininisa impendulo ephezulu yale genotype kwi-inoculating sex.I-83A: i-476 ibonise i-GO ebaluleke kakhulu: 0055114 DEG kwi-6 hpi (503 genes), ngelixa ezinye imigca kwi-48 hpi (i-Boregine, i-31 genes; i-Mandelup, i-85 genes; kunye ne-Population 22660, i-78 genes)).Kwiintlobo ezininzi ze-GO: intsapho ye-0055114, iintlobo ezimbini zeempendulo zokugonywa (ukusebenza kunye nokuthintela) zabonwa.Kuyathakazelisa ukuba ukuya kuthi ga kwi-97.6% yee-DEGs ezichongiweyo kwigama elithi GO: 0055114 eMandelupe kwi-48 hp Olu qwalaselo lubonisa ukuba, nangona umlinganiselo omncinci kakhulu (oko kukuthi, inani le-redox eguquguqukayo, i-85 kunye ne-503), iphethini yokulibaziseka kweempendulo ze-transcriptome kwi-6 impendulo ye-mandeloup yokuqala ye-mandeloup ye-6 ye-mandeloup ye-8.Kwi-Boregine kunye ne-Population 22660, le ngqungquthela iphantsi kwi-51.6% kunye ne-75.6%, ngokulandelanayo.
Iipateni zokubonakaliswa kwezinto eziphambili zexesha lenkqubo yezinto eziphilayo "GO: 0055114 inkqubo ye-Redox" yatyhilwa.Isikali se-Log2 simele amaxabiso e-log2 (utshintsho olusongekileyo) phakathi kokutofa (i-Colletotrichum lupini, i-strain Col-08, efunyenwe kumasimi e-lupine, e-Wizhenica, e-Poland, ngo-1999) kunye nokulawula (i-sham-inoculated) izityalo ngexesha elifanayo.Le migca ilandelayo ye-lupine yamagqabi ihlalutyiweyo: 83A: 476 (i-resistant, ethwele i-homozygous Lanr1 allele), i-Boregine (i-resistant, imvelaphi yofuzo engaziwayo), i-Mandelup (ixhathise ngokuphakathi, ithwele i-homozygous AnMan allele), kunye ne-Population 22660 (inokuchaphazeleka).
I-83A: 476 Iimpendulo ze-Transcriptomic kwi-inoculation kunye ne-C. lupini (i-strain Col-08) iphinde ibandakanye ukuthuliswa okucwangcisiweyo kweejeni ezibhekiselele kwigama elithi GO: 0015979 "photosynthesis" kunye nezinye iinkqubo ze-biological ezinxulumene (FIG. 5).Le GO: 0015979 DEG iseti iqulethe i-105 yezakhi zofuzo eziye zacinezelwa kakhulu kwi-6 hpi kwi-83A: 476.Kule subset, i-37 genes nayo yathotyelwa phantsi e-Mandelup kwi-48 HPI kunye ne-35 ngexesha elifanayo kwi-22660 yabantu, kubandakanywa ne-19 DEGs eziqhelekileyo kuzo zombini i-genotypes.Akukho zi-DEGs ezinxulumene ne-GO: i-0015979 isebenze kakhulu kuyo nayiphi na indibaniselwano (ixesha lomgca x).
Iipateni zokubonakaliswa kwezinto eziphambili zexesha lenkqubo yezinto eziphilayo "GO: 0015979 Photosynthesis" yatyhilwa.Isikali se-Log2 simele amaxabiso e-log2 (utshintsho olusongekileyo) phakathi kokutofa (i-Colletotrichum lupini, i-strain Col-08, efunyenwe kumasimi e-lupine, e-Wizhenica, e-Poland, ngo-1999) kunye nokulawula (i-sham-inoculated) izityalo ngexesha elifanayo.Le migca ilandelayo ye-lupine yamagqabi ihlalutyiweyo: 83A: 476 (i-resistant, ethwele i-homozygous Lanr1 allele), i-Boregine (i-resistant, imvelaphi yofuzo engaziwayo), i-Mandelup (ixhathise ngokuphakathi, ithwele i-homozygous AnMan allele), kunye ne-Population 22660 (inokuchaphazeleka).
Ngokusekwe kwiziphumo zohlalutyo lokubonisa umahluko kunye nokubandakanyeka ekucingeni ukuba kubandakanyeke kwiimpendulo ezichasene nomngundo we-pathogenic, le seti yeejini ezisixhenxe zikhethelwe ubungakanani beeprofayili zokubonisa nge-PCR yexesha lokwenyani (iTheyibhile eyoNgezelelweyo ye-S9).
I-puative protein gene i-TanjilG_10657 yakhuthazwa kakhulu kuyo yonke imigca efundiweyo kunye namanqaku exesha xa kuthelekiswa nolawulo (ukulinganisa) izityalo (Iitheyibhile ezongezelelweyo S10, S11).Ukongeza, inkangeleko yenkcazo ye-TanjilG_10657 ibonise indlela eyandayo ngexesha lovavanyo kuyo yonke imigca.I-Population 22660 ibonise uvakalelo oluphezulu lwe-TanjilG_10657 kwi-inoculation kunye ne-activation ye-114-fold kunye nenqanaba eliphezulu lokubonisa isihlobo (4.4 ± 0.4) kwi-24 HPI (Fig. 6a).I-PR10 LlR18A i-protein gene TanjilG_27015 nayo ibonise ukusebenza kuyo yonke imigca kunye namanqaku exesha, ngokubaluleka kwezibalo kwiindawo ezininzi zedatha (umzobo 6b).Ngokufana ne-TanjilG_10657, inqanaba eliphezulu lokubonisa isalamane se-TanjilG_27015 yabonwa kwi-22660 yabantu abangenayo kwi-24 HPI (19.5 ± 2.4).I-asidi ye-endochitinase gene TanjilG_04706 yayilawulwa kakhulu kuyo yonke imigca kwaye ngamaxesha onke amanqaku ngaphandle kwe-Boregine 6 hpi (Umfanekiso 6c).Kwanyanzeliswa ngamandla kwinqanaba lokuqala (6 HPI) kwi-83A: 476 (ngamaxesha angama-10.5) kwaye yanda ngokulinganayo kwezinye imigca (ngamaxesha angama-6.6-7.5).Ngexesha lovavanyo, intetho ye-TanjilG_04706 yahlala kumanqanaba afanayo kwi-83A: 476 kunye ne-Boregine, ngelixa eMandelup kunye nabemi be-22660 yanda kakhulu, yafikelela 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 ezimbini zokuqala (i-6 kunye ne-12 hpi) kuyo yonke imigca ngaphandle kwabemi be-22660 (umzobo 6d).Awona manqanaba aphezulu abonisa ukunxibelelana kwe-TanjilG_23384 abonwa kwinqanaba lesibini (12 hpi) eMandelup (2.7 ± 0.3) kunye ne-83A: 476 (1.5 ± 0.1).Kwi-24 HPI, i-TanjilG_23384 intetho yayiphantsi kakhulu kuyo yonke imigca efundiweyo (ukusuka kwi-0.04 ± 0.009 ukuya kwi-0.44 ± 0.12).
Iiprofayili zokuvakalisa iigenes ezikhethiweyo (ag) ezivezwe ngobungakanani bePCR.Amanani 6, 12 kunye ne-24 amele iiyure emva kokugonywa.I-LanDExH7 kunye ne-LanTUB6 yofuzo zisetyenziselwe uhlengahlengiso kwaye i-LanTUB6 isetyenziselwe ulungelelwaniso phakathi kothotho.Imivalo yempazamo imele ukutenxa okusemgangathweni okusekwe kwiikopi ezintathu zebhayoloji, nganye kuzo ingumndilili wokuphindaphindwa kwezobugcisa ezintathu. Ukubaluleka kwamanani okungafaniyo kumanqanaba okuvakalisa phakathi kwe-inoculated (Colletotrichum lupini, i-strain Col-08, efunyenwe ngo-1999 kwintsimi ye-lupine e-Wierzenica, ePoland) kunye nezityalo zokulawula (i-mock-inoculated) ziphawulwe ngaphezu kwamanqaku edatha (*P ixabiso <0.05, **Ixabiso le-P ≤0 0.0 P). Ukubaluleka kwamanani okungafaniyo kumanqanaba okuvakalisa phakathi kwe-inoculated (Colletotrichum lupini, i-strain Col-08, efunyenwe ngo-1999 kwintsimi ye-lupine e-Wierzenica, ePoland) kunye nezityalo zokulawula (i-mock-inoculated) ziphawulwe ngaphezu kwamanqaku edatha (*P ixabiso <0.05, **Ixabiso le-P ≤0 0.0 P). Статистическая значимость различий в уровнях экспрессии между инокулированными (Colletotrichum lupini, штамм Col-08, получен в 1999 Плавы, получен) а) и контрольными (ложно инокулированными) растениями отмечена над точками данных (*значение P < 0,05, **значение P ≤ 0,01, ***◄10 0,01, *** значение 0). Umahluko obonakalayo kumanqanaba enkcazo phakathi kwe-inoculated (i-Colletotrichum lupini, i-strain Col-08, efunyenwe kwi-1999 kwintsimi ye-lupine e-Wierzhenice, ePoland) kunye nezityalo zokulawula (i-sham-inoculated) ziphawulwe ngaphezu kwamanqaku edatha (*P ixabiso <0.05, **ixabiso le-P ≤ ≤ 0 01 ≤ ≤ 0 01 ≤ 0.01 ≤ 0.01 ≤ 0.01 ≤ 0.0 1.接种（Colletotrichum lupini，Col-08株，1999年从波兰Wierzenica的羽扇豆田获得）和对照（模拟接种种）植异的统计学显着性标记在数据点上方（*P值< 0.05, **P 值≤ 0.01, ***P 值≤ 0.001).接种 （colletorichum lupini , color-08 株 ， 1999 年 波兰 波兰 wierzenica的 羽扇 获得） 和对照 鼈接种种 对照 鼈接种种的 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 9 г.) и контрольными (ложно инокулированными) растениями отмечены над точками данных (* значение P < 0,05, ** P-значение ≤ 0,010, ***P-0,01, ***P-0,01, ***P-0,01, Ulwahlulo oluphawulekayo lwezibalo kumanqanaba okubonakalisa phakathi kwe-inoculated (i-Colletotrichum lupini, i-strain Col-08, efunyenwe kwiindawo ze-lupine e-Verzhenice, e-Poland, ngo-1999) kunye nezityalo zokulawula (i-sham-inoculated) ziphawulwe ngaphezu kwamanqaku edatha (*P ixabiso <0.05, **P-value ≤ ≤ 0-10 ≤ 0-11 ≤ 0-11 ≤ 0-11 ≤ 0.0 1).Imigca ye-NLL ehlalutyiweyo yayiyi: 83A: 476 (i-resistant, ethwele i-homozygous Lanr1 allele), i-Mandelup (i-moderal resistant, ithwele i-homozygous AnMan allele), i-Boregine (i-resistant, imvelaphi engaziwayo yofuzo) kunye nabemi be-22660 (i-susceptible).
Umviwa we-gene TanjilG_05042 kwindawo ye-Lanr1 ubonise ipateni yokubonakalisa eyahlukileyo ngokuphawulekayo kwiiprofayili ezifunyenwe kwizifundo ze-RNA-seq (umzobo 6e).Ukusebenza okubalulekileyo kolu hlobo lofuzo kwabonwa eMandelup kunye nabemi be-22660 (ukuya kwi-39.7 kunye ne-11.7 amaxesha, ngokulandelanayo), okukhokelela kumanqanaba aphezulu okuthetha (ukuya kwi-1.4 ± 0.14 kunye ne-7.2 ± 1.3, ngokulandelanayo).I-83A: i-476 iphinde ibonise ukunyuswa kwe-TanjilG_05042 gene (ukuya kuthi ga kwi-3.8-fold), nangona kunjalo, amanqanaba okubonakalisa okuhambelanayo aphunyeziweyo (0.044 ± 0.002) ayengaphezulu kwe-30 ephantsi kunabo babonwa eMandelup kunye ne-22660 yabemi.ihlalutywe yi-qPCR ibonise ulwahlulo olubonakalayo kumanqanaba okubonakalisa phakathi kwe-genotypes kwi-mock-vaccinated (control) ezahlukeneyo, ukufikelela kwi-58-fold difference phakathi kwabantu be-22660 kunye ne-83A: 476, kunye naphakathi kwabemi be-22660 kunye ne-22660. Umahluko ophindwe kabini wafunyanwa phakathi kwe-Boregine ne-Mandalup.
Ufuzo lomviwa kwindawo ye-AnMan, TanjilG_12861, lwenziwa lwasebenza ukuphendula kugonyo olukwi-83A:476 naseMandelup, lwalungathathi hlangothi kuluntu lwama-22660, kwaye lwathotywa eBoregine (Fig. 6f).Ukubonakaliswa okuzalanayo kwe-TanjilG_12861 gene yayiyeyona iphezulu kwi-83A efakwe kwi-inoculated: 476 (0.14±0.01).I-17.4 kDa iklasi I-protein ye-heat shock gene i-TanjilG_05080 HSP17.4 ibonise amanqanaba aphantsi okuchaza amanqanaba kuzo zonke iingxaki ezifundiweyo kunye namanqaku exesha (Fig. 6g).Ixabiso eliphezulu libonwe kwi-24 HPI kubantu be-22660 (0.14 ± 0.02, ukunyuka okuphindwe kasibhozo ekuphenduleni ukugonywa).
Ukuthelekiswa kweeprofayili zokubonisa i-gene (umzobo 7) ubonise ulungelelwaniso oluphezulu phakathi kweTanjilG_10657 kunye nezinye iijene ezine: TanjilG_27015 (r = 0.89), TanjilG_05080 (r = 0.85), TanjilG_05042 (r = 0.00G = 0.80) = 0.80;Iziphumo ezinjalo zingabonisa ukulungelelaniswa kwezi geni ngexesha lokuphendula lokukhusela.I-TanjilG_12861 kunye ne-TanjilG_23384 yofuzo ibonise iiprofayili zokubonisa ezahlukeneyo ezinexabiso eliphantsi le-Pearson coefficient coefficient (ukusuka ku-0.08 ukuya ku-0.43 kunye -0.19 ukuya ku-0.28, ngokulandelelana) xa kuthelekiswa nezinye iijini.
Unxulumano phakathi kweeprofayile zokuchazwa kofuzo zichongiwe kusetyenziswa ubungakanani bePCR.Kwahlalutywa le migcana ilandelayo yamagqabi elupine: 83A:476 (ixhathisa, ithwele i-homozygous Lanr1 allele), iMandelup (ixhathise ngokwemodareyitha, ithwele i-homozygous AnMan allele), iBoregine (iyaxhathisa, imvelaphi yofuzo ayaziwa), kunye nabemi abangama-22660 (abanokuchaphazeleka).Amanqaku exesha amathathu abalwe (i-6, i-12 kunye ne-24 iiyure emva kokugonywa), kubandakanywa ne-inoculated (i-Colletotrichum lupini, i-strain Col-08, efunyenwe kwiindawo ze-lupine e-Wierzhenice, ePoland, ngo-1999) kunye nokulawula (i-sham-inoculated) izityalo.Umlinganiselo ubonisa ixabiso le-Pearson coefficient coefficient.
Ngokusekelwe kwidatha efunyenwe kwi-6 horsepower nge-intshi nganye, i-WGCNA yenziwe kwi-9981 DEG ichongiwe ngokuthelekisa izityalo ezifakwe kwi-inoculated kunye nokulawula ukugxila kwiimpendulo zokukhusela kwangaphambili (i-Supplementary Table S12).Iimodyuli zofuzo ezingamashumi amabini anesibini (amaqela) zifunyenwe zineeprofayili zokubonisa ezihambelanayo (ezilungileyo okanye ezimbi) phakathi kwe-genotypes kunye nokwahluka kovavanyo. Ngokomyinge, amanqanaba embonakalo yemfuza aye ehla ngokolandelelwano 83A:476 > Mandelup > Boregine > Population 22660 (kuzo zombini iindidi, nangona kunjalo, lo mkhwa wawunamandla kwizityalo zolawulo). Ngokomyinge, amanqanaba embonakalo yemfuza aye ehla ngokolandelelwano 83A:476 > Mandelup > Boregine > Population 22660 (kuzo zombini iindidi, nangona kunjalo, lo mkhwa wawunamandla kwizityalo zolawulo). В среднем уровни экспрессии генов снижались в порядке 83A:476 > Mandelup > Boregine > Population 22660 растений). Ngokomyinge, amanqanaba embonakalo yemfuza ehlile ngokolandelelwano 83A:476 > Mandelup > Boregine > Population 22660 (kuzo zombini iindidi, nangona kunjalo, lo mkhwa wawunamandla kwizityalo zokulawula).平均而言，基因表达水平按83A:476 > Mandelup > Boregine > Population 22660的顺序下降（然而，在两种中种种种种种种种种变公物。更强）.平均 而 言 , 基因 水平 按 83a: 476> mandelup> boregine> labemi 22660 的顺序 下降 （，在 种 中 （，在 种 中 （，在种 中 （，在种 中 （，在种 中 （，在种 中（ ........ ........ В среднем уровни экспрессии генов снижались в ряду 83A:476 Mandelup > Boregine > Population 22660 (однако в обоих вариантах эта тенденция ) hayi). Ngokomndilili, amanqanaba embonakalo yemfuza ehlile kuthotho 83A:476 > Mandelup > Boregine > Population 22660 (nangona kunjalo, kuzo zombini iindidi, lo mkhwa wawunamandla kwizityalo zolawulo).Ukugonywa kubangele ukulawulwa kwe-gene expression, ngakumbi kwiimodyuli ze-18, i-19, i-14, i-6 kunye ne-1 (ngokwehla komyalelo wesiphumo), umgaqo ongalunganga (umz. iimodyuli ze-9 kunye ne-20) okanye ngemiphumo engathathi hlangothi (umz. iimodyuli 11, 22, 8 kunye ne-13).Uhlalutyo lwexesha le-GO lokutyebisa (iTheyibhile eyoNgezelelweyo ye-S13) lutyhile "GO: 0006952 Iimpendulo zoKhuseleko" kwimodyuli efakwe i-inoculated (18) kunye nokusebenza okuphezulu, kubandakanywa nofuzo oluhlalutywe yi-qPCR (TanjilG_04706, TanjilG_23384, TanjilG_1015G photo as most as well as TanjilG_1065G) iimodyuli ze-synthesis (9).Imodyuli ye-18 ye-concentrator (umzobo 8) ichongiwe njenge-TanjilG_26536 gene encoding iprotheni ye-PR-10-efana ne-LlR18B, kunye ne-module ye-9 ye-concentrator ichongiwe njenge-TanjilG_28955 gene encoding iprotein ye-photosystem II PsbQ.Ijene yokumelana ne-anthracnose yomviwa i-Lanr1, i-TanjilG_05042, ifunyenwe kwimodyuli 22 (umzobo 9) kwaye inxulunyaniswe namagama athi "GO:0044260 Cellular macromolecular metabolic processes" kunye ne "GO:0006355 Transcriptional regulation, DNA templating2G_01 ithwele iTac.i-gene ifaka ikhowudi yoxinzelelo lobushushu kwi-A-4a (HSFA4a).
Uhlalutyo lwenethiwekhi olulinganisiweyo lwe-gene co-expression yeemodyuli ezinemigaqo yenkqubo yezinto eziphilayo echazwe ngokugqithisileyo "GO: 0006952 iimpendulo zoKhuselo".I-Ligation yenziwe lula ukuqaqambisa imfuzo emine ehlalutywe yi-qPCR (TanjilG_04706, TanjilG_23384, TanjilG_10657 kunye neTanjilG_27015).
Uhlalutyo lothungelwano olulinganiselweyo lwe-gene co-expression yemodyuli enenkqubo yebhayoloji echazwe ngokugqithisileyo "HAMBA: 0006355: Ummiselo wokuBhala, i-DNA templating" kunye nokuthwala umviwa wokumelana ne-anthracnose gene Lanr1 TanjilG_05042.I-Ligation yenziwe lula ukwahlula i-TanjilG_05042 gene kunye nombindi we-TanjilG_01212 gene.
Uvavanyo lokumelana ne-anthracnose oluqokelelwe e-Australia lubonise ukuba uninzi lwemihlanganiselwa ekhutshiweyo kwangoko yayisesichengeni;I-Kalya, i-Coromup kunye ne-Mandelup zichazwe njengezixhathisa ngokuphakathi, ngelixa i-Wonga, i-Tanjil kunye ne-83A: i-476 zichazwe njenge-resistance ephezulu26,27,31.yayine-allele efanayo yokumelana, i-Lanr1 ekhethiweyo, kunye ne-Coromup kunye ne-Mandelup yayine-allele eyahlukileyo, echongiweyo i-AnMan10, i-26, i-39, ngelixa u-Kalya edlula kwi-allele eyahlukileyo., Lanr2.Uvavanyo lokumelana ne-anthracnose eJamani kubangele ukuchongwa komgca ochasayo we-Bo7212 kunye nomgqatswa we-allele ngaphandle kwe-Lanr1, ekhethiweyo i-LanrBo36.
Uphononongo lwethu lubonakalise i-frequency ephantsi kakhulu (malunga ne-6%) ye-Lanr1 allele kwi-germplasm evavanyiweyo.Olu qwalaselo luhambelana neziphumo zokuhlola i-germplasm yaseMpuma yaseYurophu usebenzisa i-Anseq3 kunye ne-Anseq4 abamakishi, ebonisa ukuba i-Lanr1 allele ikhona kwimigca emibini kuphela yaseBelarusian.Oku kubonisa ukuba i-allele ye-Lanr1 ayikasetyenziswa ngokubanzi ngeenkqubo zokuzalanisa zendawo, ngokungafaniyo ne-Australia, apho yenye yezona zinto zibalulekileyo zokumakisha-ukuncedisa ukuzala.Oku kunokuba ngenxa yezinga eliphantsi lokuchasana elinikezelwa yiLanr1 allele kwiimeko zentsimi yaseYurophu xa kuthelekiswa nengxelo yase-Australia.Ukongezelela, izifundo ze-anthracnose kwiindawo eziphakamileyo zemvula e-Australia zibonise ukuba iimpendulo zokumelana ne-Lanr1 allele zingenakusebenza kwiimeko zemozulu ezithanda ukukhula kunye nokuphuhliswa ngokukhawuleza kwe-pathogen19,42.Enyanisweni, kuphononongo lwangoku, ezinye iimpawu ze-anthracnose nazo zabonwa kwi-genotypes ephethe i-Lanr1 allele, ebonisa ukuba ukuchasana kunokunyamalala phantsi kweemeko ezifanelekileyo zokuphuhliswa kwe-C. lupini.Ukongeza, ukutolika okungalunganga kobukho be-Anseq3 kunye ne-Anseq4 abamakishi, abamalunga ne-1 cm ukusuka kwi-locus ye-Lanr1, kunokwenzeka ukuba yi-28,30,43.
Uphononongo lwethu lubonise ukuba i-83A: i-476, ephethe i-Lanr1 allele, iphendule kwi-C. lupini inoculation kunye ne-reprogramming enkulu ye-transcriptome kwixesha lokuqala elihlalutyiweyo (6 hpi), ngelixa e-Mandelup, ephethe i-AnMan allele, iimpendulo ze-transcriptomic zabonwa kamva.(ukusuka kwi-24 ukuya kwi-48 hp).Ezi zintlukwano zexeshana kwiimpendulo zokukhusela zihambelana nokungafani kweempawu zesifo, zigxininisa ukubaluleka kokuqatshelwa kwangaphambili kwe-pathogen ukuze kuphendule ngempumelelo ukuchasana.Ukosulela izicubu zezityalo, iimbewu ze anthrax kufuneka zidlule kumanqanaba ahlukeneyo okukhula kumphezulu womkhosi, kubandakanywa ukuntshula, ukwahlukana kweeseli, kunye nokwakhiwa kwendawo yokusebenzela.I-appendage yisakhiwo esichaphazelayo esinamathele kumphezulu womkhosi kwaye siququzelele ukungena kwizicubu zomkhosi.Ngaloo ndlela, i-spores ye-C. gloeosporioides kwi-extract ye-pea ibonise ulwahlulo lokuqala lwe-nucleus emva kwemizuzu engama-75-90 ye-incubation, ukubunjwa kwebhubhu yentsholongwane emva kwemizuzu engama-90-120, kunye nokunyanzeliswa emva kweeyure ezingama-45.IMango C. gloeosporioides ibonise ngaphezulu kwe-40% yokuntshula kwe-condial emva kweeyure ezi-3 zokufukamela kunye malunga ne-20% yokwakheka kwe-appressors emva kweeyure ezi-4.I-CAP20 enxulumene ne-virulence yejini ye-C. gloeosporioides ibonise umsebenzi wokubhaliweyo kwi-epiphyte-forming conidia emva kwe-3.5 h incubation kwi-avocado surface wax kunye nokugxila okuphezulu kwe-CAP20 protein emva kwe-4 h 46 min.Ngokufanayo, umsebenzi we-melanin biosynthesis genes kwi-C. trifolii wanyanzelwa ngexesha le-2-incubation yeeyure ezilandelwa kukwenziwa kwe-appressorium emva kweyure eli-1.Izifundo zamagqabi amaqabunga zibonise ukuba i-strawberries efakwe kwi-C. acutatum inoxinzelelo lokuqala kwi-8 hpi, ngelixa iitamatato ezifakwe kwi-C. coccodes zinoxinzelelo lokuqala kwi-4 hpi48,49.ubukhulu becala iyahambelana nesikali sexesha se-Colletotrichum spp.inkqubo yosulelo.Iimpendulo ezikhawulezayo zokukhusela kwi-83A: 476 zibonisa ukubandakanyeka kokuchasana kwezityalo kunye ne-etiper-triggered immunity (ETI) kulo mgca, ngelixa iimpendulo zokulibaziseka kweMandelup zixhasa i-micro-associated molecular pattern-triggered immunity (MTI) hypothesis 50. Iimpendulo zangaphambili kwi-83A: 476 kunye neMandelup.Ukugqithwa ngokuyinxenye phakathi kwee-genes eziphantsi okanye ezisezantsi kwimpendulo yokulibaziseka iphinda ixhase le ngcamango, njengoko i-ETI ihlala ithathwa njengempendulo ye-MTI ekhawulezileyo kunye neyomeleziweyo efikelela ekugqibeleni kwiprogram yokufa kweeseli kwindawo yokusuleleka, eyaziwa ngokuba yi-anaphylactic shock 51,52.
Uninzi lwezakhi zofuzo zibalelwa kwigama elimele ngokugqithisileyo i-Gene Ontology GO: 0006952 "Impendulo yoKhuselo" yi-homologues ye-11 yomyalezo wokuzila ukutya obangelwa uxinzelelo lwe-22 protein (efana ne-SAM22) kunye nesixhenxe esikhulu se-latex protein-likes (MLPs).-njengeeprotheyini ze-31, i-34, i-43 kunye ne-423 ibonise ukulandelelana ukufana.Izakhi zofuzo ezifana ne-SAM22 zibonise ukusebenza okuphawulekayo okuhlala ixesha elide, kubonisa ukunyuka kwamanqanaba okumelana ne-anthracnose (83A: 476 kunye ne-Boregine).Nangona kunjalo, i-MLP-like genes yathotywa phantsi kuphela kwimigca ethwele ukuchasana komviwa (83A: 476 / Lanr1 kwi-6 hpi kunye ne-Mandelup / AnMan kwi-24 hpi).Kufuneka kuqatshelwe ukuba zonke ii-homologues ezichongiweyo ezifana ne-SAM22 zisuka kwi-gene cluster ethatha malunga ne-105 kb, ngelixa iijini ezifana ne-MLP zisuka kwimimandla eyahlukeneyo ye-genome.Ukulungelelaniswa kokulungelelaniswa kwezakhi zofuzo ezifana ne-SAM22 nazo zifunyenwe kwisifundo sethu sangaphambili sokumelana ne-NLL kwi-Diaporthetoxica inoculation, ebonisa ukuba babandakanyeka kumacandelo anqamlekileyo empendulo yokukhusela.Esi sigqibo sikwaxhaswa neengxelo zempendulo efanelekileyo ye-SAM22-efana ne-genenes kwingozi okanye unyango nge-salicylic acid, i-inducers ye-fungal, okanye i-hydrogen peroxide.
Ufuzo olufana ne-MLP luye lwaboniswa ukuba luphendule kwiintlobo ezahlukeneyo ze-abiotic kunye ne-biotic, kubandakanywa ne-bacterial, i-viral kunye ne-pathogenic fungal infections kwiintlobo ezininzi zezityalo55.Izalathiso zokusabela kwintsebenziswano ethile phakathi kwezityalo kunye neentsholongwane ezibangela izifo zisusela ekunyukeni ngamandla (oko kukuthi, ngexesha lohlaselo lomqhaphu kunye neVerticillium dahliae) ukuya ekwehleni kakhulu (oko kukuthi, emva kosulelo lomthi we-apile nge-Alternaria spp.)56,57.Ukuthotywa okubalulekileyo kwe-MLP-like 423 gene kuye kwabonwa ngexesha lokukhusela i-avokhado kwi-F. niger usulelo kwaye ngexesha lokusuleleka komthi we-apula i-Botryosphaeria berengeriana f.cn.I-piricola kunye ne-Alternaria alternata zi-apile pathotypes58,59.Ukongeza, i-apple calli ebonisa i-MLP-efana ne-423 gene yayinembonakalo esezantsi yemfuza eyayanyaniswa noxhathiso kwaye yayisengozini yosulelo lomngundo59.Ukulandela i-Fusarium oxysporum f, i-MLP-efana ne-423 gene nayo yacinezelwa kwi-germplasm yembotyi eqhelekileyo.cn.Usulelo lweembotyi 60.
Amanye amalungu eentsapho ze-PR-10 ezichongiweyo kwisifundo sethu se-RNA-seq yayiyi-LlR18A kunye ne-LlR18B izakhi zofuzo ekuphenduleni ukulawulwa, kunye nokulawulwa (i-1 gene) okanye i-gene ye-3 ye-gene ye-lipid transfer protein DIR1..Ukongeza, i-WGCNA iqaqambisa ijini ye-LlR18B njenge hub kule modyuli, esengozini kakhulu ekugonyweni kwaye ithwala iintlobo ezininzi zokuphendula ezikhuselayo.I-LlR18A kunye ne-LlR18B izakhi zofuzo zenziwa kumagqabi e-lupine aphuzi ekuphenduleni ibhaktheriya ye-pathogenic, kunye ne-NLL iziqu emva kwe-D. toxica inoculation, ngelixa i-rice homologue yale genes, i-RSOsPR10, yabangelwa ngokukhawuleza ukusuleleka kwefungal ekucingelwa ukuba ibandakanyeke kwi-jasmonic acid, i-non-signing ye-162 ye-DIncodes162, i-DIncodes, i-DIncodes16, indlela ye-DIncodes162 ye-dincodes. iiprotheni zokuthutha i-lipid ezifunekayo ukuqala kwenkqubo yokufumana ukuchasana (SAR).Ngophuhliso lweendlela zokukhusela, iprotheni ye-DIR1 ithuthwa ekugxilweni kokusuleleka nge-phloem ukukhupha i-SAR kwizitho ezikude.Okubangel 'umdla kukuba, i-TanjilG_02313 DIR1 i-gene yanyanzeliswa kakhulu ngexesha lokuqala kwimigca ye-84A: 476 kunye ne-Population 22660, kodwa ukuxhathisa kwe-anthracnose kuphuhliswe ngempumelelo kuphela kumgca we-84A: 476.Oku kungabonisa ukunyanzeliswa kwe-DIR1 gene kwi-NLL, ekubeni i-homologues ezintathu ezisele ziphendule kwi-inoculation kuphela kwi-83A: umgca we-476 kwi-6 hpi, kwaye le mpendulo iqondiswe phantsi.
Kwisifundo sethu, amacandelo aqhelekileyo ahambelana nenkqubo yezinto eziphilayo ebizwa ngokuthi "GO: 0055114 inkqubo yeRedox" yayiyiprotheni ye-cytochrome P450, i-peroxidase, i-linoleic acid 9S-/13S-lipoxygenase, kunye ne-1-aminocyclopropane-1-carboxylic acid oxidase.Ukongeza, i-WGCNA yethu ichaza i-homologue ye-HSFA4a njenge-hub ephethe iimodyuli ezifana ne-Lanr1 yokumelana nomgqatswa we-TanjilG_05042.I-HSFA4a licandelo lolawulo oluxhomekeke kwi-redox yoshicilelo lwenyukliya kwizityalo.
Iiprotheyini ze-Cytochrome P450 ziyi-oxidoreductases eyenza i-NADPH kunye / okanye i-O2-exhomekeke kwi-hydroxylation reactions kwi-metabolism ephambili kunye neyesibini, kubandakanywa ne-metabolism ye-xenobiotics, kunye ne-hormone, i-fatty acids, i-sterols, i-cell wall components, i-biopolymers, kunye ne-biosynthesis ye-functional compounds kwi-Pyromyto ye-60 encitshisiweyo kwi-40 yesityalo esincitshisiweyo kwi-60 ye-protein ye-protectors. I-10.6 log2 (utshintsho olutshintshileyo) ukuya kwi-5.7 ngenxa yenani elikhulu lee-homologues ezitshintshiweyo (37) kunye nokwahlukana kweepatheni zokuphendula phakathi kweejeni ezithile, ezibonisa ukuhlaziywa okuphezulu..Ukusebenzisa idatha ye-RNA-seq kuphela ukucacisa umsebenzi webhayoloji we-NLL kusapho olukhulu lweprotheyini kuya kuba yintelekelelo ephezulu.Nangona kunjalo, kuyafaneleka ukuba uqaphele ukuba ezinye ii-cytochrome P450 genes zidibene nokunyuka kokuchasana kwe-pathogenic fungi okanye ibhaktheriya, kubandakanywa negalelo kwi-allergies reactions69,70,71.
I-peroxidases ye-Class III yi-enzymes yezityalo ezininzi ezibandakanyekayo kwiinkqubo ezininzi ze-metabolic ngexesha lokukhula kwezityalo kunye nophuhliso, kunye nokuphendula uxinzelelo lwendalo olufana nosawoti, isomiso, ukukhanya okuphezulu, kunye nokuhlaselwa kwe-pathogen72.I-Peroxidases ibandakanyeka ekusebenzisaneni kweentlobo ezininzi zezityalo kunye ne-Anthracis, kuquka i-Stylosanthes humilis kunye ne-C. gloeosporioides, i-Lens culinaris kunye ne-C. truncatum, i-Phaseolus vulgaris kunye ne-C.Impendulo ikhawuleza kakhulu, ngamanye amaxesha nakwi-HPI ye-4, ngaphambi kokuba i-fungus ingene kwizicubu zesityalo73.I-peroxidase gene nayo yasabela kwi-inoculation ye-D. toxica NLL.Ukongeza kwimisebenzi yabo eqhelekileyo yokulawula ukuqhuma kwe-oxidative okanye ukuphelisa uxinzelelo lwe-oxidative, i-peroxidases inokuphazamisa ukukhula kwe-pathogen ngokudala izithintelo zomzimba ezisekelwe ekuqinisweni kodonga lweseli ngexesha lokudibanisa, i-subunit okanye ukudibanisa i-cross-link ye-compounds ethile.Lo msebenzi unokuthiwa kwi-silico kwi-TanjilG_03329 gene encoding i-puative lignin-forming anion peroxidase eyayilawulwa kakhulu kwisifundo sethu kwi-83A: umgca we-476 ochasayo kwi-6 HPI, kodwa kungekhona kwezinye iintlobo kunye namanqaku exesha angazange aphendule.
I-9S-/13S-lipoxygenase ye-linoleic acid yinyathelo lokuqala kwindlela ye-oxidative ye-lipid biosynthesis78.Iimveliso zale ndlela zinemisebenzi emininzi ekukhuseleni izityalo, kubandakanywa ukuqiniswa kodonga lweseli ngokuqulunqwa kwe-callose kunye ne-pectin deposits, kunye nokulawulwa koxinzelelo lwe-oxidative ngokuveliswa kweentlobo ze-oksijeni ezisebenzayo79,80,81,82,83.Kuphononongo lwangoku, inkcazo ye-linoleic acid 9S-/13S-lipoxygenase yatshintshwa kuzo zonke iintlobo, kodwa kubantu abathintekayo be-22660, ukulawulwa kwahlula ngamaxesha ahlukeneyo, ngelixa kwiintlobo ezithwala i-Lanr1 kunye ne-AnMan allele, igxininisa ukuhlukana kwe-oxylipinthrection ye-alectrotype ye-oxylipinnotype.
I-1-aminocyclopropane-1-carboxylate oxidase (ACO) i-homologue yayilawulwa kakhulu (i-9 genes) okanye iyancipha (i-2 genes) xa ifakwe kwi-lupine.Ngaphandle kwezinto ezimbini, zonke ezi mpendulo zenzeke kwi-6 hp.ngo83A:476.I-reaction ye-enzymatic edityaniswe neeprotheni ze-ACO yinqanaba lokunciphisa izinga kwimveliso ye-ethylene kwaye ngoko ilawulwa kakhulu84.I-Ethylene yi-hormone yesityalo edlala indima ehlukeneyo ekulawuleni ukukhula kwezityalo kunye nokuphendula kwiimeko ze-abiotic kunye ne-biotic stress.Ukufakwa kwe-ACO yokubhala kunye nokusebenza kwendlela yokubonakalisa i-ethylene kubandakanyeka ekwandiseni ukuchasana kwerayisi kwi-hemibiotrophic fungus oryzae oryzae ngokulawula ukuveliswa kweentlobo ze-oksijini ezisebenzayo kunye ne-phytoalexins.Inkqubo yokusuleleka kweqabunga efana kakhulu efunyenwe phakathi kwe-M. oryzae kunye ne-C. lupini88,89, ngokubhekiselele kwi-backdrop ye-upregulation ephawulekayo ye-ACO homologues kwi-83A: i-476 line echazwe kolu cwaningo, iguqula ithuba lokunika ukuchasana kwe-NLL i-anthracnose I-Ethylene inyathelo eliphambili lokubonisa kwiindlela ze-molecular.
Kuphononongo lwangoku, ukunyanzeliswa okukhulu kweejeni ezininzi ezinxulumene ne-photosynthesis kwabonwa kwi-6 hpi kwi-83A: 476 kunye ne-48 hpi e-Mandeloop kunye ne-22660 yabemi.Ubungakanani kunye nokuqhubela phambili kolu tshintsho luhambelana nenqanaba.Ukuxhatshazwa kwe-anthracnose kwabonwa kolu vavanyo.Kungekudala, ukunyanzeliswa okunamandla kunye nokukhawuleza kwemibhalo ehambelana ne-photosynthesis kuye kwabikwa kwiimodeli ezininzi ze-plant-pathogen interactions, kubandakanywa ibhaktheriya ye-pathogenic kunye nefungi.Ukukhawuleza (ukusuka kwi-2 HPI kwezinye intsebenziswano) kunye nokunyanzeliswa kwehlabathi jikelele kwezakhi zofuzo ezihambelana ne-photosynthesis ekuphenduleni ukusuleleka kwintsholongwane kunokubangela ukukhuseleka kwezityalo ngokusekelwe ekuhanjisweni kweentlobo ze-oksijini ezisebenzayo kunye nokusebenzisana kwazo kunye nomzila we-salicylic acid ukulungelelanisa ukusabela kwe-90,94.
Ukuqukumbela, iindlela zokuphendula ezikhuselayo ezicetywayo kumgca ochasayo (83A: 476) zibandakanya ukuqaphela ngokukhawuleza kwe-pathogen yi-R gene (mhlawumbi i-TIR-NBS-LRR TanjilG_05042) kunye ne-allergic response-mediated salicylic acid kunye ne-ethylene signaling, elandelwa ngokusekwa kwe-SAR yobude obude.isenzo sixhaswa yi-DIR-1 protein.Kufuneka kuqatshelwe ukuba ixesha le-biotrophic losulelo lwe-C. lupini lifutshane kakhulu (malunga neentsuku ezi-2), lilandelwa yi-necrotic growth95.Utshintsho phakathi kwezi zigaba lunokuthi ludityaniswe ne-necrosis kunye nokubonakaliswa kweeprotheni ze-ethylene-inducible ezisebenza njengezinto ezibangela ukusabela kwe-hypersensitivity kwizityalo ezibambayo.Ngoko ke, ifestile yexesha lokubamba ngempumelelo i-C. lupini kwinqanaba le-biotrophic lincinci kakhulu.Ukucwangciswa ngokutsha kwezakhi zofuzo ezinxulumene ne-redox kunye ne-photosynthesis eqatshelwe kwi-83A: i-476 kwi-6 hpi ihambelana nokuqhubela phambili kwe-fungal hyphae kwaye ivakalisa ukuphuhliswa kwempendulo eyimpumelelo yokukhusela kwinqanaba le-biotrophic.Iimpendulo ze-transcriptomic ze-Mandelup kunye ne-22660 yabemi inokulibaziseka kakhulu ukuba ibambe i-fungus ngaphambi kokutshintshela ekukhuleni kwe-necrotic, nangona kunjalo, i-Mandelup inokusebenza ngakumbi kunabantu be-22660 ngenxa yokuba ukulawulwa ngokukhawuleza kweprotheni ye-PR-10 ikhuthaza ukuxhathisa okuthe tye.
I-ETI, eqhutywa yi-canonical gene ye-R, ibonakala iyindlela eqhelekileyo yokumelana neembotyi kwi-anthracnose.Ngaloo ndlela, kwimodeli ye-legume ye-Medicago truncatula, ukuchasana ne-anthracnose kunikwa i-RCT1 gene, ilungu le-TIR-NBS-LRR97 yesityalo se-R gene class.Lo mfuza ukwanikezela ukuxhathisa okubanzi kwe-anthracnose kwi-alfalfa xa idluliselwe kwizityalo ezisengozini.Kwimbotyi eqhelekileyo (P. vulgaris), ngaphezulu kweshumi elinesibini leejeni zokumelana ne-anthracnose zichongiwe ukuza kuthi ga ngoku.Ezinye zezi ntlobo zofuzo zifumaneka kwimimandla engenazo naziphi na iijini ze-R ze-canonical, nangona kunjalo ezinye ezininzi zifumaneka kwimida yeechromosomes ezithwele i-NBS-LRR gene cluster, kuquka i-TIR-NBS-LRRs99.Uphando lwe-SSR olubanzi lwe-genome luphinde lwaqinisekisa umbutho we-NBS-LRR gene kunye nokumelana ne-anthracnose kwimbotyi eqhelekileyo.Ijini ye-R ye-canonical nayo yafunyanwa kwingingqi ye-genomic ethwele eyona ndawo inkulu yokumelana ne-anthracnose kwi-lupine emhlophe 101.
Umsebenzi wethu ubonisa ukuba ukusabela okukhawulezileyo kokuxhathisa, okwenziwe kwasekuqaleni kosulelo lwezityalo (ngokukhethekileyo kungadlulanga i-12 hpi), ikhusela ngokufanelekileyo i-lupine enamagqabi anqabileyo kwi-anthracnose ebangelwa yi-pathogenic fungus Collelotrichum lupini.Sisebenzisa ulandelelwano lwe-throughput ephezulu, sibonise iiprofayili zokubonisa umahluko we-anthracnose yokumelana ne-anthracnose kwizityalo ze-NLL ezilamlwa yi-Lanr1 kunye ne-AnMan yokumelana nofuzo.Ukhuseleko oluyimpumelelo lubandakanya ukuyila ngononophelo iijini zeeproteni ezibandakanyeka kwi-redox, photosynthesis, kunye ne-pathogenesis kwiiyure nje ezimbalwa zokudibana kwesityalo ne-pathogen.Izithintelo ezifanayo zokhuselo, kodwa zilibazisekile ngexesha, zingasebenzi kakhulu ekukhuseleni izityalo kwizifo.Ukuxhathisa i-anthrax ehlanganiswe yi-Lanr1 gene ifana nempendulo ekhawulezayo ye-R gene (i-effector-triggered immunity), ngelixa i-AnMan gene inokwenzeka ukuba inike impendulo ethe tye (i-immunity ebangelwa yi-microbe-associated molecular pattern), inika umgangatho ophakathi wozinzo.
Imigca ye-215 ye-NLL esetyenziselwa ukukhuselwa iziphawuli ze-anthracnose yayiquka imihlanganisela ye-74, imigca engama-60 efunyenwe ngokuwela okanye ukuzala, i-5 i-mutants, kunye ne-76 ye-germplasms yasendle okanye yokuqala.Imigca yavela kumazwe angama-17, ikakhulukazi ePoland (58), eSpain (47), eJamani (27), e-Australia (26), eRashiya (19), eBelarus (7), e-Italy (5) kunye neminye imigca.ukusuka 10 kumazwe.Iseti ikwabandakanya imigca echasayo yereferensi: 83A:476, Tanjil, Wonga ethwele iLanr1 allele, kunye neMandelup ethwele iAnMan allele.Imizila ifunyenwe kwi-European Lupine Genetic Resource Database egcinwe yi-Poznań Plant Breeding Ltd., Wiatrowo, Poland (iTheyibhile eyoNgezelelweyo ye-S1).
Izityalo zakhuliswa phantsi kweemeko ezilawulwayo (iiyure ze-16 zefoto, ubushushu be-25 ° C emini kunye ne-18 ° C ​​ebusuku).Iikopi ezimbini zebhayoloji zahlalutywa.I-DNA yahlukaniswa kumagqabi eeveki ezintathu ubudala kusetyenziswa i-DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) ngokweprotocol.Umgangatho kunye nokugxininiswa kwe-DNA eyodwa yavavanywa ngeendlela ze-spectrophotometric (NanoDrop 2000; Thermo Fisher Scientific, Waltham, MA, USA).Isiphawuli se-AnManM1 esiphawula ijini yokuxhathisa i-anthracnose i-AnMan (ephuma kwi-cv. Mandelup) kunye neziphawuli i-Anseq3 kunye ne-Anseq4 esecaleni kohlobo lwe-Lanr1 (ephuma kwi-cv. Tanjil) zahlalutywa 11,26,28.I-Homozygotes ye-allele exhathisayo ifunyenwe njenge-"1", inokuba lula - njenge "0", kunye ne-heterozygotes - njenge-0.5.
Ngokusekelwe kwiziphumo zovavanyo lwabamakishi i-AnManM1, i-AnSeq3 kunye ne-AnSeq4 kunye nokufumaneka kwembewu kwiimvavanyo zokugqibela zokulandela, imigca ye-50 ye-NLL ikhethwe kwi-phenotyping yokumelana ne-anthracnose.Uhlalutyo lwenziwa ngokuphindwe kabini kwikhompyutheni elawulwa yi-greenhouse ene-photoperiod yeeyure ze-14 kunye neqondo lokushisa le-22 ° C emini kunye ne-19 ° C ebusuku.Imbewu iyakrwelwa (kunqunyulwa idyasi yembewu kwelinye icala le-embryo ngencakuba ebukhali) phambi kokuhlwayelwa ukunqanda ukubambezeleka kwembewu ngenxa yokuqina kwembewu kwaye kuqinisekiswe ukuntshula okufanayo.Izityalo zakhuliswa kwiimbiza (11 × 11 × 21 cm) kunye nomhlaba oyinyumba (TS-1 REC 085 Medium Basic, Klasmann-Deilmann Polska, Warsaw, Poland).Ukuchaneka kwaqhutywa nge-Colletotrichum lupini Col-08 strain, ekhule ngo-1999 ukusuka kwiziqu zezityalo ze-lupine ezikhuliswe kwintsimi yaseVerzhenítsa, eGreater Poland (52° 27′ 42″ N 17° 04′ 05″ E ).Fumana indawo.Iindawo ezizimeleyo zakhuliswa kwi-SNA medium kwi-20 ° C. phantsi kokukhanya okumnyama iintsuku ezingama-21 ukuze kubangele i-sporulation.Iiveki ezine emva kokuhlwayela, xa izityalo zifikelele kwinqanaba le-4-6 leqabunga, i-inoculation yenziwa ngokutshiza ngokumiswa kwe-conidia kwi-concentration ye-0.5 x 106 conidia nge-ml.Emva kokugonywa, izityalo zigcinwe ebumnyameni iiyure ezingama-24 kumswakama omalunga nama-98% kunye nobushushu obungama-25°C ukuze kube lula ukuntshula kwe-conidia kunye nenkqubo yokusuleleka.Izityalo zaye zakhuliswa phantsi kwe-14-hour photoperiod kwi-22 ° C imini / 19 ° C ebusuku kunye ne-70% yokufuma.Inqaku lesifo lenziwe emva kweentsuku ezingama-22 emva kokugonywa kwaye lisuka kwi-0 (i-immune) ukuya kwi-9 (ichaphazeleka kakhulu) kuxhomekeke kubukho okanye ukungabikho kwezilonda ze-necrotic kwiziqu kunye namagqabi.Ukongezelela, emva kokufaka amanqaku, ubunzima bezityalo bulinganiswa.Ubudlelwane phakathi kwe-marker genotypes kunye ne-phenotypes yesifo zibalwe njengenqaku lokulandelelana kolandelelwano lwesibini (ukungabikho kwabamakishi be-heterozygous kwisethi yemigca yohlalutyo lwe-anthracnose resistance phenotype).