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Angustifolius lupine (NLL, Lupinus angustifolius L.) shuka ce mai ganye da ake amfani da ita don samar da abinci da inganta ƙasa. Faɗaɗar NLL a duniya a matsayin amfanin gona ya jawo hankalin fungi da yawa masu cutarwa, ciki har da lupine anthracnose, wanda ke haifar da mummunar cutar anthracnose. An yi amfani da kwayoyin halitta guda biyu, Lanr1 da AnMan, waɗanda ke ba da ƙarin juriya, a cikin kiwo na NLL, amma har yanzu ba a san hanyoyin kwayoyin halitta ba. A cikin wannan binciken, an yi amfani da alamun Lanr1 da AnMan don tantance samfuran NLL na Turai. Gwajin allurar rigakafi a cikin yanayi mai sarrafawa ya tabbatar da ingancin masu ba da gudummawa masu juriya. An yi bayanin bayyanar kwayoyin halitta daban-daban akan layuka masu juriya da masu saurin kamuwa. Juriyar Anthracnose tana da alaƙa da yawan bayyana kalmomin ontology na kwayoyin halitta "GO:0006952 Response Defense", "GO:0055114 Redox Process", da "GO:0015979 Photosynthesis". Bugu da ƙari, layin Lanr1(83A:476) ya nuna gagarumin sake fasalin transcription da sauri bayan allurar riga-kafi, yayin da sauran layukan suka nuna jinkiri a cikin wannan martanin da kimanin awanni 42. Amsoshin tsaro suna da alaƙa da kwayoyin halittar TIR-NBS, CC-NBS-LRR da NBS-LRR, sunadarai 10 da ke da hannu a cikin pathogenesis, sunadaran canja wurin lipid, endoglucan-1,3-β-glucosidase, sunadaran bangon tantanin halitta masu arzikin glycine, da kwayoyin halitta daga hanyar amsawa ta iskar oxygen. Amsoshin farko ga 83A:476, gami da danne kwayoyin halitta da ke da alaƙa da photosynthesis, sun yi daidai da nasarar kariya a lokacin matakin girma na tsirrai na ilmin halittar fungal, wanda ke nuna cewa mai tasiri yana haifar da rigakafi. Amsar Mandeloop tana raguwa, kamar yadda ake jan hankalin gabaɗaya a kwance.
Lupine mai ganye mai ɗanɗano (NLL, Lupinus angustifolius L.) hatsi ne mai yawan furotin wanda ya samo asali daga yankin yammacin Bahar Rum1,2. A halin yanzu ana noma shi a matsayin abincin dabbobi da mutane. Haka kuma ana ɗaukarsa taki kore a cikin tsarin juyawar amfanin gona saboda daidaita nitrogen ta hanyar ƙwayoyin cuta masu daidaita nitrogen da kuma inganta tsarin ƙasa gabaɗaya. NLL ta fuskanci tsarin kiwon dabbobi cikin sauri a ƙarni na ƙarshe kuma har yanzu tana ƙarƙashin matsin lamba mai yawa na kiwo3,4,5,6,7,8,9,10,11,12. Tare da yaduwar noman NLL, jerin fungi masu cutarwa sun haɓaka sabbin wurare na noma kuma sun haifar da sabbin cututtuka masu lalata amfanin gona. Abin da ya fi burgewa ga manoma da masu kiwon lupine shine bayyanar anthracnose, wanda naman gwari mai cutarwa, Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13, ya haifar. Abin da ya fi burgewa ga manoma da masu kiwon lupine shine bayyanar anthracnose, wanda naman gwari mai cutarwa, Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13, ya haifar. Наиболее примечательным для фермеров и селекционеров. Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13. Mafi shahara ga manoma da masu kiwon lupine shine bayyanar anthracnose wanda naman gwari mai cutarwa Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13 ya haifar.对于羽扇豆农民和饲养者来说，最引人注目的是炭疽病的出现，它是由病原真語最引人注目的是炭疽病的出现。 Hagedorn13 引起的.对于羽扇豆农民和饲养者来说，最引人注目的是炭疽病的出现，它是由养者来说，最引人注目的是炭疽病的出现。 Наиболее поразительным для фермеров и селекционеров люпина грибком Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13. Abin da ya fi burge manoma da masu kiwon lupine shi ne bayyanar anthracnose wanda naman gwari mai cutarwa Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13 ke haifarwa.Rahotannin farko game da cutar sun fito ne daga Brazil da Amurka, inda alamun cutar suka bayyana a shekarar 1912 da 1929 bi da bi. Duk da haka, bayan kimanin shekaru 30, an sanya wa cutar suna Colletotrichum gloeosporioides (Penz.) Penz. & Sacc., teleomorph Glomerella cingulata (Stoneman) Spauld. & Sacc., teleomorph Glomerella cingulata (Stoneman) Spauld. & Sacc., телеоморф Glomerella cingulata (Stoneman) Spauld. & Sacc., teleomorph na 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 a cikin Tsarin Halittar da Aka Yi Niyya. & H. Schrenk,. & H. Schrenk,.da kuma H. ​​Schrenk. & H.施伦克,. & H.施伦克,.da kuma H. ​​Schlenk,.Binciken farko na cututtuka da aka yi a tsakiyar karni na 20 ya nuna wasu juriya a cikin NLL da kuma rawaya lupine (L. luteus L.), amma duk wani gwajin farin lupine (L. albus L.) da aka gwada yana da matukar saukin kamuwa da shi15,16. Bincike ya nuna cewa ci gaban anthracnose yana da alaƙa da ƙaruwar ruwan sama (danshin iska) da zafin jiki (a cikin kewayon 12-28°C), wanda ya haifar da keta juriya a yanayin zafi mafi girma17, 18. A zahiri, lokacin da ake buƙata don conidia ta tsiro da cutar ta fara, ya ninka sau huɗu a 24°C (awanni 4) fiye da a 12°C (awanni 16) a ƙarƙashin yanayin zafi mai yawa19. Don haka, ci gaba da ɗumamar yanayi ya haifar da yaɗuwar anthracnose. Duk da haka, an lura da cutar a Faransa (1982) da Ukraine (1983) a matsayin alamar barazanar da ke tafe, amma a bayyane yake masana'antar lupine ta yi watsi da ita a lokacin20,21. Bayan 'yan shekaru, wannan mummunar cuta ta bazu a duk faɗin duniya kuma ta shafi manyan ƙasashe masu samar da lupine kamar Ostiraliya, Poland da Jamus22,23,24. Bayan barkewar cutar anthracnose a tsakiyar shekarun 1990, an gudanar da cikakken bincike wanda ya haifar da gano masu ba da gudummawa da dama masu juriya a cikin samfuran NLL19. Juriyar NLL ga anthracnose tana ƙarƙashin ikon alleles guda biyu daban-daban da aka samo a cikin tushen germplasm daban-daban: Lanr1 a cikin nau'in Tanjil da Wonga da AnMan a cikin nau'in. Mandalay 25, 26. Waɗannan alleles suna ƙara wa alamun kwayoyin halitta waɗanda ke tallafawa zaɓin germplasm masu juriya a cikin shirye-shiryen kiwo25, 26, 27, 28, 29, 30. An haɗa layin kiwo mai jurewa 83A:476 wanda ke ɗauke da allele na Lanr1 tare da layin daji mai saurin kamuwa da cutar P27255 don samun yawan RIL wanda ke raba juriyar anthracnose, wanda ya ba da damar sanya wurin Lanr1 zuwa chromosome NLL-1131, 32, 33. Daidaita alamun taswirar haɗin gwiwa daga wurin juriya na gefe zuwa anthracnose tare da tsarin kwayoyin halitta, NLL ya bayyana wurin da dukkan allele uku ke kan chromosome ɗaya (NLL-11), amma a wurare daban-daban29,34,35. Duk da haka, saboda ƙarancin adadin RILs da babban tazara tsakanin alamomi da allele masu dacewa, babu wani tabbataccen ƙarshe da za a iya yankewa game da kwayoyin halittarsu da ke ƙasa. A gefe guda kuma, amfani da kwayoyin halitta na baya a cikin lupins yana da wahala saboda ƙarancin damar sake farfaɗowa, wanda ke sa sarrafa kwayoyin halitta ya zama mai wahala37.
Ci gaban ƙwayoyin cuta masu rai da ke ɗauke da allele da ake so a yanayin homozygous, kamar 83A:476 (Lanr1) da Mandelup (AnMan), ya buɗe ƙofa don yin nazarin juriyar anthracnose a gaban kasancewar haɗuwar alleles masu adawa da juna a cikin al'ummomin daji. Yiwuwar hanyoyin ƙwayoyin cuta. Kwatanta martanin kariya da aka samar ta hanyar takamaiman nau'ikan kwayoyin halitta. Wannan binciken ya kimanta martanin farko na NLL ga allurar rigakafin C. lupini. Da farko, an tantance wani kwamitin germplasm na Turai NLL wanda ke ɗauke da layuka 215 ta amfani da alamun kwayoyin halitta waɗanda ke nuna alleles na Lanr1 da AnMan. Daga nan aka yi gwajin anthracnose akan layukan NLL 50, waɗanda aka riga aka zaɓa don alamun kwayoyin halitta, a ƙarƙashin yanayi mai sarrafawa. Dangane da waɗannan gwaje-gwajen, an zaɓi layuka huɗu da suka bambanta a juriyar anthracnose da kuma tsarin allelic na Lanr1/AnMan don bayanin bayyanar kwayoyin halitta na kariya ta hanyar amfani da hanyoyi biyu masu dacewa: jerin RNA mai ƙarfi da kuma ƙididdigar PCR na ainihin lokaci.
Binciken saitin ƙwayoyin halittar NLL (N = 215) tare da alamomin Lanr1 (Anseq3 da Anseq4) da AnMan (Anseq4) da AnMan (AnManM1) ya nuna cewa layi ɗaya kawai (95726, kusa da Salamanca-b) yana ƙara yawan allele na "juriya" ga duk masu alama, yayin da "Kasancewar ƙwayoyin halittar 'masu saurin kamuwa'" ya sami rabon dukkan masu alama a cikin layuka 158 (~73.5%). Layuka goma sha uku sun samar da allele biyu na "juriya" na alamar Lanr1, kuma layuka 8 sun samar da allele "juriya" na alamar Lanr1. Allele na "juriya" na alamar AnMan (Karin Tebur S1). Layuka biyu sun kasance heterozygous don alamar Anseq3 da heterozygous ɗaya don alamar AnManM1. Layuka 42 (19.5%) sun ɗauki matakai daban-daban na alleles na Anseq3 da Anseq4, wanda ke nuna yawan haɗuwa tsakanin waɗannan loci guda biyu. Siffofin Anthracnose a ƙarƙashin yanayin da aka sarrafa (Tebur na Ƙarin S2) ya nuna bambancin juriyar nau'ikan halittu da aka gwada, wanda aka nuna a cikin tsananin anthracnose. Bambance-bambancen matsakaicin maki sun kasance daga 1.8 (masu juriya matsakaici) zuwa 6.9 (masu juriya matsakaici) kuma bambance-bambancen nauyin tsirrai ya kasance daga 0.62 (masu juriya) zuwa 4.45 g (masu juriya). Akwai wata muhimmiyar alaƙa tsakanin dabi'un da aka lura a cikin kwafi biyu na gwajin (0.51 don ma'aunin tsananin cutar, P = 0.00017 da 0.61 don nauyin shuka, P < 0.0001) da kuma tsakanin waɗannan ma'auni guda biyu (− 0.59 da − 0.77, P < 0.0001). Akwai muhimmiyar alaƙa tsakanin dabi'un da aka lura a cikin kwafi biyu na gwajin (0.51 don ma'aunin tsananin cutar, P = 0.00017 da 0.61 don nauyin shuka, P < 0.0001) da kuma tsakanin waɗannan sigogi biyu (− 0.59 da − 0.77, P < 0.0001). Кадры из видео Комментарии к видео m, P = 0,00017 da 0,61 da sauransu, P <0,0001), da sauransu. 0,0001). An sami wata muhimmiyar alaƙa tsakanin ƙimar da aka lura a cikin maimaitawa biyu na gwajin (0.51 don ma'aunin tsananin cuta, P = 0.00017 da 0.61 don nauyin shuka, P < 0.0001), da kuma tsakanin waɗannan sigogi biyu (- 0.59 da -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，植物重量为0.61，P <0.0001）以及这两个参数之间（ - 0.59在 两 次 重复 实验 中 观察 的 值 之间 存在 相关性, p = 0.00017 ， 植物 为 为 0.61 ， p <0.0001）和 - 0.59 和 - 0.77, P <0.0001). Наблюдалась значительная коррреляция между значениями, наблюдаемыmy 0,00017 da kuma 0,61, P <0,0001), da dai sauransu (-0,59 da kuma -0,0001) 0,77, P <0,0001. Akwai muhimmiyar alaƙa tsakanin ƙimar da aka lura a cikin kwafi (maki mai tsanani na cuta 0.51, P = 0.00017 da nauyin shuka 0.61, P < 0.0001) da kuma tsakanin waɗannan sigogi biyu (-0.59 da -0 .0001) 0.77, P<0.0001. ).Alamomin da aka fi gani a cikin shuke-shuke masu saurin kamuwa da cutar sun haɗa da karkacewa da karkacewar tushe kamar tsarin "bakan makiyayi", sannan kuma raunuka masu kama da launin ruwan lemu/ruwan hoda (Hoto na 1). Haɗuwar kwayoyin halittar Lanr1 (83A:476 da Tanjil) da AnMan (Mandelup) suna da juriya mai matsakaici, 0.0331 da 0.0036). Wasu layukan da ke ɗauke da ƙwayoyin Lanr1 da/ko AnMan masu "juriya" suna nuna alamun cutar.
Abin sha'awa, wasu layukan NLL da ba su da wata kwayar halitta mai "juriya" sun nuna babban matakin juriyar anthracnose (wanda za a iya kwatantawa ko sama da na Lanr1 ko AnMan genotypes), kamar Boregine (ƙimar P < 0.0001 ga sigogi biyu), Bojar (ƙimar P < 0.0001 ga maki da 0.001 ga nauyin shuka) da Population B-549/79b (ƙimar P < 0.0001 ga maki kuma ba ta da mahimmanci ga nauyi). Abin sha'awa, wasu layukan NLL da ba su da wata kwayar halitta mai "juriya" sun nuna babban matakin juriyar anthracnose (wanda za a iya kwatantawa ko sama da na Lanr1 ko AnMan genotypes), kamar Boregine (ƙimar P < 0.0001 ga sigogi biyu), Bojar (ƙimar P < 0.0001 ga maki da 0.001 ga nauyin shuka) da Population B-549/79b (ƙimar P < 0.0001 ga maki kuma ba ta da mahimmanci ga nauyi). Интересно, что несколько линий NLL, NLL устойчивости к антракнозу обоих параметров), Bojar (значение P <0,0001 для оценки и 0,001 для массы растения) и популяции B-549/79b (значение P <0,0001 для оценки и незначимо для массы). Abin sha'awa, layukan NLL da yawa waɗanda ba su da wani allele mai alamar 'mai juriya' sun nuna babban matakin juriya ga anthracnose (wanda aka kwatanta da ko sama da na Lanr1 ko AnMan genotypes), kamar Boregine (ƙimar P < 0.0001 ga sigogi biyu), Bojar (ƙimar P < 0.0001 don kimantawa da 0.001 don nauyin shuka) da yawan jama'a B-549/79b (ƙimar P < 0.0001 don kimantawa kuma ba mahimmanci ga nauyi ba).有趣的是，一些缺乏任何“抗性”标记等位基因的NLL基因型相当或更高），例如Boregine（两个参数的P 值< 0.0001）、Bojar（P 值<得分为0.0001，植物重量为0.001）和种群B-549/79b（得分P 值< 0.0001，重量不找。 Abin sha'awa ne cewa wasu tsarin NLL waɗanda ba su da alamun "antigenic" suna nuna juriya mai yawa a kwance (daidai da kwayoyin halittar Lanr1 ko AnMan ko sama da haka), kamar Boregine (duka sigogi P < 0.0001), Bojar (ƙimar P < 0.0001, nauyin shuka 0.001) da nau'in B-549/79b (ƙimar P < 0.0001, nauyi ba shi da mahimmanci). Интересно, что некоторые линии NLL, NLL устойчивости к антракнозу (сравнимые или выше, чем у генотипов Lanr1 или AnMan), такие как Boregine (значение P или вым0), <0), Bojar (значение P <0,0001, масса растения 0,001) и популяция B-549/79b (оценка P-значение) <0,0001, масса незначительна). Abin sha'awa, wasu layukan NLL waɗanda ba su da wata alama ta 'juriya' sun nuna matakan juriyar anthracnose mai yawa (wanda aka kwatanta da ko sama da nau'ikan halittar Lanr1 ko AnMan), kamar Boregine (ƙimar P ga sigogi biyu <0.0001), Bojar (ƙimar P < 0.0001, nauyin shuka 0.001) da yawan jama'a B-549/79b (ƙimar P < 0.0001, nauyi ba shi da mahimmanci).Wannan lamari yana nuna yiwuwar samun sabuwar hanyar juriya ta kwayoyin halitta, yana bayanin rashin alaƙar da aka lura tsakanin nau'ikan kwayoyin halitta masu alama da nau'ikan cututtuka (ƙimar P daga ~0.42 zuwa ~0.98). Don haka, gwajin Kolmogorov-Smirnov ya nuna cewa bayanai kan juriyar anthracnose yawanci ana rarraba su ne don maki (ƙimar P 0.25 da 0.11) da nauyin tsirrai (ƙimar P 0.47 da 0.55), yana nuna cewa I yana tsammanin cewa ƙwayoyin cuta da yawa fiye da Lanr1 da AnMan suna da hannu.
Dangane da sakamakon gwajin juriyar anthracnose, an zaɓi layuka 4 don nazarin transcriptome: 83A:476, Boregine, Mandelup, da Population 22660. An sake gwada waɗannan layukan don juriyar anthrax a cikin gwaje-gwajen allurar rigakafi ta hanyar jerin RNA, muddin sun yi daidai da gwajin da ya gabata. Ƙimar maki sune kamar haka: Boregin (1.71 ± 1.39), 83A: 476 (2.09 ± 1.38), Mandelup (3.82 ± 1.42) da yawan jama'a 22660 (6.11 ± 1.29).
Yarjejeniyar Illumina NovaSeq 6000 ta cimma matsakaicin nau'ikan Mread guda 40.5 a kowane samfuri (29.7 zuwa 54.4 Mreads) (Jadawalin Ƙarin S3). Maki na daidaitawa a cikin jerin ma'auni ya kasance daga 75.5% zuwa 88.6%. Matsakaicin alaƙar bayanan ƙidayar karatu tsakanin bambance-bambancen gwaji tsakanin kwafi na halittu ya kasance daga 0.812 zuwa 0.997 (matsakaicin 0.959). Daga cikin kwayoyin halitta 35,170 da aka yi nazari a kansu, 2917 sun nuna babu wani bayyanar cututtuka, sauran kwayoyin halitta 4785 kuma an bayyana su a matakin da ba shi da kyau (ma'aunin tushe < 5). Daga cikin kwayoyin halitta 35,170 da aka yi nazari a kansu, 2917 sun nuna babu wani bayyanar cututtuka, sauran kwayoyin halitta 4785 kuma an bayyana su a matakin da ba shi da kyau (ma'aunin tushe < 5). Иz 35 170 proanalizirovannыh genov 2917 da proyavlyaly эkspressi, a ostalnыe 4785 незначительном уровне (базовое среднее <5). Daga cikin kwayoyin halitta 35,170 da aka yi nazari a kansu, 2917 ba su nuna wata alama ba, kuma sauran kwayoyin halitta 4785 an bayyana su a wani mataki mara kyau (ma'aunin tushe <5).在分析的35,170 个基因中，2917 个没有表达，其他4785 5) .35,170 Иz 35 170 proanalizirovannыh genov 2917 da kuma ostalnыe 4785 (bazовое среднее значение <5). Daga cikin kwayoyin halitta 35,170 da aka yi nazari a kansu, ba a bayyana 2917 ba, sauran kwayoyin halitta 4785 kuma suna da rashin fahimta sosai (ma'aunin tushe <5).Saboda haka, adadin kwayoyin halittar da aka yi la'akari da su a bayyane (ma'aunin tushe ≥ 5) a lokacin gwajin shine 27,468 (78.1%) (Karin Tebur S4).
Tun daga lokacin farko, duk layukan NLL sun mayar da martani ga allurar C. lupini (nau'in Col-08) ta hanyar sake tsara tsarin rubutun (Tebur 1), duk da haka, an lura da bambance-bambance masu mahimmanci tsakanin layukan. Don haka, layin juriya 83A:476 (wanda ke ɗauke da kwayar halittar Lanr1) ya nuna babban sake tsara tsarin rubutun a lokacin farko (6 hpi) tare da ƙaruwa sau 31-69 a cikin adadin kwayoyin halitta sama da ƙasa da aka ware idan aka kwatanta da sauran lokutan lokaci a wannan lokacin. Bugu da ƙari, wannan kololuwar ba ta daɗe ba, domin bayyanar kwayoyin halitta kaɗan ne kawai suka kasance suna canzawa sosai a lokacin karo na biyu (12 hpi). Abin sha'awa, Boregine, wanda kuma ya nuna babban matakin juriya a gwajin dashen, bai yi irin wannan babban sake tsara tsarin rubutun ba yayin gwajin. Duk da haka, adadin kwayoyin halitta da aka bayyana daban-daban (DEG) iri ɗaya ne ga Boregine da 83A:476 a 12 HPI. Mandelup da kuma yawan jama'a 22660 sun nuna kololuwar DEG a lokacin ƙarshe (lita 48/s), wanda ke nuna jinkiri a martanin tsaro.
Saboda 83A:476 ya sami babban sake fasalin transcription don mayar da martani ga C. lupini a 6 HPI idan aka kwatanta da duk sauran layukan, ~91% na DEGs da aka lura a wannan lokacin sun kasance na musamman ga zuri'a (Hoto na 1). Duk da haka, akwai wasu haɗuwa a cikin martanin farko tsakanin layukan binciken, yayin da 68.5%, 50.9%, da 52.6% DEG a cikin Boregine, Mandelup, da yawan jama'a 22660, bi da bi, suka yi karo da waɗanda aka samu a cikin 83A:476 a wasu lokutan a lokaci. Duk da haka, waɗannan DEGs sun ɗauki ƙaramin juzu'i ne kawai (0.97–1.70%) na duk DEGs da aka gano a halin yanzu ta amfani da 83A:476. Bugu da ƙari, DEGs 11 daga dukkan layukan sun kasance masu daidaito a wannan lokacin (Teburin Ƙarin S4-S6), gami da abubuwan da aka saba amfani da su na martanin kare tsirrai: furotin mai canza lipid (TanjilG_32225), enzyme na endoglucan-1,3-β-glucoside (TanjilG_23384), sunadarai guda biyu masu haifar da damuwa kamar SAM22 (TanjilG_31528 da TanjilG_31531), furotin na asali na latex (TanjilG_32352), da furotin bango guda biyu masu arzikin glycine (TanjilG_19701 da TanjilG_19702). Akwai kuma babban haɗuwa a cikin martanin transcriptome tsakanin 83A:476 da Boregine a 24 HPI (jimilla 16-38% DEG) da tsakanin Mandelup da Population 22660 a 48 HPI (jimilla 14-20% DEG).
Zane na Venn wanda ke nuna adadin kwayoyin halitta da aka nuna daban-daban (DEG) a cikin layukan lupine mai ganye mai duhu (NLL) da aka yi wa allurar Colletotrichum lupini (nau'in Col-08 da aka samo daga filayen lupine a Wierzhenice, Poland, 1999). Layukan NLL da aka yi nazari a kansu sune: 83A:476 (mai juriya, yana ɗauke da allele na Lanr1), Boregine (mai juriya, ba a san asalin kwayoyin halitta ba), Mandelup (mai juriya matsakaici, yana ɗauke da allele na AnMan) da kuma yawan jama'a 22660 (mai saurin kamuwa da cutar). Takaitaccen bayanin hpi yana tsaye na tsawon awanni bayan allurar riga-kafi. An cire sifili ƙima don sauƙaƙe jadawalin.
An yi nazarin tsarin kwayoyin halittar da suka wuce gona da iri a 6 hpi don ganin kasancewar yankunan kwayoyin halittar R masu canonical (Teburin Karin Bayani S7). Wannan binciken ya nuna yadda kwayoyin halittar da ke jure wa cututtuka na gargajiya ke haifar da yankunan NBS-LRR kawai a 83A:476. Wannan saitin ya ƙunshi kwayar halittar TIR-NBS-LRR guda ɗaya (tanjilg_05042), kwayoyin halittar CC-NBS-LRR guda biyar (tanjilg_06165, tanjilg_06162, tanjilg_22773, tanjilg_22640, da tanjilg_16162), da kuma NBS-LR guda huɗu, Tanjilg_16162), da kuma NBS-LR guda huɗu (tanjilg_16162) da kuma NBS-LR guda huɗu (tanjilg_16162). Duk waɗannan kwayoyin halitta suna da yankuna masu tsari waɗanda aka tsara a cikin jerin da aka kiyaye. Baya ga kwayoyin halittar yankin NBS-LRR, an kunna kinases na RLL da yawa a 6 hpi, wato ɗaya a cikin Boregine (TanjilG_19877), biyu a cikin Mandelup (TanjilG_07141 da TanjilG_19877) da kuma a cikin yawan jama'a 22660 (TanjilG_09014 da TanjilG_10361) da biyu a cikin 83A 27:476.
An yi wa kwayoyin halitta da suka canza yanayin bayyanarsu sosai a martanin allurar rigakafin C. lupini (nau'in Col-08) nazarin wadatar Gene Ontology (GO) (Karin Tebur S8). Kalmar da aka fi nunawa fiye da kima ita ce "Amsar kariya ta GO:0006952" wadda ta bayyana a cikin haɗuwa 6 cikin 16 (lokaci × layi) tare da babban mahimmanci (ƙimar P < 0.001) (Hoto na 2). Kalmar da aka fi nunawa fiye da kima ita ce "Amsar kariya ta GO:0006952" wadda ta bayyana a cikin haɗuwa 6 cikin 16 (lokaci × layi) tare da babban mahimmanci (ƙimar P < 0.001) (Hoto na 2). Наиболее часто чрезмерно представленным термином биологического процесса был «GO: 0006952 защитой» появлялся в 6 из 16 (время × линия) комбинаций с высокой значимостью (значение P <0,001) (рис. 2). Kalmar da aka fi nunawa fiye da kima ita ce 'Amsar kariya ta GO:0006952', wadda ta bayyana a cikin haɗuwa 6 daga cikin 16 (lokaci × layi) tare da babban mahimmanci (ƙimar P < 0.001) (Hoto na 2).最常被过度代表的生物过程术语是“GO:0006952个中，具有高显着性（P 值< 0.001）（图2）。 Kalmar da ta fi wakiltar tsarin halittu ita ce "MAYAR DA HANKALI TA GO:0006952", wadda ta bayyana a cikin haɗuwa 6 daga cikin 16 (时间×线), tare da babban mahimmanci (ƙimar P < 0.001) (图2). Наиболее часто чрезмерно представленным термином биологического процесса был «GO: 0006952 Amsar Tsaro», из 16 комбинаций (время × линия) с высокой значимостью (значение P <0,001) (рис. 2). Kalmar da aka fi nuna yawan amfani da ita a fannin nazarin halittu ita ce 'GO:0006952 Defense Response', wadda ta bayyana a cikin haɗuwa 6 cikin 16 (lokaci × layi) tare da babban mahimmanci (ƙimar P < 0.001) (Hoto na 2).An wakilci wannan kalma fiye da kima a lokutan biyu a cikin 83A: 476 da Boregine (6 da 24 hpi) da kuma a wani lokaci a cikin Mandelup da Population 22660 (12 da 6 hpi, bi da bi). Wannan sakamako ne da ake tsammani, yana nuna martanin fungi na layukan da ke jure wa sinadarai. Bugu da ƙari, 83A:476 ya mayar da martani ga C. lupini ta hanyar haifar da kwayoyin halitta da suka shafi fashewar iskar oxygen da kalmar "GO:0055114 redox process" ta wakilta, yana nuna takamaiman martanin kariya, yayin da Boregine ya bayyana takamaiman martanin kariya, wanda ya shafi kalmar 'GO'. :0006950 Amsar damuwa". Population 22660 ya kunna amsawar juriya a kwance wanda ya ƙunshi metabolites na biyu, yana nuna adadin kalmomin da suka wuce gona da iri "GO:0016104 Tsarin biosynthesis na triterpene" da "GO:0006722 Tsarin metabolism na triterpene" (duka kalmomin suna cikin tsarin kwayoyin halitta iri ɗaya), idan aka yi la'akari da sakamakon nazarin wadatar da kalmar GO, kwanciyar hankali na amsawar Mandelup ya kasance tsakanin Boregine da Population 22660. Bugu da ƙari, amsawar farko 83A:476 (6 hpi) da jinkirin amsawar Mandelup da Population 22660 sun haɗa da kalmar GO:0015979 'photosynthesis' da sauran hanyoyin halittu masu alaƙa.
Kalmomin kwayoyin halittar da aka zaɓa a cikin bayanin kwayoyin halittar da aka bayyana daban-daban yayin martanin transcriptome na lupine mai ganye mai ƙunci (NLL) wanda aka yi wa allurar lupine na anthrax (nau'in Col-08 da aka samu daga filayen lupine a Wierzhenice, Poland, a 1999) an yi su da yawa. Layukan NLL da aka yi nazari a kansu sune: 83A:476 (mai juriya, yana ɗauke da allele na Lanr1 homozygous), Boregine (mai juriya, ba a san asalin kwayoyin halitta ba), Mandelup (mai juriya matsakaici, yana ɗauke da allele na AnMan homozygous) da kuma yawan jama'a 22660 (mai yiwuwa).
Saboda wannan binciken yana da nufin gano kwayoyin halittar da ke ba da gudummawa ga juriyar anthracnose, an yi nazarin kwayoyin halittar da aka sanya wa kalmomin GO “GO: 0006952 Amsoshin Kariya” da “GO: 0055114 Redox processes” tare da yanke-kashe tunda ma'anar tushe ≥ 30 tare da aƙalla layi ɗaya. × lokaci a lokaci tare da ƙimar ƙididdiga mai mahimmanci na log2 (canjin ninkawa). Adadin kwayoyin halittar da suka cika waɗannan sharuɗɗan shine 65 don GO:0006952 da 524 don GO:0055114.
83A:476 ya bayyana kololuwar DEG guda biyu da aka yi wa lakabi da GO:0006952, na farko a kwayoyin halitta 6 a kowace inci (kwayoyin halitta 64, tsarin sama da ƙasa) da na biyu a kwayoyin halitta 24 a kowace inci (kwayoyin halitta 15, tsarin sama kawai). Boregine ya kuma nuna cewa GO:0006952 ya kai kololuwa a lokaci guda, amma tare da ƙarancin DEG (11 da 8) da kuma kunna fifiko. Mandeloop ya nuna kololuwa biyu na GO:0006952 a 12 da 48 HPI, duka suna ɗauke da kwayoyin halitta 12 (na farko tare da kwayoyin halitta masu kunnawa, na biyu kuma tare da kwayoyin halitta masu hanawa kawai), yayin da yawan jama'a 22660 a 6 HPI (kwayoyin halitta 13) suna da rinjaye mafi girma na tsarin ƙara kololuwa. Ya kamata a lura cewa 96.4% na GO:0006952 DEG a cikin waɗannan kololuwa suna da irin wannan martani (sama ko ƙasa), yana nuna babban haɗuwa a cikin martanin tsaro duk da bambance-bambancen adadin kwayoyin halitta da ke ciki. Mafi girman rukuni na jerin da suka shafi kalmar GO:0006952 ya ƙunshi furotin mai alaƙa da yunwa mai damuwa 22 (SAM22-like), wanda ke cikin furotin mai alaƙa da cutarwa na aji 10 (PR-10) da kuma furotin mai kama da na MLP) (Hoto na 3). Ƙungiyoyin biyu sun bambanta a yanayin bayyanar da kuma alkiblar amsawar. Kwayoyin halittar da ke ɗauke da sunadaran SAM22 sun nuna daidaito da tasiri mai mahimmanci a farkon lokacin (6 ko 12 hpi) kuma gabaɗaya ba su amsa ba a ƙarshen gwajin (48 hpi), yayin da sunadarai masu kama da MLP sun nuna daidaito a 6 hpi. hpi. 83A:476 da Mandelup a 48 hp/in, kusan duk sauran bayanan ba su amsa ba. Bugu da ƙari, bambance-bambance a cikin bayanan bayyanar kwayoyin halittar furotin masu kama da SAM22 sun biyo bayan bambancin da aka lura a cikin juriyar anthracnose, saboda layukan da suka fi juriya suna da ƙarin maki na lokaci suna haifar da waɗannan kwayoyin halitta fiye da kwayoyin halittar da suka fi saurin kamuwa da cutar. Wata kwayar halittar PR-10 mai kama da LlR18A/B ta nuna irin wannan yanayin bayyanar kwayar halittar furotin mai kama da SAM22.
An gano manyan sassan kalmar tsarin halittu "GO:0006952 Defense Response" da kuma tsarin bayyanar kwayoyin halitta na kwayoyin halitta na Lanr1 da AnMan. Ma'aunin Log2 yana wakiltar ƙimar log2 (canjin ninkawa) tsakanin allurar rigakafi (Colletotrichum lupini, nau'in Col-08, wanda aka samo daga filayen lupine, Wizhenica, Poland, 1999) da tsire-tsire masu sarrafawa (sham-inoculated) a lokaci guda. An yi nazarin layukan lupine masu kunkuntar ganye: 83A:476 (juriya, ɗauke da allele na Lanr1 homozygous), Boregine (juriya, ba a san asalin kwayoyin halitta ba), Mandelup (mai juriya matsakaici, yana ɗauke da allele na AnMan homozygous), da Population 22660 (mai sauƙin kamuwa).
Bugu da ƙari, an kimanta bayanan bayyanar kwayoyin halittar da ke da alaƙa da RNA-seq Lanr1 (TanjilG_05042) da AnMan (TanjilG_12861) (Hoto na 3). Kwayar halittar TanjilG_05042 ta nuna wani muhimmin martani (kunnawa) a 83A:476 kawai a farkon lokacin (6 hpi), yayin da TanjilG_12861 ya kasance mai mahimmanci a Mandeloop kawai a lokutan lokaci biyu: 6 hpi (ƙasa) da 24 hpi (6 hpi). Tare da.). mai daidaitawa) ).
Kwayoyin halittar da aka fi bayyanawa a cikin kalmar "redox process" GO:0055114 sune kwayoyin halittar da ke samar da sunadaran cytochrome P450 da peroxidase (Hoto na 4). Ga samfuran da aka ware daga 83A:476 a 6 HPI, an lura da matsakaicin ko mafi ƙarancin ƙimar log2 (canjin ninkawa) (ga 86.6% na kwayoyin halittar) gabaɗaya tsakanin tsire-tsire da aka yi wa allura da kuma waɗanda ke kula da su, wanda ke nuna babban martanin wannan nau'in kwayar halittar ga jima'i da aka yi wa allura. 83A:476 ya nuna mafi mahimmancin GO: 0055114 DEG a 6 hpi (kwayoyin halittar 503), yayin da sauran layukan a 48 hpi (Boregine, kwayoyin halittar 31; Mandelup, kwayoyin halittar 85; da kuma kwayoyin halittar Population 22660, kwayoyin halittar 78)). A yawancin kwayoyin halittar dangin GO:0055114, an lura da nau'ikan martani guda biyu ga allurar rigakafi (kunnawa da hanawa). Abin sha'awa, har zuwa kashi 97.6% na DEGs da aka gano don kalmar GO: 0055114 a cikin Mandelupe a 48 hp. Waɗannan abubuwan da aka lura sun nuna cewa, duk da ƙaramin sikelin (watau, adadin kwayoyin halittar redox da aka canza, 85 idan aka kwatanta da 503), tsarin jinkirin amsawar transcriptome na mandeloup zuwa anthracnose yayi kama da farkon amsawar 83A:476. A cikin Boregine da Population 22660, wannan haɗuwa ya yi ƙasa da kashi 51.6% da 75.6%, bi da bi.
An bayyana tsarin bayyanar manyan sassan kalmar tsarin halittu "GO:0055114 Redox process". Ma'aunin Log2 yana wakiltar ƙimar log2 (canjin ninkawa) tsakanin allurar rigakafi (Colletotrichum lupini, nau'in Col-08, wanda aka samo daga filayen lupine, Wizhenica, Poland, 1999) da tsire-tsire masu sarrafawa (sham-inoculated) a lokaci guda. An yi nazarin layukan lupine masu kunkuntar ganye: 83A:476 (juriya, ɗauke da allele na Lanr1 homozygous), Boregine (juriya, ba a san asalin kwayoyin halitta ba), Mandelup (mai juriya matsakaici, yana ɗauke da allele na AnMan homozygous), da Population 22660 (mai sauƙin kamuwa).
83A:476 Amsoshin transcriptomic ga allurar rigakafi da C. lupini (nau'in Col-08) sun haɗa da rufe kwayoyin halitta da aka danganta da kalmar "photosynthesis" GO:0015979 da sauran hanyoyin halittu masu alaƙa (FIG. 5). Wannan saitin GO:0015979 DEG ya ƙunshi kwayoyin halitta 105 waɗanda aka danne sosai a 6 hpi a 83A:476. A cikin wannan rukunin, an kuma rage yawan kwayoyin halitta 37 a cikin Mandelup a 48 HPI da 35 a lokaci guda a cikin yawan jama'a 22660, gami da 19 DEGs waɗanda suka zama ruwan dare ga duka nau'ikan kwayoyin halitta. Babu DEGs da suka shafi kalmar GO: 0015979 da aka kunna sosai a cikin kowane haɗuwa (layi x lokaci).
An bayyana tsarin bayyanar manyan sassan kalmar tsarin halittu "GO:0015979 Photosynthesis". Ma'aunin Log2 yana wakiltar ƙimar log2 (canjin ninkawa) tsakanin allurar rigakafi (Colletotrichum lupini, nau'in Col-08, wanda aka samo daga filayen lupine, Wizhenica, Poland, 1999) da tsire-tsire masu sarrafawa (sham-inoculated) a lokaci guda. An yi nazarin layukan lupine masu kunkuntar ganye: 83A:476 (juriya, ɗauke da allele na Lanr1 homozygous), Boregine (juriya, ba a san asalin kwayoyin halitta ba), Mandelup (mai juriya matsakaici, yana ɗauke da allele na AnMan homozygous), da Population 22660 (mai yiwuwa).
Dangane da sakamakon nazarin bambancin magana da kuma wanda ake kyautata zaton yana da hannu a martanin kariya daga fungi masu cutarwa, an zaɓi wannan saitin kwayoyin halitta guda bakwai don ƙididdige bayanan bayyanawa ta hanyar PCR na ainihin lokaci (Tebur na Ƙarin S9).
An haifar da kwayar halittar furotin mai suna TanjilG_10657 sosai a cikin dukkan layukan da aka yi nazari a kansu da kuma lokutan lokaci idan aka kwatanta da tsire-tsire masu sarrafawa (kwaikwayo) (Tebur na Ƙarin S10, S11). Bugu da ƙari, bayanin bayyanar TanjilG_10657 ya nuna ƙaruwar yanayin a tsawon gwajin ga dukkan layukan. Yawan jama'a 22660 ya nuna mafi girman ƙarfin TanjilG_10657 ga allurar riga-kafi tare da kunnawa sau 114 da kuma mafi girman matakin bayyanar dangi (4.4 ± 0.4) a 24 HPI (Hoto na 6a). Kwayar halittar furotin ta PR10 LlR18A TanjilG_27015 ta kuma nuna kunnawa a duk layuka da wuraren lokaci, tare da mahimmancin ƙididdiga a yawancin wuraren bayanai (Hoto na 6b). Kamar TanjilG_10657, an lura da mafi girman matakin bayyanar TanjilG_27015 a cikin mutane 22660 da aka yi wa allurar rigakafi a 24 HPI (19.5 ± 2.4). An ƙara yawan kwayar halittar acid endochitinase TanjilG_04706 a duk layuka da kuma a kowane lokaci banda Boregine 6 hpi (Hoto na 6c). An haifar da shi sosai a farkon lokacin (6 HPI) a 83A:476 (da sau 10.5) kuma ya ƙaru sosai a wasu layuka (da sau 6.6-7.5). A lokacin gwajin, bayyanar TanjilG_04706 ya kasance a matakai iri ɗaya a 83A:476 da Boregine, yayin da a Mandelup da Population 22660 ya ƙaru sosai, ya kai ƙima mai girma (5.9 ± 1.5 da 6.2 ± 1.5, bi da bi). Kwayar halittar TanjilG_23384 mai kama da endoglucan-1,3-β-glucosidase ta nuna babban kunnawa a farkon maki biyu (6 da 12 hpi) a cikin dukkan layuka banda yawan jama'a 22660 (Hoto na 6d). An lura da mafi girman matakan bayyanar TanjilG_23384 a karo na biyu (12 hpi) a cikin Mandelup (2.7 ± 0.3) da 83A:476 (1.5 ± 0.1). A 24 HPI, bayyanar TanjilG_23384 ta kasance ƙasa kaɗan a cikin dukkan layukan da aka yi nazari a kansu (daga 0.04 ± 0.009 zuwa 0.44 ± 0.12).
Bayanan bayyanar kwayoyin halitta (ag) da aka bayyana ta hanyar PCR mai ƙima. Lambobi 6, 12 da 24 suna wakiltar sa'o'i bayan allurar riga-kafi. An yi amfani da kwayoyin halittar LanDExH7 da LanTUB6 don daidaita su kuma an yi amfani da LanTUB6 don daidaita su tsakanin jerin. Sandunan kuskure suna wakiltar karkacewar da aka saba bisa ga kwafi uku na halitta, kowannensu shine matsakaicin kwafi uku na fasaha. An yi wa alamun kididdigar bambance-bambancen matakan bayyanawa tsakanin allurar da aka yi wa allurar (Colletotrichum lupini, nau'in Col-08, wanda aka samu a shekarar 1999 daga filin lupin a Wierzenica, Poland) da kuma tsire-tsire masu sarrafawa (mock-inoculated) alama a sama da wuraren bayanai (*P darajar < 0.05, **P darajar ≤ 0.01, ***P darajar ≤ 0.001). An yi wa alamun kididdigar bambance-bambancen matakan bayyanawa tsakanin allurar da aka yi wa allurar (Colletotrichum lupini, nau'in Col-08, wanda aka samu a shekarar 1999 daga filin lupin a Wierzenica, Poland) da kuma tsire-tsire masu sarrafawa (mock-inoculated) alama a sama da wuraren bayanai (*P darajar < 0.05, **P darajar ≤ 0.01, ***P darajar ≤ 0.001). Сатистическая значимость различий в уровнях эkspressyy mezhdu inokulyrovannыmy (Colletotrichum lupini, shtamm Col-08, 99, поля люпина в Верженице, Польша) и контрольными 0,05, **значение P ≤ 0,01, ***значение P ≤ 0,001). An lura da bambance-bambance masu mahimmanci a cikin matakan bayyanawa tsakanin allurar rigakafi (Colletotrichum lupini, nau'in Col-08, wanda aka samo a 1999 daga filin lupine a Wierzhenice, Poland) da tsire-tsire masu sarrafawa (sham-inoculated) a sama da bayanan (*P darajar < 0.05, **P-darajar ≤ 0.01, ***P-darajar ≤ 0.001).接种 (Colletotrichum lupini, Col-08株，1999年从波兰Wierzenica的羽扇豆田获得）和对照（模拟接种）植物之间表达水平差异的统计学显着性标记在数捹疊在数捹丹丹的） 0.05, ** P 值≤ 0.01, *** P 值≤ 0.001).接种 （colletotrichum lupini ， color-08 株 ， 1999 年 波兰 波兰 wierzenica 的 羽扇 获得）水平 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). Сатически значимыe razlychyya v urovnyah эkspressyy mezhdu inokulyrovannymy (Colletotrichum lupini, shtemm Col-08, pо люпина в Верженице, Польша, в 1999 г.) и котрольныmy значение P <0,05, ** P-значение ≤ 0,01, *** P-значение ≤ 0,001). An lura da bambance-bambance masu mahimmanci a cikin matakan bayyanawa tsakanin allurar rigakafi (Colletotrichum lupini, nau'in Col-08, wanda aka samo daga filayen lupine a Verzhenice, Poland, a 1999) da tsire-tsire masu sarrafawa (sham-inoculated) a sama da bayanan (*P darajar < 0.05 , **P-darajar ≤ 0.01, ***P-darajar ≤ 0.001).Layukan NLL da aka yi nazari a kansu sune: 83A:476 (mai juriya, yana ɗauke da allele na Lanr1 homozygous), Mandelup (mai juriya matsakaici, yana ɗauke da allele na AnMan homozygous), Boregine (mai juriya, ba a san asalin kwayoyin halitta ba) da kuma yawan jama'a 22660 (mai juriya).
Kwayar halittar TanjilG_05042 da aka zaɓa a wurin Lanr1 ta nuna wata hanya ta bayyana ra'ayi daban da bayanan da aka samu daga nazarin RNA-seq (Hoto na 6e). An lura da gagarumin kunna wannan kwayar halitta a cikin Mandelup da kuma yawan jama'a 22660 (har zuwa sau 39.7 da 11.7, bi da bi), wanda ya haifar da matakan bayyana ra'ayi masu yawa (har zuwa 1.4 ± 0.14 da 7.2 ± 1.3, bi da bi). 83A:476 ya kuma nuna wasu ƙaruwar kwayar halittar TanjilG_05042 (har zuwa ninki 3.8), duk da haka, matakan bayyanar da aka samu (0.044 ± 0.002) sun fi ƙasa da waɗanda aka lura a Mandelup da yawan jama'a 22660. Binciken qPCR ya nuna bambance-bambance masu mahimmanci a matakan bayyanawa tsakanin nau'ikan halittu a cikin nau'ikan allurar rigakafi ta gwaji (control), wanda ya kai ninki 58 tsakanin al'umma 22660 da 83A:476, da kuma tsakanin al'umma 22660 da 22660. An cimma bambanci mai ninki biyu tsakanin Boregine da Mandalup.
An kunna kwayar halittar da aka zaɓa a wurin AnMan, TanjilG_12861, sakamakon allurar riga-kafi a 83A:476 da Mandelup, ba ta da tsaka-tsaki a cikin yawan jama'a 22660, kuma an rage ta a Boregine (Hoto na 6f). Bayyanar kwayar halittar TanjilG_12861 ta kasance mafi girma a cikin allurar riga-kafi 83A: 476 (0.14±0.01). Kwayar halittar furotin mai girgiza zafi ta aji I TanjilG_05080 HSP17.4 ta nuna ƙarancin matakan bayyanar dangi a cikin duk nau'ikan da aka yi nazari da lokutan lokaci (Hoto na 6g). An lura da mafi girman ƙimar a 24 HPI a cikin yawan jama'a 22660 (0.14 ± 0.02, ƙaruwa sau takwas a martanin allurar riga-kafi).
Kwatanta bayanan bayyanar kwayoyin halitta (Hoto na 7) ya nuna babban alaƙa tsakanin TanjilG_10657 da wasu kwayoyin halitta guda huɗu: TanjilG_27015 (r = 0.89), TanjilG_05080 (r = 0.85), TanjilG_05042 (r = 0.80), da TanjilG_04706 (r = 0.79). Irin waɗannan sakamakon na iya nuna haɗin kai tsakanin waɗannan kwayoyin halitta yayin martanin kariya. Kwayoyin halittar TanjilG_12861 da TanjilG_23384 sun nuna bayanan bayyanar cututtuka daban-daban tare da ƙananan ƙimar haɗin gwiwa na Pearson (daga 0.08 zuwa 0.43 da -0.19 zuwa 0.28, bi da bi) idan aka kwatanta da sauran kwayoyin halitta.
An gano alaƙa tsakanin bayanan bayyanar kwayoyin halitta ta amfani da PCR mai ƙima. An yi nazarin layukan lupine masu laushi masu zuwa: 83A:476 (mai juriya, yana ɗauke da allele na homozygous Lanr1), Mandelup (mai juriya matsakaici, yana ɗauke da allele na homozygous AnMan), Boregine (mai juriya, ba a san asalin kwayoyin halitta ba), da Population 22660 (mai saurin kamuwa da cuta). An ƙididdige maki uku na lokaci (awanni 6, 12 da 24 bayan allurar), gami da allurar (Colletotrichum lupini, nau'in Col-08, wanda aka samo daga filayen lupine a Wierzhenice, Poland, a cikin 1999) da tsire-tsire masu sarrafawa (sham-inoculated). Sikelin yana nuna ƙimar ma'aunin haɗin gwiwar Pearson.
Dangane da bayanan da aka samu a ƙarfin dawaki 6 a kowace inci, an yi WGCNA akan 9981 DEG da aka gano ta hanyar kwatanta shuke-shuken da aka yi wa allura da kuma waɗanda aka sarrafa don mai da hankali kan martanin kariya da wuri (Tebur na Ƙarin S12). An sami nau'ikan kwayoyin halitta ashirin da biyu (gungu) tare da bayanan bayyanar da suka shafi (mai kyau ko mara kyau) tsakanin nau'ikan kwayoyin halitta da bambance-bambancen gwaji. A matsakaici, matakan bayyanar kwayoyin halitta suna raguwa kamar yadda aka tsara 83A:476 > Mandelup > Boregine > Yawan jama'a 22660 (duk da haka, a cikin nau'ikan biyu, wannan yanayin ya fi ƙarfi a cikin tsire-tsire masu sarrafawa). A matsakaici, matakan bayyanar kwayoyin halitta suna raguwa kamar yadda aka tsara 83A:476 > Mandelup > Boregine > Yawan jama'a 22660 (duk da haka, a cikin nau'ikan biyu, wannan yanayin ya fi ƙarfi a cikin tsire-tsire masu sarrafawa). 83A: 476 > Mandelup > Boregine > Yawan Jama'a 22660 была сильнее у контрольных растений). A matsakaici, matakan bayyanar kwayoyin halitta sun ragu a cikin tsari 83A:476 > Mandelup > Boregine > Yawan jama'a 22660 (duk da haka, a cikin nau'ikan biyu, wannan yanayin ya fi ƙarfi a cikin tsire-tsire masu sarrafawa).平均而言，基因表达水平按83A:476 > Mandelup > Boregine > Yawan jama'a 22660的顺序下降（然而，在两种变体中，这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> yawan jama'a 22660 的 顺序中更） . . . . . . . 83A: 476 > Mandelup > Boregine > Yawan Jama'a 22660 сильнее у контрольных растений). A matsakaici, matakan bayyanar kwayoyin halitta sun ragu a cikin jerin 83A:476 > Mandelup > Boregine > Yawan jama'a 22660 (duk da haka, a cikin nau'ikan biyu, wannan yanayin ya fi ƙarfi a cikin tsire-tsire masu sarrafawa).Allurar riga-kafi ta haifar da ƙaruwar bayyanar kwayoyin halitta, musamman a cikin sassan 18, 19, 14, 6 da 1 (a cikin tsarin raguwar tasirin), ƙa'idojin rashin kyau (misali sassan 9 da 20) ko tare da tasirin tsaka-tsaki (misali sassan 11, 22, 8 da 13). Binciken wadatar da lokaci na GO (Tebur na Ƙarin S13) ya bayyana "GO: 0006952 Amsoshin Kariya" ga sashin allurar (18) tare da matsakaicin kunnawa, gami da kwayoyin halittar da qPCR ya bincika (TanjilG_04706, TanjilG_23384, TanjilG_10657 da TanjilG_27015), da kuma yawancin sassan photosynthesis da aka danne (9). An gano mai tattara bayanai na Module 18 (Hoto na 8) a matsayin kwayar halittar TanjilG_26536 wacce ke tattara sunadaran LlR18B masu kama da PR-10, kuma an gano mai tattara bayanai na Module 9 a matsayin kwayar halittar TanjilG_28955 wacce ke tattara sunadaran photosystem II PsbQ. An sami kwayar halittar da ke da juriya ga anthracnose Lanr1, TanjilG_05042, a cikin module 22 (Hoto na 9) kuma an danganta ta da kalmomin "GO:0044260 Tsarin metabolism na ƙwayoyin halitta" da "GO:0006355 Tsarin Transcriptional, DNA templating" wanda ke ɗauke da cibiyar TanjilG_01212. kwayar halittar tana tattara bayanai na yanayin zafi A-4a (HSFA4a).
Binciken hanyar sadarwa mai ma'auni na bayyana haɗin gwiwar kwayoyin halitta na na'urori tare da kalmomin tsarin halittu da aka wakilta da yawa "GO: 0006952 Amsoshin Tsaro". An sauƙaƙe ligation don haskaka kwayoyin halitta guda huɗu da qPCR ya bincika (TanjilG_04706, TanjilG_23384, TanjilG_10657 da TanjilG_27015).
Binciken hanyar sadarwa mai ma'auni na bayyanuwar kwayoyin halitta tare da kalmar da aka fi wakilta ta hanyar kwayoyin halitta "GO: 0006355: Tsarin kwafi, Tsarin DNA" da kuma ɗaukar kwayar halittar juriya ta anthracnose Lanr1 TanjilG_05042. An sauƙaƙa ligation don ware kwayar halittar TanjilG_05042 da kwayar halittar TanjilG_01212 ta tsakiya.
Binciken juriyar Anthracnose da aka tattara a Ostiraliya ya nuna cewa yawancin nau'ikan da aka fitar da su da wuri suna da sauƙin kamuwa da su; Kalya, Coromup da Mandelup an bayyana su a matsayin masu juriya matsakaici, yayin da Wonga, Tanjil da 83A:476 an bayyana su a matsayin masu juriya sosai26,27,31. suna da juriya iri ɗaya, Lanr1, kuma Coromup da Mandelup suna da kwayar halitta daban-daban, an sanya AnMan10, 26, 39, yayin da Kalya ta wuce wata kwayar halitta daban., Lanr2. Binciken juriyar anthracnose a Jamus ya haifar da gano layin juriya na Bo7212 tare da kwayar halitta daban-daban banda Lanr1, wanda aka sanya LanrBo36.
Bincikenmu ya nuna ƙarancin mitoci (kimanin kashi 6%) na allele na Lanr1 a cikin germplasm da aka gwada. Wannan lura ya yi daidai da sakamakon tantance germplasm na Gabashin Turai ta amfani da alamun Anseq3 da Anseq4, wanda ya nuna cewa allele na Lanr1 yana cikin layuka biyu na Belarus. Wannan yana nuna cewa allele na Lanr1 ba a amfani da shi sosai a shirye-shiryen kiwo na gida ba, ba kamar a Ostiraliya ba, inda yake ɗaya daga cikin manyan allele don kiwo da aka taimaka wa alama. Wannan na iya zama saboda ƙarancin matakin juriya da allele na Lanr1 ke bayarwa a cikin yanayin filin Turai idan aka kwatanta da rahoton Ostiraliya. Bugu da ƙari, nazarin anthracnose a cikin yankunan da ruwan sama ya yi yawa a Ostiraliya ya nuna cewa martanin juriya da allele na Lanr1 ke shiga tsakani bazai yi tasiri ba a cikin yanayin yanayi wanda ke fifita girma da haɓaka saurin pathogen19,42. A gaskiya ma, a cikin wannan binciken, an kuma lura da wasu alamun anthracnose a cikin genotypes ɗauke da allele na Lanr1, yana nuna cewa juriya na iya ɓacewa a ƙarƙashin yanayi mafi kyau don ci gaban C. lupini. Bugu da ƙari, fassarar ƙarya mai kyau game da kasancewar alamun Anseq3 da Anseq4, waɗanda suke kusan 1 cM daga wurin Lanr1, yana yiwuwa 28, 30, 43.
Bincikenmu ya nuna cewa 83A:476, wanda ke ɗauke da allele na Lanr1, ya amsa allurar C. lupini tare da babban tsarin sake fasalin transcriptome a lokacin farko da aka bincika (6 hpi), yayin da a Mandelup, wanda ke ɗauke da allele na AnMan, an lura da martanin transcriptomic daga baya. (daga 24 zuwa 48 hp). Waɗannan bambance-bambancen lokaci a cikin martanin kariya suna da alaƙa da bambance-bambancen alamun cututtuka, suna nuna mahimmancin gane ƙwayoyin cuta da wuri don samun nasarar amsawa ga juriya. Don kamuwa da ƙwayar shuka, ƙwayoyin anthrax dole ne su bi matakai da yawa na ci gaba a saman mai masaukin baki, gami da tsiro, rarrabuwar ƙwayoyin halitta, da samuwar appressorium. Wani abu mai kama da ƙari tsari ne mai kamuwa da cuta wanda ke manne da saman mai masaukin baki kuma yana sauƙaƙa shiga cikin kyallen mai masaukin baki. Don haka, ƙwayoyin C. gloeosporioides a cikin cirewar wake sun nuna rabuwar farko na tsakiya bayan mintuna 75-90 na haɗuwa, samuwar bututun ƙwaya bayan mintuna 90-120, da kuma dannewa bayan awanni 4 45. Mango C. gloeosporioides ya nuna fiye da kashi 40% na tsirowar conidial bayan sa'o'i 3 na shukawa da kuma kusan kashi 20% na samuwar appressors bayan sa'o'i 4. Kwayar halittar CAP20 ta C. gloeosporioides mai alaƙa da kamuwa da cuta ta nuna aikin kwafi a cikin conidia mai samar da epiphyte bayan sanyawa a cikin kakin zuma na sama na awanni 3.5 tare da yawan furotin CAP20 mai yawa bayan sa'o'i 4 da mintuna 46. Hakazalika, aikin kwayoyin halittar melanin biosynthesis a cikin C. trifolii ya faru ne yayin sanyawa na awanni 2 sannan kuma samar da appressorium bayan sa'a 1. Nazarin kyallen ganye ya nuna cewa strawberries da aka yi wa allurar C. acutatum suna da damƙar farko a 8 hpi, yayin da tumatir da aka yi wa allurar C. coccodes suna da damƙar farko a 4 hpi48,49. galibi ya yi daidai da lokacin da Collettrichum spp. kamuwa da cuta ke yaduwa. Amsoshin kariya cikin sauri ga 83A:476 sun nuna cewa akwai haɗarin juriyar tsirrai da kwayoyin halittar rigakafi (ETI) a cikin wannan layin, yayin da martanin da aka jinkirta na Mandelup ya goyi bayan hasashen ƙwayoyin cuta masu alaƙa da tsarin rigakafi (MTI) 50. Amsoshin farko ga 83A: 476 da Mandelup. Haɗuwa tsakanin kwayoyin halitta masu tasowa ko waɗanda aka rage a cikin jinkirin amsawa shima yana goyan bayan wannan ra'ayi, kamar yadda sau da yawa ana ɗaukar ETI a matsayin martanin MTI mai sauri da haɓaka wanda ke haifar da mutuwar ƙwayoyin halitta da aka tsara a wurin kamuwa da cuta, wanda aka sani da girgizar anaphylactic 51,52.
Yawancin kwayoyin halittar da aka danganta da kalmar da aka fi wakilta ta Gene Ontology GO:0006952 "Amsa ta Tsaro" sune homologues 11 na furotin azumi mai saurin damuwa wanda damuwa ta haifar (kamar SAM22) da kuma manyan sunadaran latex guda bakwai masu kama da na latex (MLPs). masu kama da na latex 31, 34, 43 da 423 sun nuna kamanceceniya a jere. Kwayoyin halittar SAM22 sun nuna gagarumin kunnawa wanda ya daɗe, wanda ke nuna ƙaruwar matakan juriyar anthracnose (83A:476 da Boregine). Duk da haka, kwayoyin halittar MLP masu kama da na MLP an rage su ne kawai a cikin layukan da ke ɗauke da allele na juriya (83A:476/Lanr1 a 6 hpi da Mandelup/AnMan a 24 hpi). Ya kamata a lura cewa duk homologues masu kama da SAM22 da aka gano sun samo asali ne daga tarin kwayoyin halitta da ke kimanin 105 kb, yayin da kwayoyin halittar MLP masu kama da na MLP suka samo asali ne daga yankuna daban-daban na kwayoyin halittar. An kuma gano yadda aka tsara kunna irin waɗannan kwayoyin halittar SAM22 a cikin bincikenmu na baya game da juriyar NLL ga allurar Diaportetoxica, yana nuna cewa suna da hannu a cikin sassan kwance na martanin kariya. Wannan ƙarshe kuma yana da goyan bayan rahotannin martani mai kyau na kwayoyin halittar SAM22 ga rauni ko magani da salicylic acid, abubuwan da ke haifar da fungal, ko hydrogen peroxide.
An nuna cewa kwayoyin halittar MLP suna mayar da martani ga matsalolin abiotic da biotic daban-daban, gami da cututtukan fungal na ƙwayoyin cuta, ƙwayoyin cuta da kuma cututtuka a cikin nau'ikan tsire-tsire da yawa55. Umarnin martani ga wasu hulɗa tsakanin tsire-tsire da ƙwayoyin cuta sun kama daga ƙaruwa sosai (watau, yayin kamuwa da auduga tare da Verticillium dahliae) zuwa raguwa sosai (watau, bayan kamuwa da itacen apple tare da Alternaria spp.)56,57. An lura da raguwar kwayar halittar MLP mai kama da 423 a lokacin karewar avocado ga kamuwa da F. niger da kuma lokacin kamuwa da bishiyar apple Botryosphaeria berengeriana f. cn. piricola da Alternaria alternata sune cututtukan apple58,59. Bugu da ƙari, apple calli da ke nuna kwayar halittar MLP mai kama da 423 fiye da kima yana da ƙarancin bayyanar kwayoyin halittar da ke da alaƙa da juriya kuma sun fi saurin kamuwa da cututtukan fungal59. Bayan Fusarium oxysporum f, kwayar halittar MLP mai kama da 423 kuma an danne ta a cikin ƙwayar wake mai jure wari. cn. Cutar wake 60.
Sauran membobin dangin PR-10 da aka gano a cikin bincikenmu na RNA-seq sune kwayoyin halittar LlR18A da LlR18B don mayar da martani ga haɓaka, da kuma kwayar halittar da aka haɓaka (ƙwayar halitta 1) ko kuma kwayar halittar da aka rage (ƙwayar halitta 3) don furotin mai canza lipid DIR1. . Bugu da ƙari, WGCNA ta nuna kwayar halittar LlR18B a matsayin cibiya a cikin wannan sashin, wanda ke da matukar saurin kamuwa da rigakafi kuma yana ɗauke da kwayoyin halittar da ke kare shi. Kwayoyin halittar LlR18A da LlR18B an haifar da su a cikin ganyen lupine masu launin rawaya saboda martani ga ƙwayoyin cuta masu cutarwa, da kuma a cikin tushen NLL bayan allurar D. toxica, yayin da haɗin gwiwar shinkafa na waɗannan kwayoyin halitta, RSOsPR10, ya haifar da sauri ta hanyar kamuwa da cuta ta fungal da ake tsammanin yana da hannu a cikin hanyar siginar jasmonic acid53,61, 62. Kwayar halittar DIR1 tana ɓoye sunadaran jigilar lipid marasa takamaiman waɗanda ake buƙata don fara juriya ta tsarin (SAR). Tare da ci gaban halayen kariya, ana jigilar furotin DIR1 daga wurin kamuwa da cuta ta hanyar phloem don haifar da SAR a cikin gabobin da ke nesa. Abin sha'awa, kwayar halittar TanjilG_02313 DIR1 ta haifar da gagarumin ci gaba a farkon lokaci a cikin layuka 84A:476 da Population 22660, amma juriyar anthracnose ta bunƙasa cikin nasara ne kawai a cikin layi 84A:476. Wannan na iya nuna wasu ƙananan ayyukan kwayar halittar DIR1 a cikin NLL, tunda sauran homologues guda uku sun amsa allurar ne kawai a cikin layin 83A:476 a 6 hpi, kuma an mayar da wannan martanin zuwa ƙasa.
A cikin bincikenmu, abubuwan da suka fi dacewa da tsarin halittu da ake kira "GO:0055114 Redox process" sune furotin cytochrome P450, peroxidase, linoleic acid 9S-/13S-lipoxygenase, da 1-aminocyclopropane-1-carboxylic acid oxidase. Bugu da ƙari, WGCNA ɗinmu ya bayyana homologue na HSFA4a a matsayin cibiyar ɗaukar kayayyaki kamar ɗan takarar kwayar halittar Lanr1 juriya TanjilG_05042. HSFA4a wani ɓangare ne na tsarin redox-dependent na kwafi na nukiliya a cikin tsire-tsire.
Sunadaran Cytochrome P450 sune oxidoreductases waɗanda ke haɓaka halayen hydroxylation na NADPH da/ko O2 da suka dogara da O2 a cikin metabolism na farko da na sakandare, gami da metabolism na xenobiotics, da kuma hormones, fatty acids, sterols, sassan bangon tantanin halitta, biopolymers, da kuma biosynthesis na mahadi masu kariya 69. A cikin wani bincike, an rage bambancin aikin cytochrome P450 na shuka daga -10.6 log2 (canjin ninka) zuwa 5.7 saboda yawan adadin homologues da aka canza (37) da bambance-bambance a cikin tsarin amsawa tsakanin takamaiman kwayoyin halitta, yana nuna sake dubawa sama. . Amfani da bayanan RNA-seq kawai don bayyana aikin ilimin halittar kwayoyin halitta na NLL a cikin irin wannan babban furotin superfamily zai zama babban hasashe. Duk da haka, ya kamata a lura cewa wasu kwayoyin halittar cytochrome P450 suna da alaƙa da ƙaruwar juriya ga fungi ko ƙwayoyin cuta masu cutarwa, gami da gudummawa ga halayen rashin lafiyan69,70,71.
Aji na III peroxidases enzymes ne masu aiki da yawa waɗanda ke da hannu a cikin nau'ikan hanyoyin rayuwa daban-daban yayin girma da haɓaka shuka, da kuma martani ga matsalolin muhalli kamar gishiri, fari, yawan haske, da harin ƙwayoyin cuta72. Peroxidases suna da hannu a cikin hulɗar nau'ikan tsire-tsire da yawa tare da Anthracis, gami da Stylosanthes humilis da C. gloeosporioides, Lens culinaris da C. truncatum, Phaseolus vulgaris da C. lindemuthianum, Cucumis sativus da C. lagenarium73,74,75,76. Amsar tana da sauri sosai, wani lokacin har ma a 4 HPI, kafin naman gwari ya shiga cikin ƙwayar shuka73. Kwayar halittar peroxidase kuma ta mayar da martani ga allurar D. toxica NLL. Baya ga ayyukansu na yau da kullun don daidaita fashewar oxidative ko kawar da damuwa na oxidative, peroxidases na iya tsoma baki tare da haɓakar ƙwayoyin cuta ta hanyar ƙirƙirar shinge na zahiri bisa ga ƙarfafa bangon tantanin halitta yayin lignification, subunit ko haɗin gwiwa na takamaiman mahadi. Ana iya danganta wannan aikin a cikin silico da kwayar halittar TanjilG_03329 wacce ke ɗauke da anion peroxidase mai samar da lignin wanda aka inganta sosai a cikin bincikenmu a cikin layin juriya na 83A:476 a 6 HPI, amma ba a cikin wasu nau'ikan da lokutan lokaci waɗanda ba su amsa ba.
9S-/13S-lipoxygenase na linoleic acid shine mataki na farko a cikin hanyar oxidative na biosynthesis na lipid78. Kayayyakin wannan hanyar suna da ayyuka da yawa a cikin kare tsirrai, gami da ƙarfafa bangon tantanin halitta ta hanyar samar da callose da pectin adibas, da kuma daidaita damuwa ta oxidative ta hanyar samar da nau'ikan iskar oxygen masu amsawa79,80,81,82,83. A cikin wannan binciken, an canza yanayin linoleic acid 9S-/13S-lipoxygenase a cikin dukkan nau'ikan, amma a cikin yawan masu rauni 22660, haɓaka tsari ya mamaye a lokutan lokaci daban-daban, yayin da a cikin nau'ikan da ke ɗauke da Lanr1 mai juriya da allele na AnMan, yana jaddada bambancin layin oxylipin a cikin halayen anthrax masu kariya tsakanin waɗannan nau'ikan kwayoyin halitta.
An ƙara yawan haɗin gwiwar 1-aminocyclopropane-1-carboxylate oxidase (ACO) sosai (ƙwayoyin halitta 9) ko kuma an rage yawansu (ƙwayoyin halitta 2) lokacin da aka yi musu allurar lupine. Banda waɗannan biyu, duk waɗannan martanin sun faru ne a 6 hp. a 83A:476. Haɗarin enzymatic da furotin ACO ke jagoranta shine matakin iyakance ƙimar samar da ethylene kuma saboda haka ana tsara shi sosai84. Ethylene wani sinadari ne na shuka wanda ke taka rawa iri-iri wajen daidaita ci gaban shuka da kuma martani ga yanayin damuwa na abiotic da biotic. Shigar da kwafi na ACO da kunna hanyar siginar ethylene suna da hannu wajen ƙara juriyar shinkafa ga naman gwari na hemibiotrophic oryzae oryzae ta hanyar daidaita samar da nau'ikan iskar oxygen masu amsawa da phytoalexins. Wani tsari makamancin wannan da aka samu tsakanin M. oryzae da C. lupini88,89, a kan asalin karuwar yawan kwayoyin halittar ACO a cikin layin 83A:476 da aka ruwaito a cikin wannan binciken, ya canza yiwuwar samar da juriya ga anthracnose na NLL Ethylene a matsayin matakin tsakiya mai nuna alama a cikin hanyoyin kwayoyin halitta.
A cikin wannan binciken, an lura da babban danne kwayoyin halitta da yawa da ke da alaƙa da photosynthesis a 6 hpi a cikin 83A:476 da kuma 48 hpi a cikin Mandeloop da kuma yawan jama'a 22660. Girman da ci gaban waɗannan canje-canjen sun yi daidai da matakin. An lura da juriyar Anthracnose a cikin wannan gwajin. Kwanan nan, an ba da rahoton ƙarfi da farko na danne bayanan da suka shafi photosynthesis a cikin samfuran hulɗar tsirrai da ƙwayoyin cuta, gami da ƙwayoyin cuta masu cutarwa da fungi. Sauri (daga 2 HPI a wasu hulɗa) da danne kwayoyin halitta na duniya da ke da alaƙa da photosynthesis don mayar da martani ga kamuwa da cuta na iya haifar da rigakafi ga tsirrai bisa ga tura nau'ikan iskar oxygen masu amsawa da hulɗarsu da hanyar salicylic acid don magance halayen rashin lafiyan 90,94.
A ƙarshe, hanyoyin da aka gabatar don kare kai daga kamuwa da cuta sun haɗa da saurin gane ƙwayoyin cuta ta hanyar kwayar halittar R (mai yiwuwa TIR-NBS-LRR TanjilG_05042) da kuma salicylic acid da ethylene siginar amsawar rashin lafiyan, sannan kuma kafa aikin SAR mai tsayi yana samun goyon bayan furotin DIR-1. Ya kamata a lura cewa lokacin biotrophic don kamuwa da cutar C. lupini yana da ɗan gajeren lokaci (kimanin kwana 2), sannan kuma ci gaban necrotic95. Sauye-sauye tsakanin waɗannan matakai na iya dangantawa da necrosis da bayyanar sunadaran ethylene waɗanda ke aiki a matsayin abubuwan da ke haifar da halayen rashin lafiyar jiki a cikin tsire-tsire masu masaukin baki. Saboda haka, lokacin da za a yi nasarar kama C. lupini a matakin biotrophic yana da matuƙar kunkuntar. Sake tsara kwayoyin halitta da ke da alaƙa da redox da photosynthesis da aka lura a cikin 83A:476 a 6 hpi ya yi daidai da ci gaban fungal hyphae kuma yana shelar ci gaban nasarar amsawar kariya a matakin biotrophic. Amsoshin da Mandelup da mutanen 22660 suka bayar na iya zama da jinkiri sosai don kama naman gwari kafin ya koma ga ci gaban ƙwayoyin cuta, duk da haka, Mandelup na iya zama mafi tasiri fiye da mutanen 22660 saboda saurin sarrafa furotin PR-10 yana haɓaka juriya a kwance.
ETI, wanda kwayar halittar R mai kama da ta canonical ke jagoranta, ya bayyana a matsayin wata hanya gama gari ta juriya ga wake ga anthracnose. Don haka, a cikin samfurin legume Medicago truncatula, juriya ga anthracnose yana samuwa ne ta hanyar kwayar halittar RCT1, memba na ajin kwayar halittar R shuka TIR-NBS-LRR97. Wannan kwayar halittar kuma tana ba da juriya ga anthracnose mai fadi a cikin alfalfa lokacin da aka canja ta zuwa ga tsirrai masu saurin kamuwa. A cikin wake na gama gari (P. vulgaris), an gano fiye da kwayoyin halittar anthracnose guda biyu zuwa yau. Wasu daga cikin waɗannan kwayoyin halittar ana samun su a yankuna da ba su da kowace kwayar halittar R mai kama da ta canonical, duk da haka wasu da yawa suna nan a gefunan chromosomes dauke da rukunin kwayar halittar NBS-LRR, gami da TIR-NBS-LRRs99. Binciken SSR na fadin kwayar halitta ya kuma tabbatar da alaƙar kwayar halittar NBS-LRR da juriya ga anthracnose a cikin wake na gama gari. An kuma sami kwayar halittar R mai kama da canonical a yankin kwayoyin halitta da ke ɗauke da babban wurin juriya ga anthracnose a cikin farin lupine 101.
Aikinmu ya nuna cewa amsawar juriya nan take, wadda aka kunna a farkon matakin kamuwa da cuta a tsirrai (zai fi kyau ba fiye da 12 hpi ba), tana kare lupine mai ganye mai ƙunci daga anthracnose wanda naman gwari mai cutarwa Collelotrichum lupini ke haifarwa. Ta amfani da jerin abubuwan da aka samar da su sosai, mun nuna bambance-bambancen bayyanar kwayoyin halittar juriyar anthracnose a cikin tsire-tsire na NLL waɗanda kwayoyin halittar juriyar Lanr1 da AnMan ke jagoranta. Nasarar kariya ta ƙunshi tsara kwayoyin halittar da kyau don sunadaran da ke da hannu a cikin redox, photosynthesis, da pathogenesis cikin sa'o'i bayan kamuwa da farko da shukar da wani cuta. Irin waɗannan halayen kariya, amma an jinkirta su cikin lokaci, ba su da tasiri sosai wajen kare tsire-tsire daga cututtuka. Juriyar Anthrax da kwayar halittar Lanr1 ke jagoranta tana kama da amsawar sauri ta kwayar halittar R (jini da ke haifar da tasirin), yayin da kwayar halittar AnMan wataƙila tana samar da martani a kwance (jini da tsarin kwayoyin halitta da ke da alaƙa da ƙwayoyin cuta ke haifarwa), yana samar da matsakaicin matakin dorewa.
Layukan NLL guda 215 da aka yi amfani da su wajen tantance alamun anthracnose sun ƙunshi nau'ikan iri 74, layuka 60 da aka samu ta hanyar ketarewa ko kiwo, sauye-sauye 5, da kuma ƙwayoyin cuta na daji ko na asali guda 76. Layukan sun fito ne daga ƙasashe 17, galibi daga Poland (58), Spain (47), Jamus (27), Ostiraliya (26), Rasha (19), Belarus (7), Italiya (5) da sauran layuka. Daga ƙasashe 10. Saitin ya kuma haɗa da layukan da ba su da juriya ga ma'ana: 83A:476, Tanjil, Wonga ɗauke da allele na Lanr1, da Mandelup wanda ke ɗauke da allele na AnMan. An samo layukan ne daga Bayanan Albarkatun Halitta na Lupine na Turai wanda Poznań Plant Breeding Ltd., Wiatrowo, Poland ke kula da su (Karin Tebur S1).
An shuka shuke-shuke a ƙarƙashin yanayi mai kulawa (hotoperiod na awanni 16, zafin jiki na 25°C a rana da kuma 18°C ​​da dare). An yi nazarin kwafi biyu na halittu. An ware DNA daga ganyen makonni uku da suka wuce ta amfani da DNeasy Plant Mini Kit (Qiagen, Hilden, Jamus) bisa ga ka'idar. An tantance inganci da yawan DNA da aka ware ta hanyar hanyoyin spectrophotometric (NanoDrop 2000; Thermo Fisher Scientific, Waltham, MA, Amurka). An yi nazarin alamar AnManM1 mai alamar kwayar halittar juriyar anthracnose AnMan (wanda aka samo daga cv. Mandelup) da kuma alamomin Anseq3 da Anseq4 da ke gefen kwayar halittar Lanr1 (wanda aka samo daga cv. Tanjil) 11,26,28. An sami Homozygotes don allele masu juriya a matsayin "1″, mai sauƙin kamuwa - kamar "0″, da heterozygotes - kamar 0.5.
Dangane da sakamakon tantance alamun AnManM1, AnSeq3 da AnSeq4 da kuma samuwar iri don gwaje-gwajen ƙarshe na bibiya, an zaɓi layukan NLL guda 50 don yin gwajin juriya ga anthracnose. An yi nazarin a cikin kwafi a cikin gidan kore mai sarrafa kwamfuta tare da lokacin ɗaukar hoto na awanni 14 tare da kewayon zafin jiki na 22°C a rana da 19°C da dare. Ana goge iri (ana yanke gashin iri a gefen tayin da wuka mai kaifi) kafin a shuka don hana kwanciya saboda gashin iri ya yi tauri da kuma tabbatar da cewa iri ɗaya ne. An shuka tsire-tsire a cikin tukwane (11 × 11 × 21 cm) tare da ƙasa mara tsabta (TS-1 REC 085 Medium Basic, Klasmann-Deilmann Polska, Warsaw, Poland). An yi allurar rigakafin ta hanyar amfani da nau'in Colletotrichum lupini Col-08, wanda aka girma a shekarar 1999 daga tushen shuke-shuken lupine masu ganye da aka shuka a wani fili a Verzhenitsa, Greater Poland (52° 27′ 42″ N 17° 04′ 05″ E). Sami yanki. An noma shuke-shuken a cikin matsakaicin SNA a 20°C a ƙarƙashin hasken baƙi na tsawon kwanaki 21 don haifar da sporulation. Makonni huɗu bayan shuka, lokacin da shuke-shuken suka kai matakin ganye 4-6, an yi allurar rigakafin ta hanyar fesawa da dakatarwar conidia a yawan conidia 0.5 x 106 a kowace ml. Bayan allurar rigakafin, an ajiye shuke-shuken a cikin duhu na tsawon awanni 24 a yanayin zafi kusan 98% da zafin jiki na 25°C don sauƙaƙe bayyanar conidia da tsarin kamuwa da cuta. Daga nan aka shuka shuke-shuken a ƙarƙashin lokacin daukar hoto na awanni 14 a zafin rana na 22°C/19°C da daddare da kuma zafi na kashi 70%. An yi gwajin cutar kwana 22 bayan an yi allurar kuma ta kama daga 0 (mai kariya daga cututtuka) zuwa 9 (mai saurin kamuwa da cutar) ya danganta da kasancewar ko rashin raunukan necrotic a kan tushe da ganye. Bugu da ƙari, bayan an yi gwaji, an auna nauyin shuke-shuken. An ƙididdige alaƙar da ke tsakanin nau'ikan alamun da kuma siffofin cututtuka a matsayin alaƙar maki biyu (rashin alamun heterozygous a cikin layukan da aka tsara don nazarin yanayin juriyar anthracnose).