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ʻO Angustifolius lupine (NLL, Lupinus angustifolius L.) kahi mea kanu leguminous i hoʻohana ʻia no ka hana ʻana i ka meaʻai a me ka hoʻomaikaʻi ʻana i ka lepo. ʻO ka hoʻonui ʻia ʻana o ka NLL ma ke ʻano he mea kanu ua huki i nā fungi pathogenic he nui, me ka lupine anthracnose, ka mea e hoʻoulu ai i ka maʻi anthracnose weliweli. ʻElua mau alleles, ʻo Lanr1 lāua ʻo AnMan, e hāʻawi ana i ka hoʻonui ʻia o ke kūʻē ʻana, ua hoʻohana ʻia i ka hoʻoulu ʻana o NLL, akā ʻaʻole ʻike ʻia nā ʻano hana molekala ma lalo. Ma kēia haʻawina, ua hoʻohana ʻia nā māka Lanr1 lāua ʻo AnMan e nānā i nā laʻana NLL ʻEulopa. Ua hōʻoia ka hoʻāʻo ʻana i ka lāʻau lapaʻau ma kahi ʻano i kāohi ʻia i ka pono o nā mea hāʻawi kūʻē ʻelua. Ua hana ʻia ka profiling hōʻike gene ʻokoʻa ma nā laina kūʻē a me nā laina maʻalahi. Ua pili ka kūʻē ʻana o Anthracnose me ka overexpression o nā huaʻōlelo ontology gene "GO:0006952 Defense Response", "GO:0055114 Redox Process", a me "GO:0015979 Photosynthesis". Eia kekahi, ua hōʻike ka laina Lanr1 (83A:476) i ka hoʻololi hou ʻana o ka transcriptome ma hope o ka hoʻopili ʻana, ʻoiai ua hōʻike nā laina ʻē aʻe i ka lohi o kēia pane ma kahi o 42 mau hola. Pili nā pane pale me nā genes TIR-NBS, CC-NBS-LRR a me NBS-LRR, 10 mau protein i komo i ka pathogenesis, nā protein hoʻoili lipid, endoglucan-1,3-β-glucosidase, nā protein paia cell waiwai glycine, a me nā genes mai ke ala reactive o ka oxygen. ʻO nā pane mua i ka 83A:476, me ka hoʻopaʻa pono ʻana i nā genes e pili ana me ka photosynthesis, ua kūlike me ka pale kūleʻa i ka wā ulu vegetative o ka biology fungal, e hōʻike ana he mea hoʻokō e hoʻoulu ai i ka palekana. Ua lohi ka hopena Mandeloop, e like me ke kauō ākea holoʻokoʻa.
ʻO ka lupine lau haiki (NLL, Lupinus angustifolius L.) he cereal kiʻekiʻe ka protein i hoʻomaka mai ma ka ʻāina komohana o ke Kaiwaenahonua1,2. Ke ulu nei ia i kēia manawa ma ke ʻano he meaʻai no nā holoholona a me nā kānaka. Ua manaʻo ʻia hoʻi he manure ʻōmaʻomaʻo i nā ʻōnaehana hoʻololi mea kanu ma muli o ka hoʻopaʻa ʻana o ka nitrogen e nā bacteria hoʻopaʻa nitrogen symbiotic a me ka hoʻomaikaʻi holoʻokoʻa o ke ʻano o ka lepo. Ua hala ʻo NLL i kahi kaʻina hana wikiwiki o ka domestication i ke kenekulia i hala a ke kau nei nō ma lalo o ke kaomi hoʻoulu kiʻekiʻe3,4,5,6,7,8,9,10,11,12. Me ka ulu nui ʻana o NLL, ua hoʻomohala ka hoʻomau ʻana o nā fungi pathogenic i nā niches mahiʻai hou a ua hoʻoulu i nā maʻi hou e luku ai i nā mea kanu. ʻO ka mea kupanaha loa no nā mahiʻai lupin a me nā mea hānai, ʻo ia ka ʻike ʻia ʻana o ka anthracnose, i hoʻokumu ʻia e ka fungi pathogenic, ʻo Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13. ʻO ka mea kupanaha loa no nā mahiʻai lupin a me nā mea hānai, ʻo ia ka ʻike ʻia ʻana o ka anthracnose, i hoʻokumu ʻia e ka fungi pathogenic, ʻo Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13. Наиболее примечательным для фермеров и селекционеров люпина было появление антракноза, вызванного памогинкриного памогинкиного (Bondar) Nirenberg, Feiler & Hagedorn13. ʻO ka mea i ʻike nui ʻia e nā mahiʻai lupine a me nā mea hānai, ʻo ia ka puka ʻana mai o ka anthracnose i hoʻokumu ʻia e ka fungus pathogenic ʻo Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13.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，它是由病原真由病原真菌 Lunenbergi Lui 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，它是由病原真菌lu。Cotric菌 Наиболее поразительным для фермеров и селекционеров люпина является появление антракноза, вызываемогего пtricмато lupini (Bondar) Nirenberg, Feiler & Hagedorn13. ʻO ka mea i ʻike nui ʻia e nā mahiʻai lupine a me nā mea hānai, ʻo ia ka puka ʻana mai o ka anthracnose i hoʻokumu ʻia e ka fungus pathogenic ʻo Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13.Ua hele mai nā hōʻike mua loa o ka maʻi mai Brazil a me ʻAmelika Hui Pū ʻIa mai, me nā hōʻailona maʻamau i ʻike ʻia i ka makahiki 1912 a me 1929. Eia naʻe, ma hope o kahi 30 mau makahiki, ua kapa ʻia ka pathogen ʻo Colletotrichum gloeosporioides (Penz.) Penz. & Sacc., teleomorph Glomerella cingulata (Stoneman) Spauld. & Sacc., teleomorph Glomerella cingulata (Stoneman) Spauld. & Sacc., телеоморф Glomerella cingulata (Stoneman) Spauld. & Sacc., teleomorph o 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 ma ke ʻano Morphology i hoʻolālā ʻia. & H. Schrenk,. & H. Schrenk, .a me H. Schrenk. & H.施伦克，。 & H.施伦克，。a me H. Schlenk, .Ua hōʻike ʻia ka phenotyping maʻi mua i hana ʻia i ka waena o ke kenekulia 20 i kekahi kūʻē i ka NLL a me nā hui lupine melemele (L. luteus L.), akā ʻo nā hui lupine keʻokeʻo a pau i hoʻāʻo ʻia he mea hiki ke hoʻopilikia ʻia15,16. Ua hōʻike ʻia nā haʻawina e pili ana ka ulu ʻana o ka anthracnose me ka hoʻonui ʻia o ka ua (ka haʻahaʻa o ka ea) a me ka mahana (ma ka pae o 12-28°C), e alakaʻi ana i ka uhaki ʻana i ke kūʻē i nā mahana kiʻekiʻe17, 18. ʻO ka ʻoiaʻiʻo, ʻo ka manawa e pono ai no ka conidia e ulu a hoʻomaka ka maʻi, ʻehā manawa pōkole ma 24°C (4 hola) ma mua o 12°C (16 hola) ma lalo o nā kūlana haʻahaʻa kiʻekiʻe19. No laila, ua alakaʻi ka hoʻomehana honua e hoʻomau nei i ka laha ʻana o ka anthracnose. Eia naʻe, ua ʻike ʻia ka maʻi ma Farani (1982) a me Ukraine (1983) ma ke ʻano he hōʻailona o kahi hoʻoweliweli e hiki mai ana, akā ua nānā ʻole ʻia e ka ʻoihana lupine i ka manawa20,21. I kekahi mau makahiki ma hope mai, ua pālahalaha kēia maʻi ʻino a puni ka honua a ua hoʻopilikia pū i nā ʻāina nui e hana ana i ka lupine e like me Australia, Polani a me Kelemania22,23,24. Ma hope o ka puka ʻana o ka anthracnose i waena o nā makahiki 1990, ua hopena ka nānā nui ʻana i ka ʻike ʻana o kekahi mau mea hāʻawi kūʻē i nā laʻana NLL19. Hoʻomalu ʻia ke kūʻē ʻana o NLL i ka anthracnose e ʻelua mau alleles dominant i loaʻa i nā kumu germplasm like ʻole: ʻo Lanr1 ma ka cultivar Tanjil a me Wonga a me AnMan ma ka cultivar. Mandalay 25, 26. Hoʻokō kēia mau alleles i nā māka molekala e kākoʻo ana i ke koho ʻana o ka germplasm kūʻē i nā papahana hoʻouluulu25,26,27,28,29,30. Ua hoʻohui ʻia ka laina hoʻouluulu kūpaʻa 83A:476 e lawe ana i ka allele Lanr1 me ka laina hihiu susceptible P27255 e loaʻa ai kahi heluna RIL e hoʻokaʻawale ana no ke kū'ē ʻana i ka anthracnose, kahi i hiki ai ke hāʻawi i ka locus Lanr1 i ka chromosome NLL-1131, 32, 33. ʻO ka hoʻonohonoho ʻana o nā māka palapala loulou mai nā loci kū'ē flanking a i ka anthracnose me kahi ʻōnaehana genomic, ua hōʻike ʻo NLL i kahi o nā alleles ʻekolu ma ka chromosome like (NLL-11), akā ma nā kūlana like ʻole29,34,35. Eia nō naʻe, ma muli o ka liʻiliʻi o nā RIL a me ka mamao genetic nui ma waena o nā māka a me nā alleles e pili ana, ʻaʻohe hopena hilinaʻi e hiki ke huki ʻia e pili ana i kā lākou mau genes ma lalo. Ma ka ʻaoʻao ʻē aʻe, he paʻakikī ka hoʻohana ʻana i nā genetics reverse i loko o nā lupins ma muli o ko lākou hiki ke hana hou haʻahaʻa loa, kahi e hoʻolilo ai i ka hoʻoponopono genetic i mea paʻakikī37.
ʻO ka hoʻomohala ʻana o ka germplasm domesticated e lawe ana i ka allele i makemake ʻia i ke kūlana homozygous, e like me 83A:476 (Lanr1) a me Mandelup (AnMan), ua wehe i ka puka no ke aʻo ʻana i ke kūʻē ʻana o ka anthracnose i ke alo o nā hui kūʻē o nā alleles i nā heluna hihiu. Nā hiki o nā ʻano hana molekala. Hoʻohālikelike i nā pane pale i hana ʻia e nā genotypes kikoʻī. Ua loiloi kēia haʻawina i ka pane transcriptome mua o NLL i ka lāʻau lapaʻau C. lupini. ʻO ka mea mua, ua nānā ʻia kahi panel germplasm NLL ʻEulopa e loaʻa ana he 215 mau laina me ka hoʻohana ʻana i nā māka molekala e hōʻailona ana i nā alleles Lanr1 a me AnMan. A laila ua hana ʻia ka phenotyping Anthracnose ma 50 mau laina NLL, i koho mua ʻia no nā māka molekala, ma lalo o nā kūlana i kāohi ʻia. Ma muli o kēia mau hoʻokolohua, ua koho ʻia ʻehā mau laina ʻokoʻa i ke kūʻē ʻana o ka anthracnose a me ka hoʻohui allelic Lanr1/AnMan no ka profiling gene pale like ʻole me ka hoʻohana ʻana i ʻelua mau ala hoʻopihapiha: ka hoʻonohonoho ʻana o ka RNA throughput kiʻekiʻe a me ka helu PCR manawa maoli.
ʻO ka nānā ʻana i kahi hui o ka germplasm NLL (N = 215) me nā māka Lanr1 (Anseq3 a me Anseq4) a me AnMan (Anseq4) a me AnMan (AnManM1) ua hōʻike ʻia hoʻokahi wale nō laina (95726, kokoke iā Salamanca-b) e hoʻonui i ka allele "resistance" no nā māka āpau, ʻoiai ʻo "Presence of 'susceptible' alleles" i loaʻa ka hapa o nā māka āpau ma 158 (~73.5%) laina. ʻUmikūmākolu laina i hana i ʻelua mau alleles "resistant" o ka māka Lanr1, a 8 mau laina i hana i nā alleles "resistant" o Lanr1. māka. ʻO ka allele "resistance" o ka māka AnMan (Papa Hoʻohui S1). ʻElua mau laina he heterozygous no ka māka Anseq3 a hoʻokahi heterozygous no ka māka AnManM1. Ua lawe ʻia e 42 mau laina (19.5%) nā pae ʻē aʻe o nā alleles Anseq3 a me Anseq4, e hōʻike ana i kahi alapine kiʻekiʻe o ka hui hou ʻana ma waena o kēia mau loci ʻelua. Ua hōʻike nā phenotypes Anthracnose ma lalo o nā kūlana i kāohi ʻia (Papa Hoʻohui S2) i ka ʻokoʻa o ke kūʻē ʻana o nā genotypes i hoʻāʻo ʻia, i hōʻike ʻia i ke koʻikoʻi o ka anthracnose. ʻO nā ʻokoʻa ma nā helu awelika mai 1.8 (kūpaʻa haʻahaʻa) a i 6.9 (hiki ke maʻalahi) a ʻo nā ʻokoʻa kaumaha o ka mea kanu mai 0.62 (hiki ke maʻalahi) a i 4.45 g (kūpaʻa). Aia kahi pilina koʻikoʻi ma waena o nā waiwai i ʻike ʻia ma nā hana hou ʻelua o ka hoʻokolohua (0.51 no nā helu koʻikoʻi o ka maʻi, P = 0.00017 a me 0.61 no ke kaumaha o ka mea kanu, P < 0.0001) a me waena o kēia mau palena ʻelua (− 0.59 a me − 0.77, P < 0.0001). Aia kahi pilina koʻikoʻi ma waena o nā waiwai i ʻike ʻia ma nā hana hou ʻelua o ka hoʻokolohua (0.51 no nā helu koʻikoʻi o ka maʻi, P = 0.00017 a me 0.61 no ke kaumaha o ka mea kanu, P < 0.0001) a me waena o kēia mau palena ʻelua (− 0.59 a me − 0.77, P < 0.0001). Выявлена ​​достоверная корреляция между значениями, наблюдаемыми в двух повторностях эксперимента (0,51 для тля = 0,00017 и 0,61 для массы растения, P < 0,0001), a также между этими двумя параметрами (-0,59 и -0,77, Р00 < 0,0). Ua loaʻa kahi pilina koʻikoʻi ma waena o nā waiwai i ʻike ʻia i ʻelua mau hana hou ʻana o ka hoʻokolohua (0.51 no nā helu koʻikoʻi o ka maʻi, P = 0.00017 a me 0.61 no ke kaumaha o ka mea kanu, P < 0.0001), a ma waena o kēia mau palena ʻelua (- 0.59 a me -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. 0.00017，植物重量为0.61，P < 0.0001）以及这两个参数之间（- 0.59 和- 0.77，P < 0.0001).在 两 次 重复 实验 中 观察 的 值 之间 存在 相关性 （疾病 严重 程度 了了 评 度 了0.51 ， p = 0.00017 ， 植物 为 为 0.61 ， p <0.0001） 以及 两 个 参数 之间 （（（（ 和 9.–5 . 0.59 和– 0.59 和- 0.77, P < 0.0001). Наблюдалась значительная корреляция между значениями, наблюдаемыми в двух повторностях (оценка тяжести,10 зал 0,0 за 1 и масса растения 0,61, P <0,0001), и между этими двумя параметрами (-0,59 и -0,0001) 0,77, P <0,0001. Aia kahi pilina koʻikoʻi ma waena o nā waiwai i ʻike ʻia ma ka pālua (helu koʻikoʻi o ka maʻi 0.51, P = 0.00017 a me ke kaumaha o ka mea kanu 0.61, P < 0.0001) a ma waena o kēia mau palena ʻelua (-0.59 a me -0 .0001) 0.77, P<0.0001. ).ʻO nā hōʻailona maʻamau i ʻike ʻia i nā mea kanu palupalu e komo pū ana me ka wili ʻana a me ka wili ʻana o ke kumu e like me ke ʻano o ke kakaka o ke kahu hipa, a ukali ʻia e nā ʻeha oval me nā sporozoites ʻalani/pink (Kiʻi Hoʻohui 1). ʻO nā ʻāpana ʻAukekulelia e lawe ana i nā genes Lanr1 (83A:476 a me Tanjil) a me AnMan (Mandelup) he kūpaʻa iki, 0.0331 a me 0.0036). ʻO kekahi mau laina e lawe pū ana i nā alleles Lanr1 "kūpaʻa" a/a i ʻole AnMan e hōʻike ana i nā hōʻailona o ka maʻi.
ʻO ka mea hoihoi, ua hōʻike kekahi mau laina NLL i nele i kekahi allele māka "kūpaʻa" i kahi kiʻekiʻe o ke kū'ē ʻana i ka anthracnose (hoʻohālikelike a ʻoi aku paha ma mua o nā genotypes Lanr1 a i ʻole AnMan), e like me Boregine (waiwai P < 0.0001 no nā palena ʻelua), Bojar (waiwai P < 0.0001 no ka helu a me 0.001 no ke kaumaha o ka mea kanu) a me Population B-549/79b (waiwai P < 0.0001 no ka helu a ʻaʻole koʻikoʻi no ke kaumaha). ʻO ka mea hoihoi, ua hōʻike kekahi mau laina NLL i nele i kekahi allele māka "kūpaʻa" i kahi kiʻekiʻe o ke kū'ē ʻana i ka anthracnose (hoʻohālikelike a ʻoi aku paha ma mua o nā genotypes Lanr1 a i ʻole AnMan), e like me Boregine (waiwai P < 0.0001 no nā palena ʻelua), Bojar (waiwai P < 0.0001 no ka helu a me 0.001 no ke kaumaha o ka mea kanu) a me Population B-549/79b (waiwai P < 0.0001 no ka helu a ʻaʻole koʻikoʻi no ke kaumaha). Интересно, что несколько линий NLL, лишенных какого-либо «резистентного» маркерного аллеля, показали высокний урость антракнозу (сопоставимый или более высокий, чем для генотипов Lanr1 или AnMan), таких как Boregine (значение P <0,0001 для проба), Boregine (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). ʻO ka mea hoihoi, ua hōʻike kekahi mau laina NLL i nele i kekahi allele māka 'kū'ē' i kahi kiʻekiʻe o ke kū'ē ʻana i ka anthracnose (hoʻohālikelike ʻia a ʻoi aku paha ma mua o nā genotypes Lanr1 a i ʻole AnMan), e like me Boregine (waiwai P < 0.0001 no nā palena ʻelua), Bojar (waiwai P < 0.0001 no ka loiloi a me 0.001 no ke kaumaha o ka mea kanu) a me ka heluna kanaka B-549/79b (waiwai P < 0.0001 no ka loiloi a ʻaʻole koʻikoʻi no ke kaumaha).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他们基因型相当或更高），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 伏< 0.0001，重量不显。 He mea hoihoi ia e hōʻike ana kekahi mau ʻōnaehana NLL ʻaʻohe ona māka "antigenic" i ke kū'ē ākea kiʻekiʻe (like me nā genes Lanr1 a i ʻole AnMan a ʻoi aku paha), e like me Boregine (nā palena ʻelua P < 0.0001), Bojar (waiwai P < 0.0001, kaumaha mea kanu 0.001) a me ke ʻano B-549/79b (waiwai P < 0.0001, ʻaʻole koʻikoʻi ke kaumaha). Интересно, что некоторые линии NLL, лишенные каких-либо маркерных аллелей «резистентности», показали высокие уроикич антракнозу (сравнимые или выше, чем у генотипов Lanr1 или AnMan), такие как Boregine (значение P для обоих параметров <0,0001, Bojar <0,0001), Boregine масса растения 0,001) и популяция B-549/79b (оценка P-значение <0,0001, масса незначительна). ʻO ka mea hoihoi, ua hōʻike kekahi mau laina NLL i nele i nā alleles māka 'kū'ē' i nā pae kiʻekiʻe o ke kū'ē ʻana i ka anthracnose (hoʻohālikelike ʻia a ʻoi aku paha ma mua o nā genotypes Lanr1 a i ʻole AnMan), e like me Boregine (waiwai P no nā palena ʻelua <0.0001), Bojar (waiwai P <0.0001, kaumaha mea kanu 0.001) a me ka heluna kanaka B-549/79b (waiwai P <0.0001, ʻaʻole koʻikoʻi ke kaumaha).Hōʻike kēia hanana i ka hiki ke loaʻa kahi kumu kūʻē genetic hou, e wehewehe ana i ka nele o ka pilina ma waena o nā genotypes marker a me nā phenotypes maʻi (nā waiwai P mai ~0.42 a ~0.98). No laila, ua hōʻike ka hoʻāʻo Kolmogorov-Smirnov ua like ka ʻikepili ma ke kūʻē anthracnose me ka maʻamau no nā helu (nā waiwai P 0.25 a me 0.11) a me ka nui o nā mea kanu (nā waiwai P 0.47 a me 0.55), e hōʻike ana ua kuhi wau he nui aku nā alleles ma mua o Lanr1 a me AnMan.
Ma muli o nā hopena o ka nānā ʻana i ke kūʻē ʻana i ka anthracnose, ua koho ʻia he 4 mau laina no ka loiloi transcriptome: 83A:476, Boregine, Mandelup, a me Population 22660. Ua hoʻāʻo hou ʻia kēia mau laina no ke kūʻē ʻana i ka anthrax i nā hoʻokolohua inoculation e ka RNA sequencing, inā like lākou me ka hoʻāʻo ma mua. ʻO nā waiwai helu penei: Boregin (1.71 ± 1.39), 83A: 476 (2.09 ± 1.38), Mandelup (3.82 ± 1.42) a me ka heluna kanaka 22660 (6.11 ± 1.29).
Ua hoʻokō ke kaʻina hana Illumina NovaSeq 6000 i ka awelika o 40.5 mau hui Mread no kēlā me kēia laʻana (29.7 a 54.4 Mreads) (Papa Hoʻohui S3). ʻO nā helu hoʻonohonoho ma ke kaʻina kuhikuhi mai 75.5% a 88.6%. ʻO ka pilina awelika o ka ʻikepili helu heluhelu ma waena o nā ʻano hoʻokolohua ma waena o nā replicates biological mai 0.812 a 0.997 (awelika 0.959). Mai loko mai o nā genes 35,170 i kālailai ʻia, ʻaʻohe hōʻike o 2917, a ʻo nā genes ʻē aʻe 4785 i hōʻike ʻia ma ka pae liʻiliʻi (ʻawelike kumu < 5). Mai loko mai o nā genes 35,170 i kālailai ʻia, ʻaʻohe hōʻike o 2917, a ʻo nā genes ʻē aʻe 4785 i hōʻike ʻia ma ka pae liʻiliʻi (ʻawelike kumu < 5). Из 35 170 проанализированных генов 2917 не проявляли экспрессии, a остальные 4785 генов экспресиронвались темирон (базовое среднее <5). No nā genes 35,170 i kālailai ʻia, ʻaʻole i hōʻike ʻia he 2917 ka hōʻike ʻana, a ʻo ke koena o 4785 mau genes i hōʻike ʻia ma kahi pae liʻiliʻi (kumu awelika <5).35,170 5）.35,170 Из 35 170 проанализированных генов 2917 не экспрессировались, а остальные 4785 генов имели незначительнсубю среднее значение <5). No nā genes 35,170 i kālailai ʻia, ʻaʻole i hōʻike ʻia he 2917 a ʻo ke koena o nā genes 4785 he mea liʻiliʻi ka hōʻike ʻana (kumu awelika <5).No laila, ʻo ka helu o nā genes i manaʻo ʻia i hōʻike ʻia (kumu awelika ≥ 5) i ka wā o ka hoʻokolohua he 27,468 (78.1%) (Papa Hoʻohui S4).
Mai ka manawa mua, ua pane nā laina NLL āpau i ka hoʻopili ʻia ʻana o C. lupini (ʻano Col-08) ma o ka hoʻoponopono hou ʻana i ka transcriptome (Papa 1), akā naʻe, ua ʻike ʻia nā ʻokoʻa koʻikoʻi ma waena o nā laina. No laila, ua hōʻike ka laina kūʻē 83A:476 (e lawe ana i ka gene Lanr1) i ka hoʻoponopono hou ʻana o ka transcriptome i ka manawa mua (6 hpi) me ka piʻi ʻana o 31-69-fold i ka nui o nā genes i luna a i lalo i hoʻohālikelike ʻia me nā manawa ʻē aʻe i kēia manawa. Eia kekahi, ua pōkole kēia piko, ʻoiai ua loli nui ka hōʻike ʻana o kekahi mau genes i ka lua o ka manawa (12 hpi). ʻO ka mea hoihoi, ʻo Boregine, ka mea i hōʻike pū i kahi kiʻekiʻe o ke kūʻē ʻana i ka hoʻāʻo graft, ʻaʻole i hana i kēlā ʻano hoʻoponopono hou ʻana i ka transcriptional i ka wā o ka hoʻokolohua. Eia naʻe, ua like ka nui o nā genes i hōʻike ʻia (DEG) no Boregine a me 83A:476 ma 12 HPI. Ua hōʻike ʻo Mandelup a me ka heluna kanaka 22660 i nā piko DEG i ka manawa hope loa (48 l/s), e hōʻike ana i ka lohi pili i nā pane pale.
Ma muli o ka hoʻololi hou ʻana o ka transcriptome o 83A:476 i ka pane ʻana iā C. lupini ma 6 HPI i hoʻohālikelike ʻia me nā laina ʻē aʻe a pau, ~91% o nā DEG i ʻike ʻia i kēia manawa he mea pili i ka moʻokūʻauhau (Kiʻi 1). Eia nō naʻe, aia kekahi mau ʻano like ʻole i nā pane mua ma waena o nā laina noiʻi, ʻoiai ʻo 68.5%, 50.9%, a me 52.6% DEG ma Boregine, Mandelup, a me ka heluna kanaka 22660, kēlā me kēia, ua like me nā mea i loaʻa ma 83A:476 i kekahi mau manawa. Eia nō naʻe, ua helu kēia mau DEG i kahi hapa liʻiliʻi wale nō (0.97–1.70%) o nā DEG a pau i ʻike ʻia i kēia manawa me ka hoʻohana ʻana iā 83A:476. Eia kekahi, ua kūlike nā DEG he 11 mai nā laina āpau i kēia manawa (Nā Papa Hoʻohui S4-S6), me nā ʻāpana maʻamau o nā pane pale o nā mea kanu: ka protein hoʻoili lipid (TanjilG_32225), ka enzyme endoglucan-1,3-β-glucoside (TanjilG_23384), ʻelua mau protein stress-inducible e like me SAM22 (TanjilG_31528 a me TanjilG_31531), ka protein latex kumu (TanjilG_32352), a me ʻelua mau protein paia cell structural waiwai glycine (TanjilG_19701 a me TanjilG_19702). Aia kekahi overlap kiʻekiʻe ma nā pane transcriptome ma waena o 83A:476 a me Boregine ma 24 HPI (huina 16-38% DEG) a ma waena o Mandelup a me Population 22660 ma 48 HPI (huina 14-20% DEG).
ʻO ke kiʻikuhi Venn e hōʻike ana i ka helu o nā genes i hōʻike ʻokoʻa ʻia (DEG) i nā laina lupine lau nawaliwali (NLL) i hoʻopili ʻia me Colletotrichum lupini (ʻano Col-08 i loaʻa mai nā kahua lupine ma Wierzhenice, Polani, 1999). ʻO nā laina NLL i kālailai ʻia: 83A:476 (kūpaʻa, e lawe ana i ka allele Lanr1), Boregine (kūpaʻa, ʻike ʻole ʻia ke kumu genetic), Mandelup (kūpaʻa haʻahaʻa, e lawe ana i ka allele AnMan) a me ka heluna kanaka 22660 (hiki ke maʻalahi loa). ʻO ka pōkole hpi ke kū nei no nā hola ma hope o ka hoʻopili ʻana. Ua wehe ʻia nā waiwai zero e hoʻomaʻalahi i ka pakuhi.
Ua kālailai ʻia ka pūʻulu o nā genes i hoʻonui nui ʻia ma 6 hpi no ke alo o nā kikowaena gene R canonical (Papa Hoʻohui S7). Ua hōʻike kēia haʻawina i ka hoʻokomo ʻana o ka transcriptome o nā genes pale maʻi maʻamau me nā kikowaena NBS-LRR ma 83A:476 wale nō. Aia kēia pūʻulu i hoʻokahi gene TIR-NBS-LRR (tanjilg_05042), ʻelima mau genes CC-NBS-LRR (tanjilg_06165, tanjilg_06162, tanjilg_22773, tanjilg_22640, a me tanjilg_16162), a me ʻehā NBS-LR, Tanjilg_16162), a me ʻehā NBS-LRRE (tanjilg_16162) a me ʻehā NBS-Lrr (tanjilg_16162) a me ʻehā NBS-LRR (TANJILG_16162). Loaʻa i kēia mau genes āpau nā kikowaena canonical i hoʻonohonoho ʻia i nā kaʻina i mālama ʻia. Ma waho aʻe o nā genes kikowaena NBS-LRR, ua hoʻāla ʻia kekahi mau RLL kinases ma 6 hpi, ʻo ia hoʻi hoʻokahi ma Boregine (TanjilG_19877), ʻelua ma Mandelup (TanjilG_07141 a me TanjilG_19877) a ma ka heluna kanaka 22660 (TanjilG_09014 a me TanjilG_10361) a ʻelua ma 83A 27:476.
Ua hoʻokomo ʻia nā genes me ka hoʻololi nui ʻana i ka hōʻike ʻana i ka pane ʻana i ka hoʻopili ʻana me C. lupini (strain Col-08) i ka loiloi hoʻonui Gene Ontology (GO) (Papa Hoʻohui S8). ʻO ka huaʻōlelo hana olaola i hōʻike pinepine ʻia ʻo "GO:0006952 defence response" i ʻike ʻia ma 6 o 16 mau hui pū ʻana (manawa × laina) me ke koʻikoʻi kiʻekiʻe (P value < 0.001) (Kiʻi 2). ʻO ka huaʻōlelo hana olaola i hōʻike pinepine ʻia ʻo "GO:0006952 defence response" i ʻike ʻia ma 6 o 16 mau hui pū ʻana (manawa × laina) me ke koʻikoʻi kiʻekiʻe (P value < 0.001) (Kiʻi 2). LIKE LIKE LIKE 6 из 16 (время × линия) комбинаций с высокой значимостью (значение P <0,001) (рис. 2). ʻO ka huaʻōlelo hana olaola i hōʻike pinepine ʻia ʻo 'GO:0006952 defence response', i ʻike ʻia ma 6 o 16 mau hui pū ʻana (manawa × lineage) me ke koʻikoʻi kiʻekiʻe (P value < 0.001) (Kiʻi 2).最常被过度代表的生物过程术语是“GO:0006952 防御反应”，它出现在16 个（时间答中文他们中文中文个中，具有高显着性（P ‼< 0.001）（图2）。 ʻO ka huaʻōlelo hana olaola i hōʻike nui ʻia ʻo "GO:0006952 defence response", ka mea i ʻike ʻia ma 6 o nā hui pū ʻana o 16 (时间 ×线), me ke koʻikoʻi kiʻekiʻe (waiwai P < 0.001) (图2). Наиболее часто чрезмерно представленным термином биологического процесса был «GO: 0006952 Defence Response», котовлыл 6 поского комбинаций (время × линия) с высокой значимостью (значение P <0,001) (рис. 2). ʻO ka huaʻōlelo hana olaola i hōʻike pinepine ʻia ʻo 'GO:0006952 Defense Response', i ʻike ʻia ma 6 o 16 mau hui pū ʻana (manawa × laina) me ke koʻikoʻi kiʻekiʻe (waiwai P < 0.001) (Kiʻi 2).Ua hōʻike nui ʻia kēia huaʻōlelo ma nā manawa ʻelua ma 83A: 476 a me Boregine (6 a me 24 hpi) a ma kekahi manawa ma Mandelup a me Population 22660 (12 a me 6 hpi, kēlā me kēia). He hopena i manaʻo ʻia kēia, e hōʻike ana i ka pane antifungal o nā laina kū'ē. Eia kekahi, ua pane ʻo 83A:476 iā C. lupini ma o ka hoʻoulu wikiwiki ʻana i nā genes e pili ana i ka pahū oxidative i hōʻike ʻia e ka huaʻōlelo "GO:0055114 redox process", e hōʻike ana i kahi pane pale kikoʻī, ʻoiai ʻo Boregine i hōʻike i nā pane pale kikoʻī, e pili ana i ka huaʻōlelo 'GO'. :0006950 Pane koʻikoʻi”. Ua hoʻāla ka heluna kanaka 22660 i ka pane kū'ē ākea e pili ana i nā metabolites lua, e hōʻike ana i ka nui o nā huaʻōlelo "GO:0016104 Kaʻina hana o ka biosynthesis triterpene" a me "GO:0006722 Kaʻina hana o ka metabolism triterpene" (no nā huaʻōlelo ʻelua i ka hoʻonohonoho like o nā genes), e noʻonoʻo ana i nā hopena o ka loiloi hoʻonui huaʻōlelo GO, ʻo ke kūpaʻa o ka hopena Mandelup ma waena o Boregine a me Population 22660. Eia kekahi, ʻo ka hopena mua 83A:476 (6 hpi) a me ka hopena lohi ʻo Mandelup a me Population 22660 e komo pū me ka huaʻōlelo GO:0015979 'photosynthesis' a me nā kaʻina hana olaola e pili ana.
Ua hoʻonui nui ʻia nā huaʻōlelo ontology gene bioprocess i koho ʻia ma ka annotation o nā genes i hōʻike ʻokoʻa ʻia i ka wā o nā pane transcriptome o ka lupine lau nawaliwali (NLL) i hoʻokomo ʻia me ka anthrax lupine (ʻano Col-08 i loaʻa mai nā kahua lupine ma Wierzhenice, Polani, i ka makahiki 1999). ʻO nā laina NLL i kālailai ʻia: 83A:476 (kūpaʻa, e lawe ana i ka allele Lanr1 homozygous), Boregine (kūpaʻa, ʻike ʻole ʻia ke kumu genetic), Mandelup (kūpaʻa haʻahaʻa, e lawe ana i ka allele AnMan homozygous) a me ka heluna kanaka 22660 (hiki ke hoʻopilikia).
Ma muli o ka manaʻo o kēia haʻawina e ʻike i nā genes e kōkua ana i ke kū'ē ʻana i ka anthracnose, ua kālailai ʻia nā genes i hāʻawi ʻia i nā huaʻōlelo GO "GO: 0006952 Defensive responses" a me "GO: 0055114 Redox processes" me nā ʻokiʻoki ʻoiai ʻo ke kumu kumu he ≥ 30 me ka liʻiliʻi o hoʻokahi laina. × kiko i ka manawa e hoʻohui ana i nā waiwai koʻikoʻi o ka log2 (fold change). ʻO ka helu o nā genes e hoʻokō ana i kēia mau pae hoʻohālikelike he 65 no GO:0006952 a me 524 no GO:0055114.
Ua hōʻike ʻo 83A:476 i ʻelua mau piko DEG i hoʻopaʻa ʻia me ka huaʻōlelo GO:0006952, ʻo ka mua ma 6 mau genes i kēlā me kēia ʻīniha (64 mau genes, hoʻoponopono i luna a i lalo) a ʻo ka lua ma 24 mau genes i kēlā me kēia ʻīniha (15 mau genes, hoʻoponopono i luna wale nō). Ua hōʻike pū ʻo Boregine ua piʻi ka piko o GO:0006952 i ka manawa like, akā me ka emi ʻana o DEG (11 a me 8) a me ka hoʻāla makemake. Ua hōʻike ʻo Mandeloop i ʻelua mau piko o GO:0006952 ma 12 a me 48 HPI, ʻelua e lawe ana i 12 mau genes (ʻo ka mua me nā genes hoʻāla, a ʻo ka lua me nā genes hoʻopau wale nō), ʻoiai ʻo ka heluna kanaka 22660 ma 6 HPI (13 mau genes) he nui ka nui o ka hoʻonui ʻana i ka piko hoʻoponopono. Pono e hoʻomaopopo ʻia he 96.4% o GO:0006952 DEG i loko o kēia mau piko i loaʻa ke ʻano pane like (i luna a i lalo paha), e hōʻike ana i kahi overlap koʻikoʻi i nā pane pale me ka ʻokoʻa o ka helu o nā genes i komo. ʻO ka hui nui loa o nā kaʻina e pili ana i ka huaʻōlelo GO:0006952 e hoʻopili ana i ka Starvation Stress-Associated Message Protein 22 (SAM22-like), nona ka clade protein pathogenesis-associated protein (PR-10) papa 10 a me ka protein core latex. like (MLP-like) protein) protein (Kiʻi 3). Ua ʻokoʻa nā hui ʻelua i ke ʻano o ka hōʻike ʻana a me ke kuhikuhi o ka pane. Ua hōʻike nā genes e hoʻopili ana i nā protein SAM22-like i ka hoʻokomo mau a koʻikoʻi i nā manawa mua (6 a i ʻole 12 hpi) a ʻaʻole i pane pinepine i ka hopena o ka hoʻokolohua (48 hpi), ʻoiai ua hōʻike nā protein MLP-like i ka hoʻohui ʻana ma 6 hpi. hpi. 83A:476 a me Mandelup ma 48 hp/in, aneane ʻaʻole i pane nā kikoʻī ʻikepili ʻē aʻe. Eia kekahi, ua ukali ʻia nā ʻokoʻa i nā ʻano hōʻike o nā genes protein SAM22-like i ka loli i ʻike ʻia i ke kūʻē ʻana o ka anthracnose, ʻoiai ʻoi aku ka nui o nā manawa o nā laina kūpaʻa e hoʻoulu nui ana i kēia mau genes ma mua o nā genes i maʻalahi. Ua hōʻike kekahi gene PR-10 like LlR18A/B i kahi ʻano hoʻopuka like loa me ka gene protein like SAM22.
Ua ʻike ʻia nā ʻāpana nui o ka huaʻōlelo hana olaola "GO:0006952 Defense Response" a me nā ʻano hōʻike o nā genes moho o nā alleles Lanr1 a me AnMan. Hōʻike ka unahi Log2 i nā waiwai log2 (hoʻololi pelu) ma waena o nā mea kanu i hoʻopili ʻia (Colletotrichum lupini, strain Col-08, i loaʻa mai nā kahua lupine, Wizhenica, Polani, 1999) a me nā mea kanu kaohi (sham-inoculated) i ka manawa like. Ua kālailai ʻia nā laina lupine narrowleaf ma lalo nei: 83A:476 (kūpaʻa, e lawe ana i ka allele Lanr1 homozygous), Boregine (kūpaʻa, ʻike ʻole ʻia ke kumu genetic), Mandelup (kūpaʻa haʻahaʻa, e lawe ana i ka allele AnMan homozygous), a me ka Population 22660 (hiki ke hoʻopilikia).
Eia kekahi, ua loiloi ʻia nā ʻano hōʻike o nā genes moho RNA-seq ʻo Lanr1 (TanjilG_05042) a me AnMan (TanjilG_12861) (Kiʻi 3). Ua hōʻike ka gene TanjilG_05042 i kahi pane koʻikoʻi (hoʻāla ʻana) ma 83A:476 wale nō i ka manawa mua (6 hpi), ʻoiai ʻo TanjilG_12861 he koʻikoʻi ma Mandeloop wale nō i ʻelua mau manawa: 6 hpi (hoʻohaʻahaʻa i lalo) a me 24 hpi (6 hpi). Me.). hiki ke hoʻololi ʻia) ).
ʻO nā genes i hoʻonui nui ʻia ma ka huaʻōlelo GO:0055114 "redox process" he mau genes e hoʻopili ana i nā protein cytochrome P450 a me ka peroxidase (Kiʻi 4). No nā laʻana i hoʻokaʻawale ʻia mai 83A:476 ma 6 HPI, ʻike nui ʻia nā waiwai log2 kiʻekiʻe a i ʻole ka liʻiliʻi (fold change) (no 86.6% o nā genes) ma waena o nā mea kanu i hoʻopili ʻia a me nā mea kanu kaohi, e hōʻike ana i ka pane kiʻekiʻe o kēia genotype i ka moekolohe i hoʻopili ʻia. Ua hōʻike ʻo 83A:476 i ka GO koʻikoʻi loa: 0055114 DEG ma 6 hpi (503 genes), ʻoiai ke koena o nā laina ma 48 hpi (Boregine, 31 genes; Mandelup, 85 genes; a me Population 22660, 78 genes)). I ka hapa nui o nā genes o ka ʻohana GO:0055114, ua ʻike ʻia ʻelua ʻano pane i ka lāʻau pale (hoʻāla a me ka pale ʻana). ʻO ka mea hoihoi, a hiki i ka 97.6% o nā DEG i ʻike ʻia no ka huaʻōlelo GO: 0055114 ma Mandelupe ma 48 hp Hōʻike kēia mau ʻike, ʻoiai ka liʻiliʻi o ka pae (ʻo ia hoʻi, ka helu o nā genes redox mutated, 85 versus 503), ʻo ke ʻano o nā pane transcriptome lohi o mandeloup i ka anthracnose ua like ia me ka pane mua o 83A:476. Ma Boregine a me Population 22660, ʻoi aku ka haʻahaʻa o kēia hui ʻana ma 51.6% a me 75.6%, kēlā me kēia.
Ua hōʻike ʻia nā ʻano o ka hōʻike ʻana o nā ʻāpana nui o ka huaʻōlelo o ke kaʻina hana olaola "GO:0055114 Redox process". Hōʻike ka unahi Log2 i nā waiwai log2 (hoʻololi pelu) ma waena o ka hoʻopili ʻia (Colletotrichum lupini, strain Col-08, i loaʻa mai nā kahua lupine, Wizhenica, Polani, 1999) a me nā mea kanu kaohi (sham-inoculated) i ka manawa like. Ua kālailai ʻia nā laina lupine narrowleaf ma lalo nei: 83A:476 (kūpaʻa, e lawe ana i ka allele homozygous Lanr1), Boregine (kūpaʻa, ʻike ʻole ʻia ke kumu genetic), Mandelup (kūpaʻa haʻahaʻa, e lawe ana i ka allele homozygous AnMan), a me ka Population 22660 (hiki ke hoʻopilikia).
83A:476 ʻO nā pane transcriptomic i ka inoculation me C. lupini (strain Col-08) ua komo pū me ka hoʻopau ʻana i nā genes i hāʻawi ʻia i ka huaʻōlelo GO:0015979 "photosynthesis" a me nā kaʻina hana olaola ʻē aʻe e pili ana (FIG. 5). Aia kēia GO:0015979 DEG set i 105 mau genes i kāohi nui ʻia ma 6 hpi ma 83A:476. Ma kēia subset, ua hoʻohaʻahaʻa ʻia hoʻi he 37 mau genes ma Mandelup ma 48 HPI a me 35 i ka manawa like ma ka heluna kanaka 22660, me 19 DEGs i maʻamau i nā genotypes ʻelua. ʻAʻohe DEGs e pili ana i ka huaʻōlelo GO: 0015979 i hoʻāla nui ʻia i kekahi hui pū ʻana (laina x manawa).
Ua hōʻike ʻia nā ʻano o ka hōʻike ʻana o nā ʻāpana nui o ka huaʻōlelo o ke kaʻina hana olaola "GO:0015979 Photosynthesis". Hōʻike ka unahi Log2 i nā waiwai log2 (hoʻololi pelu) ma waena o ka hoʻopili ʻia (Colletotrichum lupini, ʻano Col-08, i loaʻa mai nā kahua lupine, Wizhenica, Polani, 1999) a me nā mea kanu kaohi (sham-inoculated) i ka manawa like. Ua kālailai ʻia nā laina lupine narrowleaf ma lalo nei: 83A:476 (kūpaʻa, e lawe ana i ka allele Lanr1 homozygous), Boregine (kūpaʻa, ʻike ʻole ʻia ke kumu genetic), Mandelup (kūpaʻa haʻahaʻa, e lawe ana i ka allele AnMan homozygous), a me ka Population 22660 (hiki ke hoʻopilikia).
Ma muli o nā hopena o ka loiloi hōʻike ʻokoʻa a me ka manaʻo e pili ana i nā pane pale kūʻē i nā fungi pathogenic, ua koho ʻia kēia hoʻonohonoho o ʻehiku mau genes no ka helu ʻana o nā ʻikepili hōʻike e ka PCR manawa maoli (Papa Hoʻohui S9).
Ua hoʻoulu nui ʻia ka gene protein putative ʻo TanjilG_10657 i nā laina āpau i aʻo ʻia a me nā manawa i hoʻohālikelike ʻia me nā mea kanu kaohi (mimic) (Nā Papa Hoʻohui S10, S11). Eia kekahi, ua hōʻike ka ʻano hōʻike o TanjilG_10657 i kahi ʻano e piʻi nei ma luna o ka holo ʻana o ka hoʻokolohua no nā laina āpau. Ua hōʻike ka heluna kanaka 22660 i ka ʻike kiʻekiʻe loa o TanjilG_10657 i ka inoculation me ka hoʻāla ʻana he 114-fold a me ka pae hōʻike pili kiʻekiʻe loa (4.4 ± 0.4) ma 24 HPI (Kiʻi 6a). Ua hōʻike pū ka gene protein PR10 LlR18A ʻo TanjilG_27015 i ka hoʻāla ʻana ma nā laina āpau a me nā manawa, me ke koʻikoʻi helu ma ka hapa nui o nā kiko ʻikepili (Kiʻi 6b). E like me TanjilG_10657, ua ʻike ʻia ka pae hōʻike pili kiʻekiʻe loa o TanjilG_27015 i loko o ka heluna kanaka i hoʻokomo ʻia he 22660 ma 24 HPI (19.5 ± 2.4). Ua hoʻonui nui ʻia ka gene endochitinase acid ʻo TanjilG_04706 ma nā laina āpau a ma nā manawa āpau koe wale nō ʻo Boregine 6 hpi (Kiʻi 6c). Ua hoʻoulu ikaika ʻia i ka manawa mua (6 HPI) ma 83A:476 (ma 10.5 mau manawa) a ua hoʻonui iki ʻia ma nā laina ʻē aʻe (ma 6.6-7.5 mau manawa). I ka wā o ka hoʻokolohua, ua noho like ka hōʻike ʻana o TanjilG_04706 ma 83A:476 a me Boregine, ʻoiai ma Mandelup a me Population 22660 ua hoʻonui nui ʻia, e hōʻea ana i nā waiwai kiʻekiʻe (5.9 ± 1.5 a me 6.2 ± 1.5, kēlā me kēia). Ua hōʻike ka gene endoglucan-1,3-β-glucosidase-like ʻo TanjilG_23384 i ka hoʻāla kiʻekiʻe ma nā manawa mua ʻelua (6 a me 12 hpi) ma nā laina āpau koe wale nō ka heluna kanaka 22660 (Kiʻi 6d). Ua ʻike ʻia nā pae hōʻike pili kiʻekiʻe loa o TanjilG_23384 ma ka lua o ka manawa (12 hpi) ma Mandelup (2.7 ± 0.3) a me 83A:476 (1.5 ± 0.1). Ma 24 HPI, ua haʻahaʻa iki ka hōʻike ʻana o TanjilG_23384 ma nā laina āpau i aʻo ʻia (mai 0.04 ± 0.009 a i 0.44 ± 0.12).
Nā ʻikepili hōʻike o nā genes i koho ʻia (ag) i hōʻike ʻia e ka PCR quantitative. Hōʻike nā helu 6, 12 a me 24 i nā hola ma hope o ka hoʻopili ʻana. Ua hoʻohana ʻia nā genes LanDExH7 a me LanTUB6 no ka normalization a ua hoʻohana ʻia ʻo LanTUB6 no ka calibration inter-series. Hōʻike nā kaha hewa i ka ʻokoʻa maʻamau e pili ana i ʻekolu mau replicates biological, ʻo kēlā me kēia o ia ka awelika o ʻekolu mau replicates loea. Ua hōʻailona ʻia ke koʻikoʻi helu o nā ʻokoʻa ma nā pae hōʻike ma waena o nā mea kanu i hoʻopili ʻia (Colletotrichum lupini, ʻano Col-08, i loaʻa i ka makahiki 1999 mai ke kahua lupin ma Wierzenica, Polani) a me nā mea kanu kaohi (hoʻohālike ʻia) ma luna o nā kiko ʻikepili (*P value < 0.05, **P value ≤ 0.01, ***P value ≤ 0.001). Ua hōʻailona ʻia ke koʻikoʻi helu o nā ʻokoʻa ma nā pae hōʻike ma waena o nā mea kanu i hoʻopili ʻia (Colletotrichum lupini, ʻano Col-08, i loaʻa i ka makahiki 1999 mai ke kahua lupin ma Wierzenica, Polani) a me nā mea kanu kaohi (hoʻohālike ʻia) ma luna o nā kiko ʻikepili (*P value < 0.05, **P value ≤ 0.01, ***P value ≤ 0.001). Статистическая значимость различий в уровнях экспрессии между инокулированными (Colletotrichum lupini, штамм Col-08, получен гля 1 999 г. в Верженице, Польша) и контрольными (ложно инокулированными) растениями отмечена отмечена точками данных (*знач0,ние P. 0,01, ***значение P ≤ 0,001). Ua ʻike ʻia nā ʻokoʻa koʻikoʻi ma nā pae hōʻike ma waena o nā mea kanu i hoʻopili ʻia (Colletotrichum lupini, ʻano Col-08, i loaʻa i ka makahiki 1999 mai kahi kahua lupine ma Wierzhenice, Polani) a me nā mea kanu kaohi (sham-inoculated) ma luna o nā kiko ʻikepili (*P value < 0.05, **P-value ≤ 0.01, ***P-value ≤ 0.001).接种（Colletotrichum lupini，Col-08株，1999年从波兰Wierzenica的羽扇豆田获得）和对照（模拟接种）植物之间表达水平差异的统计学显着性标记在数据為0.05, **P ≤ 0.01, ***P ≤ 0.001).接种 （colletotrichum lupini ， color-08 株 ， 1999 年 波兰 波兰 wierzenica 的 羽扇 获得） 和 对照 （ 接秉水平 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). Статистически значимые различия в уровнях экспрессии между инокулированными (Colletotrichum lupini, штамм Col-08, полученплый спо Верженице, Польша, в 1999 г.) и контрольными (ложно инокулированными) растениями отмечены над точками (*, 0 ных для инокулированными) P-значение ≤ 0,01, ***P-значение ≤ 0,001). Ua ʻike ʻia nā ʻokoʻa koʻikoʻi ma nā pae hōʻike ma waena o nā mea kanu i hoʻopili ʻia (Colletotrichum lupini, ʻano Col-08, i loaʻa mai nā mahina ʻai lupine ma Verzhenice, Polani, i ka makahiki 1999) a me nā mea kanu kaohi (sham-inoculated) ma luna o nā kiko ʻikepili (*P value < 0.05, **P-value ≤ 0.01, ***P-value ≤ 0.001).ʻO nā laina NLL i kālailai ʻia: 83A:476 (kūpaʻa, e lawe ana i ka allele homozygous Lanr1), Mandelup (kūpaʻa haʻahaʻa, e lawe ana i ka allele homozygous AnMan), Boregine (kūpaʻa, ʻike ʻole ʻia ke kumu genetic) a me ka heluna kanaka 22660 (hiki ke hoʻopilikia ʻia).
Ua hōʻike ka gene moho ʻo TanjilG_05042 ma ka locus Lanr1 i kahi ʻano hōʻike ʻokoʻa loa mai nā ʻano i loaʻa mai nā haʻawina RNA-seq (Kiʻi 6e). Ua ʻike ʻia ka hoʻoulu nui ʻana o kēia gene ma Mandelup a me ka heluna kanaka 22660 (a hiki i ka 39.7 a me 11.7 mau manawa, kēlā me kēia), e hopena ana i nā pae hōʻike kiʻekiʻe (a hiki i ka 1.4 ± 0.14 a me 7.2 ± 1.3, kēlā me kēia). Ua hōʻike pū ʻo 83A:476 i kekahi piʻi ʻana o ka gene TanjilG_05042 (a hiki i ka 3.8-fold), akā naʻe, ʻoi aku ka haʻahaʻa o nā pae hōʻike pili i loaʻa (0.044 ± 0.002) ma mua o 30-fold ma mua o nā mea i ʻike ʻia ma Mandelup a me ka heluna kanaka 22660. ua hōʻike ʻia i kālailai ʻia e qPCR nā ʻokoʻa koʻikoʻi i nā pae hōʻike ma waena o nā genotypes i nā ʻano hoʻohālikelike i hoʻopā ʻia (mana), e hōʻea ana i kahi ʻokoʻa 58-fold ma waena o nā heluna kanaka 22660 a me 83A:476, a ma waena o nā heluna kanaka 22660 a me 22660. Ua hoʻokō ʻia kahi ʻokoʻa pālua ma waena o Boregine lāua ʻo Mandalup.
Ua hoʻāla ʻia ke gene moho ma ka locus AnMan, ʻo TanjilG_12861, ma muli o ka hoʻopili ʻana ma 83A:476 a me Mandelup, he kūʻokoʻa i ka heluna kanaka 22660, a ua hoʻohaʻahaʻa ʻia ma Boregine (Kiʻi 6f). ʻO ka hōʻike pili o ka gene TanjilG_12861 ka mea kiʻekiʻe loa ma 83A: 476 i hoʻopili ʻia (0.14 ± 0.01). ʻO ka gene protein haʻalulu wela 17.4 kDa papa I ʻo TanjilG_05080 HSP17.4 i hōʻike i nā pae hōʻike pili haʻahaʻa i nā ʻano āpau i aʻo ʻia a me nā manawa (Kiʻi 6g). Ua ʻike ʻia ka waiwai kiʻekiʻe loa ma 24 HPI i ka heluna kanaka 22660 (0.14 ± 0.02, he piʻi ʻewalu manawa i ka pane ʻana i ka hoʻopili ʻana).
Ua hōʻike ka hoʻohālikelike ʻana o nā ʻikepili hōʻike gene (Kiʻi 7) i kahi pilina kiʻekiʻe ma waena o TanjilG_10657 a me ʻehā mau gene ʻē aʻe: TanjilG_27015 (r = 0.89), TanjilG_05080 (r = 0.85), TanjilG_05042 (r = 0.80), a me TanjilG_04706 (r = 0.79). Hiki i kēlā mau hopena ke hōʻike i ka hoʻoponopono like ʻana o kēia mau gene i ka wā o nā pane pale. Ua hōʻike nā genes TanjilG_12861 a me TanjilG_23384 i nā ʻikepili hōʻike like ʻole me nā waiwai coefficient correlation Pearson haʻahaʻa (mai 0.08 a 0.43 a me -0.19 a 0.28, kēlā me kēia) i hoʻohālikelike ʻia me nā genes ʻē aʻe.
Ua ʻike ʻia nā pilina ma waena o nā ʻano hōʻike gene me ka hoʻohana ʻana i ka PCR quantitative. Ua kālailai ʻia nā laina lupine narrowleaf ma lalo nei: 83A:476 (kūpaʻa, e lawe ana i ka allele Lanr1 homozygous), Mandelup (kūpaʻa iki, e lawe ana i ka allele AnMan homozygous), Boregine (kūpaʻa, ʻike ʻole ʻia ke kumu genetic), a me Population 22660 (hiki ke hoʻopilikia). Ua helu ʻia ʻekolu mau manawa (6, 12 a me 24 mau hola ma hope o ka hoʻopili ʻana), me ka hoʻopili ʻia (Colletotrichum lupini, strain Col-08, i loaʻa mai nā kahua lupine ma Wierzhenice, Polani, i ka makahiki 1999) a me nā mea kanu kaohi (sham-inoculated). Hōʻike ka unahi i ka waiwai o ka coefficient correlation Pearson.
Ma muli o ka ʻikepili i loaʻa ma 6 horsepower no ka ʻīniha, ua hana ʻia ʻo WGCNA ma 9981 DEG i ʻike ʻia ma ka hoʻohālikelike ʻana i nā mea kanu i hoʻopili ʻia a me nā mea kanu kaohi e kālele i nā pane pale mua (Papa Hoʻohui S12). Ua loaʻa he iwakāluakūmālua mau modula gene (nā hui) me nā ʻaoʻao hōʻike pili (maikaʻi a maikaʻi ʻole paha) ma waena o nā genotypes a me nā ʻano hoʻokolohua. Ma ka awelika, ua iho nā pae hōʻike gene ma ke kaʻina 83A:476 > Mandelup > Boregine > Helu kanaka 22660 (ma nā ʻano ʻelua, eia naʻe, ua ikaika kēia ʻano i nā mea kanu kaohi). Ma ka awelika, ua iho nā pae hōʻike gene ma ke kaʻina 83A:476 > Mandelup > Boregine > Helu kanaka 22660 (ma nā ʻano ʻelua, eia naʻe, ua ikaika kēia ʻano i nā mea kanu kaohi). Всреднем уровни экспрессии генов снижались в порядке 83A:476 > Mandelup > Boregine > Population 22660 сильнее у контрольных растений). Ma ka awelika, ua emi nā pae hōʻike gene ma ke kauoha 83A:476 > Mandelup > Boregine > Population 22660 (ma nā ʻano ʻelua, eia naʻe, ua ikaika kēia ʻano i nā mea kanu kaohi).平均而言，基因表达水平按83A:476 > Mandelup > Boregine > Ka heluna kanaka 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> heluna kanaka 22660 的 顺序 下降 （， 在 种 中植物 中 更） . . . . . . . . . . . . . . . . В среднем уровни экспрессии генов снижались в ряду 83A:476 > Mandelup > Boregine > Population 22660 контрольных растений). Ma ka awelika, ua emi nā pae hōʻike gene ma ka moʻo 83A:476 > Mandelup > Boregine > Population 22660 (eia naʻe, ma nā ʻano ʻelua, ua ikaika kēia ʻano i nā mea kanu kaohi).Ua hopena ka lāʻau pale ʻana i ka hoʻonui ʻia ʻana o ka hōʻike ʻana o nā genes, ʻoiai ma nā modules 18, 19, 14, 6 a me 1 (ma ke ʻano iho o ka hopena), ka hoʻoponopono maikaʻi ʻole (e laʻa me nā modules 9 a me 20) a i ʻole me nā hopena kūlike ʻole (e laʻa me nā modules 11, 22, 8 a me 13). Ua hōʻike ka loiloi hoʻonui huaʻōlelo GO (Papa Hoʻohui S13) i ka "GO: 0006952 Nā pane pale" no ka module i hoʻokomo ʻia (18) me ka hoʻāla nui loa, me nā genes i kālailai ʻia e qPCR (TanjilG_04706, TanjilG_23384, TanjilG_10657 a me TanjilG_27015), a me nā modules photosynthesis i hoʻopau nui ʻia e Inoculate (9). Ua ʻike ʻia ka mea hoʻohuihui Module 18 (Kiʻi 8) ʻo ia ka gene TanjilG_26536 e hoʻopili ana i ka protein PR-10-like LlR18B, a ua ʻike ʻia ka mea hoʻohuihui module 9 ʻo ia ka gene TanjilG_28955 e hoʻopili ana i ka protein photosystem II PsbQ. Ua loaʻa kahi gene kū'ē anthracnose Lanr1, ʻo TanjilG_05042, ma ka module 22 (Kiʻi 9) a ua pili pū me nā huaʻōlelo "GO:0044260 Cellular macromolecular metabolic processes" a me "GO:0006355 Transcriptional regulation, DNA templating" e lawe ana i ka TanjilG_01212 hub. hoʻopili ka gene i ka heat stress transcription factor A-4a (HSFA4a).
ʻO ka loiloi pūnaewele kaupaona o ka hoʻopuka like ʻana o nā genes o nā modula me nā huaʻōlelo kaʻina hana olaola i hōʻike nui ʻia "GO: 0006952 Defense responses". Ua hoʻomaʻalahi ʻia ka ligation e hōʻike i nā genes ʻehā i kālailai ʻia e qPCR (TanjilG_04706, TanjilG_23384, TanjilG_10657 a me TanjilG_27015).
ʻO ka loiloi pūnaewele kaupaona o ka hoʻopuka like ʻana o nā genes o kahi module me kahi huaʻōlelo hana olaola i hōʻike nui ʻia "GO: 0006355: Hoʻoponopono Transcriptional, DNA templating" a me ka lawe ʻana i kahi gene kū'ē anthracnose moho ʻo Lanr1 TanjilG_05042. Ua hoʻomaʻalahi ʻia ka ligation e hoʻokaʻawale i ka gene TanjilG_05042 a me ke gene TanjilG_01212 waena.
Ua hōʻike ʻia ka nānā ʻana i ke kūʻē ʻana o ka Anthracnose i hōʻiliʻili ʻia ma Australia ua hiki ke hoʻopilikia i ka hapa nui o nā ʻano kanu mua i hoʻokuʻu ʻia; Ua wehewehe ʻia ʻo Kalya, Coromup lāua ʻo Mandelup he kūpaʻa iki, ʻoiai ʻo Wonga, Tanjil lāua ʻo 83A:476 ua wehewehe ʻia he kūpaʻa kiʻekiʻe26,27,31. he allele kūʻē like, i kapa ʻia ʻo Lanr1, a he allele ʻokoʻa ko Coromup lāua ʻo Mandelup, i kapa ʻia ʻo AnMan10, 26, 39, ʻoiai ua hoʻoili ʻo Kalya i kahi allele ʻokoʻa., Lanr2. ʻO ka nānā ʻana no ke kūʻē ʻana o ka anthracnose ma Kelemania ua hopena i ka ʻike ʻana o kahi laina kūʻē Bo7212 me kahi allele moho ʻē aʻe ma mua o Lanr1, i kapa ʻia ʻo LanrBo36.
Ua hōʻike kā mākou noiʻi i kahi alapine haʻahaʻa loa (ma kahi o 6%) o ka allele Lanr1 i loko o ka germplasm i hoʻāʻo ʻia. Kūlike kēia ʻike me nā hopena o ka nānā ʻana i ka germplasm Hikina ʻEulopa me ka hoʻohana ʻana i nā māka Anseq3 a me Anseq4, kahi i hōʻike ai aia ka allele Lanr1 i ʻelua wale nō laina Belarusian. Hōʻike kēia ʻaʻole i hoʻohana nui ʻia ka allele Lanr1 e nā papahana hoʻoulu kūloko, ʻaʻole like me Australia, kahi i lilo ai ia i kekahi o nā alleles koʻikoʻi no ka hoʻoulu ʻana i kōkua ʻia e nā māka. Hiki paha kēia ma muli o ka haʻahaʻa o ke kū'ē i hāʻawi ʻia e ka allele Lanr1 i nā kūlana kahua ʻEulopa i hoʻohālikelike ʻia me ka hōʻike Australia. Eia kekahi, ua hōʻike nā noiʻi o ka anthracnose ma nā wahi ua nui ma Australia ʻaʻole paha e kūpono nā pane kū'ē i hoʻopili ʻia e ka allele Lanr1 i nā kūlana o ka wā e makemake ai i ka ulu ʻana a me ka ulu wikiwiki ʻana o ka pathogen19,42. ʻO kaʻoiaʻiʻo, i loko o ke aʻo ʻana o kēia manawa, ua ʻike ʻia kekahi mau hōʻailona o ka anthracnose i nā genotypes e lawe ana i ka allele Lanr1, e hōʻike ana e nalowale paha ke kū'ē ma lalo o nā kūlana kūpono no ka ulu ʻana o C. lupini. Eia kekahi, hiki ke wehewehe ʻia nā wehewehena wahaheʻe maikaʻi ʻole o ke alo o nā māka Anseq3 a me Anseq4, aia ma kahi o 1 cM mai ka locus Lanr1, 28,30,43.
Ua hōʻike kā mākou noiʻi ua pane ʻo 83A:476, e lawe ana i ka allele Lanr1, i ka hoʻomake ʻana o C. lupini me ka hoʻolālā hou ʻana o ka transcriptome nui i ka manawa mua i kālailai ʻia (6 hpi), ʻoiai ma Mandelup, e lawe ana i ka allele AnMan, ua ʻike ʻia nā pane transcriptomic ma hope loa. (mai 24 a 48 hp). Pili kēia mau ʻokoʻa manawa i nā pane pale me nā ʻokoʻa o nā hōʻailona maʻi, e hōʻike ana i ke koʻikoʻi o ka ʻike mua ʻana i ka pathogen no ka pane kūleʻa i ke kūʻē. No ka hoʻopili ʻana i nā ʻiʻo mea kanu, pono nā spores anthrax e hele i kekahi mau pae hoʻomohala ma ka ʻili o ka mea hoʻokipa, me ka germination, ka mahele ʻana o ke kelepona, a me ka hoʻokumu ʻana o kahi appressorium. ʻO kahi appendage kahi ʻano hoʻopili e hoʻopili i ka ʻili o ka mea hoʻokipa a hoʻomaʻamaʻa i ke komo ʻana i loko o nā ʻiʻo o ka mea hoʻokipa. No laila, ua hōʻike nā spores o C. gloeosporioides i loko o ka pea extract i ka mahele mua o ka nucleus ma hope o 75-90 mau minuke o ka incubation, ka hoʻokumu ʻana o kahi tube germ ma hope o 90-120 mau minuke, a me ka suppression ma hope o 4 mau hola 45. Ua hōʻike ʻo Mango C. gloeosporioides i ka ulu ʻana o ka conidial ma mua o 40% ma hope o 3 mau hola o ka hoʻoulu ʻana a ma kahi o 20% o ka hoʻokumu ʻana o nā appressors ma hope o 4 mau hola. Ua hōʻike ka gene CAP20 e pili ana i ka virulence o C. gloeosporioides i ka hana transcriptional i loko o nā conidia e hana ana i ka epiphyte ma hope o 3.5 h incubation i loko o ka avocado surface wax me nā kiʻekiʻe kiʻekiʻe o ka protein CAP20 ma hope o 4 h 46 min. Pēlā nō, ua hoʻoulu ʻia ka hana o nā genes biosynthesis melanin ma C. trifolii i ka wā o ka incubation 2-hola a ukali ʻia e ka hoʻokumu ʻana o kahi appressorium ma hope o 1 hola. Ua hōʻike ʻia nā haʻawina o nā ʻiʻo lau he hoʻopau mua nā strawberries i hoʻokomo ʻia me C. acutatum ma 8 hpi, ʻoiai ʻo nā tōmato i hoʻokomo ʻia me C. coccodes he hoʻopau mua ma 4 hpi48,49. kūlike nui me ka manawa o ke kaʻina hana hoʻopili Colletotrichum spp. Hōʻike nā pane pale wikiwiki i ka 83A:476 i ke komo ʻana o ke kūʻē ʻana o nā mea kanu a me nā genes immunity i hoʻoulu ʻia e ka effector (ETI) i loko o kēia laina, ʻoiai ʻo nā pane lohi a Mandelup e kākoʻo ana i ka micro-associated molecular pattern-triggered immunity (MTI) hypothesis 50. Nā pane mua i ka 83A: 476 a me Mandelup. ʻO ka overlap hapa ma waena o nā genes i hoʻonui ʻia a i ʻole i hoʻohaʻahaʻa ʻia i ka pane lohi e kākoʻo pū i kēia manaʻo, ʻoiai ua manaʻo pinepine ʻia ʻo ETI he pane MTI wikiwiki a hoʻonui ʻia e hoʻopau ana i ka make ʻana o nā cell i hoʻonohonoho ʻia ma kahi o ka maʻi, i ʻike ʻia ʻo ka anaphylactic shock 51,52.
ʻO ka hapa nui o nā genes i hāʻawi ʻia i ka huaʻōlelo i hōʻike nui ʻia ʻo Gene Ontology GO:0006952 "Defense Response" ʻo ia nā homologues 11 o ka protein 22 i hoʻoulu ʻia e ka stress (like me SAM22) a me nā ʻehiku latex protein-likes nui (MLPs). Ua hōʻike nā protein like 31, 34, 43 a me 423 i ke ʻano like o ke kaʻina. Ua hōʻike nā genes like SAM22 i ka hoʻoulu nui ʻana i lōʻihi ka lōʻihi, e hōʻike ana i ka piʻi ʻana o nā pae o ke kūʻē ʻana o ka anthracnose (83A:476 a me Boregine). Eia nō naʻe, ua hoʻohaʻahaʻa ʻia nā genes like MLP i nā laina e lawe ana i ka allele kūʻē moho (83A:476/Lanr1 ma 6 hpi a me Mandelup/AnMan ma 24 hpi). Pono e hoʻomaopopo ʻia ʻo nā homologues like SAM22 i ʻike ʻia a pau mai kahi hui gene e uhi ana ma kahi o 105 kb, ʻoiai ʻo nā genes like MLP e puka mai ana mai nā wahi kaʻawale o ka genome. Ua ʻike ʻia hoʻi ka hoʻāla ʻana i hoʻonohonoho ʻia o ia mau ʻano genes like SAM22 i kā mākou noiʻi mua o ke kūʻē ʻana o NLL i ka hoʻopili ʻana o Diaporthetoxica, e hōʻike ana ua pili lākou i nā ʻāpana ākea o ka pane pale. Kākoʻo ʻia kēia hopena e nā hōʻike o kahi pane maikaʻi o nā ʻano genes like SAM22 i ka hōʻeha a i ʻole ka mālama ʻana me ka waikawa salicylic, nā mea hoʻoulu fungal, a i ʻole hydrogen peroxide.
Ua hōʻike ʻia nā genes like MLP e pane i nā pilikia abiotic a me biotic like ʻole, me nā maʻi bacteria, viral a me nā maʻi fungal pathogenic i nā ʻano mea kanu he nui55. ʻO nā kuhikuhi o ka pane ʻana i kekahi mau pilina ma waena o nā mea kanu a me nā pathogens mai ka hoʻonui nui ʻana (ʻo ia hoʻi, i ka wā o ka infestation o ka pulupulu me Verticillium dahliae) a hiki i ka emi nui ʻana (ʻo ia hoʻi, ma hope o ka maʻi ʻana o ka lāʻau ʻāpala me Alternaria spp.)56,57. Ua ʻike ʻia ka emi nui ʻana o ka gene MLP-like 423 i ka wā o nā pale avocado i ka maʻi F. niger a i ka wā o ka maʻi ʻana o ka lāʻau ʻāpala Botryosphaeria berengeriana f. cn. piricola a me Alternaria alternata he mau pathotypes apple58,59. Eia kekahi, ʻo ka apple calli e overexpressing ana i ka gene MLP-like 423 he haʻahaʻa ka hōʻike ʻana o nā genes e pili ana i ke kūʻē a ʻoi aku ka maʻalahi i ka maʻi fungal59. Ma hope o Fusarium oxysporum f, ua kāohi ʻia hoʻi ka gene MLP-like 423 i loko o ka germplasm bean maʻamau kūpaʻa. cn. Maʻi bean 60.
ʻO nā lālā ʻē aʻe o ka ʻohana PR-10 i ʻike ʻia ma kā mākou noiʻi RNA-seq ʻo ia nā genes LlR18A a me LlR18B i ka pane ʻana i ka upregulation, a me ka gene upregulated (1 gene) a i ʻole downregulated (3 genes) no ka protein hoʻoili lipid DIR1. . Eia kekahi, hōʻike ʻo WGCNA i ka gene LlR18B ma ke ʻano he kikowaena i loko o kēia module, kahi mea maʻalahi loa i ka lāʻau pale a lawe i kekahi mau genes pane pale. Ua hoʻokomo ʻia nā genes LlR18A a me LlR18B i loko o nā lau lupine melemele i ka pane ʻana i nā bacteria pathogenic, a me nā kumu NLL ma hope o ka hoʻopili ʻana o D. toxica, ʻoiai ʻo ka homologue laiki o kēia mau genes, ʻo RSOsPR10, ua hoʻokomo koke ʻia e kahi maʻi fungal i manaʻo ʻia e pili ana i ke ala hōʻailona jasmonic acid53,61, 62. Hoʻopili ka gene DIR1 i nā protein lawe lipid nonspecific e pono ai no ka hoʻomaka ʻana o ke kū'ē i loaʻa i ka systemic (SAR). Me ka hoʻomohala ʻana o nā hopena pale, ua lawe ʻia ka protein DIR1 mai ke kikowaena o ka maʻi ma o ka phloem e hoʻoulu iā SAR i nā ʻōpū mamao. ʻO ka mea hoihoi, ua hoʻoulu nui ʻia ka gene TanjilG_02313 DIR1 i ka manawa mua ma nā laina 84A:476 a me ka Population 22660, akā ua ulu pono ke kūʻē ʻana o ka anthracnose ma ka laina 84A:476 wale nō. Hōʻike paha kēia i kekahi subfunctionalization o ka gene DIR1 ma NLL, ʻoiai ua pane nā homologues ʻekolu i koe i ka inoculation ma ka laina 83A:476 wale nō ma 6 hpi, a ua kuhikuhi ʻia kēia pane i lalo.
I kā mākou noiʻi ʻana, ʻo nā ʻāpana maʻamau e pili ana i ke kaʻina hana olaola i kapa ʻia ʻo "GO:0055114 Redox process" ʻo ia ka cytochrome P450 protein, peroxidase, linoleic acid 9S-/13S-lipoxygenase, a me 1-aminocyclopropane-1-carboxylic acid oxidase. Eia kekahi, ua wehewehe kā mākou WGCNA i ka homologue HSFA4a ma ke ʻano he kikowaena e lawe ana i nā modules e like me ke moho gene kū'ē Lanr1 ʻo TanjilG_05042. ʻO HSFA4a kahi ʻāpana o ka hoʻoponopono redox-dependent o ka nuclear transcription i nā mea kanu.
ʻO nā protein Cytochrome P450 he mau oxidoreductases e hoʻoulu ai i nā hopena hydroxylation NADPH a/a i ʻole O2-dependent i ka metabolism mua a me ka lua, me ka metabolism o xenobiotics, a me nā hormones, nā waikawa momona, nā sterols, nā ʻāpana paia cell, biopolymers, a me ka biosynthesis o nā hui pale 69. I loko o kā mākou Ma kahi noiʻi, ua hoʻemi ʻia ka loli o ka hana cytochrome P450 mea kanu mai -10.6 log2 (hoʻololi fold) a i 5.7 ma muli o ka nui o nā homologues i hoʻololi ʻia (37) a me nā ʻokoʻa i nā ʻano pane ma waena o nā genes kikoʻī, e hōʻike ana i kahi hoʻoponopono i luna. . ʻO ka hoʻohana ʻana i ka ʻikepili RNA-seq wale nō e wehewehe i ka hana biological putative o nā genes NLL i loko o ia ʻano protein superfamily nui e kuhi nui ʻia. Eia nō naʻe, he mea kūpono ke hoʻomaopopo ʻana ua pili kekahi mau genes cytochrome P450 me ka hoʻonui ʻia o ke kūʻē ʻana i nā fungi pathogenic a i ʻole bacteria, me ka hāʻawi ʻana i nā hopena allergic69,70,71.
ʻO nā peroxidases Papa III he mau enzymes mea kanu multifunctional i komo i loko o nā ʻano hana metabolic like ʻole i ka wā o ka ulu ʻana a me ka hoʻomohala ʻana o nā mea kanu, a me ka pane ʻana i nā pilikia kaiapuni e like me ka salinity, ka maloʻo, ka ikaika o ka mālamalama kiʻekiʻe, a me ka hoʻouka kaua pathogen72. Pili nā Peroxidases i ka launa pū ʻana o kekahi mau ʻano mea kanu me Anthracis, me Stylosanthes humilis a me C. gloeosporioides, Lens culinaris a me C. truncatum, Phaseolus vulgaris a me C. lindemuthianum, Cucumis sativus a me C. lagenarium73,74,75,76. He wikiwiki loa ka pane, i kekahi manawa ma 4 HPI, ma mua o ke komo ʻana o ka fungus i loko o ka ʻiʻo mea kanu73. Ua pane pū ka gene peroxidase i ka hoʻomake ʻana o D. toxica NLL. Ma waho aʻe o kā lākou mau hana maʻamau e hoʻoponopono i ka pahū oxidative a hoʻopau paha i ke kaumaha oxidative, hiki i nā peroxidases ke hoʻopilikia i ka ulu ʻana o nā pathogen ma o ka hoʻokumu ʻana i nā pale kino e pili ana i ka hoʻoikaika ʻana o ka paia cell i ka wā o ka lignification, subunit a i ʻole ke keʻa ʻana o nā hui kikoʻī. Hiki ke hoʻopili ʻia kēia hana in silico i ka gene TanjilG_03329 e hoʻopili ana i kahi anion peroxidase lignin-forming putative i hoʻonui nui ʻia i kā mākou noiʻi ma ka laina kūpaʻa 83A:476 ma 6 HPI, akā ʻaʻole i nā ʻano ʻē aʻe a me nā manawa i pane ʻole.
ʻO 9S-/13S-lipoxygenase o ka waikawa linoleic ka hana mua ma ke ala oxidative o ka biosynthesis lipid78. He nui nā hana a nā huahana o kēia ala i ka pale ʻana i nā mea kanu, me ka hoʻoikaika ʻana i ka paia cell ma o ka hoʻokumu ʻana o nā waihona callose a me pectin, a me ka hoʻoponopono ʻana i ke kaumaha oxidative ma o ka hana ʻana o nā ʻano oxygen reactive79,80,81,82,83. Ma ke aʻo ʻana i kēia manawa, ua loli ka hōʻike ʻana o ka waikawa linoleic 9S-/13S-lipoxygenase ma nā ʻano āpau, akā i ka heluna kanaka susceptible 22660, ua lanakila ka upregulation ma nā manawa like ʻole, ʻoiai i loko o nā ʻano e lawe ana i ka Lanr1 kūpaʻa a me ka allele AnMan, hoʻoikaika ia i ka diversification o ka papa oxylipin i nā hopena anthrax pale ma waena o kēia mau genotypes.
Ua hoʻonui nui ʻia ka homologue 1-aminocyclopropane-1-carboxylate oxidase (ACO) (9 mau genes) a i ʻole i hoʻohaʻahaʻa ʻia (2 mau genes) i ka wā i hoʻokomo ʻia ai me ka lupine. Me ʻelua mau ʻokoʻa, ua hana ʻia kēia mau pane āpau ma 6 hp. ma 83A:476. ʻO ka hopena enzymatic i hoʻopili ʻia e nā protein ACO ke kaʻina hana palena wikiwiki i ka hana ethylene a no laila ua hoʻoponopono nui ʻia84. ʻO Ethylene kahi hormone mea kanu e pāʻani ana i nā ʻano hana like ʻole i ka hoʻoponopono ʻana i ka ulu ʻana o nā mea kanu a me ka pane ʻana i nā kūlana koʻikoʻi abiotic a me biotic. ʻO ka hoʻoulu ʻana o ka palapala ACO a me ka hoʻāla ʻana o ke ala hōʻailona ethylene e pili ana i ka hoʻonui ʻana i ke kūʻē ʻana o ka laiki i ka fungus hemibiotrophic oryzae oryzae ma o ka hoʻoponopono ʻana i ka hana ʻana o nā ʻano oxygen reactive a me nā phytoalexins. ʻO kahi kaʻina hana maʻi lau like loa i loaʻa ma waena o M. oryzae lāua ʻo C. lupini88,89, e kūʻē i ke kua o ka hoʻonui nui ʻana o nā homologues ACO ma ka laina 83A:476 i hōʻike ʻia ma kēia noiʻi, e hoʻololi i ka hiki ke hāʻawi i ke kūʻē i ka NLL anthracnose Ethylene kahi ʻanuʻu hōʻailona waena i nā ala molekala.
Ma ke aʻo ʻana o kēia manawa, ua ʻike ʻia ka hoʻopau nui ʻana o nā genes he nui e pili ana i ka photosynthesis ma 6 hpi ma 83A:476 a ma 48 hpi ma Mandeloop a me ka heluna kanaka 22660. ʻO ka nui a me ka holomua o kēia mau loli e pili ana i ka pae. Ua ʻike ʻia ke kūʻē ʻana o Anthracnose ma kēia hoʻokolohua. I kēia manawa, ua hōʻike ʻia ka hoʻopau ikaika a me ka mua o nā transcripts e pili ana i ka photosynthesis i kekahi mau hiʻohiʻona o nā pilina mea kanu-pathogen, me nā bacteria pathogenic a me nā fungi. ʻO ka wikiwiki (mai 2 HPI i kekahi mau pilina) a me ka hoʻopau honua ʻana o nā genes e pili ana i ka photosynthesis i ka pane ʻana i ka maʻi hiki ke hoʻoulu i ka pale ʻana o ka mea kanu ma muli o ka hoʻolaha ʻana o nā ʻano oxygen reactive a me kā lākou pilina me ke ala waikawa salicylic e hoʻoponopono i nā hopena allergic 90,94.
I ka hopena, ʻo nā ʻano hana pane pale i hāpai ʻia no ka laina kūpaʻa loa (83A:476) e komo pū ana me ka ʻike wikiwiki ʻana o ka pathogen e ka gene R (manaʻo ʻia ʻo TIR-NBS-LRR TanjilG_05042) a me ka waikawa salicylic a me ka ethylene signaling i hoʻopili ʻia e ka pane allergic, a ukali ʻia e ka hoʻokumu ʻana o ka SAR lōʻihi. kākoʻo ʻia ka hana e ka protein DIR-1. Pono e hoʻomaopopo ʻia he pōkole loa ka manawa biotrophic no ka maʻi C. lupini (ma kahi o 2 mau lā), a ukali ʻia e ka ulu ʻana o ka necrotic95. Hiki ke pili ka hoʻololi ʻana ma waena o kēia mau pae me ka necrosis a me ka hōʻike ʻana o nā protein ethylene-inducible e hana ma ke ʻano he mea hoʻoulu no nā hopena hypersensitivity i nā mea kanu host. No laila, he haiki loa ka puka makani no ka hopu holomua ʻana o C. lupini ma ke kahua biotrophic. ʻO ka hoʻonohonoho hou ʻana o nā genes e pili ana me ka redox a me ka photosynthesis i ʻike ʻia ma 83A:476 ma 6 hpi e kūlike me ka holomua o ka fungal hyphae a hōʻike i ka hoʻomohala ʻana o kahi pane pale holomua ma ke kahua biotrophic. ʻO nā pane transcriptomic o Mandelup a me ka heluna kanaka 22660 ua lohi loa paha e hopu i ka fungus ma mua o ka hoʻololi ʻana i ka ulu necrotic, akā naʻe, ʻoi aku ka maikaʻi o Mandelup ma mua o ka heluna kanaka 22660 no ka mea ʻo ka hoʻoponopono wikiwiki ʻana o ka protein PR-10 e paipai i ke kū'ē ākea.
ʻO ETI, i hoʻokele ʻia e ka gene canonical R, me he mea lā he ʻano maʻamau no ke kūʻē ʻana i ka pī i ka anthracnose. No laila, i loko o ke kumu hoʻohālike legume Medicago truncatula, hāʻawi ʻia ke kūʻē ʻana i ka anthracnose e ka gene RCT1, he lālā o ka papa gene mea kanu TIR-NBS-LRR97. Hāʻawi pū kēia gene i ke kūʻē ʻana i ka anthracnose ākea i ka alfalfa i ka wā e hoʻoili ʻia ai i nā mea kanu palupalu. I loko o ka pī maʻamau (P. vulgaris), ua ʻike ʻia ma mua o ʻelua kakini mau genes kūʻē anthracnose a hiki i kēia lā. Loaʻa kekahi o kēia mau genes ma nā wahi i nele i nā genes R canonical, akā naʻe aia nā mea ʻē aʻe he nui ma nā kihi o nā chromosomes e lawe ana i ka hui gene NBS-LRR, me TIR-NBS-LRRs99. Ua hōʻoia pū ka haʻawina SSR genome-ākea i ka pilina o ka gene NBS-LRR me ke kūʻē ʻana i ka anthracnose i ka pī maʻamau. Ua loaʻa pū ka gene canonical R ma ka ʻāpana genomic e lawe ana i ka locus kūʻē anthracnose nui ma ka lupine keʻokeʻo 101.
Hōʻike kā mākou hana i kahi hopena kū'ē koke, i hoʻāla ʻia i ka wā mua o ka maʻi o ka mea kanu (ʻoi aku ka maikaʻi ma mua o 12 hpi), pale pono i ka lupine lau haiki mai ka anthracnose i hoʻokumu ʻia e ka fungus pathogenic Collelotrichum lupini. Ma ka hoʻohana ʻana i ka hoʻonohonoho ʻana o ka throughput kiʻekiʻe, ua hōʻike mākou i nā ʻano hōʻike like ʻole o nā genes kū'ē anthracnose i nā mea kanu NLL i hoʻopili ʻia e nā genes kū'ē Lanr1 a me AnMan. ʻO ka pale kūleʻa e pili ana i ka hoʻolālā pono ʻana i nā genes no nā protein e pili ana i ka redox, photosynthesis, a me ka pathogenesis i loko o nā hola o ka launa mua ʻana o ka mea kanu me kahi pathogen. ʻO nā hopena pale like, akā i lohi i ka manawa, ʻaʻole iʻoi aku ka maikaʻi o ka pale ʻana i nā mea kanu mai nā maʻi. ʻO ke kū'ē Anthrax i hoʻopili ʻia e ka gene Lanr1 e like me ka pane wikiwiki maʻamau o ka gene R (effector-triggered immunity), ʻoiai ʻo ka gene AnMan e hāʻawi pinepine i kahi pane ākea (immunity i hoʻoulu ʻia e kahi ʻano molekala e pili ana i ka microbe), e hāʻawi ana i kahi pae kaulike o ka hoʻomau.
ʻO nā laina NLL he 215 i hoʻohana ʻia e nānā no nā māka anthracnose he 74 mau ʻano, 60 mau laina i loaʻa ma ke keʻa ʻana a i ʻole ka hoʻoulu ʻana, 5 mau mutants, a me 76 mau germplasm hihiu a kumu paha. Ua hele mai nā laina mai 17 mau ʻāina, ʻo ka hapa nui mai Polani (58), Sepania (47), Kelemānia (27), Australia (26), Rusia (19), Belarus (7), Italia (5) a me nā laina ʻē aʻe. mai 10 mau ʻāina. Hoʻokomo pū ka set i nā laina kū'ē i ka kuhikuhi: 83A:476, Tanjil, Wonga e lawe ana i ka allele Lanr1, a me Mandelup e lawe ana i ka allele AnMan. Ua loaʻa nā laina mai ka European Lupine Genetic Resource Database i mālama ʻia e Poznań Plant Breeding Ltd., Wiatrowo, Polani (Papa Hoʻohui S1).
Ua ulu ʻia nā mea kanu ma lalo o nā kūlana i kāohi ʻia (photoperiod 16 mau hola, mahana 25°C i ke ao a me 18°C ​​i ka pō). Ua kālailai ʻia ʻelua mau replicates biological. Ua hoʻokaʻawale ʻia ka DNA mai nā lau ʻekolu pule me ka hoʻohana ʻana i ka DNeasy Plant Mini Kit (Qiagen, Hilden, Kelemānia) e like me ke kaʻina hana. Ua loiloi ʻia ka maikaʻi a me ka nui o ka DNA i hoʻokaʻawale ʻia e nā ʻano spectrophotometric (NanoDrop 2000; Thermo Fisher Scientific, Waltham, MA, USA). Ua kālailai ʻia ka māka AnManM1 e hōʻailona ana i ka gene kūʻē anthracnose ʻo AnMan (i loaʻa mai cv. Mandelup) a me nā māka Anseq3 a me Anseq4 e pili ana i ka gene Lanr1 (i loaʻa mai cv. Tanjil) 11,26,28. Ua helu ʻia nā Homozygotes no ka allele kūʻē ma ke ʻano he "1", susceptible - ma ke ʻano he "0", a me nā heterozygotes - ma ke ʻano he 0.5.
Ma muli o nā hopena o ka nānā ʻana no nā māka ʻo AnManM1, AnSeq3 a me AnSeq4 a me ka loaʻa ʻana o nā hua no nā hoʻokolohua hope loa, ua koho ʻia he 50 mau laina NLL no ka phenotyping kū'ē i ka anthracnose. Ua hana ʻia ke kālailai ʻana ma ka pālua i loko o kahi hale kōmahana i kāohi ʻia e ke kamepiula me kahi photoperiod 14 hola me kahi pae mahana o 22°C i ke ao a me 19°C i ka pō. Ua kahakaha ʻia nā hua (e ʻoki ana i ka ʻili hua ma ka ʻaoʻao ʻē aʻe o ka embryo me kahi pahi ʻoi) ma mua o ka lūlū ʻana e pale aku i ka dormancy o ka hua ma muli o ka paʻakikī o ka ʻili hua a e hōʻoia i ka germination like. Ua ulu ʻia nā mea kanu i loko o nā ipuhao (11 × 11 × 21 cm) me ka lepo sterile (TS-1 REC 085 Medium Basic, Klasmann-Deilmann Polska, Warsaw, Polani). Ua hoʻokō ʻia ka hoʻopili ʻana me ka ʻano Colletotrichum lupini Col-08, i ulu ʻia i ka makahiki 1999 mai nā kumu o nā mea kanu lupine lau haiki i ulu ʻia ma kahi mahinaʻai ma Verzhenitsa, Polani Nui (52° 27′ 42″ N 17° 04′ 05″ E). E kiʻi i kahi ʻāpana. Ua mahi ʻia nā isolates i loko o ka medium SNA ma 20° C. ma lalo o ka mālamalama ʻeleʻele no 21 mau lā e hoʻoulu ai i ka sporulation. ʻEhā pule ma hope o ka lūlū ʻana, i ka wā i hiki ai i nā mea kanu i ke kahua lau 4-6, ua hoʻokō ʻia ka hoʻopili ʻana ma ka pīpī ʻana me kahi hoʻokuʻu ʻana o nā conidia ma kahi nui o 0.5 x 106 conidia no kēlā me kēia ml. Ma hope o ka hoʻopili ʻana, ua mālama ʻia nā mea kanu i ka pouli no 24 mau hola ma kahi haʻahaʻa o kahi 98% a me ka mahana o 25°C e hoʻomaʻamaʻa i ka germination o nā conidia a me ke kaʻina hana maʻi. A laila ua ulu ʻia nā mea kanu ma lalo o kahi photoperiod 14-hola ma 22°C lā/19°C pō a me 70% haʻahaʻa. Ua hana ʻia ka helu maʻi 22 mau lā ma hope o ka hoʻopili ʻana a ua hoʻonohonoho ʻia mai 0 (kūpaʻa) a i 9 (hiki ke maʻalahi loa) ma muli o ke alo a i ʻole ka ʻole o nā ʻeha necrotic ma nā kumu a me nā lau. Eia kekahi, ma hope o ka helu ʻana, ua ana ʻia ke kaumaha o nā mea kanu. Ua helu ʻia nā pilina ma waena o nā genotypes marker a me nā phenotypes maʻi ma ke ʻano he mau pilina ʻelua-moʻo (ʻaʻohe o nā māka heterozygous i loko o ka hoʻonohonoho o nā laina no ka nānā ʻana i ka phenotype kūʻē anthracnose).