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ʻO Angustifolius lupine (NLL, Lupinus angustifolius L.) he mea kanu leguminous i hoʻohana ʻia no ka hana ʻai a me ka hoʻomaikaʻi ʻana i ka lepo.ʻO ka hoʻonui honua ʻana o NLL ma ke ʻano he mea kanu ua hoʻowalewale i nā haʻuki pathogenic, me ka lupine anthracnose, ka mea e hoʻoulu ai i ka maʻi anthracnose pōʻino.Ua hoʻohana ʻia ʻelua alleles, Lanr1 a me AnMan, ka mea e hoʻonui ai i ke kūʻē ʻana, i ka hoʻoulu ʻana o NLL, akā ʻaʻole ʻike ʻia nā ʻano molekala lalo.Ma kēia haʻawina, ua hoʻohana ʻia nā māka Lanr1 a me AnMan e nānā i nā laʻana NLL ʻEulopa.ʻO ka hoʻāʻo ʻana i ke kano i loko o kahi kaiapuni i hoʻopaʻa ʻia i hōʻoia i ka pono o nā mea hāʻawi kūʻē ʻelua.Hana ʻia ka hoʻolaha ʻana o nā ʻano genes like ʻole ma luna o nā laina kūpaʻa a me ka maʻalahi.Hoʻopili ʻia ke kūpaʻa Anthracnose me ka overexpression o nā huaʻōlelo gene ontology "GO: 0006952 Defense Response", "GO: 0055114 Redox Process", a me "GO: 0015979 Photosynthesis".Eia kekahi, ua hōʻike ka laina Lanr1(83A:476) i ka hoʻoponopono hou ʻana i ka transcriptome nui ma hope o ka inoculation, aʻo nā laina ʻē aʻe i hōʻike i kahi lohi i kēia pane ma kahi o 42 mau hola.Hoʻopili ʻia nā pane pale me ka TIR-NBS, CC-NBS-LRR a me NBS-LRR genes, 10 protein i komo i ka pathogenesis, lipid transfer proteins, endoglucan-1,3-β-glucosidase, glycine-rich cell wall proteins, 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 i ka photosynthesis, i hui pū me ka pale kūleʻa i ka wā o ka ulu ʻana o nā mea kanu o ka biology fungal, e manaʻo ana e hoʻāla ka hopena i ka palekana.Hoʻolohi ʻia ka pane ʻana o Mandeloop, e like me ka huki ākea holoʻokoʻa.
ʻO ka lupine lau liʻiliʻi (NLL, Lupinus angustifolius L.) he kīʻaha protein kiʻekiʻe e puka mai ana ma ke komohana Mediterranean region1,2.Ke ulu nei ia i mea ʻai no nā holoholona a me nā kānaka.Manaʻo ʻia ka manure ʻōmaʻomaʻo i nā ʻōnaehana hoʻololi hua ma muli o ka hoʻopaʻa ʻana o ka nitrogen e ka symbiotic nitrogen fixing bacteria a me ka hoʻomaikaʻi holoʻokoʻa o ke ʻano o ka lepo.Ua hana ʻo NLL i kahi kaʻina wikiwiki o ka home i ka makahiki i hala a aia nō ia ma lalo o ke kaomi kiʻekiʻe kiʻekiʻe3,4,5,6,7,8,9,10,11,12.Me ka ulu nui ʻana o NLL, ua hoʻomohala ka holomua o nā fungi pathogenic i nā niches mahiʻai hou a hoʻoulu i nā maʻi luku ʻai hou. ʻO ka mea kupanaha loa no ka poʻe mahiʻai lupine a me nā mea hānai, ʻo ia ke ʻano o ka anthracnose, i kumu ʻia e ka haʻo pathogenic, Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13. ʻO ka mea kupanaha loa no ka poʻe mahiʻai lupine a me nā mea hānai, ʻo ia ke ʻano o ka anthracnose, i kumu ʻia e ka haʻo pathogenic, Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13. Наиболее примечательным для фермеров и селекционеров люпина было появление антракноза, вызванного памогенкринборский , Feiler & Hagedorn13. ʻO ka mea kaulana loa i ka poʻe mahiʻai lupine a me nā mea hānai ʻo ia ka puka ʻana mai o ka anthracnose i hoʻokumu ʻia e ka fungus pathogenic 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，它是由病原真中是菌它是由病原真Botric菌&1pinnbergi (Feederi Boi) Lunenberg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 прикломтор Nirenberg, Feiler & Hagedorn13. ʻO ka mea kaulana loa i ka poʻe mahiʻai lupine a me nā mea hānai ʻo ka puka ʻana mai o ka anthracnose i hoʻokumu ʻia e ka fungus pathogenic Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13.ʻO nā hōʻike mua loa o ka maʻi mai Brazil a me ʻAmelika Hui Pū ʻIa, me nā hōʻailona maʻamau i ʻike ʻia ma 1912 a me 1929.Eia naʻe, ma hope o 30 mau makahiki, ua koho 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 in Targeted Morphology. & H. Schrenk,. & H. Schrenk, .a me H. Schrenk. & H.施伦克，。 & H.施伦克，。a me H. Schlenk, .ʻO ka phenotyping maʻi mua i hana ʻia ma waena o ke kenekulia 20th i hōʻike ʻia i ke kūʻē ʻana i ka NLL a me ka lupine melemele (L. luteus L.) komo, akā ʻo nā lupine keʻokeʻo āpau (L. albus L.) komo i hoʻāʻo ʻia he susceptible loa15,16.Ua hōʻike nā haʻawina e pili ana ka ulu ʻana o ka anthracnose me ka piʻi ʻana o ka ua (ea humidity) a me ka mahana (ma ke ʻano o 12-28 ° C), e alakaʻi ana i ka hōʻeha ʻana i ke kū'ē i nā kiʻekiʻe kiʻekiʻe 17, 18. ʻO ka ʻoiaʻiʻo, ʻo ka manawa i koi ʻia no ka conidia e germinate a me ka hoʻomaka ʻana o ka maʻi , ʻehā mau manawa pōkole ma 24 ° C (12 mau hola) ma mua o 9 mau hola.No laila, ke hoʻomau nei ka hoʻomehana honua i alakaʻi i ka laha ʻana o ka anthracnose.Eia nō 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 ʻike ʻole ʻia e ka ʻoihana lupine i ka manawa20,21.I kekahi mau makahiki ma hope mai, ua laha kēia maʻi pōʻino ma ka honua holoʻokoʻa a ua hoʻopilikia pū i nā ʻāina hana lupine nui e like me Australia, Polani a me Kelemānia22,23,24.Ma hope o ka puka ʻana o ka anthracnose i ka waena o 1990s, ua hōʻike ʻia ka nānā ʻana i ka ʻike ʻana o nā mea hāʻawi kūʻokoʻa i nā laʻana NLL19.Hoʻomalu ʻia ke kūʻē ʻana o NLL i ka anthracnose e ʻelua mau allele mana ʻokoʻa i loaʻa i nā kumu germplasm like ʻole: Lanr1 i loko o ka cultivar Tanjil a me Wonga a me AnMan i ka cultivar.Mandalay 25, 26. Hoʻopiha kēia mau alleles i nā māka molekala e kākoʻo ana i ke koho ʻana i ka germplasm kūpaʻa i nā papahana hānau25,26,27,28,29,30.ʻO ka laina hoʻohua kūʻokoʻa 83A: 476 e lawe ana i ka Lanr1 allele i kāʻei ʻia me ka susceptible wild line P27255 no ka loaʻa ʻana o ka heluna RIL e hoʻokaʻawale ana no ke kū'ē anthracnose, i hiki ai ke hoʻokaʻawale i ka Lanr1 locus i ka chromosome NLL-1131, 32, 33. Aligners o ka hoʻopili ʻana i ka hoʻopaʻa ʻana o ka hoʻopaʻa ʻana a me ka hoʻopaʻa ʻana o ka hoʻopaʻa ʻana me ka hoʻopaʻa ʻana o ka hoʻopaʻa ʻia ʻana o ka NLL-1131, 32, 33. ua hōʻike ʻia kahi o nā allele ʻekolu ma ka chromosome hoʻokahi (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 nui ma waena o nā māka a me nā alleles pili, ʻaʻole hiki ke huki ʻia nā hopena hilinaʻi e pili ana i kā lākou mau genes lalo.Ma ka ʻaoʻao ʻē aʻe, paʻakikī ka hoʻohana ʻana i nā genetics i loko o nā lupins ma muli o ko lākou haʻahaʻa haʻahaʻa haʻahaʻa haʻahaʻa, kahi e paʻakikī ai ka manipulation genetic37.
ʻO ka hoʻomohala ʻana o ka germplasm domesticated e lawe ana i ka allele i makemake ʻia i ka mokuʻāina homozygous, e like me 83A: 476 (Lanr1) a me Mandelup (AnMan), ua wehe i ka puka e aʻo ai i ke kūʻē anthracnose i mua o ke alo o nā hui kūʻē o nā alleles i nā lāhui hihiu.Hiki i nā mīkini 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 panela germplasm European NLL i loaʻa nā laina 215 me ka hoʻohana ʻana i nā māka molekala e hōʻailona ana i nā alleles Lanr1 a me AnMan.Hana ʻia ka Anthracnose phenotyping ma nā laina 50 NLL, i koho mua ʻia no nā māka molekala, ma lalo o nā kūlana hoʻomalu.Ma muli o kēia mau hoʻokolohua, ua koho ʻia nā laina ʻehā i ʻokoʻa i ke kūʻē anthracnose a me Lanr1/AnMan allelic haku mele no ka hoʻohālikelike ʻana i ka hōʻike ʻana o ke ʻano pale ʻokoʻa me ka hoʻohana ʻana i ʻelua ala hoʻohui: kiʻekiʻe-throughput RNA sequencing a me PCR quantification manawa maoli.
ʻO ka nānā ʻana i kahi pūʻulu o ka germplasm NLL (N = 215) me nā māka Lanr1 (Anseq3 a me Anseq4) a me AnMan (Anseq4) a me AnMan (AnManM1) i hōʻike ʻia hoʻokahi wale nō laina (95726, kokoke i Salamanca-b) e hoʻonui i ka "kū'ē" allele no nā māka āpau, ʻoiai ʻo "Presence 8 ~ allelesus" o nā mea āpau. .5%) laina.He ʻumikūmākolu laina i hana i ʻelua mau alale "kūʻokoʻa" o ka māka Lanr1, a ʻo 8 mau laina i hoʻopuka i nā allele "kūʻokoʻa" o Lanr1.hoailona.ʻO ka "kū'ē" allele o ka hōʻailona AnMan (Supplementary Table S1).ʻElua laina heterozygous no ka hōʻailona Anseq3 a me hoʻokahi heterozygous no ka māka AnManM1.42 laina (19.5%) i lawe i nā ʻāpana kūʻē o ka Anseq3 a me Anseq4 alleles, e hōʻike ana i ke alapine kiʻekiʻe o ka hui hou ʻana ma waena o kēia mau loci.ʻO nā phenotypes Anthracnose ma lalo o nā kūlana i hoʻomaluʻia (Supplementary Table S2) i hōʻike i ka loli i ke kū'ēʻana o nā genotypes i ho'āʻoʻia, i hōʻikeʻia i ka paʻakikī o ka anthracnose.ʻO nā ʻokoʻa o nā helu mean mai ka 1.8 (kūpono kūpono) a hiki i ka 6.9 (susceptible) a ʻo nā ʻokoʻa kaumaha o nā mea kanu mai ka 0.62 (susceptible) a hiki i ka 4.45 g (kū'ē). Aia kekahi pilina koʻikoʻi ma waena o nā waiwai i ʻike ʻia ma nā replications ʻelua o ka hoʻokolohua (0.51 no ka nui o ka maʻi, P = 0.00017 a me 0.61 no ka paona mea kanu, P <0.0001) a ma waena o kēia mau ʻāpana ʻelua (- 0.59 a me - 0.77, P <0.00). Aia kahi pilina koʻikoʻi ma waena o nā waiwai i ʻike ʻia i ʻelua replication o ka hoʻokolohua (0.51 no ka nui o ka maʻi, P = 0.00017 a me 0.61 no ka paona mea kanu, P <0.0001) a ma waena o kēia mau ʻāpana ʻelua (-0.59 a me -0.77, P <0.77). Выявлена ​​достоверная корреляция между значениями, наблюдаемыми в двух повторностях эксперимента (0,51 для,0 стля бля) 0017 и 0,61 для массы растения, P <0,0001), а также между этими двумя параметрами (-0,59 и -0,77, Р < 0,0001). Ua ʻike ʻia kahi pilina koʻikoʻi ma waena o nā waiwai i ʻike ʻia i ʻelua hana hou ʻana o ka hoʻokolohua (0.51 no ka nui o ka maʻi, P = 0.00017 a me 0.61 no ka paona mea kanu, P <0.0001), a ma waena o kēia mau ʻāpana ʻelua (- 0.59 a me -0.77, P <0.0001)E量为0.61，P < 0.0001）以及这两个参数之间（- 0.59 a 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 评 . ， p = 0.00017 ， 植物 为 为 0.61 ， p <0.0001） 以及 两 个 参数 之间 （（（（- 5.59 . 9 和- 0.77，P < 0.0001). Наблюдалась значительная корреляция между значениями, наблюдаемыми в двух повторностях (оценка тяжести за1,0 мести за 1,0 мыми сса 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 nānā ʻia ma ke kope (ka helu o ka maʻi maʻi 0.51, P = 0.00017 a me ke kaumaha o nā mea kanu 0.61, P <0.0001) a ma waena o kēia mau ʻāpana ʻelua (-0.59 a me -0.0001) 0.77, P<0.0001 ).ʻO nā hōʻailona maʻamau i ʻike ʻia i nā mea kanu maʻalahi, ʻo ia ka kinking a me ka wili ʻana o ke kumu e like me ke ʻano o ke ʻano "kakaka kahu hipa", a ukali ʻia e nā ʻōpala oval me nā sporozoites ʻalani/ʻulaʻula (Hoʻohui Fig. 1).ʻO ke komo ʻana o Australia e lawe ana i nā genes Lanr1 (83A:476 a me Tanjil) a me AnMan (Mandelup) me ke kūpaʻa haʻahaʻa, 0.0331 a me 0.0036).ʻO kekahi mau laina e lawe pū ana i "kū'ē" Lanr1 a / a i ʻole AnMan alleles e hōʻike i nā hōʻailona o ka maʻi.
ʻO ka mea e mahalo ai, ʻo kekahi mau laina NLL i nele i kekahi "kūʻokoʻa" marker allele i hōʻike i kahi kiʻekiʻe o ke kū'ē anthracnose (hoʻohālikelike a ʻoi aku paha ma mua o Lanr1 a i ʻole AnMan genotypes), e like me Boregine (P value <0.0001 no nā ʻāpana ʻelua), Bojar (P value <0.0001 no ka helu a me 0.001 no ka helu helu a me 0.001 no ka waiwai kanu B9-5) helu a me ka mea nui ʻole no ke kaumaha). ʻO ka mea e mahalo ai, ʻo kekahi mau laina NLL i nele i kekahi "kūʻokoʻa" marker allele i hōʻike i kahi kiʻekiʻe o ke kū'ē anthracnose (hoʻohālikelike a ʻoi aku paha ma mua o Lanr1 a i ʻole AnMan genotypes), e like me Boregine (P value <0.0001 no nā ʻāpana ʻelua), Bojar (P value <0.0001 no ka helu a me 0.001 no ka helu helu a me 0.001 no ka waiwai kanu B9-5) helu a me ka mea nui ʻole no ke kaumaha). Интересно, что несколько линий NLL, лишенных какого-либо «резистентного» маркерного аллеля, показали высокний утурский у (сопоставимый или более высокий, чем для генотипов Lanr1 или AnMan), таких как Boregine (значение P <0,0001 для обои (х парад), Bojar 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 e mahalo ai, he nui nā laina NLL i nele i ka 'resistant' marker allele i hōʻike i kahi kiʻekiʻe o ke kū'ē i ka anthracnose (hoʻohālikelike a ʻoi aku paha ma mua o Lanr1 a i ʻole AnMan genotypes), e like me Boregine (P waiwai <0.0001 no nā ʻāpana ʻelua), Bojar (P waiwai <0.0001 no ka loiloi a me 0.0001 no ka loiloi a me 0.0001 no ka waiwai o nā mea kanu a me 0.0001 no ke kaumaha o ka mea kanu. 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中文型相当或更高），例如Boregine（两个参数的P 值< 0.0001）、Bojar（P 值<得分为0.0001，不礍片B-549/79b（得分P ‼< 0.0001，重量不显着）。 He mea hoihoi i kekahi mau ʻōnaehana NLL i loaʻa ʻole nā ​​māka "antigenic" e hōʻike ana i ke kūpaʻa kiʻekiʻe kiʻekiʻe (e like me Lanr1 a i ʻole AnMan genes a ʻoi aku paha), e like me Boregine (nā ʻāpana ʻelua P <0.0001), Bojar (P waiwai <0.0001, paona mea kanu 0.001) a me ke kānana B-549/70b (Pono nui, ʻaʻole P00). Интересно, что некоторые линии NLL, лишенные каких-либо маркерных аллелей «резистентности», показали высокие уростикраности внимые или выше, чем у генотипов Lanr1 a i AnMan), такие как Boregine (значение P для обоих параметров <0,0001), Bojar (значение P 1,0,мые P 0,001 P ,0,м популяция B-549/79b (оценка P-значение <0,0001, масса незначительна). ʻO ka mea e mahalo ai, ʻo kekahi mau laina NLL i nele i kekahi 'kū'ē' marker alleles i hōʻike i nā pae kiʻekiʻe o ke kū'ē anthracnose (hoʻohālikelike a ʻoi aku paha ma mua o Lanr1 a i ʻole AnMan genotypes), e like me Boregine (P waiwai no nā ʻāpana ʻelua <0.0001), Bojar (P-value <0.0001, ke kaumaha o nā mea kanu 0.0001) a me ka nui o nā mea kanu 0.0001 (P 0.001) a me ka heluna kanaka. ʻaʻole nui ke kaumaha).Hōʻike kēia hanana i ka hiki ke loaʻa kahi kumu genetic hou o ke kū'ē, e wehewehe ana i ka ʻike ʻole ʻia o ka correlation ma waena o nā genotypes marker a me nā phenotypes maʻi (P values ​​​​mai ~0.42 a ~0.98).No laila, ua hōʻike ʻia ka hōʻike ʻo Kolmogorov-Smirnov i ka ʻikepili e pili ana i ke kū'ē anthracnose e like me ka puʻunaue maʻamau no nā helu (P-values ​​0.25 a me 0.11) a me ka nui o nā mea kanu (P-values ​​0.47 a me 0.55), ke manaʻo nei au ke kuhi nei au he nui aku nā alleles ma mua o Lanr1 a me AnMan.
Ma muli o nā hopena o ka nānā ʻana i ka anthracnose resistance, ua koho ʻia nā laina 4 no ka kānana transcriptome: 83A: 476, Boregine, Mandelup, a me ka heluna kanaka 22660. Ua hoʻāʻo hou ʻia kēia mau laina no ka pale ʻana i ka anthrax i nā hoʻokolohua inoculation e RNA sequencing, inā ua like lākou me ka hoʻāʻo mua.ʻO nā helu helu 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 loaʻa i ka protocol Illumina NovaSeq 6000 ka awelika o 40.5 Mread pairs i kēlā me kēia laʻana (29.7 a i 54.4 Mreads) (Paʻi ʻĀpana S3).Mai ka 75.5% a hiki i ka 88.6% nā helu hoʻohālikelike i ka papa kuhikuhi.ʻO ka pilina maʻamau o ka ʻikepili helu heluhelu ma waena o nā ʻano hoʻokolohua ma waena o nā hoʻopiʻi olaola mai ka 0.812 a i ka 0.997 (mean 0.959). Mai loko mai o nā genes 35,170 i kālailai ʻia, 2917 i hōʻike ʻole ʻia, a ua hōʻike ʻia nā genes 4785 ʻē aʻe ma ka pae haʻahaʻa (base mean <5). Mai loko mai o nā genes 35,170 i kālailai ʻia, 2917 i hōʻike ʻole ʻia, a ua hōʻike ʻia nā genes 4785 ʻē aʻe ma ka pae haʻahaʻa (base mean <5). Из 35 170 проанализированных генов 2917 не проявляли экспрессии, а остальные 4785 генов экспресиронвались на проявляли экспрессии е среднее <5). ʻO nā genes 35,170 i kālailai ʻia, ʻaʻole hōʻike ʻia ʻo 2917, a ʻo ke koena 4785 genes i hōʻike ʻia ma kahi pae haʻahaʻa (base mean <5).35,17035,170 Из 35 170 проанализированных генов 2917 не экспрессировались, а остальные 4785 генов имели незначительнсубю значение <5). No nā genes 35,170 i kālailai ʻia, ʻaʻole i hōʻike ʻia ʻo 2917 a ʻo ke koena 4785 genes i hōʻike ʻole ʻia (base mean <5).No laila, ʻo ka helu o nā genes i manaʻo ʻia i hōʻike ʻia (base mean ≥ 5) i ka wā o ka hoʻokolohua ʻo 27,468 (78.1%) (Paʻa S4 Pākuʻi).
Mai ka manawa mua, ua pane nā laina NLL āpau i ka inoculation o C. lupini (strain Col-08) ma ka hoʻonohonoho hou ʻana i ka transcriptome (Table 1), akā naʻe, ʻike ʻia nā ʻokoʻa nui ma waena o nā laina.No laila, ʻo ka laina kū'ē 83A: 476 (e lawe ana i ka Lanr1 gene) hōʻike i ka reprogramming transcriptome koʻikoʻi i ka manawa mua (6 hpi) me ka piʻi ʻana o 31-69-fold i ka helu o nā genes i hoʻokaʻawale ʻia i luna a me lalo i hoʻohālikelike ʻia me nā wahi manawa ʻē aʻe i kēia manawa.Eia kekahi, ua pōkole kēia kiʻekiʻe, no ka mea, ua hoʻololi nui ʻia ka hōʻike ʻana o kekahi mau genes i ka lua o ka manawa (12 hpi).ʻO ka mea e mahalo ai, ʻo Boregine, ka mea i hōʻike i kahi kiʻekiʻe o ke kūʻē ʻana i ka hoʻāʻo ʻana, ʻaʻole i hoʻopaʻa ʻia i ka reprogramming transcriptional nui i ka wā o ka hoʻokolohua.Eia naʻe, ua like ka helu o nā genes i wehewehe 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 kahi lohi pili i nā pane pale.
Ma muli o ka 83A:476 i hana hou i ka transcriptome reprogramming ma 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 laina-specific (Fig. 1).Eia nō naʻe, aia kekahi overlap i nā pane mua ma waena o nā laina haʻawina, ʻoiai ʻo 68.5%, 50.9%, a me 52.6% DEG ma Boregine, Mandelup, a me ka heluna kanaka 22660, kēlā me kēia, i uhi ʻia me nā mea i loaʻa ma 83A: 476 i kekahi mau wahi i ka manawa.Eia nō naʻe, he hapa liʻiliʻi wale nō kēia mau DEG (0.97–1.70%) o nā DEG a pau i ʻike ʻia i kēia manawa me 83A:476.Eia kekahi, ʻo 11 DEG mai nā laina āpau i hui pū ʻia i kēia manawa (Supplementary Tables S4-S6), me nā mea maʻamau o nā pane pale o ka mea kanu: lipid transfer protein (TanjilG_32225), endoglucan-1,3-β-glucoside enzyme (TanjilG_23384), ʻelua mau protein-inducible e like me SAM22 (TanjilG13 protein) 32352), a me ʻelua mau pūmua paʻa paʻa o ka glycine waiwai nui (TanjilG_19701 a me TanjilG_19702).Aia kekahi ʻano kiʻekiʻe o nā pane transcriptome ma waena o 83A:476 a me Boregine ma 24 HPI (ka huina 16-38% DEG) a ma waena o Mandelup a me ka heluna kanaka 22660 ma 48 HPI (ka huina 14-20% DEG).
ʻO ke kiʻi Venn e hōʻike ana i ka helu o nā genes i hōʻike ʻokoʻa ʻia (DEG) ma nā laina lupine haiki (NLL) i hoʻopaʻa ʻia me Colletotrichum lupini (koko Col-08 i loaʻa mai nā māla lupine ma Wierzhenice, Polani, 1999).ʻO nā laina NLL i kālailai ʻia ʻo: 83A:476 (kūʻokoʻa, e lawe ana i ka Lanr1 allele), Boregine (kūʻokoʻa, ʻike ʻole ʻia ke ʻano genetic), Mandelup (kūʻokoʻa kūʻokoʻa, e lawe ana i ka Allele AnMan) a me ka heluna kanaka 22660 (maʻalahi loa).Kū ka pōkole hpi no nā hola ma hope o ka hoʻopaʻa ʻana.Ua wehe ʻia nā waiwai Zero e hoʻomaʻamaʻa i ka pakuhi.
ʻO ka hoʻonohonoho o nā genes overexpressed ma 6 hpi i nānā ʻia no ka hele ʻana o nā kāʻei kikowaena R canonical (Supplementary Table S7).Ua hōʻike ʻia kēia haʻawina i ka transcriptome induction o nā genes resistance maʻi maʻamau me nā kāʻei NBS-LRR wale nō ma 83A: 476.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-LR16jilg2, a me ʻEhā NBS-LR6162, a me ʻEhā NBS-LR16jilg2. ) a me ʻEhā NBS-Lrr (tanjilg_16162) a me ʻEhā NBS-LRR (TANJILG_16162).Loaʻa i kēia mau genes nā kikowaena canonical i hoʻonohonoho ʻia i nā kaʻina i mālama ʻia.Ma kahi o nā genes domain NBS-LRR, ua hoʻāla ʻia kekahi mau kinase RLL ma 6 hpi, ʻo ia hoʻi ma Boregine (TanjilG_19877), ʻelua ma Mandelup (TanjilG_07141 a me TanjilG_19877) a ma ka heluna kanaka 22660 (TanjilG_09014 a me TanjilG_103 a me 72.
ʻO nā Genes me ka'ōlelo hoʻololi nui i ka paneʻana i ka inoculation me C. lupini (strain Col-08) i hoʻokomoʻia i ka Gene Ontology (GO) ka hoʻonuiʻana i ka loiloi (Supplementary Table S8). ʻO ka hua'ōlelo kaʻina hana biological i hōʻike pinepine ʻia ʻo "GO: 0006952 pane pale" i ʻike ʻia ma 6 o 16 (manawa × laina) hui pū me ke koʻikoʻi kiʻekiʻe (P value <0.001) (Fig. 2). ʻO ka hua'ōlelo kaʻina hana biological i hōʻike pinepine ʻia ʻo "GO: 0006952 pane pale" i ʻike ʻia ma 6 o 16 (manawa × laina) hui pū me ke koʻikoʻi kiʻekiʻe (P value <0.001) (Fig. 2). LIKE LIKE LIKE 16 (время × линия) комбинаций с высокой значимостью (значение P <0,001) (рис. 2). ʻO ka huaʻōlelo kaʻina hana biological i hōʻike pinepine ʻia ʻo 'GO: 0006952 pane pale', i ʻike ʻia ma 6 o 16 (manawa × lineage) hui pū me ke koʻikoʻi kiʻekiʻe (P value <0.001) (Fig. 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（时闐（时闐）中中中可一一一一一上一一一一上一为具有高显着性（P 值< 0.001）（图2）。 ʻO ka huaʻōlelo kaʻina hana olaola ʻoi loa ka "GO: 0006952 pane pale", i ʻike ʻia ma 6 o nā hui 16 (时间×线), me ke koʻikoʻi nui (P value <0.001) (图2). Наиболее часто чрезмерно представленным термином биологического процесса был «GO: 0006952 Defence Response», котовлый 6 поского ий (время × линия) с высокой значимостью (значение P <0,001) (рис. 2). ʻO ka huaʻōlelo kaʻina hana biological i hōʻike pinepine ʻia ʻo 'GO:0006952 Defense Response', i ʻike ʻia ma 6 o 16 hui pū ʻana (manawa × laina) me ke koʻikoʻi kiʻekiʻe (P value <0.001) (Fig. 2).Ua hōʻike nui ʻia kēia huaʻōlelo i ʻelua mau manawa ma 83A: 476 a me Boregine (6 a me 24 hpi) a ma kahi manawa ma Mandelup a me ka heluna kanaka 22660 (12 a me 6 hpi, kēlā me kēia).He hopena kēia i manaʻo ʻia, e hōʻike ana i ka pane antifungal o nā laina kūpaʻa.Eia hou, 83A: 476 pane iā C. lupini ma ka hoʻoulu wikiwikiʻana i nā genes e pili ana i ka oxidative burst i hōʻikeʻia e ka hua'ōlelo "GO: 0055114 redox process", e hōʻike ana i kahi pane pale kūikawā,ʻoiaiʻo Boregine i hōʻike i nā pane pale kūikawā, pili i ka hua'ōlelo 'GO'.: 0006950 pane pilikia”.Ua ho'āla 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 hua'ōlelo "GO: 0016104 Kaʻina o ka biosynthesis triterpene" a me "GO: 0006722 Kaʻina o ka triterpene metabolism" (ʻo nā hua'ōleloʻelua no ka hoʻonohonoho like o nā genes), me ka noʻonoʻo i nā hopena o ka hopena o ka hua'ōlelo GO. Eia kekahi, ʻo ka pane mua ʻana 83A:476 (6 hpi) a me ka hopena lohi ʻo Mandelup a me ka heluna kanaka 22660 ka huaʻōlelo GO: 0015979 'photosynthesis' a me nā kaʻina hana olaola e pili ana.
ʻO nā huaʻōlelo bioprocess gene ontology i koho ʻia ma ka hōʻike ʻana o nā ʻano genes i hōʻike ʻia i ka wā o nā pane transcriptome o ka narrow-leaved lupine (NLL) inoculated me anthrax lupine (Col-08 strain i loaʻa mai nā māla lupine ma Wierzhenice, Polani, i ka makahiki 1999) ua hoʻonui nui ʻia.ʻO nā laina NLL i kālailai ʻia ʻo: 83A:476 (kūʻokoʻa, e lawe ana i ka homozygous Lanr1 allele), Boregine (kūʻokoʻa, ʻike ʻole ʻia ke ʻano genetic), Mandelup (kūʻokoʻa kūʻokoʻa, e lawe ana i ka homozygous AnMan allele) a me ka heluna kanaka 22660 (susceptible).
Ma muli o ka manaʻo o kēia noiʻi e ʻike i nā ʻano genes e kōkua i ka pale ʻana i ka anthracnose, ua ʻike ʻia nā genes i hāʻawi ʻia i nā huaʻōlelo GO "GO: 0006952 Defensive responses" a me "GO: 0055114 Redox process" i kālai ʻia me nā ʻoki ʻoki ʻia ʻoiai ʻo ke kumu kumu ʻo ≥ 30 me ka liʻiliʻi o hoʻokahi laina.× helu i ka manawa e hui pū ana i nā waiwai koʻikoʻi o ka log2 (hoʻololi hoʻololi).ʻO ka helu o nā genes e kūpono ana i kēia mau koina he 65 no GO:0006952 a me 524 no GO:0055114.
83A:476 ua hōʻike ʻia ʻelua mau piko DEG i hōʻike ʻia me ka huaʻōlelo GO:0006952, ʻo ka mua ma 6 genes no ka ʻīniha (64 genes, up and down regulation) a ʻo ka lua ma 24 genes no ka iniha (15 genes, up regulation only).Ua hōʻike pū ʻo Boregine i ka piʻi ʻana o GO:0006952 i ka manawa like, akā me ka liʻiliʻi 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, e lawe ana i nā genes 12 (ʻo ka mea mua me nā genes hoʻāla, a ʻo ka lua me nā genes suppressive wale nō), ʻoiai ʻo ka heluna 22660 ma 6 HPI (13 genes) i ʻoi aku ka nui o ka piʻi kiʻekiʻe.hooponopono.Pono e hoʻomaopopo ʻia ʻo 96.4% o GO: 0006952 DEG i kēia mau piko i like ke ʻano o ka pane (i luna a i lalo paha), e hōʻike ana i kahi overlap nui i nā pane pale ʻoiai nā ʻokoʻa o ka helu o nā genes i komo.ʻO ka pūʻulu nui loa o nā kaʻina e pili ana i ka huaʻōlelo GO: 0006952 hoʻopili i ka Starvation Stress-Associated Message Protein 22 (SAM22-like), nona ka papa 10 pathogenesis-associated protein (PR-10) protein clade a me ka core protein latex.like (MLP-like) protein) protein (Fig. 3).Ua ʻokoʻa nā hui ʻelua i ke ʻano o ka ʻōlelo a me ke kuhikuhi o ka pane.ʻO nā genes e hoʻopili ana i nā protein like SAM22 i hōʻike i ka hoʻokomo like ʻana a me ka nui i ka manawa mua (6 a i ʻole 12 hpi) a ʻaʻole i pane ma ka hopena o ka hoʻokolohua (48 hpi), ʻoiai ʻo nā protein e like me MLP i hōʻike i ka hoʻonohonoho ʻana ma 6 hpi.hpi.83A: 476 a me Mandelup ma 48 hp/in, aneane pau ʻole nā ​​helu ʻikepili ʻē aʻe.Eia kekahi, ua ʻike ʻia nā ʻokoʻa o nā moʻolelo hōʻike o SAM22-like protein genes i ʻike ʻia i ka ʻokoʻa ʻana o ka pale ʻana i ka anthracnose, no ka mea, ʻoi aku ka nui o nā laina kūʻokoʻa i ʻoi aku ka nui o ka manawa e hoʻoulu ai i kēia mau genes ma mua o nā genes maʻalahi.ʻO kekahi ʻano LlR18A/B-like PR-10 i hōʻike i kahi hiʻohiʻona hōʻike like loa me ka SAM22-like protein gene.
Ua ʻike ʻia nā mea nui o ke kaʻina hana olaola "GO: 0006952 Defence Response" a me nā hiʻohiʻona hōʻike o nā genes moho o ka Lanr1 a me AnMan alleles.Hōʻike ka pālākiō Log2 i nā waiwai log2 (fold change) ma waena o ka inoculated (Colletotrichum lupini, strain Col-08, i loaʻa mai nā māla lupine, Wizhenica, Polani, 1999) a me nā mea kanu (sham-inoculated) i ka manawa like.Ua nānā ʻia nā laina lupine liʻiliʻi: 83A: 476 (kūʻokoʻa, e lawe ana i ka homozygous Lanr1 allele), Boregine (kūʻokoʻa, ʻike ʻole ʻia ke ʻano genetic), Mandelup (kūʻokoʻa kūʻokoʻa, e lawe ana i ka homozygous AnMan allele), a me Population 22660 (susceptible).
Eia kekahi, ua loiloi ʻia nā ʻōlelo hōʻike o nā moho moho RNA-seq Lanr1 (TanjilG_05042) a me AnMan (TanjilG_12861) (Fig. 3).Ua hōʻike ka gene TanjilG_05042 i ka pane koʻikoʻi (hoʻoulu) ma 83A: 476 wale nō i ka manawa mua (6 hpi), ʻoiai ʻo TanjilG_12861 ka mea nui ma Mandeloop wale nō i ʻelua mau manawa: 6 hpi (lalo hoʻoponopono) a me 24 hpi (6 hpi).Me.).hiki ke hoʻololi ʻia)).
ʻO nā genes i hōʻike nui ʻia ma ka huaʻōlelo GO: 0055114 "redox process" he mau genes e hoʻopili ana i nā protein cytochrome P450 a me ka peroxidase (Fig. 4).No nā laʻana i hoʻokaʻawale ʻia mai 83A: 476 ma 6 HPI, ʻike ʻia nā helu kiʻekiʻe a i ʻole ka liʻiliʻi loa o ka log2 (no ka 86.6% o nā genes) ma waena o nā mea kanu i hoʻopaʻa ʻia a mālama ʻia, e hōʻike ana i ka pane kiʻekiʻe o kēia genotype i ka inoculating sex.Ua hōʻike ʻo 83A:476 i ka GO nui loa: 0055114 DEG ma 6 hpi (503 genes), aʻo ke koena o nā laina ma 48 hpi (Boregine, 31 genes; Mandelup, 85 genes; a me Population 22660, 78 genes)).I ka nui o nā genes o ka ʻohana GO: 0055114, ʻelua ʻano pane i ka lāʻau lapaʻau (hoʻoulu 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 Ke hōʻike nei kēia mau ʻike, ʻoiai ka liʻiliʻi liʻiliʻi loa (ʻo ia hoʻi, ka helu o nā genes redox mutated, 85 versus 503), ʻo ke ʻano o ka hoʻopaneʻe transcriptome pane o ka mandeloup 8 e like me 8.Ma Boregine a me ka heluna kanaka 22660, ua emi keia hui ana ma 51.6% a me 75.6%.
Ua hōʻikeʻia nā hiʻohiʻona o ka'ōlelo o nā mea nui o ka hua'ōlelo o ke kaʻina hana olaola "GO: 0055114 Redox process".Hōʻike ka pālākiō Log2 i nā waiwai log2 (fold change) ma waena o ka inoculated (Colletotrichum lupini, strain Col-08, i loaʻa mai nā māla lupine, Wizhenica, Polani, 1999) a me nā mea kanu (sham-inoculated) i ka manawa like.Ua nānā ʻia nā laina lupine liʻiliʻi: 83A: 476 (kūʻokoʻa, e lawe ana i ka homozygous Lanr1 allele), Boregine (kūʻokoʻa, ʻike ʻole ʻia ke ʻano genetic), Mandelup (kūʻokoʻa kūʻokoʻa, e lawe ana i ka homozygous AnMan allele), a me Population 22660 (susceptible).
83A:476 Hoʻopili pū ʻia nā pane transcriptomic i ka inoculation me C. lupini (strain Col-08) i ka hoʻopaʻa ʻana i nā genes i pili i ka huaʻōlelo GO: 0015979 "photosynthesis" a me nā kaʻina hana olaola e pili ana (FIG. 5).ʻO kēia GO:0015979 DEG set he 105 genes i hoʻopaʻa nui ʻia ma 6 hpi ma 83A:476.Ma kēia ʻāpana, ua hoʻohaʻahaʻa ʻia nā genes 37 ma Mandelup ma 48 HPI a me 35 i ka manawa like i ka heluna 22660, me 19 DEG maʻamau i nā genotype ʻelua.ʻAʻohe DEG e pili ana i ka huaʻōlelo GO: 0015979 i hoʻoikaika nui ʻia i kekahi hui (laina x manawa).
Ua hōʻike ʻia nā hiʻohiʻona o ka hōʻike ʻana i nā mea nui o ka huaʻōlelo o ke kaʻina olaola "GO: 0015979 Photosynthesis".Hōʻike ka pālākiō Log2 i nā waiwai log2 (fold change) ma waena o ka inoculated (Colletotrichum lupini, strain Col-08, i loaʻa mai nā māla lupine, Wizhenica, Polani, 1999) a me nā mea kanu (sham-inoculated) i ka manawa like.Ua nānā ʻia nā laina lupine liʻiliʻi: 83A: 476 (kūʻokoʻa, e lawe ana i ka homozygous Lanr1 allele), Boregine (kūʻokoʻa, ʻike ʻole ʻia ke ʻano genetic), Mandelup (kūʻokoʻa kūʻokoʻa, e lawe ana i ka homozygous AnMan allele), a me Population 22660 (susceptible).
Ma muli o nā hopena o ka nānā ʻana i ka ʻōlelo ʻokoʻa a me ka manaʻo e pili ana i nā pane pale e kūʻē i nā fungi pathogenic, ua koho ʻia kēia pūʻulu o ʻehiku mau genes no ka helu ʻana i nā profiles hōʻike e ka PCR manawa maoli (Supplementary Table S9).
ʻO ka putative protein gene TanjilG_10657 i hoʻoikaika nui ʻia i nā laina aʻo a me nā manawa manawa i hoʻohālikelike ʻia i nā mea kanu (mimic) (Supplementary Tables S10, S11).Eia kekahi, ua hōʻike ka ʻōlelo hoʻolaha o TanjilG_10657 i ka piʻi ʻana o ke ʻano o ka hoʻokolohua no nā laina āpau.Ua hōʻike ka heluna kanaka 22660 i ka naʻau kiʻekiʻe loa o TanjilG_10657 i ka inoculation me ka 114-fold activation a me ka kiʻekiʻe kiʻekiʻe o ka ʻōlelo pili (4.4 ± 0.4) ma 24 HPI (Fig. 6a).Ua hōʻike pū ka PR10 LlR18A protein gene TanjilG_27015 i ka hoʻōla ʻana ma nā laina āpau a me nā manawa manawa, me ka nui o nā helu ʻikepili (Fig 6b).E like me TanjilG_10657, ʻike ʻia ka pae kiʻekiʻe kiʻekiʻe loa o TanjilG_27015 i ka heluna kanaka 22660 inoculated ma 24 HPI (19.5 ± 2.4).ʻO ka acid endochitinase gene TanjilG_04706 ua hoʻonui nui ʻia i nā laina āpau a i nā manawa āpau koe wale no Boregine 6 hpi (Fig. 6c).Ua hoʻoikaika ikaika ʻia i ka manawa mua (6 HPI) ma 83A:476 (e 10.5 manawa) a hoʻonui haʻahaʻa i nā laina ʻē aʻe (e 6.6-7.5 manawa).I ka wā o ka hoʻokolohua, ua mau ka ʻōlelo o TanjilG_04706 i nā pae like ma 83A: 476 a me Boregine, ʻoiai ma Mandelup a me ka heluna kanaka 22660 ua hoʻonui nui ʻia, a hiki i nā waiwai kiʻekiʻe (5.9 ± 1.5 a me 6.2 ± 1.5, kēlā me kēia).ʻO ka endoglucan-1,3-β-glucosidase-like gene TanjilG_23384 i hōʻike i ka hoʻoulu kiʻekiʻe i nā manawa mua ʻelua (6 a me 12 hpi) ma nā laina āpau koe wale nō ka heluna kanaka 22660 (Fig. 6d).Ua ʻike ʻia nā kiʻekiʻe kiʻekiʻe e pili ana i ka 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, haʻahaʻa loa ka ʻōlelo TanjilG_23384 i nā laina aʻo a pau (mai 0.04 ± 0.009 a i 0.44 ± 0.12).
Hōʻike ʻia nā ʻōlelo kikoʻī o nā genes i koho ʻia (ag) e ka quantitative PCR.Hōʻike nā helu 6, 12 a me 24 i nā hola ma hope o ke kanu ʻana.Ua hoʻohana ʻia nā genes LanDExH7 a me LanTUB6 no ka hoʻoponopono maʻamau a ua hoʻohana ʻia ʻo LanTUB6 no ka calibration inter-series.Hōʻike nā pahu kuhi i ka hoʻokaʻawale maʻamau e pili ana i ʻekolu hoʻopiʻi koʻikoʻi, ʻo kēlā me kēia o ka awelika o ʻekolu hoʻopiʻi ʻenehana. ʻO keʻano helu helu o nāʻokoʻa i nā pae'ōlelo ma waena o nā mea i hoʻopiliʻia (Colletotrichum lupini, strain Col-08, i loaʻa i ka makahiki 1999 mai ka māla lupine ma Wierzenica, Polani) a me ka mana (mock-inoculated) nā mea kanu i kahaʻia ma luna o nā heluʻikepili (*P waiwai <0.05, **P waiwai ≤ 0.01, ***P 0.01). ʻO keʻano helu helu o nāʻokoʻa i nā pae'ōlelo ma waena o nā mea i hoʻopiliʻia (Colletotrichum lupini, strain Col-08, i loaʻa i ka makahiki 1999 mai ka māla lupine ma Wierzenica, Polani) a me ka mana (mock-inoculated) nā mea kanu i kahaʻia ma luna o nā heluʻikepili (*P waiwai <0.05, **P waiwai ≤ 0.01, ***P 0.01). Статистическая значимость различий в уровнях экспрессии между инокулированными (Colletotrichum lupini, штамм Col-08, получен в г.999 г. женице, Польша) и контрольными (ложно инокулированными) растениями отмечена над точками данных (*значение P, **, значение P < 0,05ч, **, значение P < 0,05ч P ≤ 0,001). ʻO nāʻokoʻa koʻikoʻi o ka helu helu ma waena o nā pae i hoʻopaʻa ʻia (Colletotrichum lupini, strain Col-08, i loaʻa i ka makahiki 1999 mai kahi māla lupine ma Wierzhenice, Polani) a me ka mana (sham-inoculated) nā mea kanu i ʻike ʻia ma luna o nā helu data (*P waiwai <0.05, **P-value ≤ 0.01, ***P-0.接种（Colletotrichum lupini，Col-08株，1999年从波兰Wierzenica的羽扇豆田获得）和对照（模拟接种義耶和華差异的统计学显着性标记在数据点上方（*P值< 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, полученнлий спо, полученпина спол. Польша, в 1999 г.) и контрольными (ложно инокулированными) растениями отмечены над точками данных (* знач,0ниех (* Pз, ч-0ние х5) ***P-значение ≤ 0,001). Ua ʻike ʻia nā ʻokoʻa nui o ka helu ʻana ma waena o nā mea i hoʻopaʻa ʻia (Colletotrichum lupini, strain Col-08, i loaʻa mai nā māla lupine ma Verzhenice, Polani, i ka makahiki 1999) a me nā mea kanu hoʻomalu (sham-inoculated) ma luna o nā helu data (*P waiwai <0.05 , **P-value ≤ 0.01, 0.0-1).ʻO nā laina NLL i kālailai ʻia ʻo: 83A:476 (kūʻokoʻa, e lawe ana i ka homozygous Lanr1 allele), Mandelup (kūpaʻa haʻahaʻa, e lawe ana i ka homozygous AnMan allele), Boregine (kūʻokoʻa, ʻike ʻole ʻia ka genetic background) a me ka heluna kanaka 22660 (susceptible).
ʻO ka moho gene TanjilG_05042 ma ka Lanr1 locus i hōʻike i kahi hiʻohiʻona hōʻike ʻokoʻa loa mai nā kiʻi i loaʻa mai nā haʻawina RNA-seq (Fig. 6e).Ua ʻike ʻia ka hana nui o kēia ʻano ma Mandelup a me ka heluna kanaka 22660 (a hiki i 39.7 a me 11.7 manawa, i kēlā me kēia), e hopena i nā pae kiʻekiʻe kiʻekiʻe (a hiki i 1.4 ± 0.14 a me 7.2 ± 1.3, kēlā me kēia).83A:476 hōʻike pū kekahi i ka hoʻoponopono ʻana o ka TanjilG_05042 gene (a hiki i ka 3.8-fold), akā naʻe, ʻoi aku ka nui o nā pae hōʻike pili i loaʻa (0.044 ± 0.002) ma mua o ka 30-paʻi haʻahaʻa ma mua o nā mea i ʻike ʻia ma Mandelup a me ka heluna 22660.i kālailai ʻia e qPCR ua hōʻike i nā ʻokoʻa koʻikoʻi i nā pae hōʻike ma waena o nā genotypes i nā ʻano ʻokoʻa hoʻohenehene (mana), hiki i kahi ʻokoʻa 58 ma waena o nā heluna 22660 a me 83A: 476, a ma waena o nā heluna 22660 a me 22660. Ua loaʻa kahi ʻokoʻa ʻelua ma waena o Boregine a me Mandalup.
Ka moho gene ma ka AnMan locus, TanjilG_12861, ua ho'ā 'ia i ka pane i ka vaccination ma 83A:476 a me Mandelup, ua kūʻole i ka 22660 heluna kanaka, a ua downregulated ma Boregine (Fig. 6f).ʻO ka ʻōlelo pili o ka TanjilG_12861 gene ka mea kiʻekiʻe loa i loko o ka inoculated 83A: 476 (0.14±0.01).ʻO ka 17.4 kDa papa I heat shock protein gene TanjilG_05080 HSP17.4 hōʻike haʻahaʻa haʻahaʻa haʻahaʻa haʻahaʻa pili i nā mea a pau i aʻo ʻia a me nā helu manawa (Fig. 6g).Ua ʻike ʻia ka waiwai kiʻekiʻe loa ma 24 HPI i ka heluna kanaka 22660 (0.14 ± 0.02, he piʻi ʻewalu paʻi i ka pane ʻana i ka lāʻau lapaʻau).
ʻO ka hoʻohālikelike ʻana i nā moʻolelo hōʻike gene (Fig. 7) ua hōʻike ʻia kahi pilina kiʻekiʻe ma waena o TanjilG_10657 a me ʻehā mau genes ʻē aʻe: TanjilG_27015 (r = 0.89), TanjilG_05080 (r = 0.85), TanjilG_05042 (r = 0.80), a me Tanjil6_9.Hiki i ia mau hopena ke hōʻike i ka hoʻoponopono pū ʻana o kēia mau genes i ka wā o nā pane pale.Ua hōʻike nā genes TanjilG_12861 a me TanjilG_23384 i nā ʻano hōʻike like ʻole me ka haʻahaʻa haʻahaʻa Pearson correlation coefficient (mai 0.08 a 0.43 a me -0.19 a i 0.28, kēlā me kēia) i hoʻohālikelike ʻia me nā genes ʻē aʻe.
Ua ʻike ʻia ka hoʻoponopono ʻana ma waena o nā profile expression gene me ka hoʻohana ʻana i ka PCR quantitative.Ua kālailai ʻia nā laina lupine liʻiliʻi: 83A:476 (kūʻokoʻa, e lawe ana i ka homozygous Lanr1 allele), Mandelup (kūpaʻa haʻahaʻa, e lawe ana i ka homozygous AnMan allele), Boregine (kūʻokoʻa, ʻike ʻole ʻia ke ʻano genetic), a me ka heluna kanaka 22660 (susceptible).ʻEkolu mau helu manawa i helu ʻia (6, 12 a me 24 mau hola ma hope o ka inoculation), me ka inoculated (Colletotrichum lupini, strain Col-08, i loaʻa mai nā māla lupine ma Wierzhenice, Polani, i ka makahiki 1999) a me nā mea kanu (sham-inoculated).Hōʻike ka pālākiō i ka waiwai o ka coefficient correlation Pearson.
Ma muli o ka ʻikepili i loaʻa ma 6 horsepower i kēlā me kēia ʻīniha, ua hana ʻia ʻo WGCNA ma 9981 DEG i ʻike ʻia ma ka hoʻohālikelike ʻana i nā mea kanu inoculated a me ka mana e kālele ana i nā pane pale mua (Supplementary Table S12).Ua ʻike ʻia he iwakāluakūmālua mau ʻano moʻomeheu (clusters) me nā ʻōlelo hoʻohālikelike i hoʻopili ʻia (maikaʻi a maikaʻi ʻole paha) ma waena o nā genotypes a me nā ʻano hoʻokolohua. Ma ka awelika, e iho ana nā pae hōʻike gene ma ke ʻano 83A:476 > Mandelup > Boregine > Ka heluna kanaka 22660 (ma nā ʻano ʻelua, akā naʻe, ʻoi aku ka ikaika o kēia ʻano i nā mea kanu hoʻomalu). Ma ka awelika, e iho ana nā pae hōʻike gene ma ke ʻano 83A:476 > Mandelup > Boregine > Ka heluna kanaka 22660 (ma nā ʻano ʻelua, akā naʻe, ʻoi aku ka ikaika o kēia ʻano i nā mea kanu hoʻomalu). Всреднем уровни экспрессии генов снижались в порядке 83A:476 > Mandelup > Boregine > Population 22660 контрольных растений). Ma ka awelika, ua emi nā pae hōʻike gene ma ke kauoha 83A:476 > Mandelup > Boregine > Ka heluna kanaka 22660 (ma nā ʻano ʻelua, akā, ʻoi aku ka ikaika o kēia ʻano i nā mea kanu hoʻomalu).平均而言，基因表达水平按83A:476 > Mandelup > Boregine > Population 22660 的顺序下降（然而，在两种变作耶和华照植物中更强）。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 在 种 中中 更） . . . . . . . . . . . . . . . . . . . . . В среднем уровни экспрессии генов снижались в ряду 83A:476 > Mandelup > Boregine > Population 22660 рольных растений). Ma ka awelika, ua emi nā pae hōʻike gene i ka moʻo 83A:476 > Mandelup > Boregine > Population 22660 (eia naʻe, ma nā ʻano ʻelua, ʻoi aku ka ikaika o kēia ʻano i nā mea kanu hoʻomalu).ʻO ka loaʻa ʻana o ka hoʻopaʻa ʻana i ka hoʻoponopono ʻana i ka hōʻike gene, ʻoi aku ka nui o nā modules 18, 19, 14, 6 a me 1 (ma ke ʻano iho o ka hopena), hoʻoponopono maikaʻi ʻole (e laʻa i nā modules 9 a me 20) a i ʻole me nā hopena kūʻokoʻa (e laʻa i nā modules 11, 22, 8 a me 13).E hele i ka hōʻike hōʻike inoa inoaʻo GO TER TOP. . ^.Ua ʻike ʻia ʻo Module 18 concentrator (Fig. 8) ma ke ʻano he TanjilG_26536 gene e hoʻopili ana i ka protein PR-10-like LlR18B, a ua ʻike ʻia ka module 9 concentrator ma ke ʻano he TanjilG_28955 gene e hoʻopili ana i ka protein photosystem II PsbQ.Loaʻa ka moho anthracnose resistance gene Lanr1, TanjilG_05042, ma ka module 22 (Fig. 9) a pili pū me nā huaʻōlelo "GO: 0044260 Cellular macromolecular metabolic process" a me "GO: 0006355 Transcriptional regulation, DNA templating" e lawe ana i ka TanjilG_01212 hub.Hoʻopili ka gene i ka helu A-4a (HSFA4a).
ʻO ka nānā ʻana o ka pūnaewele paona o ka hōʻike pū ʻana o nā modules me nā huaʻōlelo kaʻina biological overrepresented "GO: 0006952 Defense pane".Ua maʻalahi ka ligation e hōʻike i nā ʻehā ʻehā i kālai ʻia e qPCR (TanjilG_04706, TanjilG_23384, TanjilG_10657 a me TanjilG_27015).
ʻO ka nānā ʻana o ka pūnaewele paona o ka hōʻike pū ʻana o kahi module me kahi huaʻōlelo kaʻina biological overrepresented "GO: 0006355: Transcriptional regulation, DNA templating" a me ka lawe ʻana i kahi moho anthracnose resistance gene Lanr1 TanjilG_05042.Ua maʻalahi ka ligation no ka hoʻokaʻawale ʻana i ka gene TanjilG_05042 a me ka gene TanjilG_01212 kikowaena.
Ua hōʻike ʻia ka nānā ʻana i ke kūʻē anthracnose i hōʻiliʻili ʻia ma Australia i ka hapa nui o nā cultivars i hoʻokuʻu mua ʻia ua maʻalahi;Ua hōʻike ʻia ʻo Kalya, Coromup a me Mandelup he kūpaʻa kūʻokoʻa, ʻoiai ʻo Wonga, Tanjil a me 83A: 476 i hōʻike ʻia he kūpaʻa loa26,27,31.ua like ka allele kū'ē, i koho ʻia ʻo Lanr1, a ua loaʻa iā Coromup a me Mandelup kekahi allele ʻokoʻa, i kapa ʻia ʻo AnMan10, 26, 39, ʻoiai ua hala ʻo Kalya ma kahi alahele ʻokoʻa., Lanr2.ʻO ka nānā ʻana no ka pale ʻana i ka anthracnose ma Kelemania i ʻike ʻia ai kahi laina kūʻokoʻa Bo7212 me kahi allele moho ma waho o Lanr1, i koho ʻia ʻo LanrBo36.
Ua hōʻike kā mākou noiʻi i kahi alapine haʻahaʻa loa (ma kahi o 6%) o ka allele Lanr1 i ka germplasm i hoʻāʻo ʻia.Ua kūlike kēia ʻike me nā hopena o ka nānā ʻana i ka germplasm ʻEulopa Hikina me ka hoʻohana ʻana i nā kaha Anseq3 a me Anseq4, i hōʻike ʻia aia ka Lanr1 allele i ʻelua mau laina Belarusian.Hōʻike kēia ʻaʻole i hoʻohana nui ʻia ka Lanr1 allele e nā papahana hoʻohua kūloko, ʻaʻole like ma Australia, kahi ʻo ia kekahi o nā alleles nui no ka hoʻoulu ʻana i ka marker-assisted breeding.Hiki paha kēia ma muli o ka haʻahaʻa haʻahaʻa o ke kū'ē i hāʻawi ʻia e ka Lanr1 allele i nā kūlana kahua ʻEulopa i hoʻohālikelike ʻia i ka hōʻike Australian.Eia kekahi, ua hōʻike ʻia nā haʻawina o ka anthracnose ma nā wahi ua nui ma Australia e hiki ʻole i nā pane kū'ē i hoʻopili ʻia e ka Lanr1 allele ʻaʻole maikaʻi i nā kūlana o ka wā e makemake ai i ka ulu a me ka ulu wikiwiki ʻana o ka pathogen19,42.ʻO kaʻoiaʻiʻo, ma ka haʻawina i kēia manawa, uaʻikeʻia kekahi mau hōʻailona o ka anthracnose i nā genotypes e lawe ana i ka Lanr1 allele, e hōʻike ana e nalowale ke kū'ē ma lalo o nā kūlana kūpono no ka uluʻana o C. lupini.Eia kekahi, ʻo nā wehewehe maikaʻi ʻole o ka hele ʻana o nā māka Anseq3 a me Anseq4, aia ma kahi o 1 cM mai ka Lanr1 locus, hiki ke 28,30,43.
Ua hōʻike kā mākou haʻawina i ka 83A: 476, e lawe ana i ka Lanr1 allele, pane i ka inoculation C. lupini me ka reprogramming transcriptome nui i ka helu manawa i kālailai mua ʻia (6 hpi), ʻoiai ma Mandelup, e lawe ana i ka AnMan allele, ʻike ʻia nā pane transcriptomic ma hope.(mai 24 a 48 hp).ʻO kēia mauʻokoʻa kino i nā pane pale e pili ana i nāʻokoʻa o nā hōʻailona maʻi, e hōʻike ana i ke koʻikoʻi o kaʻike muaʻana i ka pathogen no ka pane maikaʻiʻana i ke kū'ē.No ka hoʻopili ʻana i ka ʻiʻo o ka mea kanu, pono e hele nā ​​spores anthrax i kekahi mau pae hoʻomohala ma ka ʻili o ka mea hoʻokipa, me ka germination, ka māhele cell, a me ka hoʻokumu ʻana i kahi apppressorium.ʻO kahi appendage he hale maʻi e pili ana i ka ʻili o ka mea hoʻokipa a hoʻomaʻamaʻa i ke komo ʻana i loko o nā ʻiʻo host.No laila, spores o C. gloeosporioides i ka pea extract i hōʻike i ka mahele mua o ka nucleus ma hope o 75-90 minuke o ka incubation, ka hookumu ana o ka germ tube ma hope o 90-120 minuke, a suppression ma hope o 4 hola 45.Ua hōʻike ʻo Mango C. gloeosporioides ma mua o 40% conidial germination ma hope o 3 mau hola o ka incubation a ma kahi o 20% ka hoʻokumu ʻana o nā apppressors ma hope o 4 mau hola.Ka virulence-pili CAP20 gene o C. gloeosporioides hoike transcriptional hana i loko o epiphyte-hoʻokumu conidia ma hope o 3.5 h incubation ma avocado ili wax me ka kiʻekiʻe o ka CAP20 protein ma hope o 4 h 46 min.Pēlā nō, ua hoʻoulu ʻia ka hana o ka melanin biosynthesis genes i C. trifolii i ka wā o ka incubation 2-hola i ukali ʻia e ka hoʻokumu ʻana o kahi appressorium ma hope o 1 hola.Ua hōʻike ʻia nā haʻawina o nā ʻiʻo lau ʻo nā strawberry i hoʻopaʻa ʻia me C. acutatum ua hoʻopaʻa mua ʻia ma 8 hpi, ʻoiai ʻo nā tōmato i hoʻopaʻa ʻia me C. coccodes i hoʻopaʻa mua ʻia ma 4 hpi48,49.kūlike loa me ka nui o ka manawa o Colletotrichum spp.kaʻina maʻi maʻi.ʻO nā pane pale wikiwiki i ka 83A: 476 hōʻike i ke komo ʻana o ke kūpaʻa mea kanu a me nā genes effector-triggered immunity (ETI) ma kēia laina, ʻoiai nā pane lohi o Mandelup e kākoʻo i ka micro-associated molecular pattern-triggered immunity (MTI) hypothesis 50. Nā pane mua i 83A: 476.ʻO ka ʻāpana ʻāpana ma waena o nā genes i hoʻoponopono ʻia i luna a i lalo paha i ka pane lohi e kākoʻo i kēia manaʻo, no ka mea, ua manaʻo pinepine ʻia ʻo ETI he pane MTI wikiwiki a hoʻonui ʻia e hoʻopau i ka make cell programmed ma kahi o ka maʻi, i kapa ʻia ʻo anaphylactic shock 51,52.
ʻO ka hapa nui o nā genes i pili i ka huaʻōlelo overrepresented Gene Ontology GO: 0006952 "Defense Response" ʻo ia ka 11 homologues o ka memo hoʻokēʻai i hoʻoulu ʻia i ke koʻikoʻi 22 protein (e like me SAM22) a me nā mea nui ʻehiku latex protein-likes (MLPs).-like nā proteins 31, 34, 43 a me 423 i hōʻike i ke ʻano like.ʻO nā genes like SAM22 i hōʻike i ka hoʻoikaika nui ʻana i lōʻihi loa, e hōʻike ana i ka piʻi ʻana o ka pae o ke kū'ē anthracnose (83A:476 a me Boregine).Eia naʻe, ua hoʻohaʻahaʻa ʻia nā genes like MLP ma nā laina e lawe ana i ka allele kū'ē moho (83A:476/Lanr1 ma 6 hpi a me Mandelup/AnMan ma 24 hpi).Pono e hoʻomaopopo ʻia ʻo nā ʻano homologues like me SAM22 i ʻike ʻia mai loko mai o kahi puʻupuʻu gene e pili ana ma kahi o 105 kb, aʻo nā genes like me MLP mai nā ʻāpana ʻokoʻa o ka genome.Ua ʻike ʻia ka hoʻoulu ʻia ʻana o ia mau genes like SAM22 i kā mākou noiʻi mua ʻana o ke kū'ē ʻana o NLL i ka inoculation Diaporthetoxica, e hōʻike ana ua pili lākou i nā ʻāpana ākea o ka pane pale.Kākoʻo pū ʻia kēia hopena e nā hōʻike no ka pane maikaʻi ʻana o nā genes like SAM22 i ka hōʻeha a i ʻole ka mālama ʻana me ka salicylic acid, fungal inducers, a i ʻole hydrogen peroxide.
Ua hōʻike ʻia nā genes e like me MLP e pane i nā ʻano koʻikoʻi abiotic a me biotic, me nā maʻi bacterial, viral a pathogenic fungal maʻi i loko o 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 piʻi nui ʻana (ʻo ia hoʻi, i ka wā o ka hoʻoulu ʻana 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 hoʻohaʻahaʻa nui ʻana o ka MLP-like 423 gene i ka wā o ka pale ʻana i ka avocado i ka maʻi F. niger a i ka wā maʻi o ka lāʻau ʻāpala Botryosphaeria berengeriana f.cn.piricola a me Alternaria alternata he apple pathotypes58,59.Eia kekahi, ʻo ka apple calli e hōʻike nui ana i ka MLP-like 423 gene he haʻahaʻa haʻahaʻa o nā genes pili i ke kū'ē a ʻoi aku ka maʻalahi i ka maʻi fungal59.Ma hope o Fusarium oxysporum f, ua kāohi pū ʻia ke ʻano MLP-like 423 i loko o ka germplasm piʻi maʻamau.cn.ʻO ka maʻi pīni 60.
ʻO nā lālā ʻē aʻe o ka ʻohana PR-10 i ʻike ʻia ma kā mākou haʻawina RNA-seq ʻo nā genes LlR18A a me LlR18B i pane i ka upregulation, a me ka upregulated (1 gene) a i ʻole downregulated (3 genes) genes no ka lipid transfer protein DIR1..Eia kekahi, hōʻike ʻo WGCNA i ka gene LlR18B ma ke ʻano he hub i loko o kēia module, hiki ke maʻalahi i ka lāʻau lapaʻau a lawe i kekahi mau genes pane pale.Ua hoʻokomoʻia nā genes LlR18A a me LlR18B i nā lau lupine melemele i pane i nā bacteria pathogenic, a me nā kumu NLL ma hope o D. toxica inoculation, aʻo ka homologue raiki o kēia mau genes, RSOsPR10, ua hoʻokomo wikiwikiʻia e kahi maʻi fungal i komo i loko o ka jasmonic acid signaling genes 1, 62 lipid transport genes. i koi 'ia no ka ho'omaka 'ana o ka systemic acquired resistance (SAR).Me ka hoʻomohala ʻana o nā hopena pale, lawe ʻia ka protein DIR1 mai ke kikowaena o ka maʻi ma o ka phloem e hoʻoulu i ka SAR ma nā ʻāpana mamao.ʻO ka mea e mahalo ai, ua hoʻoikaika nui ʻia ka gene TanjilG_02313 DIR1 i ka manawa mua ma nā laina 84A:476 a me ka heluna kanaka 22660, akā ua hoʻomohala maikaʻi ʻia ke kūʻē anthracnose ma ka laina 84A:476 wale nō.Hōʻike paha kēia i kekahi subfunctionalization o ka DIR1 gene ma NLL, no ka mea, ua pane nā homologues ʻekolu i ka inoculation wale nō ma ka laina 83A:476 ma 6 hpi, a ua kuhikuhi ʻia kēia pane i lalo.
I kā mākou noiʻi ʻana, ʻo nā mea maʻamau e pili ana i ke kaʻina olaola i kapa ʻia ʻo "GO: 0055114 Redox process" he protein cytochrome P450, 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 hub e lawe ana i nā modula e like me ka Lanr1 resistance gene candidate TanjilG_05042.ʻO ka HSFA4a kahi mea o ka redox-dependent regulation o ka nuklear transcription i nā mea kanu.
Cytochrome P450 proteins he oxidoreductases e catalyze NADPH a me / a O2-hilinaʻi hydroxylation reactions i ka mea mua a me ka lua metabolism, me ka metabolism o xenobiotics, a me nā hormones, fatty acids, sterols, cell wall components, biopolymers, a me ka biosynthesis o ka mea pale 69. I loko o kā mākou P40. fold change) i ka 5.7 ma muli o ka nui o nā homologues i hoʻololi ʻia (37) a me nā ʻokoʻa o 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 olaola putative o nā genes NLL i loko o kahi superfamily protein nui e manaʻo nui ʻia.Eia nō naʻe, pono e hoʻomaopopo ʻia e pili ana kekahi mau genes cytochrome P450 me ka hoʻonui ʻana i ke kū'ē i nā fungi pathogenic a i ʻole bacteria, me ka hāʻawi ʻana i nā hopena maʻi69,70,71.
ʻO ka papa III peroxidases he mau enzymes mea kanu multifunctional i komo i loko o kahi ākea o nā kaʻina metabolic i ka wā o ka ulu ʻana a me ka ulu ʻana o ka mea kanu, a me ka pane ʻana i nā koʻikoʻi o ke kaiapuni e like me ka paʻakai, ka maloʻo, ka ikaika o ka māmā, a me ka hoʻouka pathogen72.Hoʻopiliʻia nā peroxidases i ka pilina o nāʻano mea kanu me Anthracis, e like 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 haʻuki i ka ʻiʻo mea kanu73.Ua pane pū ka gene peroxidase i D. toxica NLL inoculation.Ma waho aʻe o kā lākou mau hana maʻamau e hoʻoponopono i ka oxidative burst a i ʻole ka hoʻopau ʻana i ke koʻikoʻi oxidative, hiki i nā peroxidases ke hoʻopilikia i ka ulu ʻana o ka pathogen ma o ka hana ʻana i nā pale kino e pili ana i ka hoʻoikaika ʻana i ka paia o ka cell i ka wā lignification, subunit a i ʻole cross-linking o nā pūhui kikoʻī.Hiki ke hoʻopili ʻia kēia hana ma ka silico i ka TanjilG_03329 gene e hoʻopili ana i kahi anion peroxidase i hoʻokumu ʻia i ka lignin i hoʻonui nui ʻia i kā mākou noiʻi ʻana ma ka 83A:476 laina kūpaʻa ma 6 HPI, ʻaʻole naʻe i nā mea ʻē aʻe a me nā manawa manawa i pane ʻole.
ʻO 9S-/13S-lipoxygenase o ka linoleic acid ka hana mua i ke ala oxidative o lipid biosynthesis78.ʻO nā huahana o kēia alahele he nui nā hana i ka pale ʻana i nā mea kanu, me ka hoʻoikaika ʻana i ka paia o ke kelepona ma o ka hoʻokumu ʻana i nā waihona callose a me pectin, a me ka hoʻoponopono ʻana i ke koʻikoʻi oxidative ma o ka hana ʻana i nā ʻano oxygen reactive79,80,81,82,83.Ma ka haʻawina i kēia manawa, ua hoʻololi ʻia ka ʻōlelo o ka linoleic acid 9S-/13S-lipoxygenase i nā ʻano āpau, akā i ka heluna kanaka maʻalahi 22660, ua lanakila ka upregulation i nā manawa manawa like ʻole, ʻoiai i nā ʻano e lawe ana i ka Lanr1 kūpaʻa a me ka AnMan allele, hoʻoikaika ʻo ia i ka hoʻololi ʻana o kēia mau ʻano o ka oxylipinx i ka pale pale.
ʻO ka 1-aminocyclopropane-1-carboxylate oxidase (ACO) homologue i hoʻonui nui ʻia (9 genes) a i ʻole downregulated (2 genes) i ka wā i hoʻopaʻa ʻia me ka lupine.Me nā ʻokoʻa ʻelua, ua loaʻa kēia mau pane āpau ma 6 hp.ma 83A:476.ʻO ka hopena enzymatic i hoʻopili ʻia e nā protein ACO ʻo ia ke kaʻina palena palena i ka hana ʻana i ka ethylene a no laila ua hoʻoponopono nui ʻia84.He hormone mea kanu ka Ethylene 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 hoʻokomo ʻana o ka ACO transcription 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 hemibiotrophic fungus 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 a me C. lupini88,89, e kū'ē i ke kua o ka upregulation nui o ACO homologues ma ka 83A: 476 laina i hōʻike ʻia i loko o kēia haʻawina, hoʻololi i ka hiki ke hāʻawi i ke kū'ē iā NLL anthracnose Ethylene kahi hōʻailona kikowaena i nā ala mole.
Ma ke aʻo ʻana i kēia manawa, ʻike ʻia ka hoʻopau nui ʻana o nā genes e pili ana i ka photosynthesis ma 6 hpi ma 83A:476 a ma 48 hpi ma Mandeloop a me ka heluna 22660.ʻO ka nui a me ka holomua o kēia mau hoʻololi e like me ka pae.Ua ʻike ʻia ka pale ʻana o Anthracnose i kēia hoʻokolohua.I kēia mau lā, ua hōʻike ʻia ka paʻi ikaika a me ka hoʻomaka mua ʻana o nā transcripts pili i ka photosynthesis i kekahi mau hiʻohiʻona o ka launa pū ʻana o nā mea kanu-pathogen, me ka bacteria pathogenic a me nā haʻi.ʻO ka wikiwiki (mai ka 2 HPI i kekahi mau pilina) a me ka hoʻopau honua i nā genes e pili ana i ka photosynthesis i ka paneʻana i ka maʻi hiki ke hoʻoulu i ka palekana o ka mea kanu ma muli o ka hoʻokomoʻana o nāʻano oxygen reactive a me kā lākou pilina me ke ala salicylic acid e hoʻoponopono i nā hopena maʻi 90,94.
I ka hopena, ʻo nā hana pane pale i manaʻo ʻia no ka laina kūʻokoʻa loa (83A:476) e komo pū me ka hoʻomaopopo ʻana i ka pathogen wikiwiki e ka R gene (ʻoiai paha ʻo TIR-NBS-LRR TanjilG_05042) a me ka salicylic acid a me ka hōʻailona ethylene i pane i ka maʻi, a ma hope o ka hoʻokumu ʻana i 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 necrotic95.Hiki ke hoʻopili ʻia ka hoʻololi ʻana ma waena o kēia mau ʻanuʻu me ka necrosis a me ka hōʻike ʻana o nā protein ethylene-inducible e hana ma ke ʻano he mea hoʻoulu i nā hopena hypersensitivity i nā mea kanu hoʻokipa.No laila, he haiki loa ka puka makani no ka hopu ana i ka C. lupini ma ke kahua biotrophic.ʻO ka reprogramming o nā genes e pili ana me redox a me photosynthesis i ʻike ʻia ma 83A:476 ma 6 hpi e kūlike me ka holomua o ka hyphae fungal a hōʻike i ka hoʻomohala ʻana o kahi pane pale kūleʻa i ka pae biotrophic.ʻO nā pane transcriptomic o Mandelup a me ka heluna 22660 hiki ke hoʻopaneʻe loa i ka hopu ʻana i ka haʻiki ma mua o ka hoʻololi ʻana i ka ulu necrotic, akā naʻe, ʻoi aku ka maikaʻi o Mandelup ma mua o ka heluna 22660 no ka mea ʻo ka hoʻoponopono wikiwiki ʻana o ka protein PR-10 e hoʻoikaika i ka pale ākea.
ʻO ETI, i alakaʻi ʻia e ka canonical R gene, ʻike ʻia he ʻano hana maʻamau no ka pale ʻana o ka pī i ka anthracnose.No laila, ma ka legume Medicago truncatula, ke kū'ē i ka anthracnose i hāʻawi ʻia e ka RCT1 gene, he lālā o ka TIR-NBS-LRR97 mea kanu R gene class.Hāʻawi kēia ʻano i ka pale ʻana i ka anthracnose ākea ākea i ka alfalfa ke hoʻololi ʻia i nā mea kanu maʻalahi.I loko o ka pī maʻamau (P. vulgaris), ʻoi aku ma mua o ʻelua mau kukini anthracnose genes resistance i ʻike ʻia a hiki i kēia lā.Loaʻa kekahi o kēia mau genes ma nā ʻāpana i nele i nā genes R canonical, akā naʻe, aia nā mea ʻē aʻe ma nā kihi o nā chromosomes e lawe ana i ka pūʻulu gene NBS-LRR, me TIR-NBS-LRRs99.Ua hōʻoia pū ka haʻawina SSR genome-wide i ka hui ʻana o ka gene NBS-LRR me ke kū'ē anthracnose i ka pī maʻamau.Ua ʻike ʻia ka gene canonical R ma ka ʻāina genomic e lawe ana i ka locus kūʻē anthracnose nui ma ke keʻokeʻo lupine 101.
Hōʻike kā mākou hana i ka hopena kū'ē koke, hoʻāla ʻia i ka wā mua o ka maʻi 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.Me ka hoʻohana ʻana i nā kaʻina hana kiʻekiʻe, ua hōʻike mākou i nā moʻolelo hōʻike ʻokoʻa o nā genes kū'ē anthracnose i nā mea kanu NLL i hoʻopili ʻia e nā genes resistance Lanr1 a me AnMan.ʻO ka pale kūleʻa e pili ana i ka hoʻolālā ʻana i nā genes no nā protein i komo 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 lohi i ka manawa, ʻoi aku ka maikaʻi o ka pale ʻana i nā mea kanu mai nā maʻi.ʻO ke kūpaʻa Anthrax i hoʻopili ʻia e ka Lanr1 gene e like me ka pane wikiwiki maʻamau o ka gene R (effector-triggered immunity), aʻo ka AnMan gene ka mea i hāʻawi i kahi pane ākea (immunity triggered by microbe-associated molecular pattern), e hāʻawi ana i kahi pae kūpono o ka hoʻomau.
ʻO nā laina 215 NLL i hoʻohana ʻia no ka nānā ʻana no nā hōʻailona anthracnose he 74 mau mea kanu, 60 mau laina i loaʻa ma ka hele ʻana a i ʻole ka hānau ʻana, 5 mau mutants, a me 76 mau germplasms hihiu a i ʻole kumu.Ua hele mai nā laina mai 17 mau ʻāina, mai Polani (58), Sepania (47), Kelemānia (27), Australia (26), Russia (19), Belarus (7), Italia (5) a me nā laina ʻē aʻe.mai 10 mau ʻāina.Aia pū ka pūʻulu i nā laina kūpaʻa kuhikuhi: 83A: 476, Tanjil, Wonga e lawe ana i ka Lanr1 allele, a me Mandelup e lawe ana i ka AnMan allele.Ua loaʻa nā laina mai ka European Lupine Genetic Resource Database i mālama ʻia e Poznań Plant Breeding Ltd., Wiatrowo, Polani (Supplementary Table S1).
Hoʻoulu ʻia nā mea kanu ma lalo o nā kūlana mālama ʻia (photoperiod 16 mau hola, ka mahana 25 ° C i ke ao a me 18 ° C ​​i ka pō).Ua kālailai ʻia ʻelua hoʻopiʻi olaola.Ua hoʻokaʻawale ʻia ʻo DNA mai nā lau ʻekolu pule me ka hoʻohana ʻana i ka DNeasy Plant Mini Kit (Qiagen, Hilden, Kelemānia) e like me ka protocol.Ua loiloi ʻia ka maikaʻi a me ka noʻonoʻo o ka DNA i hoʻokaʻawale ʻia e nā ʻano spectrophotometric (NanoDrop 2000; Thermo Fisher Scientific, Waltham, MA, USA).Ka AnManM1 marker marking the anthracnose resistance gene AnMan (derived from cv. Mandelup) and the markers Anseq3 and Anseq4 flanking the gene Lanr1 (derived from cv. Tanjil) were analyzed 11,26,28.Ua helu ʻia nā homozygotes no ka allele kūpaʻa ma ke ʻano he "1", hiki ke maʻalahi - e like me "0", a me heterozygotes - he 0.5.
Ma muli o nā hopena o ka nānā ʻana no nā māka AnManM1, AnSeq3 a me AnSeq4 a me ka loaʻa ʻana o nā hua no nā hoʻokolohua hope hope, ua koho ʻia nā laina 50 NLL no ka phenotyping kū'ē anthracnose.Ua hana ʻia ka ʻimi ʻana ma ke ʻano pālua i loko o kahi hale ʻōmaʻomaʻo i hoʻokele ʻia e ka lolouila me kahi kiʻi kiʻi 14 hola me kahi mahana wela o 22 ° C i ke ao a me 19 ° C i ka pō.E ʻoki ʻia nā ʻanoʻano (e ʻoki ana i ka ʻaʻahu ʻanoʻano ma ka ʻaoʻao ʻē aʻe o ka embryo me ka maka ʻoi) ma mua o ka lūlū ʻana i mea e pale ai i ka moe ʻana o nā hua ma muli o ka paʻakikī loa o ka ʻaʻahu ʻano a e hōʻoiaʻiʻo i ka ulu like ʻana.Ua ulu nā mea kanu i loko o nā ipu hao (11 × 11 × 21 cm) me ka lepo sterile (TS-1 REC 085 Medium Basic, Klasmann-Deilmann Polska, Warsaw, Polani).Ua hana ʻia ka inoculation me ka Colletotrichum lupini Col-08 strain, i ulu ʻia i ka makahiki 1999 mai nā kumu o nā mea kanu lupine haiki i ulu ʻia ma kahi māla ma Verzhenitsa, Polani Nui (52° 27′ 42″ N 17° 04′ 05″ E ).E kiʻi i kahi.Hoʻopili ʻia nā mea hoʻokaʻawale i ka SNA medium ma 20 ° C. ma lalo o ke kukui ʻeleʻele no 21 mau lā e hoʻoulu ai i ka sporulation.ʻEhā mau pule ma hope o ka lūlū ʻana, i ka wā i hōʻea ai nā mea kanu i ka pae lau 4-6, ua hana ʻia ka inoculation ma ke kāpīpī ʻana me ka hoʻokuʻu ʻana o ka conidia ma kahi ʻano o 0.5 x 106 conidia per ml.Ma hope o ka inoculation, mālama ʻia nā mea kanu i ka pōʻeleʻele no 24 mau hola ma kahi haʻahaʻa o 98% a me kahi mahana o 25 ° C e hoʻomaʻamaʻa i ka germination o ka conidia a me ke kaʻina hana maʻi.Hoʻoulu ʻia nā mea kanu ma lalo o kahi photoperiod 14-hola ma 22 ° C i ka lā / 19 ° C i ka pō a me 70% humidity.Hana ʻia ka helu maʻi ma 22 mau lā ma hope o ka hoʻopaʻa ʻia ʻana a mai ka 0 (immune) a hiki i ka 9 (maʻalahi loa) ma muli o ka loaʻa ʻana a i ʻole ka loaʻa 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.ʻO nā pilina ma waena o nā genotypes marker a me nā phenotypes maʻi i helu ʻia ma ke ʻano he helu ʻelua-sequence correlations (ka nele o nā māka heterozygous i ka hoʻonohonoho o nā laina no ka nānā ʻana i ka phenotype kū'ē anthracnose).