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Angustifolius lupine (NLL, Lupinus angustifolius L.) ndi mbewu ya nyemba zomwe zimagwiritsidwa ntchito popanga chakudya komanso kukonza nthaka.Kukula kwapadziko lonse kwa NLL ngati mbewu kwakopa mafangasi ambiri oyambitsa matenda, kuphatikiza lupine anthracnose, omwe amayambitsa matenda owopsa a anthracnose.Ma alleles awiri, Lanr1 ndi AnMan, omwe amathandizira kukana, akhala akugwiritsidwa ntchito pakuweta kwa NLL, koma njira zoyambira zama cell sizidziwika.Mu kafukufukuyu, zolembera za Lanr1 ndi AnMan zidagwiritsidwa ntchito kuyesa zitsanzo za European NLL.Kuyesedwa kwa katemera m'malo olamuliridwa kunatsimikizira mphamvu za opereka chithandizo osamva.Kufotokozera kwamitundu yosiyanasiyana kunkachitika pamizere yoyimilira komanso yosavutikira.Kukana kwa anthracnose kumalumikizidwa ndi kufotokoza mopambanitsa kwa mawu a jini ontology "GO:0006952 Defense Response", "GO:0055114 Redox Process", ndi "GO:0015979 Photosynthesis".Kuonjezera apo, mzere wa Lanr1 (83A: 476) unawonetsa kusintha kwakukulu kwa transcriptome mofulumira pambuyo pa inoculation, pamene mizere ina inasonyeza kuchedwa kwa yankho ili pafupifupi maola 42.Mayankho a chitetezo amagwirizana ndi TIR-NBS, CC-NBS-LRR ndi NBS-LRR majini, mapuloteni a 10 omwe amakhudzidwa ndi tizilombo toyambitsa matenda, mapuloteni opititsa lipid, endoglucan-1,3-β-glucosidase, mapuloteni a glycine-rich cell wall, ndi majini ochokera ku njira yowonongeka ya mpweya.Mayankho oyambilira ku 83A: 476, kuphatikiza kupondereza mosamalitsa kwa majini okhudzana ndi photosynthesis, kumagwirizana ndi chitetezo chopambana panthawi yakukula kwa zomera za fungal biology, kutanthauza kuti choyambitsa chimayambitsa chitetezo chokwanira.Mandeloop amachita pang'onopang'ono, monganso kukokera kopingasa konse.
Lupine yopapatiza (NLL, Lupinus angustifolius L.) ndi chimanga chochuluka chochokera ku chigawo chakumadzulo kwa nyanja ya Mediterranean1,2.Panopa amalimidwa ngati chakudya cha nyama ndi anthu.Amatengedwanso ngati manyowa obiriwira mu kasinthasintha wa mbewu chifukwa cha kukhazikika kwa nayitrogeni pogwiritsa ntchito mabakiteriya a symbiotic nitrogen fixing komanso kusintha kwa dothi lonse.NLL yakhala ikuchita zoweta mwachangu m'zaka zapitazi ndipo ikadali pansi pa kusweka kwakukulu3,4,5,6,7,8,9,10,11,12.Ndi kulima kofala kwa NLL, kutsatizana kwa bowa woyambitsa matenda kunapanga minda yatsopano yaulimi ndikuyambitsa matenda owononga mbewu. Chodabwitsa kwambiri kwa alimi ndi obereketsa a lupine chinali mawonekedwe a anthracnose, omwe amayamba chifukwa cha bowa woyambitsa matenda, Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13. Chodabwitsa kwambiri kwa alimi ndi obereketsa a lupine chinali mawonekedwe a anthracnose, omwe amayamba chifukwa cha bowa woyambitsa matenda, Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13. Наиболее примечательным для фермеров ndi селекционеров люпина было появление антракноза, вызванного патогенным грибком Nitrogenim Colletotrichum (3) Chodziwika kwambiri kwa alimi ndi obereketsa a lupine chinali kutuluka kwa anthracnose oyambitsidwa ndi bowa woyambitsa matenda Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13.对于羽扇豆农民和饲养者來說，最引人注目的是炭疽病的起现，它是由病原真菌Colletotrichum lupini).对于羽扇豆农民和饲养者來說，最引人注目的是炭疽病的出现，它是由病原真菌Colletotrichum lupini). Наиболее поразительным для фермеров и селекционеров люпина является появление антракноза, вызываемого патогенгеным гриtricбком Colople . Chodabwitsa kwambiri kwa alimi ndi obereketsa a lupine ndi kutuluka kwa anthracnose oyambitsidwa ndi bowa woyambitsa matenda Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13.Malipoti oyambilira kwambiri a matendawa anachokera ku Brazil ndi United States, ndipo zizindikiro zake zinaonekera mu 1912 ndi 1929 motsatira.Komabe, patapita zaka pafupifupi 30, tizilombo toyambitsa matenda tinatchedwa Colletotrichum gloeosporioides (Penz.) Penz. & Sacc., teleomorph Glomerella cingulata (Stoneman) Spauld. & Sacc., teleomorph Glomerella cingulata (Stoneman) Spauld. & Sacc., телеоморф Glomerella cingulata (Stoneman) Spauld. & Sacc., teleomorph of Glomerella cingulata (Stoneman) Spauld. & Sacc., 有目的形态的Glomerella cingulata (Stoneman) Spauld. & Sacc., 有目的形态的Glomerella cingulata (Stoneman) Spauld. & Sacc., Glomerella cingulata (Stoneman) Spauld в Целенаправленной морфологии. & Sacc., Glomerella cingulata (Stoneman) Spauld in Targeted Morphology. ndi H. Schrenk,. ndi H. Schrenk, .ndi H. Schrenk. & H.施伦克,. & H.施伦克,.ndi H. Schlenk, .Matenda oyambirira a phenotyping omwe anachitika pakati pa zaka za m'ma 1900 anasonyeza kukana kwa NLL ndi yellow lupine (L. luteus L.) accessions, koma zonse zoyera za lupine (L. albus L.) zomwe zinayesedwa zinali zovuta kwambiri15,16.Kafukufuku wasonyeza kuti chitukuko cha anthracnose chikugwirizana ndi kuwonjezereka kwa mpweya (chinyezi cha mpweya) ndi kutentha (m'kati mwa 12-28 ° C), zomwe zimayambitsa kuphwanya kukana pa kutentha kwapamwamba17, 18. Ndipotu, nthawi yofunikira kuti conidia imere ndipo matendawa ayambe , inali yofupikitsa kanayi pa 24 ° C (28 ° C) (maola a 12 ° C pamwamba pa 9 maola 9).Choncho, kutentha kwapadziko lonse kwachititsa kuti matenda a anthracnose afalikire.Komabe, matendawa adawonedwa ku France (1982) ndi Ukraine (1983) ngati chiwopsezo cha chiwopsezo chomwe chikubwera, koma mwachiwonekere adanyalanyazidwa ndi makampani a lupine panthawiyo20,21.Zaka zingapo pambuyo pake, matenda oopsawa adafalikira padziko lonse lapansi ndipo adakhudzanso mayiko akuluakulu opanga lupine monga Australia, Poland ndi Germany22,23,24.Kutsatira kufalikira kwa anthracnose chapakati pa zaka za m'ma 1990, kuyang'ana kwakukulu kunapangitsa kuti adziwike opereka angapo osamva mu zitsanzo za NLL19.Kukana kwa NLL ku anthracnose kumayendetsedwa ndi ma alleles awiri osiyana omwe amapezeka m'magwero osiyanasiyana a majeremusi: Lanr1 mu cultivar Tanjil ndi Wonga ndi AnMan mu cultivar.Mandalay 25, 26. Ma alleles awa amakwaniritsa zolembera za ma molekyulu zomwe zimathandizira kusankha majeremusi osamva m'mapulogalamu oswana25,26,27,28,29,30.Mzere wosamva kuswana 83A:476 wonyamula Lanr1 allele adawoloka ndi mzere wakuthengo wa P27255 kuti apeze anthu a RIL omwe amalekanitsa anthracnose kukana, zomwe zidapangitsa kuti malowa a Lanr1 akhale ndi chromosome NLL-1131, 32, 33 yolumikizana ndi kukana kwa mapu kuchokera kumtunda wa kukana kwa mapu kupita ku kulumikizana kwa mapu. chimango, NLL idawulula malo a alleles atatu pa chromosome imodzi (NLL-11), koma m'malo osiyanasiyana29,34,35.Komabe, chifukwa cha chiwerengero chochepa cha ma RILs ndi mtunda waukulu wa majini pakati pa zolembera ndi ma alleles ofananira, palibe mfundo zodalirika zomwe zingapangidwe ponena za majini awo.Kumbali inayi, kugwiritsa ntchito ma genetics mu lupins ndizovuta chifukwa chakuchepa kwawo kosinthika, zomwe zimapangitsa kusintha kwa majini kukhala kovuta37.
Kukula kwa majeremusi obereketsa omwe amanyamula ma aleli omwe amafunidwa m'chigawo cha homozygous, monga 83A: 476 (Lanr1) ndi Mandelup (AnMan), kwatsegula chitseko chophunzira kukana kwa anthracnose pamaso pa kukhalapo kwa mitundu yotsutsana ya alleles m'magulu akutchire.Kuthekera kwa njira zama cell.Yerekezerani mayankho achitetezo opangidwa ndi ma genotypes enaake.Kafukufukuyu adawunikira kuyankha koyambirira kwa transcriptome kwa NLL ku katemera wa C. lupini.Choyamba, gulu la majeremusi a NLL la European NLL lomwe linali ndi mizere 215 lidawunikidwa pogwiritsa ntchito zolembera zamamolekyulu zomwe zimalemba Lanr1 ndi AnMan alleles.Anthracnose phenotyping ndiye adachitidwa pamizere ya 50 NLL, yomwe idasankhidwa kale kuti ikhale yolemba ma cell, pansi pamikhalidwe yoyendetsedwa.Kutengera zoyesererazi, mizere inayi yosiyana pakukana kwa anthracnose ndi Lanr1/AnMan allelic composition idasankhidwa kuti iwonetsere mafotokozedwe amtundu wodzitchinjiriza pogwiritsa ntchito njira ziwiri zowonjezera: kutsata kwapamwamba kwa RNA ndi kuchuluka kwa PCR munthawi yeniyeni.
Kuwunika kwa gulu la majeremusi a NLL (N = 215) okhala ndi zolembera Lanr1 (Anseq3 ndi Anseq4) ndi AnMan (Anseq4) ndi AnMan (AnManM1) adawonetsa kuti mzere umodzi wokha (95726, pafupi ndi Salamanca-b) umakulitsa "kukaniza" kwa zolembera zonse, pomwe "zofunikira zonse" (~ 73.5%) mizere.Mizere khumi ndi itatu idatulutsa ma alleles awiri "osamva" a chikhomo cha Lanr1, ndipo mizere 8 idatulutsa ma alleles "osamva" a Lanr1.chikhomo.Kukaniza "kukaniza" kwa chizindikiro cha AnMan (Supplementary Table S1).Mizere iwiri inali heterozygous ya chikhomo cha Anseq3 ndi heterozygous imodzi ya chikhomo cha AnManM1.Mizere ya 42 (19.5%) inali ndi magawo osiyana a Anseq3 ndi Anseq4 alleles, kusonyeza kubwereza kwafupipafupi pakati pa ma loci awiriwa.Anthracnose phenotypes pansi pazikhalidwe zoyendetsedwa (Supplementary Table S2) adawonetsa kusiyanasiyana kwa kukana kwa ma genotypes oyesedwa, omwe adawonetsedwa ndi kuuma kwa anthracnose.Kusiyana kwa ziwerengero zapakati kumachokera ku 1.8 (kugonjetsedwa pang'ono) kufika ku 6.9 (kutengeka) ndi kusiyana kwa kulemera kwa zomera kumachokera ku 0.62 (yosavuta) kufika pa 4.45 g (yosamva). Panali kugwirizana kwakukulu pakati pa zikhalidwe zomwe zimawonedwa muzobwereza ziwiri za kuyesera (0.51 chifukwa cha zovuta za matenda, P = 0.00017 ndi 0.61 kwa kulemera kwa zomera, P <0.0001) komanso pakati pa magawo awiriwa (- 0.59 ndi - 0.77, P0100). Panali kulumikizana kwakukulu pakati pa zikhalidwe zomwe zidawonedwa muzobwereza ziwiri zoyeserera (0.51 pazovuta za matenda, P = 0.00017 ndi 0.61 ya kulemera kwa mbewu, P <0.0001) komanso pakati pa magawo awiriwa (- 0.59 ndi - 0.0.0. Выявлена ​​достоверная корреляция между значениями, наблюдаемыми в двух повторностях эксперимента (0,51 для баллов баллов тяжезд00, 1 = 1, 1 = 1 = массы растения, P < 0,0001), а также между этими двумя параметрами (-0,59 и -0,77, Р < 0,0001) 0,0001). Kulumikizana kwakukulu kunapezeka pakati pa zikhalidwe zomwe zawonedwa kubwereza kuwiri kwa kuyesako (0.51 chifukwa cha kuchuluka kwa matenda, P = 0.00017 ndi 0.61 ya kulemera kwa mbewu, P <0.0001), komanso pakati pa magawo awiriwa (- 0.59 ndi -0.77, P0 <0.0) 0.0.在两次重复实验中观察到的值之间存在显着相关性（疾病严重程度评分為0.51,P = 111,P = 70.000. 0.0001) 以及這两个参数之间（- 0.59 和- 0.77，P <0.0001).在 两 次 重复 实验 中 观察 的值 之间 存在 相关性 （疾病 严重 程度 0 0 0 0 0 0 0 0 0 0 0 0 p = 0 0 0 0 p = 0 0 0 0 p = 0 0 0 0 p = 0 0 0 0 p = 0 0 0 p = 0 0 0 p = 0 0 0 0 p = 0 0 0 0 p = 0 0 0 0 p. , 植物 為 為 0.61 , p <0.0001） 以及 两 个 参数 之间 （（（- 0.59 和– 0.59 和– 0.59 和– 0.50 ，– 0.50 ,– 0 09 .– 0 0 7). Наблюдалась значительная корреляция между значениями, наблюдаемыми в двух повторностях (оценка тяжести заболевания 0,51, 61, 600, 10 = P <0,0001), ndi между этими двумя параметрами (-0,59 ndi -0,0001) 0,77, P <0,0001. Panali kulumikizana kwakukulu pakati pa zikhalidwe zomwe zawonedwa mobwerezabwereza (kuchuluka kwa matenda 0.51, P = 0.00017 ndi kulemera kwa chomera 0.61, P <0.0001) ndi pakati pa magawo awiriwa (-0.59 ndi -0 .0001) 0.77, P <0.000 ).Zizindikiro zodziwika bwino m'zomera zomwe zitha kutengeka ndi monga kinking ndi kupindika kwa tsinde ngati mawonekedwe a "uta wa abusa", kutsatiridwa ndi zotupa zozungulira zokhala ndi malalanje / pinki sporozoite (Supplementary Fig. 1).Zolowa zaku Australia zonyamula ma jini a Lanr1 (83A:476 ndi Tanjil) ndi AnMan (Mandelup) ndizosagwirizana, 0.0331 ndi 0.0036).Mizere ina yomwe ilinso ndi "resistant" Lanr1 ndi/kapena AnMan alleles imasonyeza zizindikiro za matendawa.
Chochititsa chidwi n'chakuti mizere yochepa ya NLL yomwe ilibe "resistant" marker allele inavumbulutsa mlingo waukulu wa kukana kwa anthracnose (wofanana kapena wapamwamba kusiyana ndi Lanr1 kapena AnMan genotypes), monga Boregine (P mtengo <0.0001 pazigawo zonse ziwiri), Bojar (P mtengo <0.000100000000000000 wamtengo wapatali wa B/P9 <0.0001) ndi chiwerengero cha 4 / P9. 0.0001 ya mphambu komanso yosafunikira kulemera). Chochititsa chidwi n'chakuti mizere yochepa ya NLL yomwe ilibe "resistant" marker allele inavumbulutsa mlingo waukulu wa kukana kwa anthracnose (wofanana kapena wapamwamba kusiyana ndi Lanr1 kapena AnMan genotypes), monga Boregine (P mtengo <0.0001 pazigawo zonse ziwiri), Bojar (P mtengo <0.000100000000000000 wamtengo wapatali wa B/P9 <0.0001) ndi chiwerengero cha 4 / P9. 0.0001 ya mphambu komanso yosafunikira kulemera). Интересно, что несколько линий NLL, лишенных какого-либо «резистентного» маркерного аллеля, показали высокий уровень устойчинтного е высокий, чем для генотипов Lanr1 или AnMan), таких как Boregine (значение P <0,0001 для обоих параметров), Bojar (значение P < 0,00001,0001,0001 стения) ndi популяции B-549/79b (значение P <0,0001 для оценки и незначимо для массы). Chochititsa chidwi n'chakuti mizere ingapo ya NLL yomwe ilibe "resistant" marker allele inasonyeza kukana kwakukulu kwa anthracnose (kuyerekeza kapena kupitirira kuposa Lanr1 kapena AnMan genotypes), monga Boregine (P mtengo <0.0001 pazigawo zonse ziwiri), Bojar (P mtengo <0.0001 ndi 7b / 9 kulemera kwa 9b-04) P mtengo <0.0001 pakuwunika komanso osafunikira kulemera).有趣的是，一些缺乏任何“抗性”标记等位基因的NLL 系显示出高水平的炭疽病抗性（帎LanAn1更高），例如Boregine（两个参数的P 值< 0.0001）、Bojar（P 值<得分為0.0001，植物重和量為0/B-7161（9得（9B75）值< 0.0001, 重量不显着）. Ndizosangalatsa kuti machitidwe ena a NLL omwe alibe zizindikiro za "antigenic" amasonyeza kukana kwakukulu kopingasa (kofanana ndi Lanr1 kapena AnMan majini kapena apamwamba), monga Boregine (magawo onse a P <0.0001), Bojar (P mtengo <0.0001, kulemera kwa zomera 0.001) ndi 490 / 0 9b yofunika kwambiri <0 0001 b-0000000000000001) ndi 4 90 / 9b mtengo wofunikira . Интересно, что некоторые линии NLL, лишенные каких-либо маркерных аллелей «резистентности», показали высокие уровни устойчинистины (высокие высокие уровни устойчанистиный чем у генотипов Lanr1 или AnMan), такие как Boregine (значение P для обоих параметров <0,0001), Bojar (значение P <0,0001, масса раст00лц1b/9) енка P-значение <0,0001, масса незначительна). Chochititsa chidwi n'chakuti, mizere ina ya NLL yomwe ilibe "kukana" chizindikiro cha alleles inasonyeza kukana kwa anthracnose (kuyerekeza kapena kupitirira kuposa Lanr1 kapena AnMan genotypes), monga Boregine (P mtengo wa magawo onse <0.0001), Bojar (P-value <0.000001,0001,0001,0001,0001, P-value <0.00001,701,001,001,001,001,001,001,0001, P-value). <0.0001, kulemera kwake sikofunikira).Chodabwitsa ichi chikuwonetsa kuthekera kwa gwero lakale la kukana, kufotokozera kusowa kwa kulumikizana pakati pa ma genotypes ndi matenda a phenotypes (P values ​​​​kuchokera ~ 0.42 mpaka ~ 0.98).Chifukwa chake, mayeso a Kolmogorov-Smirnov adawonetsa kuti zomwe zakhudzana ndi kukana kwa anthracnose zimagawika pafupifupi (P-values ​​0.25 ndi 0.11) ndi misa ya zomera (P-values ​​0.47 ndi 0.55), kutanthauza kuti ndikulingalira kuti ma alleles ambiri kuposa Lanr1 ndi AnMan amakhudzidwa.
Malingana ndi zotsatira za kufufuza kwa anthracnose kukana, mizere ya 4 inasankhidwa kuti ifufuze transcriptome: 83A: 476, Boregine, Mandelup, ndi Population 22660. Mizere iyi inayesedwanso kukana kwa anthrax m'mayesero a inoculation ndi RNA sequencing, malinga ngati anali ofanana ndi mayesero apitalo.Zolembazo zinali motere: Boregin (1.71 ± 1.39), 83A: 476 (2.09 ± 1.38), Mandelup (3.82 ± 1.42) ndi anthu 22660 (6.11 ± 1.29).
The Illumina NovaSeq 6000 protocol akwaniritsa avareji 40.5 Mread awiriawiri pa chitsanzo (29.7 kuti 54.4 Mreads) (Supplementary Table S3).Zolemba zofananira pamagawo ofotokozera zidachokera ku 75.5% mpaka 88.6%.Kulumikizana kwapakati kwa data yowerengera yowerengera pakati pa zoyeserera zoyeserera pakati pa zofananira zamoyo kuyambira 0.812 mpaka 0.997 (kutanthauza 0.959). Mwa majini a 35,170 omwe adawunikidwa, 2917 sanawumbe mawu, ndipo ma gene ena a 4785 adawonetsedwa pamlingo wocheperako (kutanthauza kuti <5). Mwa majini a 35,170 omwe adawunikidwa, 2917 sanawumbe mawu, ndipo ma gene ena a 4785 adawonetsedwa pamlingo wocheperako (kutanthauza kuti <5). Из 35 170 проанализированных генов 2917 не проявляли экспрессии, а остальные 4785 генов экспрессировались на ненезнабрессии. Pamajini a 35,170 omwe adawunikidwa, 2917 sanawonetse mawu, ndipo ma gene otsala a 4785 adawonetsedwa pamlingo wocheperako (kutanthauza kuti <5).在分析的35,170 个基因中，2917 个沒有表达，其他4785 个基因的表达可以忽略不计（基本平均值。35,170 Из 35 170 проанализированных генов 2917 не экспрессировались, а остальные 4785 генов имели незначительную экспрессировались, а остальные 4785 генов имели незначительную экспрессировались (базение 5). Mwa majini a 35,170 omwe adawunikidwa, 2917 sanafotokozedwe ndipo ma gene otsala a 4785 anali ndi mawu osaneneka (matanthauzidwe oyambira <5).Choncho, chiwerengero cha majini omwe amaganiziridwa kuti afotokozedwa (chithunzi choyambira ≥ 5) panthawi yoyesera chinali 27,468 (78.1%) (Supplementary Table S4).
Kuyambira nthawi yoyamba, mizere yonse ya NLL inayankhira ku inoculation ya C. lupini (strain Col-08) mwa kukonzanso zolembazo (Table 1), komabe, kusiyana kwakukulu kunawonedwa pakati pa mizere.Choncho, mzere wotsutsa 83A: 476 (wonyamula jini la Lanr1) unawonetsa kusintha kwakukulu kwa transcriptome pa nthawi yoyamba (6 hpi) ndi kuwonjezeka kwa 31-69 kwa chiwerengero cha ma jini odzipatula ndi otsika poyerekeza ndi nthawi zina panthawiyi.Kuonjezera apo, nsonga iyi inali yaifupi, monga momwe kufotokozera kwa majini ochepa chabe kunasinthidwa kwambiri panthawi yachiwiri (12 hpi).Chochititsa chidwi n'chakuti, Boregine, yemwe adawonetsanso kukana kwakukulu mu mayesero a graft, sanakhalepo ndi kukonzanso kwakukulu kotereku panthawi yoyesera.Komabe, kuchuluka kwa majini owonetsedwa mosiyanasiyana (DEG) kunali kofanana kwa Boregine ndi 83A: 476 ku 12 HPI.Onse a Mandelup ndi 22660 adawonetsa nsonga za DEG panthawi yomaliza (48 l / s), zomwe zikuwonetsa kuchedwa kwa mayankho achitetezo.
Chifukwa 83A: 476 inakhala ndi reprogramming yaikulu ya transcriptome poyankha C. lupini pa 6 HPI poyerekeza ndi mizere ina yonse, ~ 91% ya DEGs yomwe inawonedwa panthawiyi inali yeniyeni ya mzere (mkuyu 1).Komabe, panali mayankho oyambilira pakati pa mizere yophunzirira, monga 68.5%, 50.9%, ndi 52.6% DEG ku Boregine, Mandelup, ndi kuchuluka kwa anthu 22660, motsatana, adadutsana ndi omwe amapezeka mu 83A: 476 munthawi zina.Komabe, ma DEG awa amawerengera gawo laling'ono chabe (0.97-1.70%) la ma DEG onse omwe akupezeka pano pogwiritsa ntchito 83A: 476.Kuonjezera apo, 11 DEGs kuchokera ku mizere yonse inali yogwirizana panthawiyi (Supplementary Tables S4-S6), kuphatikizapo zigawo zofala za mayankho a chitetezo cha zomera: lipid transfer protein (TanjilG_32225), endoglucan-1,3-β-glucoside enzyme (TanjilG_23384), awiri stress-inducible TaG5_1 proteins SAMG5_31 31), mapuloteni oyambira a latex (TanjilG_32352), ndi mapuloteni awiri opangidwa ndi glycine (TanjilG_19701 ndi TanjilG_19702).Panalinso kuphatikizika kwakukulu kwamayankho a transcriptome pakati pa 83A:476 ndi Boregine pa 24 HPI (total 16-38% DEG) komanso pakati pa Mandelup ndi Population 22660 pa 48 HPI (chiwerengero cha 14-20% DEG).
Chithunzi cha Venn chosonyeza kuchuluka kwa majini owonetsedwa mosiyanasiyana (DEG) mumizere yopapatiza ya lupine (NLL) yojambulidwa ndi Colletotrichum lupini (strain Col-08 yochokera kuminda ya lupine ku Wierzhenice, Poland, 1999).Mizere ya NLL yomwe idawunikidwa inali: 83A: 476 (yosagwirizana, yonyamula Lanr1 allele), Boregine (yosamva, chibadwa sichidziwika), Mandelup (yosamva bwino, yonyamula AnMan allele) ndi anthu 22660 (otengeka kwambiri).Chidule cha hpi chimayimira maola mutalandira katemera.Zero values ​​zachotsedwa kuti graph ikhale yosavuta.
Seti ya majini ochulukirachulukira pa 6 hpi adawunikidwa chifukwa cha kukhalapo kwa madera ovomerezeka a R (Supplementary Table S7).Kafukufukuyu adavumbulutsa kulowetsedwa kwa ma transcriptome amitundu yakale yolimbana ndi matenda okhala ndi madera a NBS-LRR okha pa 83A:476.Setiyi inali ndi jini imodzi ya TIR-NBS-LRR (tanjilg_05042), majini asanu a CC-NBS-LRR (tanjilg_06165, tanjilg_06162, tanjilg_22773, tanjilg_22640, ndi tanjilg_16162R), Four Tanjilg_16162-BS ndi Tanjilg_16162R RRE (tanjilg_16162) komanso Four NBS-Lrr (tanjilg_16162) ndi Four NBS-LRR (TANJILG_16162).Majini onsewa ali ndi madera ovomerezeka omwe amasanjidwa motsatizana.Kuphatikiza pa majini amtundu wa NBS-LRR, ma RLL kinase angapo adatsegulidwa pa 6 hpi, yomwe ili ku Boregine (TanjilG_19877), awiri ku Mandelup (TanjilG_07141 ndi TanjilG_19877) komanso m'gulu la anthu 22660 (TanjilG_06877) ndi awiri TanjilG_0617 ndi 2: 3701774 6.
Majini okhala ndi mawu osinthika kwambiri poyankha katemera ndi C. lupini (strain Col-08) adayesedwa ndi Gene Ontology (GO) kusanthula kopindulitsa (Supplementary Table S8). Nthawi zambiri zomwe zimawonetsedwa kwambiri pazachilengedwe zinali "GO: 0006952 yankho lachitetezo" lomwe lidawonekera mu 6 kuchokera ku 16 (nthawi × mzere) kuphatikiza kufunikira kwakukulu (P mtengo <0.001) (Mkuyu 2). Nthawi zambiri zomwe zimawonetsedwa kwambiri pazachilengedwe zinali "GO: 0006952 yankho lachitetezo" lomwe lidawonekera mu 6 kuchokera ku 16 (nthawi × mzere) kuphatikiza kufunikira kwakukulu (P mtengo <0.001) (Mkuyu 2). Наиболее часто чрезмерно представленным термином биологического процесса был «GO: 0006952 защитный отвенным термином биологического процессса был «GO: 0006952 защитный ответ», который песни 6 комбинаций с высокой значимостью (значение P <0,001) (рис. 2). Nthawi zambiri zomwe zimayimiridwa kwambiri ndi zamoyo zinali 'GO: 0006952 yankho lachitetezo', lomwe lidawonekera mu 6 ya 16 (nthawi × mzere) kuphatikiza ndi kufunikira kwakukulu (P mtengo <0.001) (mkuyu 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)着性 (P 值 <0.001) (图2). Mawu oyimira kwambiri achilengedwe ndi "GO: 0006952 yankho lachitetezo", lomwe limapezeka mu 6 mwa 16 (时间× 线) kuphatikiza, ndi kufunikira kwakukulu (P mtengo <0.001) (图2) . Наиболее часто чредставленным термином биологического процесса был «GO: 0006952 Defense Response», который появлялся 19 × 6 с высокой значимостью (значение P <0,001) (рис. 2). Nthawi zambiri zomwe zimayimiridwa kwambiri ndi biological process term inali 'GO: 0006952 Defense Response', yomwe idawonekera mu 6 mwa 16 kuphatikiza (nthawi × mzere) ndi tanthauzo lalikulu (P mtengo <0.001) (mkuyu 2).Mawu awa adawonetsedwa mopitilira muyeso mu 83A: 476 ndi Boregine (6 ndi 24 hpi) komanso nthawi ina ku Mandelup ndi Population 22660 (12 ndi 6 hpi, motsatana).Izi ndi zotsatira zoyembekezeredwa, zomwe zikuwonetsa kuyankha kwa antifungal kwa mizere yosamva.Kuonjezera apo, 83A: 476 inayankha C. lupini mwa kuyambitsa mofulumira majini okhudzana ndi kuphulika kwa okosijeni komwe kumayimiridwa ndi mawu akuti "GO: 0055114 redox process", kusonyeza yankho lapadera la chitetezo, pamene Boregine adawonetsa mayankho enieni a chitetezo, okhudzana ndi mawu akuti 'GO'.: 0006950 Kupsinjika maganizo".Chiwerengero cha anthu 22660 chinayambitsa kukana kopingasa komwe kumaphatikizapo metabolites achiwiri, kuwonetsa kuchuluka kwa mawu akuti "GO:0016104 Njira ya triterpene biosynthesis" ndi "GO:0006722 Njira ya triterpene metabolism" (mawu onsewa ali m'gulu lomwelo la majini), poganizira zotsatira za kusanthula kwa Mandel 2660. Kuphatikiza apo, kuchitapo kanthu koyambirira 83A:476 (6 hpi) ndikuchita mochedwa Mandelup ndi Population 22660 kumaphatikizapo mawu akuti GO:0015979 'photosynthesis' ndi zina zokhudzana ndi biological process.
Mawu a bioprocess gene ontology osankhidwa pofotokoza za majini owonetsedwa mosiyanasiyana panthawi ya mayankho a lupine-leved lupine (NLL) ojambulidwa ndi anthrax lupine (mtundu wa Col-08 wotengedwa kuminda ya lupine ku Wierzhenice, Poland, mu 1999) ndiwokokomeza kwambiri.Mizere ya NLL yomwe inafufuzidwa inali: 83A: 476 (yosagwirizana, yonyamula homozygous Lanr1 allele), Boregine (yosagonjetsedwa, chibadwa chosadziwika), Mandelup (yosamva bwino, yonyamula homozygous AnMan allele) ndi chiwerengero cha 22660 (chikhoza kutenga).
Chifukwa kafukufukuyu ankafuna kudziwa majini omwe amathandizira kukana kwa anthracnose, majini omwe amaperekedwa ku mawu akuti GO "PITA: 0006952 Mayankho odzitchinjiriza" ndi "GO: 0055114 njira za Redox" adawunikidwa ndi kudulidwa chifukwa zoyambira zikutanthauza ≥ 30 yokhala ndi mzere umodzi.× mfundo mu nthawi kuphatikiza zowerengera zofunika za log2 (kusintha pindani).Chiwerengero cha majini okwaniritsa izi chinali 65 pa GO:0006952 ndi 524 a GO:0055114.
83A: 476 idavumbulutsa nsonga ziwiri za DEG zotchulidwa ndi mawu akuti GO: 0006952, yoyamba pa 6 majini pa inchi (majini 64, mmwamba ndi pansi malamulo) ndipo yachiwiri pa 24 majini pa inchi (majini 15, okwera pamalamulo okha).Boregine adawonetsanso kuti GO: 0006952 idakwera nthawi yomweyo, koma ndi DEG yocheperako (11 ndi 8) komanso kuyambitsa mwamakonda.Mandeloop adawonetsa nsonga ziwiri za GO: 0006952 pa 12 ndi 48 HPI, onse atanyamula majini 12 (woyamba anali ndi ma jini oyambitsa, ndipo yachiwiri anali ndi ma jini opondereza okha), pomwe chiwerengero cha 22660 pa 6 HPI (majini 13) chinali ndi kuchulukirachulukira kwa chiwonjezeko.malamulo.Tiyenera kuzindikira kuti 96.4% ya GO: 0006952 DEG pamapiriwa anali ndi mayankho amtundu womwewo (mmwamba kapena pansi), kusonyeza kuyanjana kwakukulu mu mayankho a chitetezo ngakhale kusiyana kwa chiwerengero cha majini okhudzidwa.Gulu lalikulu kwambiri lamatsatidwe okhudzana ndi mawu akuti GO:0006952 amaikapo Starvation Stress-Associated Message Protein 22 (SAM22-like), yomwe ili mgulu la 10 pathogenesis-associated protein (PR-10) protein clade ndi core protein latex.ofanana (MLP-ngati) mapuloteni) mapuloteni (mkuyu 3).Magulu awiriwa ankasiyana malinga ndi momwe akuyankhira.Ma jini omwe amasunga mapuloteni onga SAM22 adawonetsa kukhazikika komanso kofunikira panthawi yoyambira (6 kapena 12 hpi) ndipo nthawi zambiri samayankha kumapeto kwa kuyesako (48 hpi), pomwe mapuloteni onga MLP adawonetsa kulumikizana pa 6 hpi.hpi.83A: 476 ndi Mandelup pa 48 hp / in, pafupifupi mfundo zina zonse za deta sizinayankhe.Kuonjezera apo, kusiyana kwa mafotokozedwe amtundu wa mapuloteni a SAM22 kumatsatira kusinthasintha kwa kukana kwa anthracnose, chifukwa mizere yolimba kwambiri inali ndi nthawi yochuluka yomwe imayambitsa majiniwa kusiyana ndi majini omwe amatha kutenga kachilomboka.Jini lina la LlR18A/B-ngati PR-10 linawonetsa mawonekedwe ofanana kwambiri ndi jini ya protein ya SAM22.
Zigawo zazikulu za njira yachilengedwe ya "GO:0006952 Defense Response" komanso mawonekedwe amtundu wamtundu wa Lanr1 ndi AnMan alleles adadziwika.Sikelo ya Log2 imayimira mitengo ya log2 (kusintha) pakati pa katemera (Colletotrichum lupini, strain Col-08, yotengedwa kuchokera kuminda ya lupine, Wizhenica, Poland, 1999) ndi kuwongolera (sham-inoculated) zomera nthawi yomweyo.Mizere yopapatiza ya lupine inawunikidwa: 83A:476 (yosamva, yonyamula homozygous Lanr1 allele), Boregine (yosamva, chibadwa sichidziwika), Mandelup (yosamva bwino, yonyamula homozygous AnMan allele), ndi Population 22660 (yotengeka).
Kuphatikiza apo, mafotokozedwe amtundu wa RNA-seq gene Lanr1 (TanjilG_05042) ndi AnMan (TanjilG_12861) adawunikidwa (mkuyu 3).Jini la TanjilG_05042 linawonetsa kuyankha kwakukulu (kuyambitsa) ku 83A: 476 kokha panthawi yoyamba (6 hpi), pamene TanjilG_12861 inali yofunika kwambiri ku Mandeloop pa nthawi ziwiri zokha: 6 hpi (pansi malamulo) ndi 24 hpi (6 hpi).Ndi.).chosinthika)).
Ma jini ochulukirachulukira m'mawu akuti GO: 0055114 "ndondomeko ya redox" anali ma encoding cytochrome P450 mapuloteni ndi peroxidase (mkuyu 4).Kwa zitsanzo zosiyanitsidwa ndi 83A: 476 pa 6 HPI, zochulukirapo kapena zochepa za log2 (kusintha kosintha) (kwa 86.6% ya majini) nthawi zambiri zimawonedwa pakati pa zomera zotetezedwa ndi zowongolera, kuwonetsa kuyankha kwakukulu kwa genotype iyi pakulowetsa kugonana.83A: 476 inasonyeza GO: 0055114 DEG pa 6 hpi (503 majini), pamene mizere yotsalayo inali 48 hpi (Boregine, 31 majini; Mandelup, 85 majini; ndi Population 22660, 78 majini)).Mu majini ambiri a GO: 0055114 banja, mitundu iwiri ya mayankho ku katemera (kuyambitsa ndi kuletsa) inawonedwa.Chochititsa chidwi n'chakuti, mpaka 97.6% ya ma DEGs omwe amadziwika kuti GO: 0055114 ku Mandelupe pa 48 hp Zomwe zikuwonetseratu izi zikusonyeza kuti, ngakhale kuti ndi ochepa kwambiri (ie, chiwerengero cha chibadwa cha redox chosinthika, 85 motsutsana ndi 503), chitsanzo cha kuchedwa kwa mayankho a transcriptome ku 6 7 mandeloup yofanana ndi mandeloup 6.Ku Boregine ndi Population 22660, kuphatikizika uku ndikotsika pa 51.6% ndi 75.6%, motsatana.
Mafotokozedwe a zigawo zazikulu za nthawi ya biological process "GO: 0055114 Redox process" adawululidwa.Sikelo ya Log2 imayimira mitengo ya log2 (kusintha) pakati pa katemera (Colletotrichum lupini, strain Col-08, yotengedwa kuchokera kuminda ya lupine, Wizhenica, Poland, 1999) ndi kuwongolera (sham-inoculated) zomera nthawi yomweyo.Mizere yopapatiza ya lupine inawunikidwa: 83A:476 (yosamva, yonyamula homozygous Lanr1 allele), Boregine (yosamva, chibadwa sichidziwika), Mandelup (yosamva bwino, yonyamula homozygous AnMan allele), ndi Population 22660 (yotengeka).
83A: 476 Mayankho a Transcriptomic ku inoculation ndi C. lupini (strain Col-08) adaphatikizaponso kutonthola kwa majeremusi otchedwa GO: 0015979 "photosynthesis" ndi zina zokhudzana ndi biological process (FIG. 5).GO: 0015979 DEG set ili ndi majini a 105 omwe adatsitsidwa kwambiri pa 6 hpi ku 83A: 476.M'chigawo ichi, majini 37 adatsitsidwanso ku Mandelup pa 48 HPI ndi 35 panthawi yomweyi mu chiwerengero cha 22660, kuphatikizapo 19 DEGs yodziwika kwa ma genotypes onse.Palibe ma DEG okhudzana ndi mawu akuti GO: 0015979 adayatsidwa kwambiri pakuphatikiza kulikonse (mzere x nthawi).
Mafotokozedwe a zigawo zikuluzikulu za nthawi ya biological process "GO: 0015979 Photosynthesis" adawululidwa.Sikelo ya Log2 imayimira mitengo ya log2 (kusintha) pakati pa katemera (Colletotrichum lupini, strain Col-08, yotengedwa kuchokera kuminda ya lupine, Wizhenica, Poland, 1999) ndi kuwongolera (sham-inoculated) zomera nthawi yomweyo.Mizere yopapatiza ya lupine inawunikidwa: 83A:476 (yosamva, yonyamula homozygous Lanr1 allele), Boregine (yosamva, chibadwa sichidziwika), Mandelup (yosamva bwino, yonyamula homozygous AnMan allele), ndi Population 22660 (yotengeka).
Kutengera zotsatira za kusanthula kwamafotokozedwe osiyanitsidwa komanso mwina kukhudzidwa ndi mayankho achitetezo motsutsana ndi bowa woyambitsa matenda, gululi la majini asanu ndi awiri adasankhidwa kuti awerengere mbiri yapanthawiyo PCR (Supplementary Table S9).
Pulative protein jini ya TanjilG_10657 idakopeka kwambiri m'mizere yonse yophunziridwa ndi nthawi yake poyerekeza ndi zowongolera (zotsanzira) (Matebulo Owonjezera S10, S11).Kuphatikiza apo, mawonekedwe a TanjilG_10657 adawonetsa kuchulukirachulukira pakuyesa kwa mizere yonse.Population 22660 anasonyeza tilinazo kwambiri TanjilG_10657 kuti inoculation ndi 114-khola kutsegula ndi apamwamba kwambiri wachibale mlingo (4.4 ± 0.4) pa 24 HPI (mkuyu. 6a).PR10 LlR18A jini ya puloteni TanjilG_27015 inasonyezanso kutsegula pamizere yonse ndi nthawi, ndi kufunikira kwa chiwerengero pazigawo zambiri za deta (mkuyu 6b).Mofanana ndi TanjilG_10657, chiwerengero chapamwamba kwambiri cha TanjilG_27015 chinawonedwa mu 22660 chojambulidwa pa 24 HPI (19.5 ± 2.4).The asidi endochitinase jini TanjilG_04706 kwambiri upregulated mu mizere onse ndi nthawi zonse mfundo kupatula Boregine 6 hpi (mkuyu 6c).Zinalimbikitsidwa kwambiri panthawi yoyamba (6 HPI) pa 83A: 476 (ndi nthawi za 10.5) ndipo zinawonjezeka pang'onopang'ono m'mizere ina (ndi 6.6-7.5 nthawi).Panthawi yoyeserera, mawu a TanjilG_04706 adakhalabe pamilingo yofanana mu 83A: 476 ndi Boregine, pomwe ku Mandelup ndi Population 22660 idakula kwambiri, kufika pamikhalidwe yapamwamba (5.9 ± 1.5 ndi 6.2 ± 1.5, motsatana).Endoglucan-1,3-β-glucosidase-ngati jini TanjilG_23384 inasonyeza kutsegulira kwakukulu pazigawo ziwiri zoyambirira za nthawi (6 ndi 12 hpi) m'mizere yonse kupatulapo chiwerengero cha 22660 (Mkuyu 6d).Mawonekedwe apamwamba kwambiri a TanjilG_23384 adawonedwa panthawi yachiwiri (12 hpi) ku Mandelup (2.7 ± 0.3) ndi 83A:476 (1.5 ± 0.1).Pa 24 HPI, TanjilG_23384 mawu anali otsika m'mizere yonse yophunziridwa (kuchokera 0.04 ± 0.009 mpaka 0.44 ± 0.12).
Mafotokozedwe amitundu yosankhidwa (ag) yowululidwa ndi kuchuluka kwa PCR.Nambala 6, 12 ndi 24 zimayimira maola pambuyo polandira katemera.Mitundu ya LanDExH7 ndi LanTUB6 idagwiritsidwa ntchito kuti ikhale yokhazikika ndipo LanTUB6 idagwiritsidwa ntchito poyesa ma inter-series.Mipiringidzo yolakwika imayimira kupatuka kokhazikika kutengera zofananira zamoyo zitatu, iliyonse yomwe ili avareji ya zofananira zaukadaulo zitatu. Kufunika kwachiwerengero cha kusiyana kwa mawu omwe ali pakati pa katemera (Colletotrichum lupini, strain Col-08, yomwe inapezedwa mu 1999 kuchokera kumunda wa lupine ku Wierzenica, Poland) ndi zomera zoyang'anira (mock-inoculated) zili pamwamba pa mfundo za deta (*P mtengo <0.05, **P mtengo ≤0 0.0 P mtengo ≤0 0. Kufunika kwachiwerengero cha kusiyana kwa mawu omwe ali pakati pa katemera (Colletotrichum lupini, strain Col-08, yomwe inapezedwa mu 1999 kuchokera kumunda wa lupine ku Wierzenica, Poland) ndi zomera zoyang'anira (mock-inoculated) zili pamwamba pa mfundo za deta (*P mtengo <0.05, **P mtengo ≤0 0.0 P mtengo ≤0 0. Статистическая значимость различий в уровнях экспрессии между инокулированными (Colletotrichum lupini, штам Col-08, получен в 1999 пльше, песни, песни 1999, песни, в 1999 в 1999 пльше песни. а) и контрольными (ложно инокулированными) растениями отмечена над точками данных (*значение P < 0,05, **значение P ≤ 0,01, ***значение 0, ***значение 0, 0. Kusiyanasiyana kwakukulu pamawu ofotokozera pakati pa jekeseni (Colletotrichum lupini, strain Col-08, yomwe inapezedwa mu 1999 kuchokera kumunda wa lupine ku Wierzhenice, Poland) ndi zomera zolamulira (sham-inoculated) zimatchulidwa pamwamba pa mfundo za deta (*P mtengo <0.05, **P-value ≤ 0.0 01, ***P-value ≤ 0.01 ≤ 0.01 ≤ 0.01 ≤ 0.0 0.接种（Colletotrichum lupini，Col-08株，1999年从波兰Wierzenica的羽扇豆田获得）和对照（模拟接种种种）植异的统计学显着性标记在数据点上方（*P值< 0.05, **P 值≤ 0.01, ***P 值≤ 0.001).接种 （colletorichum lupini ， color-08 株 ， 1999 年 波兰 波兰 wierzenica 的羽扇 获得的 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, штам Col-08, полученный с полей люпи, 9 9 г.) ndi контрольными (ложно инокулированными) растениями отмечены над точками данных (* значение P < 0,05, ** P-значение ≤ 0,01, ≤ 0,01, ≤ 0,01, 0, 01, 0. Kusiyanasiyana kwakukulu pamawu ofotokozera pakati pa inoculated (Colletotrichum lupini, strain Col-08, yomwe inapezedwa kuchokera ku minda ya lupine ku Verzhenice, Poland, mu 1999) ndi kulamulira (sham-inoculated) zomera zatchulidwa pamwamba pa mfundo za deta (*P mtengo <0.05 , **P-mtengo ≤ 0-0 ≤ 0-0 1 ≤ 0-0 1).Mizere ya NLL yomwe inafufuzidwa inali: 83A: 476 (yosagwirizana, yonyamula homozygous Lanr1 allele), Mandelup (yosamva bwino, yonyamula homozygous AnMan allele), Boregine (yosagonjetsedwa, chibadwa chosadziwika) ndi chiwerengero cha 22660 (chotengeka).
The phungu jini TanjilG_05042 pa Lanr1 locus anasonyeza moonekera osiyana chitsanzo chitsanzo kuchokera mbiri anapezedwa RNA-seq maphunziro (mkuyu. 6e).Kutsegulira kwakukulu kwa jini iyi kudawonedwa ku Mandelup ndi kuchuluka kwa 22660 (mpaka 39.7 ndi 11.7 nthawi, motsatana), zomwe zidapangitsa kuti pakhale mawonekedwe apamwamba (mpaka 1.4 ± 0.14 ndi 7.2 ± 1.3, motsatana).83A:476 idawululanso kuwongolera kwa jini ya TanjilG_05042 (mpaka 3.8-fold), komabe, milingo yofananira yomwe idakwaniritsidwa (0.044 ± 0.002) inali yochepera 30 kuposa yomwe idawonedwa ku Mandelup ndi anthu 22660.kufufuzidwa ndi qPCR kunawonetsa kusiyana kwakukulu kwa milingo ya mawu pakati pa ma genotypes mumitundu yonyengerera-katemera (kuwongolera), kufikira kusiyana kwa 58 pakati pa anthu 22660 ndi 83A: 476, komanso pakati pa anthu 22660 ndi 22660. Kusiyana kowirikiza kawiri kunachitika pakati pa Boregine ndi Mandalup.
Jini ya phungu ku AnMan locus, TanjilG_12861, idatsegulidwa poyankha katemera mu 83A:476 ndi Mandelup, sanalowerere m'gulu la 22660, ndipo adachepetsedwa ku Boregine (mkuyu 6f).Mafotokozedwe achibale a jini ya TanjilG_12861 anali apamwamba kwambiri mu 83A: 476 (0.14±0.01).The 17.4 kDa kalasi I kutentha mantha mapuloteni jini TanjilG_05080 HSP17.4 anasonyeza m'munsi wachibale mawu milingo mu mitundu yonse anaphunzira ndi mfundo nthawi (mkuyu. 6g).Mtengo wapamwamba kwambiri udawonedwa pa 24 HPI mwa anthu a 22660 (0.14 ± 0.02, kuwonjezeka kasanu ndi katatu poyankha katemera).
Kuyerekeza kwa mbiri ya jini (mkuyu 7) kunawonetsa kulumikizana kwakukulu pakati pa TanjilG_10657 ndi mitundu ina inayi: TanjilG_27015 (r = 0.89), TanjilG_05080 (r = 0.85), TanjilG_05042 (r = 0.80G = 0.80), ndi 0.Zotsatira zoterezi zingasonyeze kugwirizanitsa kwa majiniwa panthawi yachitetezo.Mitundu ya TanjilG_12861 ndi TanjilG_23384 idawonetsa mafotokozedwe osiyanasiyana okhala ndi ma coefficient otsika a Pearson (kuyambira 0.08 mpaka 0.43 ndi -0.19 mpaka 0.28, motsatana) poyerekeza ndi majini ena.
Kugwirizana pakati pa mbiri ya jini kunapezeka pogwiritsa ntchito kuchuluka kwa PCR.Mizere yopapatiza ya lupine yotsatizanayi idawunikidwa: 83A:476 (yosamva, yonyamula homozygous Lanr1 allele), Mandelup (yosamva bwino, yonyamula homozygous AnMan allele), Boregine (yosamva, chibadwa sichidziwika), ndi Chiwerengero cha anthu 22660 (chotengera).Nthawi zitatu zinawerengedwa (maola 6, 12 ndi 24 pambuyo pa kulowetsedwa), kuphatikizapo inshuwaransi (Colletotrichum lupini, strain Col-08, yochokera ku minda ya lupine ku Wierzhenice, Poland, mu 1999) ndi kulamulira (sham-inoculated) zomera.Sikelo ikuwonetsa mtengo wa Pearson coefficient coefficient.
Malingana ndi deta yomwe inapezedwa pa 6 horsepower pa inchi, WGCNA inachitidwa pa 9981 DEG yodziwika poyerekezera zomera zowonongeka ndi zowongolera kuti ziganizire pa mayankho oyambirira a chitetezo (Supplementary Table S12).Ma module a jini makumi awiri ndi awiri (magulu) adapezeka ndi mbiri yofananira (yabwino kapena yoyipa) pakati pa ma genotypes ndi mitundu yoyesera. Pa avareji, milingo ya ma jini inali kutsika mu dongosolo 83A:476 > Mandelup > Boregine > Population 22660 (m'mitundu yonseyi, komabe, izi zinali zamphamvu pazomera zowongolera). Pa avareji, milingo ya ma jini inali kutsika mu dongosolo 83A:476 > Mandelup > Boregine > Population 22660 (m'mitundu yonseyi, komabe, izi zinali zamphamvu pazomera zowongolera). В среднем уровни экспрессии генов снижались в порядке 83A:476 > Mandelup > Boregine > Population 22660 malo). Pa avareji, milingo ya jini idatsika mu dongosolo 83A:476 > Mandelup > Boregine > Population 22660 (m'mitundu yonseyi, komabe, izi zidali zamphamvu pazomera zowongolera).平均而言，基因表达水平按83A:476 > Mandelup > Boregine > Population 22660 的顺序下降（然而，在两种种种种中变公红，更强.平均 而 言 , 基因 水平 按 83a: 476> mandelup> boregine> anthu 22660 的顺序 下降 （，在 种 中 中 。 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... В среднем уровни экспрессии генов снижались в ряду 83A:476 > Mandelup > Boregine > Population 22660 ine). Pa avareji, milingo ya jini idatsika pamndandanda 83A:476> Mandelup> Boregine> Population 22660 (komabe, m'mitundu yonse iwiri, izi zidali zamphamvu pazomera zowongolera).Katemera adayambitsa kuwongolera kwa mawu a jini, makamaka m'magawo 18, 19, 14, 6 ndi 1 (mukutsika kwa zotsatira), malamulo olakwika (mwachitsanzo ma module 9 ndi 20) kapena osalowerera ndale (mwachitsanzo ma module 11, 22, 8 ndi 13).GO term enrichment analysis (Supplementary Table S13) inavumbulutsa "GO: 0006952 Protective responses" for the inoculated module (18) with maximum activation, including genes Analycated by qPCR (TanjilG_04706, TanjilG_23384, TanjilG_1058G zithunzi zambiri za TanjilG_1055G ndi TanjilG_10657) magawo oyambira (9).Module 18 concentrator (mkuyu. 8) adadziwika kuti ndi jini ya TanjilG_26536 yokhota puloteni ya PR-10-ngati LlR18B, ndipo gawo la 9 concentrator linadziwika kuti ndi TanjilG_28955 jini yomwe imayika puloteni ya photosystem II PsbQ.Mtundu wa anthracnose resistance gene Lanr1, TanjilG_05042, unapezeka mu gawo 22 (mkuyu 9) ndipo umalumikizidwa ndi mawu akuti "GO:0044260 Cellular macromolecular metabolic process" ndi "GO:0006355 Transcriptional regulation, DNA templating1G_01 Table.jini imayika chizindikiro cha kutentha kwa A-4a (HSFA4a).
Kusanthula kwapaintaneti kolemetsa kwa ma jini ofotokozera ma module okhala ndi mawu opitilira muyeso "GO: 0006952 Mayankho achitetezo".Ligation inasinthidwa kuti iwonetsere chibadwa chinai chomwe chinafufuzidwa ndi qPCR (TanjilG_04706, TanjilG_23384, TanjilG_10657 ndi TanjilG_27015).
Kusanthula kwa netiweki kolemetsa kwa gene co-expression ya module yokhala ndi njira yodziwika bwino yachilengedwe "GO: 0006355: Transcriptal regulation, DNA templating" komanso kunyamula mtundu wa anthracnose resistance gene Lanr1 TanjilG_05042.Ligation idasinthidwa kuti ikhale yopatula jini ya TanjilG_05042 ndi jini yapakati ya TanjilG_01212.
Kuwunika kwa anthracnose resistance komwe kunasonkhanitsidwa ku Australia kunawonetsa kuti mitundu yambiri yomwe idatulutsidwa koyambirira idayamba kutenga;Kalya, Coromup ndi Mandelup zafotokozedwa kuti ndizosamva bwino, pomwe Wonga, Tanjil ndi 83A:476 adanenedwa kuti ndi osamva26,27,31.anali ndi kukana komweko, komwe kumatchedwa Lanr1, ndipo Coromup ndi Mandelup anali ndi allele yosiyana, yotchedwa AnMan10, 26, 39, pamene Kalya adadutsa njira ina., Lanr2.Kuwunika kukana kwa anthracnose ku Germany kudapangitsa kuti kuzindikirike kwa mzere wosamva wa Bo7212 wokhala ndi allele kupatula Lanr1, wosankhidwa LanrBo36.
Kafukufuku wathu adawulula pafupipafupi (pafupifupi 6%) ya Lanr1 allele mu majeremusi oyesedwa.Kuwonetsetsa uku kumagwirizana ndi zotsatira zowunikira majeremusi a East European pogwiritsa ntchito zizindikiro za Anseq3 ndi Anseq4, zomwe zinasonyeza kuti Lanr1 allele ilipo m'mizere iwiri yokha ya Chibelarusi.Izi zikusonyeza kuti Lanr1 allele sinagwiritsidwebe ntchito kwambiri ndi mapulogalamu oweta am'deralo, mosiyana ndi ku Australia, komwe ndi imodzi mwazinthu zofunika kwambiri pakuweta mothandizidwa ndi chizindikiro.Izi zitha kukhala chifukwa cha kuchepa kwamphamvu komwe kumaperekedwa ndi Lanr1 allele m'magawo aku Europe poyerekeza ndi lipoti la Australia.Kuonjezera apo, kafukufuku wa anthracnose m'madera omwe amagwa mvula yambiri ku Australia awonetsa kuti mayankho otsutsa omwe amatsutsana ndi Lanr1 allele sangakhale othandiza pa nyengo zomwe zimakonda kukula ndi chitukuko chofulumira cha tizilombo toyambitsa matenda19,42.Ndipotu, mu phunziro lamakono, zizindikiro zina za anthracnose zinawonedwanso mu genotypes yonyamula Lanr1 allele, kutanthauza kuti kukana kungathe kutha pansi pazikhalidwe zabwino za chitukuko cha C. lupini.Kuonjezera apo, kutanthauzira kwabodza kwa kukhalapo kwa zizindikiro za Anseq3 ndi Anseq4, zomwe ziri pafupifupi 1 cm kuchokera ku malo a Lanr1, ndizotheka 28,30,43 .
Phunziro lathu linasonyeza kuti 83A: 476, yonyamula Lanr1 allele, inayankha C. lupini inoculation ndi transcriptome reprogramming yaikulu pa nthawi yoyamba kusanthula nthawi (6 hpi), pamene ku Mandelup, atanyamula AnMan allele, transcriptomic mayankho anawonedwa patapita nthawi.(kuchokera 24 mpaka 48 hp).Kusiyanasiyana kwakanthawi kochepa mu mayankho achitetezo kumalumikizidwa ndi kusiyana kwa zizindikiro za matenda, kuwonetsa kufunikira kwa kuzindikira koyambirira kwa tizilombo toyambitsa matenda kuti tiyankhe bwino pakukana.Kuti tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda, tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda, tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda tizilombo toyambitsa matenda tima timene timatulutsa timadzi timene timapanga tizilombo toyambitsa matenda.Chowonjezera ndi kachidutswa kakang'ono kamene kamamangirira pamwamba pa wolandirayo ndikuthandizira kulowa mu minyewa yolandirira.Choncho, spores wa C. gloeosporioides mu mtola Tingafinye anasonyeza kugawanika koyamba kwa phata pambuyo 75-90 mphindi makulitsidwe, mapangidwe majeremusi chubu pambuyo 90-120 mphindi, ndi kupondereza pambuyo 4 hours 45 .Mango C. gloeosporioides anasonyeza kupitirira 40% conidial kumera pambuyo 3 hours makulitsidwe ndi pafupifupi 20% mapangidwe appressors pambuyo 4 hours.Jini ya CAP20 yokhudzana ndi virulence ya C. gloeosporioides inasonyeza ntchito yolembera mu epiphyte-forming conidia pambuyo pa maola 3.5 mu incubation mu sera ya avocado yokhala ndi mapuloteni ambiri a CAP20 pambuyo pa 4 h 46 min.Mofananamo, ntchito ya majeremusi a melanin biosynthesis mu C. trifolii inayambitsidwa panthawi ya makulitsidwe a maola a 2 ndikutsatiridwa ndi mapangidwe a appressorium pambuyo pa ola la 1.Kafukufuku wa masamba a masamba asonyeza kuti strawberries opangidwa ndi C. acutatum amayamba kuponderezedwa pa 8 hpi, pamene tomato wopangidwa ndi C. coccodes amayamba kuponderezedwa pa 4 hpi48,49.zimagwirizana kwambiri ndi nthawi ya Colletotrichum spp.matenda opatsirana.Mayankho ofulumira achitetezo ku 83A: 476 akuwonetsa kukhudzidwa kwa kukana kwa mbewu ndi majini oyambitsa chitetezo chamthupi (ETI) pamzerewu, pomwe mayankho ochedwa a Mandelup amathandizira gawo la micro-associated molecular pattern-triggered immune (MTI) hypothesis 50. Mayankho oyambilira ku 83A: 476 ndi Mandelup.Kuphatikizika kwapang'onopang'ono pakati pa majeremusi okwera kapena otsika poyankha mochedwa kumathandiziranso lingaliro ili, popeza ETI nthawi zambiri imatengedwa kuti ndi njira yofulumira komanso yowonjezereka ya MTI yomwe imafika pachimake pa kufa kwa maselo pa malo a matenda, omwe amadziwika kuti anaphylactic shock 51,52.
Mitundu yambiri yomwe imatchedwa Gene Ontology GO:0006952 "Defense Response" ndi ma homologues 11 a uthenga wama protein 22 (ofanana ndi SAM22) ndi mapuloteni asanu ndi awiri akuluakulu a latex (MLPs).-monga mapuloteni 31, 34, 43 ndi 423 adawonetsa kufanana.Ma jini onga SAM22 adawonetsa kuyambitsa kwakukulu komwe kunatenga nthawi yayitali, kuwonetsa kuchuluka kwa kukana kwa anthracnose (83A: 476 ndi Boregine).Komabe, majini onga a MLP adatsitsidwa kokha m'mizere yonyamula otsutsa (83A:476/Lanr1 pa 6 hpi ndi Mandelup/AnMan pa 24 hpi).Zindikirani kuti ma homologue onse odziwika ngati SAM22 amachokera kugulu la majini lomwe limatalika pafupifupi 105 kb, pomwe majini onga MLP amachokera kumadera osiyanasiyana a genome.Kulumikizana kogwirizana kwa majini ngati SAM22 kunapezekanso mu kafukufuku wathu wam'mbuyomu wa NLL kukana Diaporthetoxica inoculation, kutanthauza kuti amakhudzidwa ndi zigawo zopingasa zachitetezo.Kutsiliza kumeneku kumathandizidwanso ndi malipoti a kuyankha kwabwino kwa jini ngati SAM22 kuvulaza kapena kuchiza ndi salicylic acid, fungal inducers, kapena hydrogen peroxide.
Mitundu yofanana ndi ya MLP yawonetsedwa kuti imayankha kupsinjika kwamitundu yosiyanasiyana ya abiotic ndi biotic, kuphatikiza mabakiteriya, ma virus ndi mafangasi amtundu wamitundu yambiri55.Njira zoyankhira ku kugwirizana kwina pakati pa zomera ndi tizilombo toyambitsa matenda zinayambira pakuchulukirachulukira (mwachitsanzo, pa nthawi ya thonje lodzala ndi Verticillium dahliae) mpaka kutsika kwambiri (ie, pambuyo pa matenda a apulosi ndi Alternaria spp.)56,57.Kutsika kwakukulu kwa jini ya MLP-like 423 kwawonedwa panthawi ya chitetezo cha avocado ku matenda a F. niger komanso panthawi ya matenda a mtengo wa apulo Botryosphaeria berengeriana f.cn.piricola ndi Alternaria alternata ndi apulo pathotypes58,59.Kuphatikiza apo, apple calli yofotokoza kwambiri jini ya MLP-ngati 423 inali ndi mawonekedwe otsika a majini okhudzana ndi kukana ndipo amatha kutenga matenda oyamba ndi fungus59.Kutsatira Fusarium oxysporum f, jini ya MLP-ngati 423 idaponderezedwanso mu majeremusi wamba omwe samva.cn.Matenda a nyemba 60.
Mamembala ena a banja la PR-10 omwe adadziwika mu phunziro lathu la RNA-seq anali ma jini a LlR18A ndi LlR18B poyankha kuwongolera, komanso jini yoyendetsedwa bwino (1 jini) kapena yocheperako (3 jini) ya lipid transfer protein DIR1..Kuphatikiza apo, WGCNA ikuwonetsa jini ya LlR18B ngati gawo la gawoli, lomwe limatha kutenga katemera ndipo limanyamula ma jini angapo oteteza.Mitundu ya LlR18A ndi LlR18B inayambitsidwa mu masamba achikasu a lupine poyankha mabakiteriya a tizilombo toyambitsa matenda, komanso mu NLL zimayambira pambuyo pa D. toxica inoculation, pamene mpunga wamtundu wa majiniwa, RSOsPR10, unayambitsidwa mofulumira ndi matenda a fungal omwe amawoneka kuti anali nawo mu jasmonic acid, 62 syringe 62, DIFIdescgene, DIFIDES 61, DIFIDE 6, DIFIDES 61. Mapuloteni oyendetsa lipid omwe amafunikira kuti ayambirenso kukana kwadongosolo (SAR).Ndi chitukuko cha machitidwe otetezera, puloteni ya DIR1 imatengedwa kuchokera kumalo okhudzidwa ndi matenda kudzera mu phloem kuti ipangitse SAR ku ziwalo zakutali.Chochititsa chidwi, jini ya TanjilG_02313 DIR1 idakopeka kwambiri nthawi yoyamba pamzere 84A:476 ndi Population 22660, koma kukana kwa anthracnose kudakula bwino pamzere wa 84A:476.Izi zikhoza kusonyeza kusinthika kwa jini ya DIR1 ku NLL, popeza otsala atatu otsalawo adayankha ku inoculation kokha mu 83A: mzere wa 476 pa 6 hpi, ndipo yankholi linalunjika pansi.
Mu phunziro lathu, zigawo zomwe zimagwirizana kwambiri ndi njira yachilengedwe yotchedwa "GO: 0055114 Redox process" inali mapuloteni a cytochrome P450, peroxidase, linoleic acid 9S-/13S-lipoxygenase, ndi 1-aminocyclopropane-1-carboxylic acid oxidase.Kuphatikiza apo, WGCNA yathu imatanthauzira HSFA4a homologue ngati malo onyamula ma module monga Lanr1 resistance gene candidate TanjilG_05042.HSFA4a ndi gawo la malamulo odalira redox a zolemba za nyukiliya muzomera.
Mapuloteni a Cytochrome P450 ndi oxidoreductases omwe amachititsa kuti NADPH ndi / kapena O2-amadalira hydroxylation reaction mu primary and secondary metabolism, kuphatikizapo kagayidwe ka xenobiotics, komanso mahomoni, mafuta acids, sterols, cell wall components, biopolymers, ndi biosynthesis of protective compounds In 60 cytochrome in 60 mu phunziro lathu la cytochrome In a9. 10.6 log2 (kusintha pindani) kupita ku 5.7 chifukwa cha kuchuluka kwa ma homologues osinthidwa (37) komanso kusiyana kwa mayankho pakati pa majini enaake, kuwonetsa kubwereza kokwera..Kugwiritsa ntchito data ya RNA-seq yokha kuti timvetsetse momwe ma jini a NLL alili m'gulu lalikulu la mapuloteni kungakhale kongoyerekeza.Komabe, ndizofunika kudziwa kuti ma jini ena a cytochrome P450 amalumikizidwa ndi kuchuluka kwa kukana kwa bowa kapena mabakiteriya oyambitsa matenda, kuphatikiza kuthandizira pakukhudzidwa ndi ziwengo69,70,71.
Class III peroxidases ndi michere yambiri ya zomera zomwe zimagwira ntchito zosiyanasiyana pakukula ndi kukula kwa zomera, komanso poyankha zovuta za chilengedwe monga salinity, chilala, kuwala kwakukulu, ndi tizilombo toyambitsa matenda72.Peroxidase imakhudzidwa ndi kuyanjana kwa mitundu ingapo ya zomera ndi Anthracis, kuphatikizapo Stylosanthes humilis ndi C. gloeosporioides, Lens culinaris ndi C. truncatum, Phaseolus vulgaris ndi C. lindemuthianum, Cucumis sativus ndi C. lagenarium73,764,75.Yankho limakhala lofulumira kwambiri, nthawi zina ngakhale pa 4 HPI, bowa lisanalowe mumtundu wa mbewu73.Jini la peroxidase linayankhanso D. toxica NLL inoculation.Kuphatikiza pa ntchito zawo zomwe zimayang'anira kuphulika kwa okosijeni kapena kuthetsa kupsinjika kwa okosijeni, ma peroxidase amatha kusokoneza kukula kwa tizilombo toyambitsa matenda popanga zotchinga zakuthupi potengera kulimba kwa khoma la cell panthawi ya lignification, subunit kapena kulumikizana kwazinthu zinazake.Ntchitoyi imatha kunenedwa kuti ili mu silico ku TanjilG_03329 jini yomwe imayika ma putative lignin-forming anion peroxidase yomwe idasinthidwa kwambiri mu phunziro lathu mu 83A:476 resistant line pa 6 HPI, koma osati mu zovuta zina ndi nthawi zomwe sizinayankhe.
9S-/13S-lipoxygenase ya linoleic acid ndi sitepe yoyamba mu njira ya okosijeni ya lipid biosynthesis78.Zogulitsa zanjirayi zimakhala ndi ntchito zambiri pachitetezo cha zomera, kuphatikizapo kulimbitsa makoma a cell kudzera pakupanga ma callose ndi pectin deposits, komanso kuwongolera kupsinjika kwa okosijeni mwa kupanga mitundu ya oxygen79,80,81,82,83.Mu phunziro lino, mawu a linoleic asidi 9S-/13S-lipoxygenase anasintha mitundu yonse, koma atengeke anthu 22660, upregulation anapambana pa nthawi zosiyanasiyana, pamene mavuto kunyamula kugonjetsedwa Lanr1 ndi AnMan allele, izo zikugogomezera kusiyanasiyana kwa oxylipinnthrec wosanjikiza mu oxylipinnotype wosanjikiza awa.
1-aminocyclopropane-1-carboxylate oxidase (ACO) homologue idasinthidwa kwambiri (majini 9) kapena kuchepetsedwa (majini a 2) atayikidwa ndi lupine.Kupatulapo ziwiri, mayankho onsewa adachitika pa 6 hp.ku 83:476.Kuchita kwa enzymatic komwe kumalumikizidwa ndi mapuloteni a ACO ndiye njira yochepetsera kuchuluka kwa ethylene ndipo chifukwa chake imayendetsedwa kwambiri84.Ethylene ndi timadzi tambiri timene timagwira ntchito zosiyanasiyana pakuwongolera kukula kwa mbewu ndikuyankha kupsinjika kwa abiotic ndi biotic.Kulowetsedwa kwa zolemba za ACO ndi kutsegula kwa njira yowonetsera ethylene kumaphatikizapo kuonjezera kukana kwa mpunga ku hemibiotrophic fungus oryzae oryzae poyang'anira kupanga mitundu yowonjezereka ya okosijeni ndi phytoalexins.Njira yofananira kwambiri ya matenda a masamba yomwe imapezeka pakati pa M. oryzae ndi C. lupini88,89, motsutsana ndi kusintha kwakukulu kwa ACO homologues mu 83A: 476 mzere wofotokozedwa mu phunziroli, amasintha mwayi wopereka kukana kwa NLL anthracnose Ethylene chizindikiro chapakati pa njira za maselo.
Mu kafukufuku wapano, kuponderezedwa kwakukulu kwa majini ambiri okhudzana ndi photosynthesis kudawonedwa pa 6 hpi mu 83A: 476 ndi 48 hpi ku Mandeloop ndi 22660 anthu.Kukula ndi kupita patsogolo kwa zosinthazi kumayenderana ndi mulingo.Kukana kwa anthracnose kunawonedwa pakuyesa uku.Posachedwapa, kuponderezedwa kwamphamvu ndi koyambirira kwa zolemba zokhudzana ndi photosynthesis zafotokozedwa m'mitundu ingapo ya kugwirizana kwa tizilombo toyambitsa matenda, kuphatikizapo mabakiteriya a pathogenic ndi bowa.Kufulumira (kuchokera ku 2 HPI muzochita zina) ndi kuponderezedwa kwapadziko lonse kwa majini okhudzana ndi photosynthesis poyankha matenda kungayambitse chitetezo cha zomera potengera kutumizidwa kwa mitundu yogwira ntchito ya okosijeni ndi kugwirizana kwawo ndi njira ya salicylic acid kuti athetseretu 90,94.
Pomaliza, njira zoyankhira zodzitchinjiriza zomwe zaperekedwa pamzera wosamva (83A: 476) zikuphatikiza kuzindikira kwapathogen mwachangu ndi jini ya R (mwina TIR-NBS-LRR TanjilG_05042) ndi allergenic reaction-mediated salicylic acid ndi chizindikiro cha ethylene, kutsatiridwa ndi kukhazikitsidwa kwa SAR yayitali.Zochita zimathandizidwa ndi DIR-1 protein.Tiyenera kuzindikira kuti nthawi ya biotrophic ya matenda a C. lupini ndi yochepa kwambiri (pafupifupi masiku a 2), ndikutsatiridwa ndi kukula kwa necrotic95.Kusintha kwapakati pazigawo izi kungagwirizane ndi necrosis ndi kufotokoza kwa mapuloteni otchedwa ethylene-inducible omwe amakhala ngati zoyambitsa zochitika za hypersensitivity mu zomera zomwe zimagwidwa.Choncho, zenera la nthawi yogwira bwino C. lupini pa siteji ya biotrophic ndi yopapatiza kwambiri.Kukonzanso kwa majini okhudzana ndi redox ndi photosynthesis komwe kumawonedwa mu 83A: 476 pa 6 hpi kumagwirizana ndi kukwera kwa fungus hyphae ndipo kumalengeza chitukuko cha kuyankhidwa kotetezedwa bwino pa biotrophic stage.Mayankho a transcriptomic a Mandelup ndi chiwerengero cha 22660 akhoza kuchedwa kwambiri kuti agwire bowa asanasinthe kukula kwa necrotic, komabe, Mandelup ikhoza kukhala yothandiza kwambiri kuposa chiwerengero cha 22660 chifukwa malamulo ofulumira a mapuloteni a PR-10 amalimbikitsa kukana kopingasa.
ETI, yoyendetsedwa ndi jini yovomerezeka ya R, ikuwoneka ngati njira yodziwika bwino yothanirana ndi nyemba ku anthracnose.Choncho, mu chitsanzo cha legume Medicago truncatula, kukana kwa anthracnose kumaperekedwa ndi RCT1 jini, membala wa gulu la TIR-NBS-LRR97 chomera R gene class.Jini imeneyi imaperekanso kukana kwa anthracnose mumtundu wa alfalfa ikasamutsidwa ku zomera zomwe zingatengeke.Mu nyemba wamba (P. vulgaris), mitundu yopitilira makumi awiri ndi iwiri ya anthracnose yadziwika mpaka pano.Ena mwa majiniwa amapezeka m'magawo opanda ma jini ovomerezeka a R, komabe ena ambiri amakhala m'mphepete mwa ma chromosome omwe amanyamula gulu la jini la NBS-LRR, kuphatikiza TIR-NBS-LRRs99.Kafukufuku wa SSR wa genome-wide adatsimikiziranso kuyanjana kwa jini ya NBS-LRR ndi kukana kwa anthracnose mu nyemba wamba.Jini yovomerezeka ya R idapezekanso m'chigawo cha genomic chomwe chili ndi malo akuluakulu olimbana ndi anthracnose mu white lupine 101.
Ntchito yathu ikuwonetsa kuti kukana kwanthawi yayitali, komwe kumayambika kuyambika kwa matenda a zomera (makamaka osapitirira 12 hpi), kumateteza bwino lupine kuchokera ku anthracnose chifukwa cha bowa Collelotrichum lupini.Pogwiritsa ntchito kutsatizana kwakukulu, tidawonetsa mafotokozedwe osiyanasiyana amitundu yolimbana ndi anthracnose muzomera za NLL zolumikizidwa ndi jini yotsutsa ya Lanr1 ndi AnMan.Kuteteza bwino kumaphatikizapo kupanga mosamala majini a mapuloteni omwe amapangidwa ndi redox, photosynthesis, ndi pathogenesis patangotha ​​​​maola ochepa chomeracho chinakhudza tizilombo toyambitsa matenda.Kutetezanso kofananako, koma kuchedwa pakapita nthawi, sikuthandiza kwambiri kuteteza mbewu ku matenda.Kukana kwa anthrax komwe kumayenderana ndi jini ya Lanr1 kumafanana ndi kuyankha mwachangu kwa jini ya R (chitetezo choyambitsa chitetezo), pomwe jini ya AnMan nthawi zambiri imapereka yankho lopingasa (chitetezo choyambitsidwa ndi mamolekyu ogwirizana ndi ma microbe), kupereka mulingo wokhazikika wokhazikika.
Mizere 215 ya NLL yomwe imagwiritsidwa ntchito poyesa zolembera za anthracnose inali ndi mitundu 74, mizere 60 yopezedwa powoloka kapena kuswana, 5 mutants, ndi 76 majeremusi amtchire kapena oyambilira.Mizere inachokera ku mayiko a 17, makamaka ochokera ku Poland (58), Spain (47), Germany (27), Australia (26), Russia (19), Belarus (7), Italy (5) ndi mizere ina.ochokera kumayiko 10.Setiyi ilinso ndi mizere yosagwira: 83A:476, Tanjil, Wonga yonyamula Lanr1 allele, ndi Mandelup yonyamula AnMan allele.Mizereyi inapezedwa kuchokera ku European Lupine Genetic Resource Database yosungidwa ndi Poznań Plant Breeding Ltd., Wiatrowo, Poland (Supplementary Table S1).
Zomera zimabzalidwa molamulidwa (nthawi yazithunzi maola 16, kutentha 25 ° C masana ndi 18 ° C usiku).Zofananira ziwiri zamoyo zidawunikidwa.DNA idasiyanitsidwa ndi masamba a masabata atatu pogwiritsa ntchito DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) malinga ndi protocol.Ubwino ndi kuchuluka kwa DNA yokhayokha idayesedwa ndi njira za spectrophotometric (NanoDrop 2000; Thermo Fisher Scientific, Waltham, MA, USA).Chizindikiro cha AnManM1 cholemba jini yolimbana ndi anthracnose AnMan (yochokera ku cv. Mandelup) ndi zolembera Anseq3 ndi Anseq4 zamtundu wa Lanr1 (zochokera ku cv. Tanjil) zidawunikidwa 11,26,28.Ma homozigote a allele osamva adagoletsa ngati "1", osavuta - ngati "0", ndi ma heterozygotes - ngati 0.5.
Kutengera zotsatira za kuwunika kwa zolembera AnManM1, AnSeq3 ndi AnSeq4 komanso kupezeka kwa mbewu zoyeserera komaliza, mizere 50 ya NLL idasankhidwa kuti ipange phenotyping ya anthracnose resistance.Kusanthula kunachitika mobwerezabwereza mu wowonjezera kutentha woyendetsedwa ndi makompyuta wokhala ndi chithunzi cha maola 14 ndi kutentha kwa 22 ° C masana ndi 19 ° C usiku.Mbewu zimakandidwa (kudula chikhoto cha mbeu kumbali ina ya mluza ndi tsamba lakuthwa) musanabzale kuti mbeu isasamale bwino chifukwa cholimba kwambiri komanso kuti mbeu zimere mofanana.Zomera zidakula mumiphika (11 × 11 × 21 cm) ndi dothi lopanda kanthu (TS-1 REC 085 Medium Basic, Klasmann-Deilmann Polska, Warsaw, Poland).Kuthirira kunachitika ndi mtundu wa Colletotrichum lupini Col-08, womwe unakula mu 1999 kuchokera kumitengo ya masamba opapatiza a lupine omwe amamera m'munda wa Verzhenítsa, Greater Poland (52° 27′ 42″ N 17° 04′ 05″ E ).Pezani malo.Zodzipatulazo zidakulitsidwa mu SNA sing'anga pa 20 ° C. pansi pa kuwala kwakuda kwa masiku 21 kuti apangitse sporulation.Patatha milungu inayi kufesa, pamene zomera zafika 4-6 tsamba siteji, inoculation inachitika ndi kupopera mbewu mankhwalawa ndi kuyimitsidwa kwa conidia pa ndende ya 0,5 x 106 conidia pa ml.Pambuyo pa katemera, mbewuzo zimasungidwa mumdima kwa maola 24 pa chinyezi pafupifupi 98% ndi kutentha kwa 25 ° C kuti zithandizire kumera kwa conidia ndi matenda.Zomerazo zidabzalidwa pansi pa chithunzi cha maola 14 pa 22°C usana/19°C usiku ndi chinyezi cha 70%.Matendawa adapangidwa patatha masiku a 22 pambuyo pa katemera ndipo adachokera ku 0 (chitetezo cha mthupi) mpaka 9 (chotengeka kwambiri) malingana ndi kukhalapo kapena kusowa kwa zilonda za necrotic pa zimayambira ndi masamba.Kuonjezera apo, atatha kugoletsa, kulemera kwa zomera kunayesedwa.Ubale pakati pa chizindikiro cha genotypes ndi matenda a phenotypes adawerengedwa ngati mfundo ziwiri zotsatizana (kusakhalapo kwa zizindikiro za heterozygous mu seti ya mizere yowunikira phenotype ya anthracnose resistance).