【作者】 邢光南；

【导师】 盖钧镒；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2007， 博士

【摘要】 随着人们对无公害绿色食品和环境保护意识的日益增强,抗虫育种逐渐被人们所重视。我国南方大豆害虫种类多,常造成严重的经济损失。豆卷叶螟、筛豆龟蝽和斜纹夜蛾是我国南方大豆上的主要害虫。目前已对大豆抗斜纹夜蛾进行了较深入的研究,继续探讨大豆对豆卷叶螟和筛豆龟蝽的抗性鉴定方法,筛选优异抗源,研究其抗性遗传机制,对开展我国南方大豆抗虫育种具有重要的理论与实践意义。本文从资源鉴定、遗传分析和QTL定位三个层次对大豆抗豆卷叶螟和筛豆龟蝽进行了研究,所获结果包括如下:1大豆抗豆卷叶螟和筛豆龟蝽的资源鉴定(1)豆卷叶螟抗性鉴定:1)治虫和品种两因子裂区试验表明豆卷叶螟危害可导致大豆品种平均减产26.27%,但受害处理的百粒重与对照无显著差异,外观品质也无显著差异;2)对虫包数、卷叶率和籽粒产量三个抗性鉴定指标进行了比较,确定在田间自然危害条件下以卷叶率为最佳抗性指标;3)2004-2006三年用卷叶率指标评价了从国内外大批资源中筛选出的58份抗、感食叶性害虫的大豆材料对豆卷叶螟的抗性稳定性,并用网室人工接虫试验验证了田间鉴定结果的可靠性,抗性鉴定结果表明,品种间、年份间、观察日期间、品种×观察日期和品种×年份都有极显著的差异;4)筛选出监利牛毛黄、吴江青豆3、沔阳白毛豆、丰平黑豆和安陆小黄豆等高抗种质,临沂糙绿豆、Morsoy、Bethol、Dare和皖82-178等高感种质,并建议把其分别作为高抗和高感标准品种;5)2006年全国抽样的387份材料抗性结果表明,地方品种和育成品种中高抗材料都有较高的频率;品种抗性程度与品种来源地的地理纬度呈显著负相关。株高高、开花期晚、成熟期晚、茸毛斜立型或紧贴型的品种较抗虫。(2)筛豆龟蝽抗性鉴定:1)确定按茎秆黑霉程度结合叶片紫斑数进行抗性分级;2)鉴定了从国内外大批资源中筛选出的58份抗、感食叶性害虫的大豆材料对筛豆龟蝽的抗性,发现品种间、观察日期间和品种×观察日期都有极显著差异;3)筛选出PI227687、安陆小黄豆、花柒黄毛豆和沔阳白毛豆等高抗种质,临沂糙绿豆、大黑豆、Morsoy、黄皮小青豆和皖82-178等高感种质,并建议把其分别作为高抗和高感标准品种。(3)豆卷叶螟、筛豆龟蝽和斜纹夜蛾抗性的相关性分析及兼抗资源鉴定:1)发现大豆对豆卷叶螟的抗性和对筛豆龟蝽的抗性高度正相关(r=0.92),而与对斜纹夜蛾的抗性负相关(r=-0.46),特别表现在豆卷叶螟和筛豆龟蝽的多数高感、高抗品种与斜纹夜蛾的正好相反;2)结合前人的研究结果筛选出具有较高的田间综合虫种抗性又具有较高的对豆卷叶螟、筛豆龟蝽和斜纹夜蛾单一虫种抗性的大豆种质PI227687、中豆14、日本、南农89-30和吴江青豆3,但除PI227687外,其它种质对三种害虫不全是高抗;3)筛选出高抗豆卷叶螟和筛豆龟蝽但高感斜纹夜蛾的大豆种质沔阳白毛豆、丰平黑豆、监利牛毛黄、枞阳猴子毛、巨县秋豆A和安陆小黄豆,同时筛选出高抗斜纹夜蛾但高感豆卷叶螟和筛豆龟蝽的大豆种质Lamar、PI171451、PI229358、矮秆黄、黄皮小青豆和山东大豆。2大豆抗豆卷叶螟和筛豆龟蝽的遗传分析(4)应用三个抗感杂交组合[科丰1号×南农1138-2(NJRIKY)、皖82-178×通山薄皮黄豆甲(NJRIWT)和苏88-M21×新沂小黑豆(NJRISX)]衍生的重组自交系群体,在田间自然虫源条件下,2004-2006年鉴定了大豆抗豆卷叶螟的植株反应:1)验证了9月上旬卷叶率为鉴定大豆对豆卷叶螟抗性的最佳指标;2)NJRIKY、NJRIWT和NJRISX三个群体抗性遗传分离分析结果,一致表明大豆对豆卷叶螟的抗性符合两对主基因+多基因的混合遗传模型,主基因遗传率分别为51.0%、80.5%和56.3%,多基因遗传率分别为39.1%、11.4%和29.1%。两对主基因的作用方式表现组合间有差异。(5)以NJRIKY和NJRIWT为材料,2004-2006三年在田间自然虫源下鉴定了筛豆龟蝽抗性,发现NJRIKY和NJRIWT两重组自交系群体对筛豆龟蝽的抗性分别由三对和两对主基因控制。3大豆抗豆卷叶螟和筛豆龟蝽的QTL分析(6)Mapmaker/Exp(3.0)在分子标记数量大时(多于500个)往往出现所绘制连锁图谱图距偏大的现象。本文从标记分群和标记排序两个遗传作图环节分析原因并概括出以下两个实施要点:1)标记分群不应强求同一LOD值,对特殊的连锁群可试用不同LOD值;2)在标记排序时,一次order命今后用ripple命令反复梳理有时并不能获得最佳排列顺序,而应多次使用order,每次order后用ripple反复梳理,经反复比较才能得出最佳的排列顺序,必要时还须结合人工调整。(7)豆卷叶螟抗性QTL定位发现:1)NJRIKY的抗性位点较多主要位于D1a、H、C2、D1b和O连锁群上,NJRIWT抗性QTL主要位于H、D1b和O连锁群上,NJRISX抗性QTL主要位于D1a连锁群上;2)抗虫QTL的抗性等位基因多数来自抗虫亲本南农1138-2、通山薄皮黄豆甲和苏88-M21;3)NJRIKY和NJRISX位于D1a连锁群上的qRLP-d1a-1处于相同区间且都是最主要的抗性QTL;4)NJRIKY和NJRIWT位于H连锁群上的qRLP-h-2处于相同区间且都是最主要的抗性QTL,NJRIKY和NJRIWT位于D1b连锁群上的qRLP-d1b-2也处于相同区间;5)大豆对豆卷叶螟抗性的QTL定位,在群体间、指标间、年度间、日期间能相互验证,说明QTL存在的真实性,QTL附近的标记有望用于标记辅助选择育种。(8)筛豆龟蝽抗性QTL定位发现:1)2004-2006三年NJRIKY群体均检测出的qRMC-d1a-1位于D1a连锁群,贡献率为7.6%-31.4%;2005和2006两年均检测出的qRMC-c2-1位于C2连锁群,与环境有互作,效应相对较小,抗性等位基因都来自南农1138-2;2)NJRIWT群体连锁群H上的qRMC-h-1在三年中都被检测到,贡献率为16.3%-36.2%,D1b连锁群上的qRMC-d1b-2在2004和2005年被检测到,效应相对较小,抗性等位基因都来自通山薄皮黄豆甲;3)本研究中两个群体抗性位点基本上是不同的。(9)豆卷叶螟抗性与筛豆龟蝽抗性QTL定位结果比较:筛豆龟蝽抗性检测出的QTL较少,但检测出的重要QTL与豆卷叶螟的重要QTL位于相近区间。NJRIKY群体中包括位于D1a连锁群上相同区间A947V-Satt482的qRMC-d1a-1和qRLP-d1a-1,位于C2连锁群上相同区间A748V-A397I的qRMC-c2-1和qRLP-c2-1;NJRIWT群体中包括位于H连锁群上相同区间Satt181-Satt434的qRMC-h-1和qRLP-h-2。(10)茸毛着生性状的基因定位及其与豆卷叶螟抗性位点的关系:1)利用重组自交系群体分别从关联分析和QTL定位两方面明确了大豆茸毛着生状态与豆卷叶螟的抗性存在密切联系,茸毛紧贴、茸毛斜立是抗性性状,而茸毛直立是感虫性状;2)茸毛紧贴、茸毛斜立比茸毛直立能提高27%-46%的抗性;3)大豆茸毛着生状态的基因在NJRIKY和NJRISX中都被定位在D1a上,且都处于qRLP-d1a-1所在的位置。更多还原

【Abstract】 As the public concern about food and environment safety growing steadily,to develop insect resistant or tolerant cultivars is considered the most economical way in avoiding yield losses due to insect pests.There were many insect pests limiting soybean production,specially in southern China,where a subtropical climate characterized by mild winters and long growing seasons harbored a complex of potentially important insect species that attack soybean,and sometimes,soybean plants are seriously damaged.Bean pyralid[Lamprosema indicata(Fabricius)],globular stink bug[Megacopta cribraria (Fabricius)]and common cutworm[Spodoptera litura(Fabricius)]were reported as the most important insects on soybean in southern China.A study of soybean resistant to S. litura have been done at Soybean Research Institute of Nanjing Agricultural University.To establish the method for evaluation of resistance,to screen for genetic sources with resistance and to reveal the genetic mechanism of resistance of soybeans to L.indicate and M.cribraria is of great significance in breeding for insect resistant of soybeans in southern China.The main results of the present study were as follows:1 Evaluation and identification of genetic sources resistant to L.indicata and M. cribraria in soybean(1) The results of evaluation and identification of genetic sources resistant to L. indicata:1) Damage to soybean infested by L.indicata larvae was estimated under with vs. without insecticide spray.The damage of L.indicata was mainly on seed yield rather than on seed quality.Yield losses caused by L.indicata infestation was averagely 26.27%.2) A set of resistance(damage) indices were designed and compared.Among them,rolled leave percentage was chosen to evaluate soybean resistance to L.indicata.3) The identification results of 58 varieties resistant or susceptible to leaf-feeding insect screened out from a total of 6724 domestic and foreign germplasm showed the existence of significant difference among years,observation dates and varieties in a three-year test during 2004-2006.There was also significant interaction between varieties and years and between varieties and dates. 4) After three years’ tests,Jianliniumaohuang,Wujiangqingdou 3,Mianyangbaimaodou, Fengpingheidou and Anluxiaohuangdou were identified to be highly resistant and Linyicaolvdou,Morsoy,Bethol,Dare and Wan 82-178 were identified to be highly susceptible,and were suggested as the standard checks in field evaluation to compensate the fluctuations in materials and environmental conditions.5) 387 varieties were screened in 2006.Breeding lines have similar resistant frequency as land race.There was significant differential geographical distribution of resistant resources among varietal eco-regions. Materials with longer days to flowering and maturity,higher heights,semi-erect or appressed pubescence orientation might be more resistant to L.indicata and vice versa.(2) The results of evaluation and identification of genetic sources resistant to M. cribraria:1) Based on the observation of occurrence and symptoms of the insect on soybeans,a set of resistance(damage) indicators were designed and compared.Among them,the grade system according to the degree of black mildew on stem and purple spots on leaves was chosen to evaluate the resistance to M.cribraria.2) The identification results of 58 accessions showed that there existed significant difference among accessions, observation dates,and accession×date.3) A group of highly resistant accessions such as PI227687,Anluxiaohuangdou,Huaqihuangmaodou,Mianyangbaimaodou,and highly susceptible accessions such as Linyicaolvdou,Daheidou,morsoy,Huangpixiaoqingdou, Wan82-178,were screened out for breeding purposes,and was suggested as the standard checks in field evaluation.(3) Comparing results of resistance to L.indicata,M.cribraria and S.litura,the following was found:1) There existed significant positive correlation between resistance to L.indicata and that to M.cribraria,significant negative correlation between resistance to S. litura and that to M.cribraria and between resistance to S.litura and that to L.indicata.2) A group of accessions with high resistance to the three major insects,such as PI227687, Zhongdou 14,Riben,Nannong 89-30 and Wujiangqingdou 3 were screened out for breeding purposes.3) The accessions highly resistant to L.indicata and M.cribraria,such as Mianyangbaimaodou,Fengpingheidou,Jianliniumaohuang,Congyanghouzimao, Juxianqiudou A and Anluxiaohuangdou were highly susceptible to S.litura.On the contrary, the accessions highly resistant to S.litura,such as Lamar,PI171451,PI229358, Aiganhuang,Huangpixiaoqingdou and Shandongdadou were highly susceptible to L. indicata and M.cribraria.2 Inheritance of resistance to L.indicata and M.cribraria in soybean(4) Three recombinant inbred line(RIL) populations derived from susceptible by resistant crosses,i.e.NJRIKY,NJRIWT and NJRISX(derived from Kefeng No.1×Nannong 1138-2,Wan 82-178×Tongshanbopihuangdoujia and Su 88-M21×Xinyixiaoheidou,respectively) were tested in field under natural infestation.1) Rolled leave percentage evaluated in early September was chosen as the best indicator of resistance to L.indicata due to its higher genetic variation,heritability value and stability, as well as higher negative correlation with seed yield in the three populations.2) The segregation analyses were performed under major gene+polygene mixed inheritance model for the three populations.The results showed that the resistance to L.indicata was controlled by two major genes plus polygenes in the three populations with their major gene heritability being 51.0%,80.5%and 56.3%,and polygene heritability being 39.1%, 11.4%and 29.1%,respectively.(5) Two RIL populations,NJRIKY and NJRIWT were tested for their resistance to M. cribraria under natural infestation according to the degree of black mildew.The results of segregation analyses showed that the resistance to M.cribraria was controlled by three major genes and two major genes in the two populations,respectively.3 Mapping QTLs of resistance to L.indieata and M.cribraria in soybean(6) It was found that the distances of the map constructed with the software Mapmaker/Exp(3.0) were often exaggerated when large number of markers(e.g.more than 500) was involved.Two innovations were suggested in the application of Method 2 of Mapmaker/Exp(3.0) as:1) Different LOD values used for some specific linkage groups in addition to a common LOD value for the others;and 2) Multiple "order" commands each followed with multiple "ripple" commands used for ordering markers in linkage groups with a window size of 5,combined with some artificial adjustments when needed,for relatively higher likelihoods of the linkage group.(7) The results of mapping QTLs conferring resistance to L.indicata in soybean:1) The QTLs on linkage group Dla,H,C2,Dlb and O were detected often associated with resistance to L.indicata in NJRIKY during the three years;the QTLs on linkage group H, D1b and O were detected often associated with resisitance to L.indicata in NJRIWT during the three years;the QTL on linkage group Dla were detected consistently associated with resistance to L.indicata in NJRISX during the three dates in 2006.2) The resistance alleles were often from Nannong 1138-2,Tongshanbopihuangdoujia and Su 88-M21.3) The qRLP-d1a-1 on linkage group D1a in the NJRIKY population also identified in NJRISX population,is an important QTL resistant to L.indicata in soybean.4) The qRLP-h-2 on linkage group H in the NJRIKY population also identified in NJRIWT population,is another important QTL resistant to L.indicata in soybean.The qRLP-d1b-2 on linkage group D1b in the NJRIKY population was also identified in NJRIWT population.5) The fact that the QTLs was repeatedly detected in different populations,different years (environments),different dates and different indices,indicated the resistance was controlled stably by the major QTLs.Based on the results,it is inferred that the markers linked to the detected QTLs should be useful for marker-assisted selection for resistance to L.indicata in soybean.(8) The results of mapping QTLs conferring resistance to M.cribraria in soybean:1) The QTL qRMC-d1a-1 on linkage group D1a was detected consistently associated with resistnace to M.cribraria in NJRIKY during the three years,which accounted for about 7.6%-31.4%of the phenotypic variation;the QTL qRMC-c2-1 on linkage group C2 was detected during 2005-2006,which accounted for less phenotypic variation than the former one;the resistance alleles were from Nannong 1138-2.2) The QTL qRMC-h-1 on linkage group H was detected also consistently associated with resistance to M.cribraria in NJRIWT during the three years,which accounted for about 16.3%-36.2%of the phenotypic variation;the QTL qRMC-d1b-2 on linkage group D1b was detected during 2004-2005, which accounted for less phenotypic variation than the former one;the resistant alleles were from Tongshanbopi- huangdoujia.3) Therefore,basically different QTLs conferred resistance to M.cribraria in NJRIKY and NJRIWT.(9) The relationship between QTLs resistant to L.indicate and to M.cribraria in soybean:The number of QTLs resistant to M.cribraria was fewer,while important QTLs were mapped in homologous regions of linkage group.Such as,qRMC-d1a-1 and qRLP-d1a-1 were mapped between A947V and Satt482 on linkage group D1a in NJRIKY; qRMC-c2-1and qRLP-c2-1 were mapped between A748V and A397I on linkage group C2 in NJRIKY;and qRMC-h-1 and qRLP-h-2 were mapped between Satt181 and Satt434 on linkage group H in NJRIWT.(10) Gene mapping of pubescence orientation and association analysis between pubescence orientation and resistance to L.indicate:1) Association anlyisis between pubescence orientation and resistance to L.indicata and QTL/gene mapping in two recombinant inbred line populations NJRIKY and NJRISX were taken,indicating genetic association of pubescences orientation and resistance to L.indicata in soybean.2) The average rolled leave percentage reduced 27-46%in pubescence appressed and semi-appressed lines in comparision with pubescence erect line.3) The pubescence orientation gene and qRLP-d1a-1 resistant to L.indicata in the two populations were mapped in a homologous region of linkage group D1a.更多还原