【作者】 石红良；

【导师】 潘光堂； 张世煌； 李新海；

【作者基本信息】 四川农业大学 ， 作物遗传育种， 2009， 博士

【摘要】 玉米(Zea mays L.)丝黑穗病是一种世界性病害,也是我国春玉米产区的主要病害之一。这种病害是由土壤或种子带菌传播的真菌病害,其病原菌丝轴黑粉菌(Sphacelotheca reiliana(K(u|¨)hn)Clint)在玉米幼苗期地下部分侵入、系统感染,成株期表现典型症状,在雄穗和雌穗上产生黑粉孢子。数量遗传学研究表明,玉米对丝黑穗病的抗性属数量性状遗传,同时受基因加性、显性和上位性效应控制,其中基因加性效应占主导作用,非加性效应作用较小,抗性能稳定遗传。随着分子生物学的发展,现代数量遗传学和生物技术手段也已应用于玉米丝黑穗病的抗性遗传研究。迄今,多位研究者在国内外玉米种质中发现了多个抗病基因位点(QTL)及其连锁分子标记,并获得了与丝黑穗病抗性相关的候选基因(TUGs,tentative unique genes)。这些研究结果为玉米丝黑穗病抗性基因的发掘奠定了良好基础,但现有研究结果还尚不能完全满足基因克隆及分子标记辅助育种的需要。本文围绕玉米丝黑穗病主效抗性基因精细定位及分子标记开发开展研究。主要研究内容、方法和结果如下:1.以玉米自交系为材料,采用BSA和AFLP标记相结合的方法,开发了2个与玉米丝穗病抗性相关SCAR标记。利用玉米10个主要抗丝黑穗病病自交系和10个主要感病自交系构建一组抗、感基因池。用这组抗、感基因池随机筛选101对AFLP引物,平均每对引物扩增46个片断,共扩增约4646个片断,其中由56对引物扩增的65个片断在抗、感基因池间呈现多态性。用筛选获得的56对AFLP引物分析组成抗、感池的20个自交系,经χ~2独立性测验,发现7个片断似乎与丝黑穗病抗性相关,分别为P54M71-148、P44M62-331、P46M37-136、P46M37-137、P44M46-147、P38M46-137和P51M38-130。将7个片断回收、克隆和测序,经同源性分析,片断P46M37-136和P46M37-137为同源序列。对6个回收差异片断的序列设计6对引物(根据2个同源序列设计ASP引物),然后利用抗、感基因池及其组成自交系的预扩产物和相应的基因组DNA分别对6对引物进行检验。检验结果表明,6对引物全部转化为SCAR标记,分别为S126、S258、AS136/A137、S98、S131和S100。进一步利用其它54个玉米丝黑穗病抗、感自交系对转化的6个SCAR标记进行验证。经χ~2独立性测验,ASP(χ_c~2=26.06)和S100(χ_c~2=4.13),表明它们与丝黑穗病抗性相关,并将其分别定位于玉米染色体的bin1.09和bin3.08区域。2.利用玉米的2个BC3回交群体,采用BSA和AFLP标记相结合的方法,开发了1个与玉米丝穗病抗性相关SCAR标记。利用2个抗玉米丝黑穗病自交系(Qi319和Mo17,供体亲本)与1个感病自交系(黄早四,轮回亲本)构建2个BC3回交群体(BC3Q群体和BC3M群体)。BC3Q群体,即齐319(高抗,供体亲本)×黄早四(高感,轮回亲本)群体;而BC3M群体,即Mo17(抗病,轮回亲本)×黄早四(高感,轮回亲本)群体。通过对2个BC3群体进行丝黑穗病抗病鉴定筛选得到抗、感极端株系,利用群体中的极端抗、感株系分别构建了两组抗、感基因池。分别利用抗、感基因池及相应的亲本自交系筛选获得了31对AFLP引物,平均每对引物扩增46个片断,共扩增约1426个片断。对于BC3Q群体,有8对引物的11个片断在抗池、抗病亲本齐319和感池、感病亲本黄早四之间呈现多态性;对于BC3M群体,有9对引物的10个片断在抗池、抗病亲本Mo17和感池、感病亲本黄早四间呈现多态性。进一步用这些多态性引物组合分析组成相应抗、感基因池的个体,经χ~2独立性测验,在两个群体中初步发现共有6个片断与丝黑穗病抗性相关,分别为P12M48-215、P13M61-152、P39M46-137、P64M47-204、P13M49-185和P64M47-170。将这6个片断分别回收、克隆及测序,但片断P13M49-185没有回收成功。将其它5个片断进行同源性分析,序列P64M47-204与玉米基因组未知功能的mRNA高度同源。对回收得到的5个序列共设计了7对引物(根据P64M47-204设计3对引物),利用相应抗、感池及组成个体的预扩产物和基因组DNA分别进行验证,只有3对引物成功转化成了SCAR标记,分别是S130、S193和S116。此后,进一步利用相应BC3回交群体中更多抗、感株系和74个玉米自交系进行验证,经χ~2独立性测验,S130与丝黑穗病抗性高度相关,并将其定位于玉米染色体bin2.09区域,即研究发现的抗玉米丝黑穗病主效QTL区域。3.采用SSR和SCAR标记定位玉米抗丝黑穗病主效QTL。利用玉米染色体bin2.09主效QTL区域的5个SSR标记和新开发的SCAR标记S130完善抗病QTL区段遗传连锁图谱,获得平均间距为4.78cM的区段加密图谱。用软件Winqtl cartographer2.5的单标记分析法分析,发现该区域QTL的连锁标记有11个,分别是bnlg1520、umc1525、p3864185、p3946135、bnlg1893、umc1207、umc2184、umc2077、S130、p5138120和p3762147;利用复合区间作图法分析,发现该区域QTL连锁标记有6个,分别为umc1207,umc2184,umc2077,S130和p5138120,它们被包含于单标记分析法获得的连锁标记。继后,以6个QTL连锁标记分析2个BC3回交群体中的抗、感株系,通过比较同一群体内抗病家系和感病家系供体抗病亲本导入片断的大小,拟将玉米丝黑穗病抗性基因定位在一定的区域内。根据BC3Q群体分析结果,将位于bin2.09区域的抗病QTL定位在标记bnlg1893附近,并由标记umc1525和umc1207界定到12cM的区域;根据BC3M群体的分析结果,同时比较QTL定位及BC3Q群体的基因定位结果,推测在玉米基因组bin2.09上可能存在另1个丝黑穗病抗性基因,位于标记S130和p5138120的右侧,暂时未能被明确界定。4.基于抗病与感病玉米自交系序列差异开发了2个STS标记。首先,通过分析www.maizegdb.org网站IBM2 2008 Neighbors Frame2图谱上抗玉米丝黑穗病QTL区域内(标记umc1525和umc1207之间)的标记及相关序列,发现RFLP位点mmp195e的2个相关序列AZ916344和AZ916345均为RGA序列ZmGsstuc11.12-04.4163.2的一部分。然后对ZmGsstuc11.12-04.4163.2序列中片断AZ916344和AZ916345分别设计引物AZ44和AZ45,扩增抗、感自交系齐319、Mo17和黄早四基因组DNA。分析表明,序列AZ916344在3个自交系中序列几乎一致,而序列AZ916345在自交系Mo17,齐319与黄早四中存在2处差异明显的In/Del位点。最后针对这2处In/Del位点再次设计引物MH-1和MH-2,经检测,MH-1和MH-2能在齐319、Mo17和黄早四间得到特异性、多态性扩增产物,随即开发成为2个STS标记,并进一步定位于玉米染色体bin1.09区域。更多还原

【Abstract】 Head smut of maize(Zea mays L.) is a kind of worldwide disease and is also an important constraint in spring maize region of China.Head smut,caused by Sphacelotheca reiliana(K(u|¨)hn) Clint,is a soil-borne or seed-borne systemic disease.The fungus invades plants during emergence or at the seedlings stage through teliospores and grows systemically with the meristem.Infection becomes visible at a late stage of plant development,when tassels and ears are partially or totally replaced by sori filled with teliospores.Quantitive genetics studies have revealed that maize resistance to S.reiliana is under polygenic control and the mode of gene action is predominantly additive or dominant.Further,molecular biology techniques were applied to study inheritance of head smut.Many resistant quantitive trait lici(QTLs) had been detected in the germplasm of China and abroad,and the linkage markers had also been indentified.Moreover,some TUGs(tentative unique genes) had been identified by the methods of SSH and cDNA chip. All the results would be useful for finding resistance genes for S.reiliana,but could not meet for propose of both marker-assisted selection(MAS) and map-based cloning.In the dissertation,we studied head smut of maize on two subjects.One was marker developing,and the other was fine mapping the major resistant gene for S.reiliana.The main methods and results are as follows:1) Basing on inbred lines,a combination of BSA with AFLP method was applied to develop SCAR primers for the detection of resistance to Sporisorium reiliana in maize:The 10 most resistant and the 10 most susceptible inbred lines for head smut of maize were used to form the resistant and the susceptible DNA bulk,respectively.Then the two bulks were analyzed with 101 AFLP primer combinations.About 4646 bands were amplified with an average of 46 bands of each AFLP primer pairs.And 65 polymorphic fragments involving in 55 AFLP primer pairs between the resistant and the susceptible bulk were identified.Further,the 65 polymorphic bands were tested by the 20 inbred lines composed of the two bulks,andχ2 test for independence was done.There were 7 candidate fragments showing association with resistance to S.reiliana,including P54M71-148, P44M62-331,P46M37-136,P46M37-137,P44M46-147,P38M46-137 and P51M38-130. Then they were all extracted,cloned,and sequenced.P46M37-136 and P46M37-137 fragments were homologous with one nucleotide InDel and one single nucleotide transversion.Then all of 6 candidate SCAR primers were designed,including l allele-specific PCR primers(ASP).Each primer was tested by the pre-selective amplification products and genomic DNA of both the two bulks and their composed inbred lines.All the 6 candidate SCAR primers were all developed into SCAR markers.They were S126、S258、AS136/A137、S98、S131and S100.Moreover,the SCAR markers were tested by other 54 inbred lines andχ2 test for independence was done.Only 2 SCAR markers,ASP and S100,were associated with resistance to S.reilian and mapped at chromosome bin1.09 and bin3.08 in maize,respectively.2) Basing on two sets of BC3 progenies,a combination of BSA with AFLP method was applied to develop SCAR primers for the detection of resistance to Sporisorium reiliana in maize:Two sets of BC3 progenies were generated with 2 resistant inbred lines(Mo17 and Qi319) and 1 susceptible inbred line(Huangzao4) of head smut of maize.One was BC3Q, derived from the cross Qi319(donor parent,highly resistant)×Huangzao4(recurrent parent,highly susceptible),the other was BC3M,derived from the cross Mo17(donor parent,highly resistant)×Huangzao4(recurrent parent,highly susceptible).Families of BC3 progenies were evaluated for resistance to S.reiliana under artificial inoculation. Then two sets of resistant and susceptible DNA bulks were prepared respectively,with QR-bulk and QS-bulk coming from BC3Q progeny and MR-bulk and MS-bulk from BC3M progeny.The DNA bulks and corresponding parental lines were analyzed with 31 AFLP primer pairs,respectively.About 1426 bands were amplified with an average of 46 bands of each AFLP primer pairs.For BC3Q,11 polymorphic fragments involving in 8 AFLP primer pairs between QR-bulk,Qi319 and QS-bulk,Huangzao4 were identified.For BC3M,10 polymorphic bands involving in 9 AFLP primer pairs between MR-bulk,Mo17 and MS-bulk,Huangzao4 were identified.Furthermore,these 21 polymorphic bands were tested by the individuals composed of the bulks,andχ2 test for independence was done. There were 6 candidate fragments showing association with resistance to S.reiliana, including P12M48-215,P13M61-152,P39M46-137,P64M47-204,P13M49-185 and P64M47-170.Then they all were extracted,cloned,and sequenced,with 5 candidate bands except P13M49-185 were re-amplified successfully.A search for sequences homologous with the AFLP sequences was conducted in Genbank and the fragment of P64M47-204 showed significant alignments with a part of EU974082.1,a Zea mays clone 439268 mRNA sequence with unknown function.All of 7 candidate SCAR primers were designed basing on the 5 sequeces.Each primer was tested by its corresponding pre-selective amplification products and genomic DNA,both corresponding bulks and their relevant individuals.Thus,3 SCAR markers,S130、S193and S116,were developed.Moreover,the 3 SCAR markers were tested by more resistant and susceptible individuals from the corresponding BC3 progeny and 74 inbred lines,andχ2 test for independence was done respectively.Only 1 SCAR marker S130 was associated with resistance to S.reilian and mapped at chromosome bin 2.09 in maize.3) Mapping of the major QTL conferring resistance to Sporisorium reiliana using SSR and anchored SCAR markers in maize:First,a relatively fine genetic linkage map of chromosome bin 2.09 was generated by adding another 5 SSR markers and 1 SCAR marker S130,with an average distance of 4.78 cM between adjacent markers.With single marker analysis methods,all of 11 markers showed linked to the major QTL resistance to S.reiliana,including bnlg1520,umc1525, p3864185,p3946135,bnlg1893,umc1207,umc2184,umc2077,S130,p5138120 and p3762147.Also,composite interval mapping were employed,and the possible linked markers were umc1207,umc2184,umc2077,S130 and p5138120,which were lying in the linked markers determined by single marker analysis method.Then the QTL linked markers were used to analyze the most resistant and susceptible families of the two sets of BC3 progenies(BC3Q and BC3M in chapter2).Analysis of genotypes between the most resistant and susceptible families of BC3Q progeny allowed delimiting the major QTL into an interval of 12 cM,flanked by the SSR marker umc1525 and umc1207.And according to the genotype analysis for the most resistant and susceptible families of BC3M progeny,we deduced that there was another resistant gene locating at right side of S130 and p5138120 in bin 2.09 except for the one located in the interval(umc1525/umc1207) by comparing the results of QTL mapping and fine mapping resulting from BC3Q progeny.4) Developing STS marker basing on the polymorphic sequences between inbred lines of maize:First,we analyzed all the loci between umc1525 and umc1207 of IBM2 2008 Neighbors Frame2 map,which were in the resistant QTL supporting interval for the head smut of maize.Then two related GSS of RFLP locus mmp195e,AZ916344 and AZ916345, were identified.They were all a part of ZmGsstuc11-12-04.4163.2,which might snythsize proteins having a region of leucine rich repeats(LRRs).Second,two primers,AZ44 and AZ45,were designed according to AZ916344 and AZ916345 fragment basing on ZmGsstuc11-12-04.4163.2 sequence.And the corresponding sequences in Qi319,Mo17 and Huangzao4 were got by PCR method and sequenced and analyzed.The sequences of AZ916344 among Qi319,Mo17 and Huangzao4 were nearly identical,while the corresponding sequences of AZ916345 among them had two In/Del loci.At last,two primers,MH-1and MH-2,were designed according to the two In/Del loci and tested by the genomic DNA of Qi319,Mo17 and Huangzao4,respectively.Polymorphic and specific bands were observed and two STS markers were developed.Further,they were all mapped at chromosome bin1.09 in maize.更多还原