【作者】 丁成龙；

【导师】 沈益新；

【作者基本信息】 南京农业大学 ， 作物栽培学与耕作学， 2005， 博士

【摘要】 多花黑麦草（Lolium multiflorum Lam．）为黑麦草属中最具利用价值的牧草之一，也是我国南方及其它冷凉地区栽培利用面积最广的冷季型牧草。病害是影响多花黑麦草饲草产量和品质的重要因素，多花黑麦草灰叶斑病是由病原真菌Pyricularia sp.引起的一种严重病害。遗传连锁图的构建及基于基因组DNA分子标记的开发将有助于多花黑麦草的遗传及育种研究。本研究以多花黑麦草为材料，利用简并引物PCR扩增法进行多花黑麦草抗病基因类似物（RGA）的克隆并根据获得的RGA序列设计STS引物；以多花黑麦草细胞质雄性不育分离群体（CMS群体）为材料，以SSR、AFLP、EST-CAPS和RGA-CAPS等分子标记构建多花黑麦草遗传连锁图；以多花黑麦草灰叶斑病抗病植株和感病植株杂交构建F₁抗、感病分离群体，分别采用RGA-CAPS、EST-CAPS以及AFLP等分子标记方法进行抗病基因的分子标记和分析，主要结果如下：1．多花黑麦草RGA的克隆利用简并引物和巢式PCR扩增克隆法从多花黑麦草基因组中获得24000个300-500bp的DNA片段，采用单通道法对所得DNA序列进行测序，对所得序列进行公共数据库的搜索。结果表明，9344个克隆的DNA序列同已知的抗病基因或从其它植物中获得的RGA序列同源性较高，根据所得克隆的核苷酸序列的相似性，通过聚类分析将其分为185个簇，每簇中选取一个具代表性克隆作为多花黑麦草的抗病基因类似物，对185个抗病基因类似物序列进行开放阅读框的检查，舍弃无效序列，最后对115个被选择的克隆序列进行STS引物设计，成功地设计RGA-STS引物113对。2．多花黑麦草遗传连锁图的构建利用多花黑麦草假测交细胞质雄性不育（CMS）分离群体（124个个体），以来自父本和母本的分离位点采用SSR、AFLP、EST-CAPS和RGA-CAPS等标记方法进行多态性的选择和并对父、母本分别构建遗传连锁图。母本连锁图包含362个标记位点，分别包含SSR标记240个、AFLP标记84个、EST-CAPS标记24个和RGA-CAPS标记14个，整个连锁图由LG1—LG7共7个连锁群组成，覆盖全长776.4cM，7个连锁群中最长的为LG3，135cM，最短的为LG5，85.8cM。相邻标记之间的平均距离为2.14cM。以来自父本的分离位点进行作图所得的LG1—LG7共7个连锁群，包含376个标记，其中SSR标记252个、AFLP标记82个、EST-CAPS标记29个和RGA-CAPS标记13个，连锁图全长710.0cM，7个连锁群中最长的为LG6，117.3cM，最短的为LG7，为80.5cM，相邻标记之间的平均距离为1.89cM。同已报道的多年生黑麦草遗传连锁图相比，本研究中所构建的遗传连锁图具有标记密度高、标记分布均匀和连锁图长度中等等特点。3．黑麦草RGA在基因组中的分布应用由RGA序列设计的113对RGA-STS引物进行CMS群体中各个体基因组DNA的PCR扩增，扩增产物分别以识别4到6对碱基的限制性内切酶MseⅠ、AluⅠ，HpyCH4Ⅳ、ScrFⅠ、Sau96Ⅰ、Epy188Ⅲ、DdeⅠ、NlaⅣ、HpyCH4Ⅲ，Fnu4HⅠ、Hpy188Ⅰ，AfaⅠ、MspⅠ、HaeⅢ、HhaⅠ、MboⅠ、HindⅢ、和HinfⅠ共18种进行酶切，酶切产物经2.0％琼脂糖凝胶电脉、显像和多态性检测，共检测到25对引物及相应的限制性内切酶酶切后产生多态性。所检测到的多态性位点经遗传作图，23个标记被标于多花黑麦草遗传连锁图中。结果表明多花黑麦草RGA分布于整个基因组中，但局部有RGA的聚集，其中标记RG124E11-2、RG001E01-2和RG088C11-1聚集于LG2，之间的遗传距离为8.5cM，标记RG192G09-2、RG073A04-3和RG119F12-1聚集于LG7，之间的遗传距离为10.2 cM，标记RG021F04-2和RG227D4-1聚集于LG3，标记间距离仅为2.0cM。RGA-CAPS标记聚集的位点可能存在多花黑麦草抗病基因。4．多花黑麦草抗灰叶斑病的分子标记以多花黑麦草灰叶斑病抗病个体（Sachiaoba）和敏感个体（Minamiaoba）杂交构建F₁分离群体，F₁群体的致病菌接鉴定结果显示多花黑麦草抗灰叶斑病由单个主效基因（LmPi1）控制。采用分群分离分析（BSA）法进行多花黑麦草抗灰叶斑病的AFLP和EST-CAPS以及RGA-CAPS标记筛选，筛选到27个标记同多花黑麦草抗灰斑病基因LmPi1紧密连锁，其中AFLP标记25个、EST-CAPS标记1个和RGA-CAPS标记1个。经连锁分析，结果表明，27个标记构成一个紧密连锁的连锁群，标记间最大遗传距离19.6 cM。其中12个标记包括1个EST-CAPS标记和11个聚集于相同的位点，经QTL分析，该连锁区间支持1-LOD，进一步证实本研究中多花黑麦草抗灰叶斑病由单基因所控制。EST-CAPS标记p56的限制性酶切片段同多花黑麦草LmPi1位点的抗病等位基因一致。参考对照群体的遗传连锁图，抗病基因LmPi1位于多花黑麦草的第五连锁群（LG5）。标记p56试用于多花黑麦草抗灰叶斑病抗病和感病品种的基因型分析结果表明，标记p56对于感病种质中引入抗病基因LmPi1以提高其抗病性具有重要作用。多花黑麦草抗病基因类似物的克隆以及遗传连锁图的构建将为多花黑麦草抗病基因的分子标记、抗病基因的图位克隆以及抗病育种提供了方便。更多还原

【Abstract】 Italian ryegrass （Lolium multiflorum Lam） is one of the most important temperate forage grasses and is widely cultivated in temperate areas of the world and also in the southern of China. Disease is one of the main factors, which affects its forage yield and quality of Italian ryegrass. Ryegrass blast, also called gray leaf spot, is caused by the fungus Pyricularia sp. It is one of the most serious diseases for Italian ryegrass in China and other temperate regions. With construction of genetic linkage map and development of molecular marker based on DNA, it would facilitate genetic research and breeding of Italian ryegrass. The present study was designed according to the NBS-LRR motif of disease resistance gene by isolating the resistance gene analogs from Italian ryegrass using PCR amplification, multiple combinations of degenerate primers and construct a high-density molecular linkage map of Italian ryegrass, one pseudo-testcross full-sib F₁ population consisting of 124 individuals was used to analyze four types of marker: （1） amplified fragment length polymorphism （AFLP） markers, （2）simple sequence repeat （SSR） markers, （3）expressed sequence tag cleaved amplification polymorphism sequence （EST-CAPS） markers and （4）resistance gene analog CAPS （RGA-CAPS） markers. An F₁ population, consisted 162 individuals, composed from a cross between resistant and susceptible cultivars, was used to screen closely linked markers to resistance gene of gray leaf spot disease by AFLP, EST-CAPS and RGA-CAPS. The main results are as follows:1. RGA isolated from the genome of Italian ryegrassThe large-scale cloning of resistance gene analogs （RGAs） was determined in Italian ryegrass. The degenerate primer designed from conserved motifs of known plant resistance gene products were used to amplify genomic DNA sequences by nested PCR amplification. Total 24000 PCR clones were selected and subjected to one pass sequencing for the sequence similarity search with public database. As a result, 9354 clones showed significantly similar levels of identity compared with known resistance genes or RGAs derived from other species. All these clones were clustered and grouped into 185 clusters based on the nucleotide sequence similarity. The representative clone was selected from each cluster and then was defined as Italian ryegrass RGAs. 150 representative clones, which contains continuous open reading frame, they were selected to covert RGAs to STS markers, 113 specific sequence tagged site （STS） primer sets were designed successfully from these RGA sequences.2. Genetic linkage map of Italian ryegrassOne pseudo-testcross full-sib F₁ population generated for the genetic analysis of cytoplasmic male sterility （CMS） analysis, consisting of 124 individuals, was used to construct a high-density linkage map of Italian ryegrass. Male and female molecular-marker linkage maps were developed using SSR, AFLP, EST-CAPS, RGA-CAPS markers, respectively. In the linkage map of female parent, a total of 362 marker loci, which was consisted of 240 SSR markers, 84 AFLP markers, 24 EST-CAPS markers and 14 RGA-CAPS markers, they were divided into seven linkage groups and covered a total length of 776.4cM. The size of the linkage groups ranged from 85.8 to 135.0 cM with an average of 2.14 cM among markers. The male linkage map was consisted of 376 marker loci, 252 SSR markers, 82 AFLP markers, 29 EST-CAPS markers and 13 RGA-CAPS markers among seven groups, which covered the map distance 710.0 cM. The longest and shortest linkage groups were LG6 and LG7, 113.7 cM and 80.5 cM, respectivcly. The average distance is 1.89 cM among markers. These linkage maps were compared with those constructed from perennial ryegrass, the map constructed in this research showed very high-density of markers, moderately in length, and markers distributed evenly.3. Distribution of RGA in the genome of Italian ryegrassCMS F₁ population （consisting 124 individuals） and 113 pairs of RGA primers were used for RGA mapping. Eighty pairs of RGA primers were amplified with one clear anticipated STS （300-600bp）, and then were selected. Using of the 80 pairs of primers to amplify the genomic DNA of two parents and individuals of F₁ population, PCR products digested with endonucleases AluⅠ, AfaⅠ, MboⅠ, MspⅠ, MseⅠ, HinfⅠ, Sau96Ⅰ, HindⅢ, SerFⅠ, HpyCH4Ⅲ, HpyCH4Ⅳ, NlaⅣ, HhaⅠ, Hpy188Ⅰ, Hpy188Ⅲ, HaeⅢ; DdeⅠand Fnu4HⅠ, respectively. Twenty-five polymorphisms were detected and 23 RGA-CAPS markers were mapped to all the seven genetic linkage groups of Italian ryegrass. RGA-CAPS markers distribute all the genome, however, clustered in some loci. Two threemarkers and one two-markers were clustered on the genetic map. Marker RG124E11-2, RG001E01-2 and RG088C11-1 clustered on LG2 within 8.5 cM, marker RG192G09-2, RG073A04-3 and RG119F 12-1 clustered on LG7 within 10.2 cM, marker RG021 F04-2 and RG227D4-1 were mapped on LG3 within 2.0 cM. The region clustered RGA-CAPS markers maybe existed disease resistance locus or loci.4. Screening molecular markers linked to resistance gene of gray leaf spot in Italian ryegrassAn F: population with a cross species from a resistant （Sachiaoba） and a susceptible （Minamiaoba） cultivar was analyzed. The disease severity distribution in the F₁ population suggested that the disease resistance was controlled by a major gene （LmPil）. Analysis of amplified fragment length polymorphisms （AFLP） with bulked segregant analysis identified several markers tightly linked to LmPil. To identify other markers linked to LmPil, expressed sequence tag cleaved amplified polymorphic sequence （EST-CAPS） markers and resistance gene analog CAPS （RGA-CAPS） markers were also screened and analyzed. One EST-CAPS marker, p56, and one EST-CAPS marker, RG036G04-1, were also found to be closely linked to LmPil locus. Twenty-seven markers were mapped to a closely linked group with 19.6 cM in length. Furthermore, twelve markers including one ESI-CAPS marker, p56, and other eleven AFLP markers clustered in one locus. QTL analysis indicated that the region clustered markers supported 1-LOD interval, and the result also indicated one major gene controlled the disease resistance. The restriction pattern of p56 amplification showed a unique fragment corresponding to the resistant allele at the LmPi1 locus. A nkage map constructed from the reference population showed that the LmPi1 locus was located in linkage group 5 （LG5） of Italian rycgrass. Genotype results obtained from resistant and susceptible cultivars indicate that the marker p56 is useful for introduction of the LmPi1 gene into susceptible germplasm in order to develop Italian ryegrass cultivars with enhanced resistance to Italian ryegrass gray leaf spot disease. The cloning of RGAs, construction of high density genetic linkage map and development of molecular markers closely linked to gray leaf spot disease resistance gene will provide for gene targeting, quantitative trait loci mapping, and marker-assisted selection in Italian ryegrass.更多还原