SSR标记在宁波地区水稻品种DNA指纹图谱构建及纯度鉴定中的应用

利用农业行业标准NY/T-1433-2007、NY/T-1433-2014中推荐的53对SSR引物,对宁波地区34个水稻品种进行DNA指纹图谱构建和遗传多样性分析。53对引物在研究材料中共获得183个等位基因,每对引物得到的等位基因数为1～7个,平均每对引物可获得3.5个等位基因。遗传相似性和聚类分析结果表明,34份水稻品种在遗传相似系数为0.5处可分为籼粳两个类群,其中,甬优系列籼粳杂交组合、6个常规粳稻及3个糯稻归为粳稻类群,6个常规籼稻和5个杂交籼稻归为籼稻类群。用RM590和RM3331对19个甬优杂交稻系列品种进行100粒种子的纯度鉴定,仅甬优2640和甬优50检测到1个单株的混杂。结果为SSR标记在宁波水稻品种的纯度和真伪鉴定方面的应用提供了一定的技术支撑。     

利用SSR分子标记构建Y58S及部分重要两系杂交水稻亲本的DNA指纹图谱

以优良光温敏核不育系Y58S及其杂交组合Y两优1号和生产中广泛应用的部分光温敏核不育系和恢复系等18个水稻品种为材料,利用SSR分子标记进行了DNA指纹图谱的构建。聚类分析表明,18个供试水稻品种的遗传相似度为0.672-0.945;利用筛选到的RM164、RM18和RM258等3对引物扩增出的14条多态性片段初步构建了18个供试水稻品种的DNA指纹图谱,每个品种的DNA指纹图谱具有特异性,可互相鉴别;引物RM164可在超级杂交稻组合Y两优1号、两优培九中扩增出父母本的互补带型,从而可鉴别其杂交种子中混杂的母本杂株。     

基于NYT 1433-2014中48对SSR引物的94份杂交稻亲本DNA分子数字指纹库研究

建立方法简便、分辨率高的水稻品种遗传多态性和真实性鉴定的分子指纹技术对于指导水稻育种和规范种子市场都具有重要意义。农业部颁布的水稻品种鉴定技术规程行业新标准是基于35个不同遗传特点的代表性水稻品种建立的SSR分子标记技术规程。本研究根据该标准方法,对94份杂交水稻亲本材料的遗传多态性和特异性进行了比较分析,结果表明,供试品种间至少具有3对以上引物扩增的DNA片段差异,即利用该标准能很好地区分供试杂交水稻亲本的遗传差异。对新标准中48对推荐引物的比较与分析表明,46对引物扩增的DNA片段多态性较高,而RM176和RM551两对引物扩增带多态性较低,因此在其染色体的其他位点可进一步研究多态性更高的分子标记。与标准中35个水稻品种的指纹库进行比较,发现了16个新的等位变异,这些位点可作为标准指纹库的信息补充,丰富标准库中的遗传信息。对94个杂交水稻亲本的分子指纹比较分析,发现23个亲本材料具有特异性分子标记,这些特异分子标记可应用于杂交组合的真实性以及杂交种子纯度的分子鉴定。根据供试亲本的数字分子指纹,构建了87个不育系与7个父本杂交的虚拟组合数字分子指纹库以及虚拟组合的真实性和纯度快速鉴定的特异数字分子标记。     

四川省主要杂交稻亲本的SSR多态性分析和指纹图谱的构建与应用

利用微卫星标记对四川省主推杂交水稻品种进行了DNA指纹图谱构建和品种鉴定研究。208对引物中具有多态性的引物共123对,占所用引物的59.13%。不同染色体的微卫星分析的多态性不同,第9、10染色体微卫星的多态性高于其他染色体,第12染色体上的微卫星标记的多态性最差,仅为46.15%。42份常用杂交水稻亲本材料聚类分析表明,恢复系和不育系的遗传基础均较狭窄,但恢复系和不育系之间的遗传距离相对较远,从一定程度上反映了遗传距离与杂种优势的正相关。筛选出的各个亲本材料的特异引物或引物组合,能够将某个亲本材料与其他材料相区分。利用这些SSR引物建立了四川省主要杂交水稻亲本的DNA指纹数据库,可以有效地解决杂交水稻及其杂交种的鉴定问题,以及有效地分析各材料间的亲缘关系。从DNA指纹数据库中筛选出D优527的特异引物RM337、RM244和RM346,可以鉴定出D优527中的纯度,与田间鉴定结果一致;在真伪性鉴定中将同一不育系配组的D优527和D优68区分开,说明微卫星鉴定结果是准确可靠的,可用于品种权保护和品种真伪性及纯度鉴定。     

利用SSR标记检测棉铃种仁DNA鉴定杂交种纯度

利用棉铃种皮DNA和种仁DNA的差异，通过Simple sequence repeats (SSR)分子鉴定技术在棉花制种后期快速检测制种质量。筛选杂交种亲本间的多态性SSR引物，应用多态性引物鉴定棉铃种仁的指纹图谱，指纹图谱与种皮一致，表明该棉铃为自交铃；种仁图谱为共显性谱带，则该棉铃为杂交铃，计算杂交铃的比例得出杂交种的制种纯度。F₂的图谱分离验证了遗传的分离规律，F₂的个体间具有不同的指纹图谱，育种过程中的杂交后代的分子检测能加速育种进程。     

滇型杂交粳稻主要亲本的SSR 指纹图谱及其遗传差异分析

用筛选的13对SSR引物,构建了41个水稻品种(包括35份滇型杂交粳稻恢复系、保持系材料、2个杂交种以及4个籼稻品系)的分子指纹图谱.聚类分析表明,41个水稻品种间遗传相似系数在0.52～0.94之间;在相似系数0.52处,可以将粳稻与籼稻区分开;在相似系数0.66处,可将杂交粳稻与其它粳稻品系区分开;在相似系数0.71处,又可将大部分恢复系与保持系区分开.生产中应用的一些强优势滇型杂交粳稻的亲本分别聚于不同的类群,表明亲本间的遗传差异较大.     

一个水稻颖壳扭曲突变体的遗传分析与基因定位

 从水稻育种后代材料中获得1个颖壳扭曲突变体Osth (twisted hull)。遗传分析结果表明,该突变性状由单核基因隐性突变造成。以突变体与颖壳正常籼稻R725杂交的F2群体为基因定位群体,利用SSR标记将突变位点定位在第2染色体上的SSR标记RM14128与RM208之间,遗传距离分别为1.4 cM 和2.7 cM。这些结果为该基因的精细定位和克隆以及研究水稻花发育的分子机理奠定了基础。     

甬优系列杂交水稻SSR标记指纹图谱和籼粳属性

用22个SSR分子标记对13份甬优系列杂交水稻组合及其亲本材料和6份籼粳对照种进行分析,共获得87个等位基因,每对引物扩增的等位基因数为2~8个。所有供试材料间均检测到了差异,从而建立起甬优1号~甬优6号杂交水稻组合及其亲本的DNA指纹图谱。聚类分析结果表明,9份供试材料与3份粳稻对照种聚于一类,属粳稻;4份供试材料与籼稻对照种IR36聚于一类,属籼稻。这与形态分类结果基本一致。从分子水平确认了杂交水稻甬优6号和甬优4号为籼粳亚种间杂交稻。     

2个杂交籼稻和2个粳稻品种SSR指纹图谱的构建及双重PCR技术的初步研究

 用460对水稻SSR引物对5个杂交籼稻及其双亲和16个粳稻品种（系）进行了PCR扩增和聚丙烯酰胺凝胶电泳,筛选出12对稳定性好、多态性高、杂带少的SSR引物作为核心引物,构建了国稻6号和两优培九的SSR指纹图谱。另外,还筛选出9对核心引物,构建了粳稻品种盐稻8号和徐稻4号的分子指纹图谱。其中,筛选出国稻6号的特异性标记5个,两优培九的特异性标记3个,盐稻8号的特异性标记1个,徐稻4号的特异性标记2个。这些特异标记可用于种子纯度的快速鉴定。另外,挑选扩增条带稳定、特异性较好的引物组合,进行了双重PCR分析。     

云南元江普通野生稻(Oryza rufipogon）群体籼粳分化的SSR分析

 利用经测验可区分籼粳品种的19对SSR特异引物对来自云南元江普通野生稻自然居群的56个个体进行了SSR分析。19对引物中，有17对（占89.47%）在所有参试个体中仅能扩增出一种带型，而RM18和RM251能扩增出多态带型。RM4等16对（84.21%）引物扩增出的带与籼稻或粳稻特征带相同，其中11个位点偏粳而4个位点偏籼；RM18、RM202、RM205等3对引物扩增出的带型不同于籼稻或粳稻带型。结果表明，云南元江普通野生稻基因组DNA在所检测的19个位点上，多数位点（89.47%）上个体间无差异，84.21%的位点已有籼粳分化，13.79%的位点仍具有原始性。说明云南元江普通野生稻主体比较纯而原始，但已开始了籼粳的分化。     

应用SSR分子标记鉴定超级杂交水稻组合及其纯度

 应用SSR分子标记技术对超级杂交稻5个组合（HYS 1/R105、培矮64S/E32、两优培九、88S/0293、J23A/Q611）及其9个亲本进行了鉴定。用144对SSR引物进行筛选，有47对能够在实验材料中显示较好的多态性，其中，RM337与RM154呈现丰富多态性，可鉴别供试组合并分别与其亲本区分开。对于水稻的每一条染色体，各筛选出两条产生多态性的引物，共24对，并提供一组作为鉴定参考的图谱；通过杂种表现为父母本互补带型的特点，找到在杂交稻组合及其亲本间具有多态性的引物，筛选出5对引物分别作为鉴定上述5个超级杂交稻组合的特异引物，进而针对杂交稻不同的纯度问题设计鉴定方法。     

Identification and Purity Test of Super Hybrid Rice with SSR Molecular Markers

Five super hybrid rice combinations, i.e. HYS-1/R105, Pei‘ai 64S/E32, Liangyoupeijiu (Pei‘ai 64S/9311), 88S/0293, and J23A/Q611, and their parental lines were tested by means of SSR analysis. A total of 144 SSR primer pairs distributed on 12 rice chromosomes were used, out of which 47 detected polymorphism among the tested rice lines. Among all these primers, RM337 and RM154 produced polymorphic patterns in four or more of the tested experimental materials respectively, and they could distinguish among most rice genotypes tested. Twenty-four primer pairs, two on each rice chromosome, were selected to make a reference SSR marker-based fingerprinting for the rice lines. For most of the primer pairs, F1 hybrids mainly showed complementary pattern of both parents, which could be very useful to distinguish the F1 from its parental lines. In addition, 5 primer pairs were selected as special primer pairs for five hybrid rice combinations respectively. By combining the rapid, simple method on DNA extraction, it is suggested that SSR technique has wide prospective in variety authentication and purity identification.     

我国常规稻主栽品种的遗传变异分析

 采用40个SSR标记,分析了329份我国近50年来常规稻主栽品种的遗传变异。结果显示,39个SSR标记具有多态性,在多态性位点共检测到223个等位基因,每个位点2～11个,平均57个;平均Nei基因多样性指数（He）为0632。籼粳亚种间的SSR变异差异明显,籼稻平均等位基因数（Na）和Nei基因多样性指数（Na = 54,He = 0440）均高于粳稻品种（Na = 44,He = 0397）。Nei遗传相似系数表明总体样本具有较小的遗传相似度（I = 0366）,而骨干亲本具有较高的遗传相似度（籼:I = 0590;粳:I = 0590）。这导致了籼粳亚种内较高的遗传一致性（籼:I = 0558;粳:I = 0600）。早、中、晚稻各类型遗传相似度差异明显,晚籼和早粳类型具有较高的遗传变异。籼粳稻品种尤其是粳稻的聚类结果显示出较强的季节型和地域特征。这些均提示育种家应选择更广泛的亲本源以拓宽选育品种的遗传基础。     

Genetic Diversity of Rice Landraces from Lowland and Upland Accessions of China

Genetic diversity of rice landraces from lowland and upland accessions of China was investigated using 66 polymorphic simple sequence repeat (SSR) markers.The total number of alleles detected from all 324 tested accessions was 555 with an average allele number (Na) of 8.409 per locus,the average effective number of alleles (Ne) of 3.574 and the average Shannon’s information index (I) of 1.378.The genetic diversity was higher for the indica landraces compared to the japonica landraces,and the upland landraces were more genetically diverse than the lowland landraces.The SSR markers,RM72,RM232,RM219,RM241,RM224 and RM3 showed the highest rates of polymorphism and these SSR markers were suitable to assess the genetic diversity of rice germplasm resources.A dendrogram of 324 accessions of lowland and upland landraces showed that all rice accessions were mainly subdivided into two groups,japonica and indica,with some being intermediate.The distribution of lowland and upland landraces among the japonica and indica rice groups was distinct,with obvious differentiation between the lowland and upland landraces in japonica rice,but no such clear distinction in indica rice.     

Genetic Analysis and Mapping of TWH Gene in Rice Twisted Hull Mutant

A mutant with twisted hulls was found in a breeding population of rice (Oryza sativa L.). The mutant shows less grain weight and inferior grain quality in addition to twisted hulls. Genetic analysis indicated that the phenotype of mutant was controlled by a single recessive gene (temporarily designated as TWH). To map the TWH gene, an F2 population was generated by crossing the twh mutant to R725, an indica rice variety with normal hulls. For bulked segregant analysis, the bulk of mutant plants was prepared by mixing equal amount of plant tissue from 10 twisted-hull plants and the bulk of normal plants was obtained by pooling equal amount tissue of 10 normal-hull plants. Two hundred and seven pairs of simple sequence repeat (SSR) primers, which are distributed on 12 rice chromosomes, were used for polymorphism analysis of the parents and the two bulks. The TWH locus was initially mapped close to the SSR marker RM526 on chromosome 2. Therefore, further mapping was performed using 50 pairs of SSR primers around the marker RM526. The TWH was delimited between the SSR markers RM14128 and RM208 on the long arm of chromosome 2 at the genetic distances of 1.4 cM and 2.7 cM, respectively. These results provide the foundation for further fine mapping, cloning and functional analysis of the TWH gene.     

A Comparative Study of SSR Diversity in Chinese Major Rice Varieties Planted in 1950s and in the Recent Ten Years （1995-2004）

Forty pairs of SSR markers were used to compare the genetic diversity changes in 151 Chinese major rice varieties planted in 1950s and in the recent ten years. Of 40 SSR loci, 39 were found to be polymorphic while one locus (RM479) monomorphic. A total of 213 alleles were identified from the 39 polymorphic loci. The average number of alleles per locus (Na) was of 5.5, ranging from 2 to 11. Nei’s gene diversity index (He) varied drastically among loci from 0.309 at RM174 to 0.869 at RM418, with an average value of 0.649. There existed significant difference in SSR allelic diversity between indica and japonica subspecies, and indica had more variation than japonica both in Na and He. By comparison with the genetic changes in Na and He, it was revealed that the varieties planted in 1950s had more alleles and higher He than those in the recent ten years both for indica and japonica rices. The difference between two subspecies for Na was significant in a tendency over time (indica: z = 2.677, P = 0.007; japonica: z = 3.441, P = 0.001), but not significant for He (indica: z = 1.471, P = 0.141; japonica: z = 1.932, P = 0.053). Analysis of molecular variance (AMOVA) indicated that there existed significant difference (P < 0.05) in genetic variation between the two periods, of which more genetic variation was contributed by indica (Fst = 0.050) and japonica (Fst = 0.082) subsets. Using locus-by-locus AMOVA procedure, significant genetic differentiations were observed in 13 loci (RM21, RM128, RM147, RM169, RM190, RM221, RM231, RM251, RM253, RM317, RM341, RM418, and RM478) for indica varieties and 11 loci (RM101, RM135, RM152, RM159, RM169, RM190, RM251, RM253, RM311, RM418, and RM478) for japonica ones between the two periods. It was found some alleles had been lost in current major rice varieties as comparing with those in 1950s. Therefore, it should be necessary to exploit more alien elite genetic resources for extension of genetic background in current rice breeding program.     

SSR Marker Analysis on indica-japonica Differentiation of Natural Population of Oryza rufipogon in Yuanjiang, Yunnan Province

It was generally recognized that the common wild rice (Oryza rufipogon Griff.) was the ancestor of cultivated rice (O. sativa L.) and there was indica-japonica differentiation in common wild rice before it evolved into cultivated rice [1-10]. The analysis…     

Genetic Diversity and Relationship of Weedy Rice in Taizhou City, Jiangsu Province, China

Microsatellite markers and morphological charactedstics were used to explore the genetic diversity and possible origin of weedy dce in Taizhou City, Jiangsu Province, China. Fifty-two weedy rice (Oryza sativa L.) accessions were compared with two wild rice, four hybdd rice and five cultivars using 22 simple sequence repeat (SSR) primer pairs. A total of 107 fragments were amplified, averaging 5.6 alleles per primer pair. The polymorphic index content (PIC) values ranged from 0.3077 to 0.7951, averaging at 0.5870. The average genetic distance of all samples ranged from 0.02 to 0.46 with an average of 0.262. The genetic distance among Taizhou weedy rice ranged from 0.03 to 0.44 with an average of 0.224. Cluster analysis showed that all the weedy rice accessions from Taizhou City were indica, and could be subdivided into different genotypes. The majority (86%) of weedy rice was most closely related to hybrid rice. The Taizhou weedy dce accessions were morphologically similar, but still could be delineated into indica or japonica group by some morphological traits. it is suggested that the levels of genetic and morphological diversities of weedy rice in Taizhou City are low and these weedy rice plants originated from the segregating progenies of hybrid rice that had naturally introgressed with cultivated rice.