Development and Application of a Whole-Genome Simple Sequence Repeat Panel for High-Throughput Genotyping in Soybean

Among commonly applied molecular markers, simple sequence repeats (SSRs, or microsatellites) possess advantages such as a high level of polymorphism and codominant pattern of inheritance at individual loci. To facilitate systematic and rapid genetic mapping in soybean, we designed a genotyping panel comprised 304 SSR markers selected for allelic diversity and chromosomal location so as to provide wide coverage. Most primer pairs for the markers in the panel were redesigned to yield amplicons of 80–600 bp in multiplex polymerase chain reaction (PCR) and fluorescence-based sequencer analysis, and they were labelled with one of four different fluorescent dyes. Multiplex PCR with sets of six to eight primer pairs per reaction generated allelic data for 283 of the 304 SSR loci in three different mapping populations, with the loci mapping to the same positions as previously determined. Four SSRs on each chromosome were analysed for allelic diversity in 87 diverse soybean germplasms with four-plex PCR. These 80 loci showed an average allele number and polymorphic information content value of 14.8 and 0.78, respectively. The high level of polymorphism, ease of analysis, and high accuracy of the SSR genotyping panel should render it widely applicable to soybean genetics and breeding.     

中国李简单重复序列(SSR)反应体系的建立

探讨中国李品种美丽李(Prunus salicina‘Beauty')简单重复序列(simple sequence repeat,SSR)反应体系中5种主要成分浓度对反应产物的影响.结果表明,引物对中国李有通用性,25μLSSR反应体系中,TaqDNA酶、Mg2 、每个引物、模板DNA和dNTPs等5种成分的适宜浓度分别是:1.5 U、2.0 mmol·L-1、0.8μmol·L-1、30～40 ng和0.16～0.24 mmol·L-1.     

玉米基因组的简单重复序列遗传研究进展

前言在真核生物基因组中重复序列占有很大比重,它的绝大部分存在于诸如间隔序列和调控序列非编码序列中,但它也分布在有些结构基因的序列中,它多为轻度重复序列。植物的基因组重复序列一般占80%左右,基因组较大其重复序列所占比重较大,如玉米基因组(ｈａｐｌｏｉｄｇｅｎｏｍｅ)大约有3×109,其中几乎80%以上是重复序列[12]。重复序列对维持染色体的空间结构、基因的表达、遗传重组都具有重要作用。重复序列单...     

Rapid Evolution of Simple Sequence Repeat Induced by Allopolyploidization

Microsatellite evolution normally occurs in diploids. Until now, there has been a lack of direct experimental evidence for microsatellite evolution following allopolyploidization. In the present study, F₁ hybrids and newly synthesized allopolyploids were derived from Triticum aestivum Chinese Spring × Secale cereale Jinzhou-heimai. One hundred and sixty-three wheat simple sequence repeat (SSR) markers were used to investigate the variation of wheat microsatellites after allopolyploidization and variation of the PCR products of 29 of the SSR markers was observed. Of these 29 SSR markers, 15 were unable to produce products from amphiploids. The other 14 SSR markers did produce products from parental wheat, F₁ hybrids and amphiploids. However, the length of the products amplified from amphiploids was different from the length of the products amplified from parental wheat and F₁ hybrids. Sequencing indicated that the length variation of the 14 microsatellites stemmed mainly from variation in the number of repeat units. The alteration of repeat units occurred in both perfect and compound repeats. In some compound SSR loci, one motif was observed to expand whereas another to contract. Almost all the microsatellite evolution observed in this study could be explained by the slipped-strand mispairing model. The results of this study seem to indicate that stress caused by allopolyploidization might be one of the factors that induce microsatellite evolution. In addition, the findings of present study provided an instance of how simple sequence repeats evolved after allopolyploidization.     

大豆SSR技术研究进展

微卫星DNA是重复单位少于6个核苷酸的简单重复序列。在大部分真核细胞的基因组中有着广泛分布,呈孟德尔式遗传。以此为基础发展起来的SSR标记是一种共显性分子标记,遗传多态性丰富。本文着重介绍近年来SSR技术应用在大豆遗传图谱构建、基因研究、品种鉴定和分子标记辅助育种等方面取得的进展,并初步预测该方法在大豆研究中的发展方向。     

大豆SSR技术研究进展

微卫星DNA是重复单位少于 6个核苷酸的简单重复序列。在大部分真核细胞的基因组中有着广泛分布 ,呈孟德尔式遗传。以此为基础发展起来的SSR标记是一种共显性分子标记 ,遗传多态性丰富。本文着重介绍近年来SSR技术应用在大豆遗传图谱构建、基因研究、品种鉴定和分子标记辅助育种等方面取得的进展 ,并初步预测该方法在大豆研究中的发展方向。     

Simple Sequence Repeat Markers Distinguish among Morphotypes of Sphaeropsis sapinea

Sphaeropsis sapinea is a fungal endophyte of Pinus spp. that can cause disease following predisposition of trees by biotic or abiotic stresses. Four morphotypes of S. sapinea have been described from within the natural range of the fungus, while only one morphotype has been identified on exotic pines in the Southern Hemisphere. The aim of this study was to develop robust polymorphic markers that could be used in both taxonomic and population studies. Inter-short-sequence-repeat primers containing microsatellite sequences and degenerate anchors at the 5′ end were used to target microsatellite-rich areas in an S. sapinea isolate. PCR amplification using an annealing temperature of 49°C resulted in profiles containing 5 to 10 bands. These bands were cloned and sequenced, and new short-sequence-repeat (SSR) primer pairs were designed that flanked microsatellite-rich regions. Eleven polymorphic SSR markers were tested on 40 isolates of S. sapinea representing different morphotypes as well as on 2 isolates of the closely related species Botryosphaeria obtusa. The putative I morphotype was found to be identical to B. obtusa. Otherwise, the markers clearly distinguished the remaining three morphotypes and, furthermore, showed that the C morphotype was more closely related to the A than the B morphotype. The B morphotype was the most genetically diverse, and the isolates could be further divided based on their geographic origins. Sequencing of different alleles from each locus showed that the most polymorphic markers had mutations within a microsatellite sequence.     

Mapping of a Wheat Resistance Gene to Yellow Mosaic Disease by Amplified Fragment Length Polymorphism and Simple Sequence Repeat Markers

Wheat （Triticum aestivum L.） yellow mosaic virus （WYMV） is transmitted by a fungal vector through soil and causes serious wheat yield losses due to yellow mosaic disease, with yellow-streaked leaves and stunted plants. In the present study, the amplified fragment length polymorphisms （AFLP） and simple sequence repeat （SSR） were used to identify the molecular linkages with the resistance gene against WYMV. Bulked segregant analysis was performed with an F2 population derived from the cross of cultivar Ningmai 9 （resistant） × cultivar Yangmai 10 （susceptible）. By screening among the resistant or susceptible parents, the F2 pools and the individuals in the F2 population with 64 combined selective AFLP primers （EcoRI/MseI） or 290 reported SSR primers, a polymorphic DNA segment （approximately 120 bp） was amplified using the primer pair E2/M5, and an SSR marker （approximately 180 bp） was located on wheat chromosome 2A using the primer Xgwm328. Analysis with MAPMAKER/Exp Version 3.0b （Whitehead institute for Biomedical Research, Cambridge, MA, USA） indicated that these two markers were dominantly associated with the resistance gene at distances of 5.4 cM or 17.6 cM, respectively. The resistance gene to WYMV derived from Ningmai 9, is temporarily named YmNM, and was mapped to wheat chromosome 2A.     

DNA导入和系选大豆品种及其亲本遗传关系的SSR标记分析

利用现有品种的变异进行系选并培育新品种是一种重要的育种方法。利用SSR标记, 对系选大豆（Glycine max）新品种和其亲本的遗传组成进行分析, 明确二者的遗传关系, 为大豆系选育种提供依据。使用48对SSR引物对22个大豆品种及其亲本进行分析, 结果表明, 由外源DNA导入育成的3个品种与其亲本的一致性为35.42%–95.83%, 虽然供体相同, 但由于导入的外源DNA不同, 故育成的品种间差异很大。另外19个系选品种与其亲本的一致性为27.08%–89.58%, 其中有6个品种与亲本间的差异小于30%。聚类分析发现, 所有参试品种分为东北春大豆及黄淮和南方大豆2类, 其中有8个品种不能与其亲本聚在一起, 说明系选品种并不完全是由于亲本通过突变获得。此外, 农艺性状与分子标记分析结果差异较大, 说明利用有限的农艺性状评价品种间的遗传关系存在一定的局限性。通过比较系选品种和亲本之间的保守位点发现, 特定等位变异出现次数≥7的位点有14个, 分布在11个连锁群, 其中有7个位点与产量和抗病性等重要农艺性状有关, 说明DNA导入和系选品种在选择变异性状的同时, 使一些与重要农艺性状有关的等位变异得到了保留。     

DNA导入和系选大豆品种及其亲本遗传关系的SSR标记分析

利用现有品种的变异进行系选并培育新品种是一种重要的育种方法。利用SSR标记, 对系选大豆(Glycine max)新品种和其亲本的遗传组成进行分析, 明确二者的遗传关系, 为大豆系选育种提供依据。使用48对SSR引物对22个大豆品种及其亲本进行分析, 结果表明, 由外源DNA导入育成的3个品种与其亲本的一致性为35.42%–95.83%, 虽然供体相同, 但由于导入的外源DNA不同, 故育成的品种间差异很大。另外19个系选品种与其亲本的一致性为27.08%–89.58%, 其中有6个品种与亲本间的差异小于30%。聚类分析发现, 所有参试品种分为东北春大豆及黄淮和南方大豆2类, 其中有8个品种不能与其亲本聚在一起, 说明系选品种并不完全是由于亲本通过突变获得。此外, 农艺性状与分子标记分析结果差异较大, 说明利用有限的农艺性状评价品种间的遗传关系存在一定的局限性。通过比较系选品种和亲本之间的保守位点发现, 特定等位变异出现次数≥7的位点有14个, 分布在11个连锁群, 其中有7个位点与产量和抗病性等重要农艺性状有关, 说明DNA导入和系选品种在选择变异性状的同时, 使一些与重要农艺性状有关的等位变异得到了保留。     

基于SSR分子标记的酸浆属植物亲缘关系研究

近年来, 由于营养价值高、果实可当水果食用且具有潜在的药用价值, 酸浆属(Physalis)植物在全球范围内受到了越来越多的关注。采用SSR分子标记技术对我国范围内主要分布的4种酸浆属植物的22份样品进行了亲缘关系研究, 结果表明, 20对SSR引物共扩增出118个位点, 其中107个(90.7%)为多态性位点; 平均种间遗传相似系数为0.501。UPGMA聚类和PCoA分析结果得出相似结论, 并且将供试酸浆属植物样品分为两大类。其中, 酸浆(P. alkekengi var. francheti)的所有样品与其它酸浆属植物的遗传距离最远, 单独聚为一类, 与前人的研究结果非常吻合。研究表明, SSR标记遗传信息丰富, 可以用于酸浆属植物的亲缘关系分析。研究结果为酸浆属种质资源保护提供了有效信息, 并且为酸浆属植物的分子辅助育种奠定了重要基础。     

SSR分子标记在牡丹亲缘关系研究中的应用与研究进展

SSR标记具有操作简便、共显性和重复性好等特点。该文总结了通过生物信息学技术、磁珠富集法及二代测序技术开发牡丹(Paeonia suffruticosa) SSR标记引物的方法, 并根据现有研究结果解析了牡丹基因组中SSR位点频率及分布, 详细阐述了SSR标记在牡丹种质资源的遗传多样性、亲缘关系和遗传关系等方面的应用概况, 旨在为今后牡丹SSR标记的研究与应用提供参考。     

SED1 Gene Length and Sequence Polymorphisms in Feral Strains of Saccharomyces cerevisiae

The SED1 gene (YDR077W), coding for the major cell wall glycoprotein of Saccharomyces cerevisiae stationary-phase cells, contains two blocks of tandem repeat units located within two distinct regions of the nucleotide sequence. A PCR survey of the SED1 open reading frames (ORFs) of 186 previously uncharacterized grape must isolates of S. cerevisiae yielded 13 PCR profiles arising from different combinations of seven SED1 length variants in individuals homozygous or heterozygous for the gene. Comparison of the nucleotide sequences of a group of representatives of each of the seven length variants with those of S288C and the type strain, CBS1171, unequivocally identified them as SED1 alleles and provided evidence for the presence of two minisatellite-like sequences, variable in length, within the ORF of an S. cerevisiae gene. The segregation analyses of the SED1 length variants and other genetic markers in 13 isolates representative of each PCR profile suggested that molecular mechanisms involved in minisatellite expansion and contraction may be responsible for SED1 heterozygosities within a population of homothallic must isolates of S. cerevisiae.     

Multilevel Control of Organelle DNA Sequence Length in Plants

We have compared the length of noncoding organelle DNA spacers in a broad sample of plant species characterized by different life history traits to test hypotheses regarding the nature of the mechanisms driving changes in their size. We first demonstrate that the spacers do not evolve at random in size but have experienced directional evolutionary trends during plant diversification. We then study the relationships between spacer lengths and other molecular features and various species attributes by taking into account population genetic processes acting within cell lineages. Comparative techniques are used to test these relationships while controlling for species phylogenetic relatedness. The results indicate that spacer length depends on mode of organelle transmission, on population genetic structure, on nucleotide content, on rates of molecular evolution, and on life history traits, in conformity with predictions based on a model of intracellular competition among replicating organelle genomes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

云南栽培稻种SSR 遗传多样性比较

采用64个SSR标记对96份云南水稻(Oryz a sativa)地方品种和选育品种的遗传多样性进行比较分析。结果发现64个标记都具有多态性, 共检测到741个等位基因, 每个多态性位点检测到的等位基因数为2-29个, 平均11.57个; Nei基因多样性指数(He)范围在0.345(RM321)-0.932(RM1)之间, 平均为0.56。水稻品种的遗传多样性并非按地理位置均匀分布, 而是在相 似系数为0.17的水平上明显分为2个不同类群, 即籼稻类群和粳稻类群, 且籼粳亚种间的SSR多样性差异不明显, 籼稻平均等位基因数(Ap)和Nei基因多样性指数(Ap=10.6, He=0.46)与粳稻品种(Ap=10.7, He=0.48)十分接近, 可能与这些品种间存在一定频率的基因交流有关。糯稻和非糯稻在籼稻群和粳稻群中都有表现, 没有特别的分布规律。云南栽培稻选育品种与地方稻亲缘关系较近, 其遗传基础可能来源于云南水稻地方品种。本研究结果表明, SSR标记能较好地区分云南栽培稻品种, 且云南水稻地方品种遗传多样性丰富, 存在大量的优质性状可供育种实践选择。     

Cultivation-Independent Analysis of Fungal Genotypes in Soil by Using Simple Sequence Repeat Markers

Cultivation-independent analyses of fungi are used for community profiling as well as identification of specific strains in environmental samples. The objective of the present study was to adapt genotyping based on simple sequence repeat (SSR) marker detection for use in cultivation-independent monitoring of fungal species or strains in bulk soil DNA. As a model system, a fungal biocontrol agent (BCA) based on Beauveria brongniartii, for which six SSR markers have been developed, was used. Species specificity of SSR detection was verified with 15 fungal species. Real-time PCR was used to adjust for different detection sensitivities of the six SSR markers as well as for different template quantities. The limit for reliable detection per PCR assay was below 2 pg target DNA, corresponding to an estimated 45 genome copies of B. brongniartii. The cultivation-independent approach was compared to cultivation-dependent SSR analysis with soil samples from a B. brongniartii BCA-treated field plot. Results of the cultivation-independent method were consistent with cultivation-dependent genotyping and allowed for unambiguous identification and differentiation of the applied as well as indigenous strains in the samples. Due to the larger quantities of soil used for cultivation-dependent analysis, its sensitivity was higher, but cultivation-independent SSR genotyping was much faster. Therefore, cultivation-independent monitoring of B. brongniartii based on multiple SSR markers represents a rapid and strain-specific approach. This strategy may also be applicable to other fungal species or strains for which SSR markers have been developed.     

云南栽培稻种SSR遗传多样性比较

采用64个SSR标记对96份云南水稻（Oryza sativa）地方品种和选育品种的遗传多样性进行比较分析。结果发现64个标记都具有多态性,共检测到741个等位基因,每个多态性位点检测到的等位基因数为2—29个,平均11．57个：Nei基因多样性指数（He）范围在0．345（RM321）-0．932（RM1）之间,平均为0．56。水稻品种的遗传多样性并非按地理位置均匀分布,而是在相似系数为0．17的水平上明显分为2个不同类群,即籼稻类群和粳稻类群,且籼粳亚种间的SSR多样性差异不明显,籼稻平均等位基因数（Ap）和Nei基因多样性指数（Ap=10．6,He=0．46）与粳稻品种（Ap=10．7,He=0．48）十分接近,可能与这些品种间存在一定频率的基因交流有关。糯稻和非糯稻在籼稻群和粳稻群中都有表现,没有特别的分布规律。云南栽培稻选育品种与地方稻亲缘关系较近,其遗传基础可能来源于云南水稻地方品种。本研究结果表明,SSR标记能较好地区分云南栽培稻品种,且云南水稻地方品种遗传多样性丰富,存在大量的优质性状可供育种实践选择。     

Semi-Automated Simple Sequence Repeat Analysis Reveals Narrow Genetic Base in Indian Potato Cultivars

A study was conducted to generate fingerprints of thirty-two Indian potato cultivars using capillary electrophoresis based semi-automated simple sequence repeat analysis. Five fluorescent-tagged primer pairs (STPOACUTR, LEGAST1, STPOAC58, STM0030 and STM0031) used in this study yielded 43 alleles at 16 different loci, showing multi-loci resolving character. The estimated similarity between the cultivars was in the range of 0.72 to 0.98 indicating narrow genetic relationship. None of the primer set alone could differentiate all 32 cultivars included in this study. However, two primer sets STM-0031 and STPOAC58 amplifying 12 and 9 polymorphic alleles, respectively, could together distinguish all of them. The results indicated usefulness of semi-automated capillary electrophoresis in quick and reproducible SSR genotyping of potato cultivars.