Development and characterisation of simple sequence repeat (SSR) markers for perennial ryegrass (Lolium perenne L.)

Enrichment methods were optimised in order to isolate large numbers of simple sequence repeat (SSR) markers for perennial ryegrass (Lolium perenne L.), with the aim of developing a comprehensive set of loci for trait mapping and cultivar identification. Two libraries were constructed showing greater than 50% enrichment for a variety of SSR-motif types. Sequence characterisation of 1853 clones identified 859 SSR-containing clones, of which 718 were unique. Truncation of flanking sequences limited potential primer design to 366 clones. One-hundred selected SSR primer pairs were evaluated for amplification and genetic polymorphism across a panel of diverse genotypes. The efficiency of amplification was 81%. A relatively high level of SSR polymorphism was detected (67%), with a range of 2–7 alleles per locus. Mendelian segregation of alleles detected by selected SSR-locus primer pairs was demonstrated in the F₁ progeny of a pair cross. Cross-species amplification was detected in a number of related pasture and turfgrass species, with high levels of transfer to other Lolium species and members of the related genus Festuca. The identity of putative SSR ortholoci in these related species was confirmed by DNA sequence analysis. These loci constitute a valuable resource of ideal markers for the molecular breeding of ryegrasses and fescues. Received: 8 May 2000 / Accepted: 13 June 2000     

Comparison of a retrotransposon-based marker with microsatellite markers for discriminating accessions of Vitis vinifera

Identification and knowledge concerning genetic diversity are fundamental for efficient management and use of grapevine germplasm. Recently, new types of molecular markers have been developed, such as retrotransposon-based markers. Because of their multilocus pattern, retrotransposon-based markers might be able to differentiate grapevine accessions with just one pair of primers. In order to evaluate the efficiency of this type of marker, we compared retrotransposon marker Tvv1 with seven microsatellite markers frequently used for genotyping of the genus Vitis (VVMD7, VVMD25, VVMD5, VVMD27, VVMD31, VVS2, and VZAG62). The reference population that we used consisted of 26 accessions of Vitis, including seven European varieties of Vitis vinifera, four North American varieties and hybrids of Vitis labrusca, and 15 rootstock hybrids obtained from crosses of several Vitis species. Individually, the Tvv1 and the group of seven SSR markers were capable of distinguishing all accessions except 'White Niagara' compared to 'Red Niagara'. Using the Structure software, the retrotransposon marker Tvv1 generated two clusters: one with V. vinifera plus North American varieties and the other comprising rootstocks. The seven SSR markers generated five clusters: V. vinifera, the North American varieties, and three groups of rootstock hybrids. The percentages of variation explained by the first two components in the principal coordinate analysis were 65.21 (Tvv1) and 50.42 (SSR markers) while the Mantel correlation between the distance matrixes generated by the two types of markers was 42.5%. We conclude that the Tvv1 marker is useful for DNA fingerprinting, but it lacks efficiency for discrimination of structured groups.     

基于简化基因组数据开发岭南青冈微卫星引物

微卫星标记（simple sequence repeat，SSR）在基因组中普遍存在，具有共显性、高多态性等特点，在群体空间遗传研究中应用广泛。随着测序技术的发展，SSR的开发方法也更加多样化。岭南青冈（Quercus championii Benth）是中国南方常绿阔叶林的珍贵材用树种，对其分子标记的开发可促进岭南青冈的育种和种质保护。本研究基于4个岭南青冈个体的简化基因组序列进行SSR引物的开发。使用pyRAD软件分析显示：①每个个体中包含微卫星重复片段的序列在46 000~84 000条；②个体内聚类后得到的位点数在5 500~24 000；③个体间聚类后获得1 158个一致性位点。在1 158个位点中获得186个侧翼序列没有变异且重复碱基位于序列中间位置的位点。使用Primer Premier 5.0设计了25对引物，在来自3个群体的36个岭南青冈个体中进行验证，结果表明17对引物能成功扩增，共获得106个等位位点，每个引物的等位基因数在2~12，平均为6.2。引物的期望杂合度和观测杂合度分别为0.19~0.88和0.11~0.76。本研究的引物开发方法具有速度快、效率高、成本低的特点，可应用于群体遗传学分子标记的开发。     

Chloroplast microsatellite polymorphisms in Vitis species.

The use of consensus chloroplast microsatellites primers for dicotyledonous chloroplast genomes revealed the existence of intra and interspecific length variation within the genus Vitis. Three chloroplast microsatellite loci were found to be polymorphic in samples of Vitis vinifera, Vitis berlandieri, Vitis riparia, and Vitis rupestris out of a total of 10 consensus primer pairs tested. These polymorphisms were always due to a variable number of mononucleotide residues within A and (or) T stretches in the amplified regions. Chloroplast microsatellite polymorphisms were used to demonstrate the maternal inheritance of chloroplast in V. vinifera and to characterise the chloroplast haplotypes present in wine grape cultivars of this species grown in Spain and Greece. The different distribution of haplotype frequencies in the two ends of the Mediterranean growth area suggests the existence of independent domestication events for grapevine.     

Molecular linkage maps of Vitis vinifera L. and Vitis riparia Mchx

Two linkage maps for grape (Vitis spp.) have been developed based on 81 F(1) plants derived from an interspecific cross between the wine cultivar Moscato bianco (Vitis vinifera L.) and a Vitis riparia Mchx. accession, a donor of pathogen resistance traits. The double pseudotest-cross mapping strategy was applied using three types of molecular markers. The efficiency of SSRs to anchor homologous linkage groups from different Vitis maps and the usefulness of AFLPs in saturating molecular linkage maps were evaluated. Moreover, the SSCP technique was developed based on sequence information in public databases concerning genes involved in flavonoid and stilbene biosynthesis. For the maternal genetic map a total of 338 markers were assembled in 20 linkage groups covering 1,639 cM, whereas 429 loci defined the 19 linkage groups of the paternal map which covers 1,518 cM. The identification of 14 linkage groups common to both maps was possible based on 21 SSR and 19 AFLP loci. The position of SSR loci in the maps presented here was consistent with other published mapping experiments in Vitis.     

Development and evaluation of microsatellite markers in Phoenix dactylifera L. and their transferability to other Phoenix species

Forty one simple sequence repeats were isolated from two microsatellite enriched libraries of date palm (Phoenix dactylifera L.). After screening, 17 selected microsatellite loci were characterized and evaluated on a set of 31 cultivars and clones from Algerian and Californian germplasm. All primer pairs produced an amplification product of the expected size and detected high polymorphism among the analysed samples. These nuclear simple sequence repeat (SSR) markers are expected to be a very effective tool for evaluating genetic diversity in date palm germplasm. Acrosstaxa amplification showed the usefulness of most SSR markers in 14 other species across the genus Phoenix.     

Mining and validating grape (<Emphasis Type="Italic">Vitis</Emphasis> L.) ESTs to develop EST-SSR markers for genotyping and mapping

Grape expressed sequence tags (ESTs) are a new resource for developing simple sequence repeat (SSR) functional markers for genotyping and genetic mapping. An integrated pipeline including several computational tools for SSR identification and functional annotation was developed to identify 6,447 EST-SSR sequences from a total collection of 215,609 grape ESTs retrieved from NCBI. The 6,447 EST-SSRs were further reduced to 1,701 non-redundant sequences via clustering analysis, and 1,037 of them were successfully designed with primer pairs flanking the SSR motifs. From them, 150 pairs of primers were randomly selected for PCR amplification, polymorphism and heterozygosity analysis in V. vinifera cvs. Riesling and Cabernet Sauvignon, and V. rotundifolia (muscadine grape) cvs. Summit and Noble, and 145 pairs of these primers yielded PCR products. Pairwise comparisons of loci between the parents Riesling and Cabernet Sauvignon showed that 72 were homozygous in both cultivars, while 70 loci were heterozygous in at least one cultivar of the two. Muscadine parents Noble and Summit had 90 homozygous SSR loci in both parents and contained 50 heterozygous loci in at least one of the two. These EST-SSR functional markers are a useful addition for grape genotyping and genome mapping.     

Development of 13 EST-SSRs for Cerasus jamasakura and their transferability for Japanese flowering cherries

Simple sequence repeat (SSR) markers were developed for the flowering cherry Cerasus jamasakura (also known as Prunus jamasakura) using 31,995 expressed sequence tags (ESTs) from the NCBI database. Out of 96 of designed primer pairs, 63 showed clear PCR amplification and 13 of these revealed polymorphism in eight individuals sampled across the species’ range. The number of alleles detected and expected heterozygosity ranged from 1 to 8 and 0.000 to 0.833, respectively, when these 13 loci were examined in 23 individuals from a single population. For all except one of the lcoi, polymorphism was also detected in at least four of six other taxa of flowering cherries examined. The results show that the developed EST-SSRs are highly transferable, and that these markers are likely to be useful in studies of the population genetics of flowering cherries.     

Thirty-four Musa (Musaceae) expressed sequence tag-derived microsatellite markers transferred to Musella lasiocarpa

We assembled 31,308 publicly available Musa EST sequences into 21,129 unigenes; 4944 of them contained 5416 SSR motifs. In all, 238 unigenes flanking SSRs were randomly selected for primer design and then tested for amplification in Musella lasiocarpa. Seventy-eight primer pairs were found to be transferable to this species, and 49 displayed polymorphism. A set of 34 polymorphic SSR markers was analyzed in 24 individuals from four wild M. lasiocarpa populations. The mean number of alleles per locus was 3.0, ranging from 2 to 7. The observed and expected heterozygosities per marker ranged from 0.087 to 0.875 (mean 0.503) and from 0.294 to 0.788 (mean 0.544), respectively. These markers will be of practical use for genetic diversity and quantitative trait loci analysis of M. lasiocarpa.     

Development of 11 EST-SSRs for Japanese white birch, Betula platyphylla var. japonica and their transferability to related species

Simple sequence repeat (SSR) markers were developed for Japanese white birch, Betula platyphylla var. japonica, using previously designed primer pairs derived from expressed sequence tags (ESTs). Out of 98 unpublished primer pairs, 35 yielded clear PCR amplification products, 11 of which revealed polymorphism in eight individuals sampled across the species’ range. The number of alleles detected and the expected heterozygosity ranged from 1 to 10 and 0.000 to 0.857, respectively, when these 11 loci were examined in 24 individuals from a single B. platyphylla var. japonica population. In cross-species transferability tests most of the 11 loci were also polymorphic in three other Betula species examined, but not B. maximowicziana. We have now developed a total of 25 polymorphic EST-SSRs for the genus Betula (including 14 we previously developed), which are likely to be highly useful in studies of various aspects of population genetics, including hybridization dynamics, in the genus.     

Isolation and characterization of new polymorphic simple sequence repeat loci in grape (Vitis vinifera L.).

Four new simple sequence repeat (SSR) loci (designated VVMD5, VVMD6, VVMD7, and VVMD8) were characterized in grape and analyzed by silver staining in 77 cultivars of Vitis vinifera. Amplification products ranged in size from 141 to 263 base pairs (bp). The number of alleles observed per locus ranged from 5 to 11 and the number of diploid genotypes per locus ranged from 13 to 27. At each locus at least 75% of the cultivars were heterozygous. Alleles differing in length by only 1 bp could be distinguished by silver staining, and size estimates were within 1 or 2 bp, depending on the locus, of those obtained by fluorescence detection at previously reported loci. Allele frequencies were generally similar in wine grapes and table grapes, with some exceptions. Some alleles were found only in one of the two groups of cultivars. All 77 cultivars were distinguished by the four loci with the exception of four wine grapes considered to be somatic variants of the same cultivar, 'Pinot noir', 'Pinot gris', 'Pinot blanc', and 'Meunier'; two table grapes that are known to be synonymous, 'Keshmesh' and 'Thompson Seedless'; and three table grapes, 'Dattier', 'Rhazaki Arhanon', and 'Markandi', the first two of which have been suggested to be synonymous. Although the high polymorphism at grape SSR loci suggests that very few loci would theoretically be needed to separate all cultivars, the economic and legal significance of grape variety identification requires the increased resolution that can be provided by a larger number of loci. The ease with which SSR markers and data can be shared internationally should encourage their broad use, which will in turn increase the power of these markers for both identification and genetic analysis of grape. Key words : grape, Vitis, microsatellite, simple sequence repeat, DNA typing, identification.     

A dense single-nucleotide polymorphism-based genetic linkage map of grapevine (Vitis vinifera L.) anchoring Pinot Noir bacterial artificial chromosome contigs

The construction of a dense genetic map for Vitis vinifera and its anchoring to a BAC-based physical map is described: it includes 994 loci mapped onto 19 linkage groups, corresponding to the basic chromosome number of Vitis. Spanning 1245 cM with an average distance of 1.3 cM between adjacent markers, the map was generated from the segregation of 483 single-nucleotide polymorphism (SNP)-based genetic markers, 132 simple sequence repeats (SSRs), and 379 AFLP markers in a mapping population of 94 F(1) individuals derived from a V. vinifera cross of the cultivars Syrah and Pinot Noir. Of these markers, 623 were anchored to 367 contigs that are included in a physical map produced from the same clone of Pinot Noir and covering 352 Mbp. On the basis of contigs containing two or more genetically mapped markers, region-dependent estimations of physical and recombinational distances are presented. The markers used in this study include 118 SSRs common to an integrated map derived from five segregating populations of V. vinifera. The positions of these SSR markers in the two maps are conserved across all Vitis linkage groups. The addition of SNP-based markers introduces polymorphisms that are easy to database, are useful for evolutionary studies, and significantly increase the density of the map. The map provides the most comprehensive view of the Vitis genome reported to date and will be relevant for future studies on structural and functional genomics and genetic improvement.     

Development of 14 EST-SSRs for Betula maximowicziana and their applicability to related species

Simple sequence repeats (SSRs) markers were developed for Betula maximowicziana using 2698 expressed sequence tags (ESTs) from the NCBI database. Out of 112 designed primer pairs, 54 showed clear PCR amplification and 14 of these revealed polymorphism in eight individuals sampled across the species’ range. The number of alleles detected and the expected heterozygosity ranged from 1 to 3 and 0.000 to 0.570, respectively, when these 14 loci were examined in 49 individuals from a single population. In the cross species transferability test, eight of the 14 loci were also polymorphic in all four of the diploid, tetraploid and hexaploid Betula species examined. These results showed high transferability of the developed EST-SSRs and that these markers are likely to be useful in studies of the population genetics of species in the genus Betula.     

Development and transferability of apricot and grape EST microsatellite markers across taxa

EST microsatellite markers were developed in apricot (Prunus armeniaca L.) and grape (Vitis vinifera L.). cDNA libraries from either apricot leaves or grape roots were used in an enrichment procedure for GA and CA repeats. The transferability of EST simple sequence repeat (SSR) markers from apricot and grapevine to other related and unrelated species was examined. Overall, grape primers amplified products in most of the Vitaceae accessions while the apricot primers amplified polymorphic alleles only in closely related species of the Rosaceae. In this taxonomic family, ten EST SSR loci were tested, and one single primer pair, PacB22, was amplified across species and sections in the Prunoideae and Maloideae. Sequencing of EST SSR loci in other species and genera confirmed a higher level of conservation in the microsatellite motif and flanking regions in the Vitaceae compared to the Rosaceae. Two distinct fragments of the PacB22 locus amplified across the Malus and Pyrus genera; however, while the coding region was highly conserved, the microsatellite repeat motif was no longer present. The banding pattern was explained by base substitution and insertion/deletion events in the intronic region of PacB22. This study includes the determination of the degree of polymorphism detected among species and genera in two unrelated taxonomic families and the evaluation of the information provided by the microsatellite repeats and the flanking regions.     

Development and characterization of polymorphic microsatellite markers for Neolitsea sericea using Illumina paired‐end draft sequencing data

Simple sequence repeat (SSR) markers were developed and characterized for Neolitsea sericea (Bl.) Koidz. (Lauraceae). Out of 196 designed primer pairs, a total of 144 pairs showed amplification, of which 44 had clear and stable chromatograms. Polymorphism of these 44 loci was tested using 32 individuals sampled from a single population of N. sericea. The number of alleles and the polymorphism information content varied from 3 to 12 and 0.271 to 0.853, respectively. A significant departure from the Hardy‐Weinberg equilibrium was observed in one of the 44 loci. These SSR markers are useful for population genetic studies and parentage analysis in N. sericea, which is one of the most common evergreen species in coastal Pinus thunbergii forests in central‐western Japan.     

Development and characterisation of simple sequence repeat (SSR) markers for white clover (Trifolium repens L.)

Highly informative molecular markers, such as simple sequence repeats (SSRs), can greatly accelerate breeding programs. The aim of this study was to develop and characterise a comprehensive set of SSR markers for white clover (Trifolium repens L.), which can be used to tag genes and quantitative trait loci controlling traits of agronomic interest. Sequence analysis of 1123 clones from genomic libraries enriched for (CA) _n repeats yielded 793 clones containing SSR loci. The majority of SSRs consisted of perfect dinucleotide repeats, only 7% being trinucleotide repeats. After exclusion of redundant sequences and SSR loci with less than 25 bp of flanking sequence, 397 potentially useful SSRs remained. Primer pairs were designed for 117 SSR loci and PCR products in the expected size range were amplified from 101 loci. These markers were highly polymorphic, 88% detecting polymorphism across seven white clover genotypes with an average allele number of 4.8. Four primer pairs were tested in an F₂ population revealing Mendelian segregation. Successful cross-species amplification was achieved in at least one out of eight legume species for 46 of 54 primer pairs. The rate of successful amplification was significantly higher for Trifolium species when compared to species of other genera. The markers developed in this study not only provide valuable tools for molecular breeding of white clover but may also have applications in related taxa. Received: 3 April 2000 / Accepted: 12 May 2000     

基于SSR分子标记分析云南月季种质资源亲缘关系(英文)

利用简单重复序列SSR(simple sequence repeat)标记技术对48份月季种质资源(包括14份野生种、20份古老月季品种和14份栽培品种)的遗传多样性和亲缘关系进行了分析.结果表明,(1)18对SSR引物在18个SSR位点上共检测到160个等位基因,每一位点的等位基因变幅为6～13个,平均8.9个,材料间遗传相似系数变化范围为0.157 8～0.754 9,表明在分子水平上云南月季种质资源具有丰富的遗传多样性.(2)聚类分析结果显示,在相似系数为0.44处,可将 14个野生种明显分为6个组,这与植物形态学分类结果上基本一致;在遗传相似系数为0.40水平上将48份供试材料分为八大组群.(3)亲缘关系分析结果显示,5个野生种和所有古老月季品种与大多数栽培品种的亲缘关系较近.     

Development of 28 EST‐SSR markers based on transcriptome sequences of Gobiobotia filifer and cross‐species amplification

Gobiobotia filifer is a small benthic fish distributed in Yangtze River Basin. The abundance of G. filifer increased after impoundment of Xiluodu Dam and Xiangjiaba Dam. The state of population structure and changes of genetic diversity before and after impoundment of Xiluodu Dam and Xiangjiaba Dam were interesting issues. However, efficient molecular markers were rare, which will limit us to solve above problems. Twenty‐eight expressed sequence tag SSRs (EST‐SSRs) were successfully identified and verified as stable amplification and polymorphic loci by polyacrylamide gel electrophoresis (PAGE) and capillary electrophoresis. The number of alleles at these EST‐SSR loci ranged from 3 to 14, the polymorphism information content values were 0.125–0.897, and the observed and expected heterozygosities were 0.0–0.857 and 0.132–0.928, respectively. Cross‐species amplification of the 28 loci developed in this study was examined in seven individuals of each of the 7 taxa. The amplification efficiency of 28 EST‐SSRs primer pairs is related to the distance of genetic relationship between cross‐species with G. filifer, and same subfamily species (Xenophysogobio boulengeri and Xenophysogobio nudicorpa) showed the highest (50%) amplification efficiency. These EST‐SSR markers could be used to analyse genetic diversity and population structure of G. filifer and related species.     

Cross-species microsatellite amplification in Vasconcellea and related genera and their use in germplasm classification.

To generate inexpensive and efficient DNA markers for addressing a number of population genetics problems and identification of wild hybrids in Vasconcellea, we have evaluated the use of simple sequence repeat (SSR) primers previously developed for other species. A set of 103 Vasconcellea accessions and some individuals of the related genera Carica and Jacaratia were analyzed with 10 primer pairs directing amplification of chloroplast microsatellites in Nicotiana tabacum and 9 nuclear SSR primer pairs recently identified in Vasconcellea x heilbornii. Heterologous amplification of chloroplast SSRs was successful for 8 of the 10 loci, of which 6 showed polymorphism. Seven of the 9 nuclear SSR primer pairs were useful in Vasconcellea and often also in Jacaratia and Carica, all revealing polymorphism. Exclusive haplotypes for each described taxon were identified based on chloroplast microsatellite data. Clustering based on separate nuclear and chloroplast data resulted in a clear grouping per taxon, but only low resolution was obtained above species level. The codominancy of nuclear SSRs and the general high polymorphism rate of SSR markers will make them more useful in future population genetics studies and diversity assessment in conservation programs.     

Development of microsatellite and amplicon length polymorphism markers for Camellia japonica L. from tea plant (Camellia sinensis) expressed sequence tags

Simple sequence repeats and amplicon length polymorphism markers for Camellia japonica were developed, based on Camellia sinensis sequences in the National Center for Biotechnology Information database. In total, 2495 gene sequences were used to design 216 primer pairs. To identify amplicon length polymorphism markers, 61 gene loci in 16 Camellia individuals were re-sequenced. In total, 10 markers (three expressed sequence tags-simple sequence repeats and seven amplicon length polymorphisms) yielded polymerase chain reaction products with clear polymorphic patterns and were used for genotyping 22 C. japonica individuals from a population. Numbers of alleles and expected heterozygosity ranged from two to 13 and from 0.28 to 0.90, respectively.