Development and characterization of microsatellite markers for a mangrove tree species Sonneratia caseolaris (L.) Engler (Lythraceae sensu lato)

Sonneratia caseolaris is a typical non-viviparous mangrove species and a key component of mangrove community in the Indo-West Pacific region. Here we isolated nine microsatellite simple sequence repeat (SSR) loci from the genome of S. caseolaris. Our isolated loci provided SSR markers with polymorphism of 2–6 alleles per locus. The expected and observed heterozygosities ranged from 0.242 to 0.745 and from 0.083 to 0.417, respectively. Cross-species amplification in S. alba and S. ovata showed that a subset of these markers holds promise for these congeneric species. These polymorphic SSR markers would be useful tools for population genetics studies on S. caseolaris as well as other congeneric species.     

Isolation and characterization of polymorphic nuclear microsatellite loci in <Emphasis Type="Italic">Taxus baccata</Emphasis> L.

Seven polymorphic nuclear microsatellite markers for Taxus baccata L. (English yew) were developed using an enriched-library method. An additional polymorphic SSR was obtained by testing eight primer pairs from the congeneric species Taxus sumatrana. Mendelian inheritance for the seven Taxus baccata SSRs was proved by genotyping 17 individuals and 124 megagametophytes (conifer seed haploid tissue). A total of 96 individuals from 5 different populations (10–26 samples per population) were used to estimate genetic diversity parameters. High levels of genetic diversity, with values ranging from 0.533 to 0.929 (6–28 alleles per SSR) were found. No linkage disequilibrium between pairs of loci was detected. All loci but one showed significant departures from Hardy–Weinberg equilibrium. Excess of homozygosity was probably due to high inbreeding in English yew populations, an outcome of low effective population size and/or fragmented distribution. Highly polymorphic SSRs will be used to conduct population genetic studies at different geographical scales and to monitor gene flow.     

Isolation and characterization of microsatellite loci in <Emphasis Type="Italic">Pedicularis verticillata</Emphasis> L. using PCR-based isolation of microsatellite arrays (PIMA)

Pedicularis verticillata L. is a highly valuable herb for traditional Chinese medical treatment. In this report, 11 microsatellite loci from P. verticillata were isolated. The simple sequence repeat (SSR) markers were screened in 23 samples of wild populations of P. verticillata, and eight samples from its sister P. ikomai. The number of alleles ranged from 4 to 13, and value of expected (H _E) and observed (H ₀) heterozygosity was 0.62609–0.89662 and 0–0.95652, respectively. All loci were significantly deviated from Hardy–Weinberg expectations due to the heterozygote deficiency, indicating a dramatic loss of genetic polymorphisms in the restrictedly distributed species. The markers amplified well in the two species are useful for examining genetic diversity and population genetic structure, which, in turn, can provide information for establishing conservation strategy for these endangered species.     

Evaluation of three molecular methods of repetitive element loci for differentiation of <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subsp. <Emphasis Type="Italic">paratuberculosis</Emphasis> (MAP)

The aim of the present study is to evaluate the efficiency of three methods to determine the molecular diversity of 34 Mycobacterium avium subsp. paratuberculosis (MAP) strains isolated from 17 cattle herds. The applied methods included the analysis of sequence polymorphism of the mononucleotide (G1 and G2) and trinucleotide sequences (GGT) of the Short Sequence Repeats (SSR) and the determination of size polymorphism of 9 different Mycobacterial Interspersed Repetitive Units (MIRU) and 6 Variable Number Tandem Repeats (VNTR). Sequence analysis of SSR of 34 isolates showed 4, 6, and 2 alleles of G1, G2, and GGT repeats, respectively. The amplification of the investigated 9 MIRU units revealed only two discriminatory genotyping systems (MIRU2 and MIRU3). Out of 6 VNTR PCR differentiation methods, only one method could be recommended for genotyping purposes. The profile 7g-12g-4ggt-II-b-2 of the combination systems G1-G2-GGT-MIRU2-MIRU3-VNTR1658 dominates among the examined isolates and was detected in 14.7% of the isolates. The use of certain repetitive loci of SSR, MIRU, and VNTR techniques in this study showed greater potential than others for the characterization of MAP isolates. The recommended loci can be used for the epidemiological tracing of MAP field strains and to determine the relationships between isolates in different herds.     

菲律宾蛤仔EST_SSR标记与生长性状的相关分析

研究利用20个微卫星标记对菲律宾蛤仔斑马蛤F2代家系107个个体进行遗传多样性分析,并对标记位点与生长相关性状进行分析。在20个微卫星位点共检测到41个等位基因,各位点等位基因数为2—3个,等位基因片段大小为109—430 bp,平均等位基因数为2.05个。平均有效等位基因数为1.71个,观测杂合度平均值为0.504,期望杂合度的平均值为0.431,平均多态信息含量为0.324。经卡方检验,3个位点SSR11,SSR164和SSR213的基因型分布显著偏离了孟德尔定律(P0.01)。运用SPSS 20.0对20个微卫星位点与菲律宾蛤仔斑马蛤家系生长性状的相关性(壳长、壳宽、壳高和体重)进行连锁显著性检验。结果表明,SSR9位点与壳高存在显著的相关关系(P0.05),SSR135和SSR164位点与壳宽呈显著相关(P0.05),SSR142位点与体重呈显著性相关(P0.05)。研究结果可为菲律宾蛤仔的分子标记辅助选育提供参考。     

Transferability of olive microsatellite loci across the genus <Emphasis Type="Italic">Olea</Emphasis>

The transferability of microsatellite markers developed for olive cultivars (Olea europaea L.) has been tested and confirmed in the Olea complex. Thirty two genotypes, belonging to different taxa of the genus Olea, have been analyzed with four olive SSRs. Positive amplifications at all loci were obtained in 13 taxa (at least one accession per species). Sixty seven different alleles have been detected at the four loci analyzed. Polymorphic products have been observed at the inter- and intra-species level. Some SSR loci have shown multiple amplification products in some species. The high number of unique alleles has allowed the unambiguous discrimination of most accessions. Similarity coefficients and relationships among the Olea taxa have been calculated based on SSR amplification results. The reliability of SSRs as markers for intra-species variability evaluation has been confirmed while their use to explore relationships at the inter-species level is discussed, being dependent on the locus analyzed.Communicated by H.F. Linskens     

Isolation and characterization of microsatellite markers in Dysosma versipellis (Berberidaceae), a rare endemic from China

Dysosma versipellis (Berberidaceae) is an important threatened medicinal plant (TMP) species. Here we isolated nine polymorphic microsatellite loci from D. versipellis using a modified biotin-capture method. Our isolated loci provided SSR markers with polymorphism of 2–7 alleles per locus. The expected and observed heterozygosities ranged from 0.507 to 0.864 and from 0.360 to 0.720, respectively. These markers would be the useful tools for analyzing questions concerning population genetic structure and mating system of D. versipellis.     

Integrative placement and orientation of non-redundant SSR loci in cotton linkage groups by deficiency analysis

A combination of previously mapped and unmapped non-redundant SSR loci, using 381 primer pairs were chromosomally and sub-chromosomally localized by deficiency analysis of two sets of quasi-isogenic interspecific Gossypium hirsutum L. hypoaneuploid F₁ hybrids involving Gossypium barbadense L. and Gossypium tomentosum (Nuttall ex Seemann). Polymorphisms were detected for 369 SSR primer pairs. A total of 318 SSR loci were rendered deficient by the available hypoaneuploid stocks, which included primary monosomics (2n = 51), monotelodisomics and duplication-deficient (segmental trisomic–monosomic) (2n = 52) types. Chromosomal associations were newly determined for 123 SSR loci, of which 90, 106 and 73 were polymorphic in G. tomentosum, G. barbadense, and both sets, respectively. The deficiency tests independently confirmed the recent identifications of linkage groups (LG) A01, A02, A03 and D08 to be chromosome (Chr)-13, Chr-8, Chr-11 and Chr-19, respectively, and collectively delimited LG D02 and D03 to Chr-21 and 24, and their homeologs to Chr-8 and 11. Segmental homeology was detected between Chr-2 and Chr-17 loci, adding to evidence of segmental homeology between Chr-2 and 3 versus Chr-14 and 17. The 318 non-redundant SSR loci localized in this study will enhance the construction of linkage maps and QTL identification in molecular marker assisted selection since the confirmed and newly discovered SSR loci can serve as anchor loci for their respective chromosomes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

基于磁珠富集法开发并筛选西川红景天的微卫星标记

利用FIASCO磁珠富集法,开发和筛选青藏高原特有珍贵植物西川红景天(Rhodiola alsia)多态性微卫星标记。结果表明:用(AG)15和(AC)15两种微卫星标记探针构建富集文库,共获得阳性克隆约2500个;从中随机挑取1200检测,发现具有多态性的阳性克隆达400个;随机挑取200多态性的阳性克隆进行测序,从中获得105个SSR位点,用在线软件primer3-2.3.4成功设计得SSR引物105对;其中45对可以成功扩增,而13对所扩增的片段在相距较远的4个自然居群的24个个体中显示较高的遗传多态性。用4个居群的80个个体检测这13对引物发现,平均等位基因数(A)约为9.192,观测杂合度(Ho)和期望杂合度(He)均值分别约为0.712和0.734,如此高的多态性已经满足后期研究的需要;数个位点在某些居群中显著偏离哈迪温伯格平衡(P0.01),这可能是实际研究的居群并不能达到哈迪—温伯格定律所假设的无限大等理想状态所致。结合此前基于表达序列标签(Expressed Sequence Tag,EST)序列开发的SSR多态性位点,该研究结果为今后利用SSR位点开展西川红景天的居群遗传结构分析和其他研究提供了一组有效工具。     

Analysis of microsatellite locus polymorphism in potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis>) cultivars of Russian breeding

Polymorphism of microsatellite loci of the nuclear genome was examined in 29 cultivars and accessions of wild potato (S. tuberosum, S. stoloniferum, S. demissum, and S. phureja). Nine SSR markers, most informative (PIC = 0.61–0.92) for genotyping of the cultivars of Russian breeding were selected. Polymorphism of the selected SSR loci was characterized, and prevailing, as well as unique SSR allele phenotypes were described. A total of 87 allele phenotypes were identified. The highest number of allele phenotypes was detected for the SSR1 (17), ST83/84 (12), and STRBCS1b (12) loci. The least numbers of allele phenotypes were typical of the ST47/48 (5) and STWIN12G (6) loci. Based on the microsatellite loci analyzed, for each of the cultivars examined, its allele formula was established. The latter can be uses as the cultivar molecular genetic passport. Diagnostic sets of most informative loci (SSR markers), enabling identification of the genotypes of all potato cultivars of Russian breeding examined, were determined     

Development of ten microsatellite markers for <Emphasis Type="Italic">Quercus mongolica</Emphasis> var. <Emphasis Type="Italic">crispula</Emphasis> by database mining

Simple sequence repeats (SSRs) in the NCBI dbEST database were surveyed to identify potential SSR markers for Quercus mongolica. In total, 2,691 gene sequences, mainly from expressed sequence tags (ESTs) for Q. robur and Q. petraea had been registered. Twenty-two PCR primers were designed for SSRs in these sequences and screened for polymorphisms in 16 Q. mongolica trees. Ten loci were easily genotyped and showed polymorphism, with numbers of alleles and expected heterozygosity ranging from 3 to 15 and 0.28 to 0.94, respectively. These EST-SSR markers should be useful for studying the genetic diversity of Quercus species.     

Isolation of microsatellite loci from the endemic and endangered Adriatic sturgeon (<Emphasis Type="Italic">Acipenser naccarii</Emphasis>)

With the aim of elaborating a breeding plan on a captive stock of the highly endangered Adriatic sturgeon (Acipenser naccarii), a total of 10 polymorphic microsatellite loci were isolated from an enriched library. The results of cross amplification of additional 8 loci previously isolated from A. oxyrinchus, A. fulvescens and Scaphyrinchus platorhynchus are also reported. Given the tetraploid condition of the species the genetic variability was estimated basing on the number of alleles per individuals and the average band sharing.     

Development and characterization of microsatellite markers in <Emphasis Type="Italic">Castanea sativa</Emphasis> (Mill.)

Thirty-three simple sequence repeat (SSR) markers were isolated andcharacterized in Castanea sativa (Mill.) from the cultivarGarrone Nero. For the identification of SSR loci, primers were designed on eachside flanking the repeat region and they were initially tested on 5 chestnutsamples using chemiluminescence detection. Twenty four loci where shown to bepolymorphic and the number of alleles detected per locus varied from 2 to 7.Fourteen loci were chosen for the analysis of 20 cultivars grown in North Italyusing the semi-automatic system ABI PRISM 377. These 14 markers showed a highlevel of genetic polymorphism with a total of 90 alleles; the number of allelesranged from 4 to 10 per locus, with an average level of 6.4. The mean expectedand observed heterozygosity were 0.724 (range: 0.649–0.835) and 0.793(range: 0.350–0.950) respectively. The estimated frequency of nullallelesshowed a positive value for 3 loci, but except for 1 locus, the values wereverylow. The total value for the probability of identity was 7.04 ×10^–11. Paternity exclusion probability was very high (0.999),sufficiently high to study pollen flow.     

Isolation and characterization of microsatellites in trembling aspen (Populus tremuloides)

 We have identified, isolated, and characterized microsatellite/simple sequence repeat (SSR) loci in trembling aspen (Populus tremuloides) by screening partial genomic libraries. We have also examined the compatibility and use of the P. tremuloides SSR primers to resolve microsatellites in other Populus species. Fourteen microsatellites were identified from 1600 clones screened. The TC/AG microsatellites were the most abundant. A total of 29 alleles were detected in 36 P. tremuloides individuals at the four SSR loci (two each of di- and tri-nucleotide repeats) characterized. The number of alleles at the SSR loci ranged from 5 to 11, with an average of 7.25 alleles per locus, and the observed heterozygosity ranged from 0.19 to 0.82, with a mean of 0.46 per locus. Although the highest polymorphism was observed for a dinucleotide SSR locus, the trinucleotide SSR loci showed substantial polymorphism. There were 34 unique multilocus genotypes among the 36 P. tremuloides individuals examined, and 89% of the individuals had unique multilocus genotypes. Two pairs of SSR primers were successful in PCR, amplifying genomic DNA and resolving microsatellites of comparable size from Populus deltoides, P. nigra, P.×canadensis, and P. maximowiczii. The microsatellite DNA markers developed could be used for clonal fingerprinting, certification of controlled crosses, genome mapping, marker-assisted early selection, genetic diversity assessments, and conservation and sustainable management of poplar genetic resources. Received: 14 November 1997 / Accepted: 17 November 1997     

An integrative genetic linkage map of winter wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

We constructed a genetic linkage map based on a cross between two Swiss winter wheat (Triticum aestivum L.) varieties, Arina and Forno. Two-hundred and forty F₅ single-seed descent (SSD)-derived lines were analysed with 112 restriction fragment length polymorphism (RFLP) anonymous probes, 18 wheat cDNA clones coding for putative stress or defence-related proteins and 179 simple-sequence repeat (SSR) primer-pairs. The 309 markers revealed 396 segregating loci. Linkage analysis defined 27 linkage groups that could all be assigned to chromosomes or chromosome arms. The resulting genetic map comprises 380 loci and spans 3,086 cM with 1,131 cM for the A genome, 920 cM for the B genome and 1,036 cM for the D genome. Seventeen percent of the loci showed a significant (P < 0.05) deviation from a 1:1 ratio, most of them in favour of the Arina alleles. This map enabled the mapping of QTLs for resistance against several fungal diseases such as Stagonospora glume blotch, leaf rust and Fusarium head blight. It will also be very useful for wheat genetic mapping, as it combines RFLP and SSR markers that were previously located on separate maps. S. Paillard and T. Schnurbusch contributed equally to the work     

基于转录组测序的枫香EST-SSR引物开发及有效性评价

枫香是广西重要的乡土树种之一，具有较高的用材、观赏及药用价值。为验证枫香SSR引物的实际应用效果，该研究基于转录组测序技术，检测枫香SSR位点并设计引物，通过PCR扩增和聚丙烯酰胺凝胶电泳筛选出具有较高多态性的枫香EST-SSR引物，并对1个枫香天然群体进行遗传多样性分析。结果表明：(1)共发掘到23 777个SSR位点，单核苷酸重复类型SSR位点占总位点比例最高(46.54%),在重复次数上5～12次之间的SSR位点占比最高(72.36%)。(2)共开发出262对SSR引物，有效扩增率为53.1%,最终筛选出扩增稳定、条带清晰的引物18对。(3)多态性检测结果显示所有位点均具有多态性，天然群体遗传多样性结果显示该天然群体中等位基因数量(Na)、有效等位基因数量(Ne)、Shannon多样性指数(I)、观测杂合度(Ho)变化范围分别为2～4、1.112 8～2.609 6、0.208 9～1.112 7和0.275 9～1.000 0,平均值分别为2.333 3、1.957 4、0.708 5和0.722 6。综上认为，枫香中占优势的SSR位点重复类型和重复基序与其他物种基本相同，所开...     

Development and characterization of microsatellite markers for <Emphasis Type="Italic">Prunus verecunda</Emphasis> and <Emphasis Type="Italic">Prunus grayana</Emphasis> (Rosaceae)

Simple sequence repeat (SSR) markers were developed for Prunus verecunda and Prunus grayana to help understand the seed dispersal pattern of each species. We isolated and characterized nine microsatellite loci (four from P. verecunda and five from P. grayana). In P. verecunda, the number of alleles detected and the expected heterozygosity of five loci ranged from 11 to 24 and 0.59 to 0.92, respectively. In P. grayana, the number of alleles detected and the expected heterozygosity of five loci ranged from 4 to 14 and 0.62 to 0.86, respectively. These results show that the markers described here will be useful in studying the seed dispersal pattern of Prunus species.     

Simple sequence repeat (SSR) markers in the ectomycorrhizal fungus Laccaria bicolor for environmental monitoring of introduced strains and molecular ecology applications

Simple sequence repeat (SSR) markers were developed from SSR-enriched genome libraries for the ectomycorrhizal basidiomycete Laccaria bicolor. Seven markers were single-locus and amplified unambiguously in L. bicolor. The seven SSR markers were further characterized using an array of 15 L. bicolor strains representative of diverse origins worldwide. The observed number of alleles per locus varied from 5–9 and the values of observed heterozygosity from 0.167 to 0.667. The seven SSR loci could be amplified from DNA extracted from root tips of L. bicolor inoculated pine seedlings. All the L. bicolor ectomycorrhizas analysed exhibited the same SSR multi-locus profile as that detected for the UAMH8232 inoculant strain. The set of markers described represents a potent tool for the monitoring of introduced strains of L. bicolor and for molecular ecology applications.     

Assessing genetic diversity in <Emphasis Type="Italic">Gossypium arboreum</Emphasis> L. cultivars using genomic and EST-derived microsatellites

The cultivated diploid, Gossypium arboreum L., (A genome) is an invaluable genetic resource for improving modern tetraploid cotton (G. hirsutum L. and G. barbadense L.) cultivars. The objective of this research is to select a set of informative and robust microsatellites for studying genetic relationships among accessions of geographically diverse G. arboreum cultivars. From more than 1,500 previously developed simple sequence repeat (SSR) markers, 115 genomic (BNL) and EST-derived (MUCS and MUSS) markers were used to evaluate the allelic diversity of a core panel of G. arboreum accessions. These SSR data enabled advanced genome analyses. A set of 25 SSRs were selected based both upon their high level of informativeness (PIC ≥ 0.50) and the production of clear PCR bands on agarose gels. Subsequently, 96 accessions representing a wide spectrum of diversity of G. arboreum cultivars were analyzed with these markers. The 25 SSR loci revealed 75 allelic variants (polymorphisms) ranging from 2 to 4 alleles per locus. The Neighborjoining (NJ) method, based on genetic dissimilarities, revealed that cultivars from geographically adjacent countries tend to cluster together. Outcomes of this research should be useful in decreasing redundancy of effort and in constructing a core collection of G. arboreum, important for efficient use of this genetic resource in cotton breeding.     

Microsatellite diversity in tetraploid <Emphasis Type="Italic">Gossypium</Emphasis> germplasm: assembling a highly informative genotyping set of cotton SSRs

A series of 320 mapped simple sequence repeats (SSRs) have been used to screen the allelic diversity of tetraploid Gossypium species. Fourty-seven genotypes were analyzed representing (i) the wide spectrum of diversity of the cultivated pool and of the primitive landraces of species G. hirsutum (‘marie-galante’, ‘punctatum’, ‘richmondi’, ‘morrilli’, ‘palmeri’, and ‘latifolium’, and ‘yucatanense’), and (ii) species G. barbadense, G. darwinii and G. tomentosum. The polymorphism of 201 SSR loci revealed 1128 allelic variants ranging from 3 to 17 per locus. Neighbor-joining (NJ) method based on genetic dissimilarities produced groupings consistent with the assignments of accessions both at species and at race level. Our data confirmed the proximity of the Galapagos endemic species G. darwinii to species G. barbadense. Within species G. hirsutum, and as compared to the other 6 races, race yucatanense appeared as the most distant from cultivated genotypes. Race yucatanense also exhibited the highest number of unique alleles. The important informative heterogeneity of the 201 SSR loci was exploited to select the most polymorphic ones that were assembled into three series of genome-wide (i.e. each homoeologous AD chromosome pair being equally represented) and mutliplexable (× 3) SSRs. Using one of these ‘genotyping set’, consisting of 39 SSRs (one 3-plex for each of the 13 AD chromosomes pairs) or 45 loci, we were able to assess the relationships between accessions and the topology in the genetic diversity sampled. Such genotyping set of highly informative SSR markers assembled in PCR-multiplex, while increasing genotyping throughput, will be applicable for molecular genetic diversity studies of large germplasm collections. Electronic Supplementary Material Supplementary material is available in the online version of this article at and is accessible for authorized users.