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.     

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.     

Characterization of chloroplast DNA microsatellites from Saccharum spp and related species

Microsatellites, or simple sequence repeats (SSRs), and their flanking regions in chloroplast genomes (plastomes) of some species of the family Poaceae were analyzed in silico to look for DNA sequence variations. Comparison of the complete chloroplast DNA sequences (cpDNAs) of sugarcane (Saccharum hybrid cv. SP-80-3280 and S. officinarum cv. NCo310) and related species, Agrostis stolonifera, Brachypodium distachyon, Hordeum vulgare subsp vulgare, Lolium perenne, Oryza nivara, O. sativa subsp indica, O. sativa subsp japonica, Sorghum bicolor, Triticum aestivum, Zea mays, and Z. mays cv. B73, allowed us to examine the organization of chloroplast SSRs (cpSSRs) in genic and intergenic regions. We identified 204 cpSSRs in the sugarcane cpDNA; 22.5% were in genic regions. The ndh, rps, trn, and rpl gene clusters of the chloroplasts had the most repeats. Mononucleotide repeats were the most abundant cpSSRs in these species; however, di-, tri-, tetra-, penta-, and hexanucleotide repeats were also identified. Many base substitutions and deletions/insertions were identified in the cpSSR loci and their flanking regions. Multiple alignments of all cpSSR sequences of Poaceae species made identification of nucleotide variability possible; repeat motifs are not uniformly distributed across the Poaceae plastomes, but are mostly confined to intergenic regions. Phylogeny was determined by maximum parsimony and neighbor-joining inference methods. The cpSSRs of these species were found to be polymorphic. It appears that individual cpSSRs in the Poaceae are stable, at least over short periods of evolutionary time. We conclude that the plastome database can be exploited for phylogenetic analysis and biotechnological development.     

Development and validation of microsatellite markers for Karnal bunt (Tilletia indica) and loose smut (Ustilago segetum tritici) of wheat from related fungal species

Simple sequence repeats (SSRs) are preferred molecular markers because of their abundance, robustness, high reproducibility, high efficiency in detecting variation and suitability for high‐throughput analysis. In this study, an attempt was made to mine and analyse the SSRs from the genomes of two seed‐borne fungal pathogens, viz Ustilago maydis, which causes common smut of maize, and Tilletia horrida, the cause of rice kernel smut. After elimination of redundant sequences, 2,703 SSR loci of U. maydis were identified. Of the remaining SSRS, 44.5% accounted for di‐nucleotide repeats followed by 29.8% and 2.7% tri‐ and tetranucleotide repeats, respectively. Similarly, 2,638 SSR loci were identified in T. horrida, of which 20.2% were di‐nucleotide, 50.4% tri‐ and 20.5% tetra‐nucleotide repeats. A set of 65 SSRs designed from each fungus were validated, which yielded 23 polymorphic SSRs from Ustilago and 21 from Tilletia. These polymorphic SSR loci were also successfully cross‐amplified with the Ustilago segetum tritici and Tilletia indica. Principal coordinate analysis of SSR data clustered isolates according to their respective species. These newly developed and validated microsatellite markers may have immediate applications for detection of genetic variability and in population studies of bunt and smut of wheat and other related host plants. Moreover, this is first comprehensive report on molecular markers suitable for variability studies in wheat seed‐borne pathogens.     

An extreme cytoplasmic bottleneck in the modern European cultivated potato (Solanum tuberosum) is not reflected in decreased levels of nuclear diversity

We have used the polymorphic chloroplast (cp) and nuclear simple sequence repeats (SSRs) to analyse levels of cytoplasmic and nuclear diversity in the gene pool of the European cultivated potato (Solanum tuberosum ssp. tuberosum). Primers designed from the complete chloroplast sequence of tobacco (Nicotiana tabacum) were used to amplify polymorphic products in a range of potato cultivars. Combining the data from seven polymorphic cpSSR loci gave 26 haplotypes, one of which (haplotype A) accounted for 151 out of the 178 individuals studied and corresponded to the T-type cytoplasm previously identified in cultivated potatoes using chloroplast restriction fragment length polymorphism analysis. Phylogenetic and diversity analyses of the relationships between cpSSR haplotypes confirmed much higher levels of cytoplasmic diversity outwith the T-type group. Diversity levels at eight nuclear SSR loci, however, were not significantly different between cytoplasmic groups, suggesting a severe maternal bottleneck in the evolution of the modern cultivated potato. These results highlight the importance in quantifying levels of cytoplasmic as well as nuclear diversity and confirm the need for a change in breeding practices to increase levels of non-T-type cytoplasm in the cultivated gene pool, thus helping reduce problems associated with pollen sterility. This may be facilitated by germplasm analysis using cpSSRs, which will allow efficient selection of diverse cytoplasm donors.     

Polymorphic dinucleotide microsatellites in tongue sole (Cynoglossus semilaevis)

The tongue sole, Cynoglossus semilaevis, is a rare marine flatfish distributed in Chinese coastal waters. From a (GT)_n‐enriched genomic library, 57 microsatellites were isolated and characterized. Seventeen of these loci were polymorphic in a test population with alleles ranging from three to 13, and observed and expected heterozygosities from 0.1613 to 1.0000 and from 0.2126 to 0.8983, respectively. Five loci deviated from the Hardy–Weinberg equilibrium in the sampled population, and linkage disequilibrium between two loci was significant after applying Bonferroni correction. Three additional fish species assessed for cross‐species amplification revealed that only one locus was also polymorphic in one species. These polymorphic microsatellite loci should provide sufficient level of genetic diversity to evaluate the breeding strategy and investigate the fine‐scale population structure in C. semilaevis.     

Characterization of comparative genome‐derived simple sequence repeats for acanthopterygian fishes

Simple sequence repeats (SSRs) have become one of the most popular molecular markers for population genetic studies. The application of SSR markers has often been limited to source species because SSR loci are too labile to be maintained in even closely related species. However, a few extremely conserved SSR loci have been reported. Here, we tested for the presence of conserved SSR loci in acanthopterygian fishes, which include over 14 000 species, by comparing the genome sequences of four acanthopterygian fishes. We also examined the comparative genome‐derived SSRs (CG‐SSRs) for their transferability across acanthopterygian fishes and their applicability to population genetic analysis. Forty‐six SSR loci with conserved flanking regions were detected and examined for their transferability among seven nonacanthopterygian and 27 acanthopterygian fishes. The PCR amplification success rate in nonacanthopterygian fishes was low, ranging from 2.2% to 21.7%, except for Lophius litulon (Lophiiformes; 80.4%). Conversely, the rate in most acanthopterygian fishes exceeded 70.0%. Sequencing of these 46 loci revealed the presence of SSRs suitable for scoring while fragment analysis of 20 loci revealed polymorphisms in most of the acanthopterygian fishes. Population genetic analysis of Cottus pollux (Scorpaeniformes) and Sphaeramia orbicularis (Perciformes) using CG‐SSRs showed that these populations did not deviate from linkage equilibrium or Hardy–Weinberg equilibrium. Furthermore, almost no loci showed evidence of null alleles, suggesting that CG‐SSRs have strong resolving power for population genetic analysis. Our findings will facilitate the use of these markers in species in which markers remain to be identified.     

Isolation and characterization of microsatellite markers for Brachiaria brizantha (Hochst. ex A. Rich.) Stap

The first set of nuclear simple sequence repeat (SSR) loci for Brachiaria brizantha (Hochst. ex A. Rich.) Stap is described. A microsatellite-enriched library was constructed and 19 loci were characterized. About 13 SSR loci were found to be polymorphic and across-taxa amplification tests showed that six of them can be transferred to four other species of Brachiaria. This new SSR resource will be a powerful tool for population genetic studies of B. brizantha, for interspecific genetic studies within the genus Brachiaria, for mapping and for marker assisted selection in breeding.     

Development of EST‐SSRs from the Alpine Lady‐fern,Athyrium distentifolium

The utility of EST‐simple sequence repeats (EST‐SSRs) was evaluated in the fern Athyrium distentifolium. From 1152 frond cDNA clones, 165 microsatellites, including di‐, tri‐, tetra and penta‐nucleotide repeat motifs, were identified. Primer design was possible for 74 of the SSRs; subsequent screening of 10 loci on 186 individuals from six natural populations revealed between two and seven alleles per locus and expected heterozygosity (H_E) estimates ranging from 0.027 to 0.809. Eight of these loci were further examined for cross‐species and cross‐generic amplification in other Woodsiaceae species, and polymorphic products were detected. EST‐derived SSRs provide robust, informative and potentially transferable polymorphic markers suitable for biodiversity research.     

Microsatellite loci for the southern pine beetle (Dendroctonus frontalis) and cross‐species amplification in Dendroctonus

We developed microsatellite loci for the southern pine beetle (Dendroctonus frontalis). Twelve microsatellite loci were identified. Eight loci were polymorphic and sufficiently variable in 62 individuals (expected heterozygosity ranged from 0.707 to 0.880) to investigate population structure. All loci conformed to HWE except Dfr‐14, which showed heterozygote excess, and no two loci deviated from linkage equilibrium. The loci were tested for cross‐species amplification in four species of Dendroctonus (D. valens, D. terebrans, D. brevicomis, and D. ponderosae). Seven loci were polymorphic in at least one of the species tested.     

Isolation and characterization of microsatellites in Chinese soft‐shelled turtle,Pelodiscus sinensis

Fifteen polymorphic microsatellite loci were developed for the Chinese soft‐shelled turtle (Pelodiscus sinensis) from the (GT)_n microsatellite‐enriched genomic library, using the fast isolation by amplified fragment length polymorphism of sequences containing repeats protocol. The polymorphism of all 15 loci ranged from two to seven alleles with observed heterozygosities ranging from 0.03 to 0.98 (mean 0.43) in one population of 40 individuals. These novel loci will be helpful for understanding the population structure at genetic level and marker‐assisted breeding of this vulnerable species.     

Tall fescue genomic SSR markers: development and transferability across multiple grass species

Simple sequence repeat (SSR) markers are highly informative and widely used for genetic and breeding studies. Currently, a very limited number of SSR markers are available for tall fescue (Festuca arundinacea Schreb.) and other forage grass species. A tall fescue genomic library enriched in (GA/CT) _n repeats was used to develop primer pairs (PPs) flanking SSRs and assess PP functionality across different forage, cereal, and turf grass species. A total of 511 PPs were developed and assessed for their utility in six different grass species. The parents and a subset of a tall fescue mapping population were used to select PPs for mapping in tall fescue. Survey results revealed that 48% (in rice) to 66% (in tall fescue) of the PPs produced clean SSR-type amplification products in different grass species. Polymorphism rates were higher in tall fescue (68%) compared to other species (46% ryegrass, 39% wheat, and 34% rice). A set of 194 SSR loci (38%) were identified which amplified across all six species. Loci segregating in the tall fescue mapping population were grouped as loci segregating from the female parent (HD28-56, 37%), the male parent (R43-64, 37%), and both parents (26%). Three percent of the loci that were polymorphic between parents were monomorphic in the pseudo F1 mapping population and the remaining loci segregated. Sequencing of amplified products obtained from PP NFFAG428 revealed a very high level of sequence similarity among the grass species under study. Our results are the first report of genomic SSR marker development from tall fescue and they demonstrate the usefulness of these SSRs for genetic linkage mapping in tall fescue and cross-species amplification.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Comparative analysis of genetic diversity in the mangrove species Avicennia marina (Forsk.) Vierh. (Avicenniaceae) detected by AFLPs and SSRs

Avicennia marina is an important mangrove species with a wide geographical and climatic distribution which suggests that large amounts of genetic diversity are available for conservation and breeding programs. In this study we compare the informativeness of AFLPs and SSRs for assessing genetic diversity within and among individuals, populations and subspecies of A. marina in Australia. Our comparison utilized three SSR loci and three AFLP primer sets that were known to be polymorphic, and could be run in a single analysis on a capillary electrophoresis system, using different- colored fluorescent dyes. A total of 120 individuals representing six populations and three subspecies were sampled. At the locus level, SSRs were considerably more variable than AFLPs, with a total of 52 alleles and an average heterozygosity of 0.78. Average heterozygosity for AFLPs was 0.193, but all of the 918 bands scored were polymorphic. Thus, AFLPs were considerably more efficient at revealing polymorphic loci than SSRs despite lower average heterozygosities. SSRs detected more genetic differentiation between populations (19 vs 9%) and subspecies (35 vs 11%) than AFLPs. Principal co-ordinate analysis revealed congruent patterns of genetic relationships at the individual, population and subspecific levels for both data sets. Mantel testing confirmed congruence between AFLP and SSR genetic distances among, but not within, population comparisons, indicating that the markers were segregating independently but that evolutionary groups (populations and subspecies) were similar. Three genetic criteria of importance for defining priorities for ex situ collections or in situ conservation programs (number of alleles, number of locally common alleles and number of private alleles) were correlated between the AFLP and SSR data sets. The congruence between AFLP and SSR data sets suggest that either method, or a combination, is applicable to expanded genetic studies of mangroves. The codominant nature of SSRs makes them ideal for further population-based investigations, such as mating-system analyses, for which the dominant AFLP markers are less well suited. AFLPs may be particularly useful for monitoring propagation programs and identifying duplicates within collections, since a single PCR assay can reveal many loci at once. Received: 3 October 2000 / Accepted: 19 February 2001     

Genetic structure of disjunct Argentinean populations of the subtropical tree Anadenanthera colubrina var. cebil (Fabaceae)

Anadenanthera colubrina var. cebil is a native South American tree species inhabiting seasonally dry tropical forests (SDTFs). Its current disjunct distribution presumably represents fragments of a historical much larger area of this forest type, which has also been highly impacted by human activities. In this way the hypothesis of this study is that the natural populations of A. colubrina var. cebil from Northern Argentina represent vestiges of ancient fragmentation, but they are additionally influenced by a certain degree of gene flow among them. We aimed to analyze the genetic structure of both nuclear and chloroplast DNA to evaluate the relative role of ancient and recent fragmentation on intraspecific diversity patterns. Sixty-nine individuals of four natural populations were analyzed using eight nuclear microsatellites (ncSSR) and four chloroplast microsatellite loci (cpSSR). The level and distribution of genetic variation were estimated by standard population genetic parameters and Neighbor Joining as well as Bayesian analyses. The eight ncSSR loci were highly polymorphic, while genetic diversity of cpSSRs was low. Nuclear SSRs displayed lower genetic differentiation among populations than cpSSR haplotypes (F _ST 0.11 and 0.95, respectively). However, high differentiation between phytogeographic provinces was observed in both genomes. The high genetic differentiation detected emphasizes the role of ancient fragmentation. However, the Paranaense province also shows the effects of recent fragmentation on genetic structure, whereas gene flow by pollen preserves the effects of genetic drift in the Yungas province.     

Development of nuclear microsatellite markers for the Japanese conifers Tsuga diversifolia and T. sieboldii (Pinaceae)

Nuclear microsatellite markers were developed for the two Tsuga species native to the Japanese Archipelago, Tsuga diversifolia and T. sieboldii, and a population with genetic affinities to T. diversifolia on Ulleung Island, Korea. Tsuga diversifolia and T. sieboldii are widespread dominant trees of temperate and subalpine forests in Japan but to date no genetic markers have been developed for these species. Fifteen polymorphic loci were developed and characterized, of which 14 are reliably amplified in each taxon. Across both species and the Ulleung Island population, the number of alleles per locus ranged from 3 to 26 (average = 13.93) and observed heterozygosity ranged from 0.005 to 0.935 (average = 0.535). In addition, all 15 loci were successfully amplified in a single accession of the Chinese species, T. chinensis. These markers will be useful for investigating the species’ biogeography, range‐wide genetic diversity, conservation genetic issues and potential for hybridisation.     

Characterization of tetranucleotide microsatellite loci and development and validation of multiplex reactions for the study of right whale species (genus Eubalaena)

A North Atlantic right whale (Eubalaena glacialis) genomic library was developed and screened with a (GATA)₈ probe to identify tetranucleotide microsatellite loci. Sixteen characterized loci were polymorphic in North Atlantic and/or South Atlantic (Eubalaena australis) right whales, 12 being polymorphic in E. glacialis, and 15 in E. australis. Fourteen of these were combined with 21 other previously identified loci for a suite of 35 loci which can be used to increase resolution of genetic analyses of these species. Multiplex reactions were developed for genotyping samples at these loci, providing a method that is rapid, reliable and cost‐effective.     

Identification and characterization of EST-SSRs and cpSSRs in endangered <Emphasis Type="Italic">Cycas hainanensis</Emphasis>

We report on the identification and characterization of six EST-linked simple sequence repeats (EST-SSRs) and chloroplast SSRs (cpSSRs) in endangered Cycas hainanensis. The number of alleles ranged from two to eight for EST-SSRs, two to three for cpSSRs. Observed and expected heterozygosities ranged from 0.042 to 0.417 and 0.042 to 0.811 for EST-SSRs, respectively. Expected heterozygosities ranged from 0.156 to 0.457 for cpSSRs. All these markers gave successful cross-species amplification in C. fairylakea. These markers will allow analyses of the baseline genetic variability and population structure of C. hainanensis to provide strategies for effective conservation and management. The experiments were carried out in South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, P.R. China.     

Development of Simple Sequence Repeat Markers from Functional Genes and Establishment of Molecular Identity for Tree Peony

Gene-derived simple sequence repeats (genic SSRs), also known as functional markers, are generally superior to random markers because they are located in genes and therefore may affect gene expression or function. However, extremely limited genic SSRs are available for tree peony. We used the functional gene sequences available from Paeonia to develop genic SSRs. A total of 132 SSR loci were identified from 35 cDNA sequences, of which trinucleotide (58, 43.9%) and hexanucleotide repeat (37, 28.0%) were dominant. Moreover, 121 primer pairs were successfully designed and synthesized, of which 49 primer pairs (40.5%) provided efficient and reliable amplification. By screening 16 tree peony varieties, we developed eight polymorphic genic SSRs with 37 alleles, ranging from 2 to 11 for each marker. Transferability analysis indicated that 100% of the genic SSRs could be amplified in eight other Paeonia samples. Based on eight polymorphic genic SSRs and 12 polymorphic EST-SSRs developed by predecessors, the molecular identity of 190 tree peony cultivars was constructed by capillary electrophoresis. The results showed that 146 alleles and 338 genotypes were detected, with 2–13 alleles and 3–36 genotypes for each marker. All cultivars were completely identified and exhibited unique DNA identity. In addition, the identification efficiency of different primers combinations was analyzed, and 190 germplasms were identified using 6 core primers. This study provides valuable genic SSR resources for marker-assisted selection breeding of the genus Paeonia. The DNA identity of cultivars is of great significance for the protection, utilization and management of tree peony resources.

     

Chloroplast DNA markers (cpSSRs,SNPs) for <Emphasis Type="Italic">Miscanthus,Saccharum</Emphasis> and related grasses (Panicoideae,Poaceae)

Miscanthus and Saccharum are closely related perennial C₄ grasses. Miscanthus has recently attracted interest as a non-food crop for energy and fibre production. However, molecular genetic tools for the selection of new Miscanthus genotypes and study of its genetic resources are limited. We have identified six chloroplast (plastid) marker loci,containing both microsatellites (cpSSRs) and single nucleotide polymorphisms (SNPs) and developed primers to amplify and sequence these regions. The primers were designed using the complete chloroplast genome sequence of sugarcane and were tested on a collection of 164 Miscanthus genotypes and 14 related species of the subfamily Panicoideae. The cpSSR markers were highly polymorphic, with the number of alleles ranging from 10 to 16 per locus. Within the six cpSSR marker loci, the hybrid M. ×giganteus exhibits virtually no cpDNA variation compared with its putative parents M. sinensis and M. sacchariflorus. These SNP markers enable the differentiation of most Miscanthus species and detect infraspecific variation suitable for defining cytoplasmic genepools of Miscanthus for breeding purposes.