Development of highly polymorphic tetranucleotide microsatellite markers in Austrocedrus chilensis

An enriched genomic library was constructed and 9 novel polymorphic tetranucleotide microsatellite markers developed for Austrocedrus chilensis, the most economically important native conifer in the Andean Patagonian region. Polymorphism was investigated for these markers in 48 individuals from two populations. Numbers of alleles ranged from 3 to 19 and levels of observed heterozygosity among the 9 loci ranged from 0.32 to 0.95. No locus combinations exhibited linkage disequilibrium. These polymorphic markers will be useful tools for the study of demography and gene flow and more in general for population and conservation genetics of this species.     

Isolation and characterization of polymorphic microsatellite loci in the freshwater fishes Telestes souffia and Telestes muticellus (Teleostei: Cyprinidae)

Ten novel polymorphic microsatellites (seven with perfect motifs) were isolated from vairone species (Telestes souffia and Telestes muticellus), which are endangered European cyprinid species. Together with 11 previously published cyprinid-specific loci, five multiplex sets were optimized, allowing the genotyping of 21 polymorphic loci. The level of genetic diversity was assessed in 97 individuals from the two species T. souffia and T. muticellus. We also successfully applied the 21 microsatellites to nine related species. These primers will thus be useful in assessing population structure of the vairone and other cyprinid species, with application for conservation issues and phylogeographical approaches.     

PERMANENT GENETIC RESOURCES: Isolation and characterization of microsatellite DNA loci from the southern flounder, Paralichthys lethostigma

Paucity of polymorphic molecular markers in southern flounder (Paralichthys lethostigma) has been a major limitation in genetic improvement of this important economic fish. Hence, we constructed a repeat-enriched genomic library from P. lethostigma. A total of 39 new microsatellites were identified, for which 33 primer pairs were designed. After validating and scoring, 10 of these loci were polymorphic in a test population with the range of alleles from two to nine per locus. The observed and expected heterozygosities ranged from 0.2500 to 0.9000 and from 0.4469 to 0.8514, respectively. These polymorphic microsatellites will be useful for genetic diversity analysis and linkage map construction for P. lethostigma.     

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.     

Isolation and characterization of polymorphic microsatellite loci for the dace complex: Leuciscus leuciscus (Teleostei: Cyprinidae)

Ten novel polymorphic microsatellites were isolated from the dace complex (Leuciscus leuciscus), which is a European cyprinid species. Four multiplex polymerase chain reaction sets were optimized in order to genotype 26 polymorphic loci in all, including 16 previously published cyprinid-specific loci. The level of genetic diversity was assessed in 142 dace individuals. We also successfully applied 26 of the microsatellites to 10 related species. These primers thus will be useful to assess population structure of the dace and other cyprinid species, with application for conservation issues and phylogeographical approaches.     

Isolation and characterization of polymorphic microsatellite loci in the green leafhopper Empoasca vitis Goethe (Homoptera)

Eight dinucleotide microsatellite loci were isolated and characterized within the green leafhopper Empoasca vitis (Goethe) using an enrichment cloning procedure. Primers were tested on 171 individuals collected in the southwest of France from the vine plants. The identified loci were polymorphic, with allelic diversity ranging from two to 18 alleles per locus. Observed heterozygosities were from 0.021 to 0.760. These microsatellite markers should prove to be a useful tool for estimating the population genetic structure, host-plant specialization and migration capacity of this insect.     

牦牛基因组微卫星富集文库的构建与分析

根据生物素与链亲和素的强亲和性原理,用链亲和素磁珠亲和捕捉与生物素标记的微卫星寡核苷酸探针(CA)12、(CCG)8、(CAG)8、(TTTC)8退火结合的含有接头和牦牛微卫星序列的单链限制性酶切片段,获得单链目的片段,经PCR扩增形成双链,然后克隆到pMD18-T载体上,转化至DH5α中,首次成功构建牦牛基因组微卫星富集文库。测序结果发现,阳性克隆率为77％(37／48),说明构建的牦牛基因组微卫星富集文库是一个高质量的文库。牦牛富集微卫星文库的建立和牦牛微卫星的筛选将为下一步进行牦牛基因组结构的分析、牦牛遗传连锁图谱的构建、分子进化和系统发育研究、标记辅助选择以及经济性状的QTL定位提供大量的微卫星标记。     

Ten polymorphic microsatellite markers for Scaphoideus titanus,the vector of flavescence dorée phytoplasma

The leafhopper Scaphoideus titanus is a vector of flavescence dorée phytoplasma, the causal agent of the most important grapevine yellow disease in European vineyards. Ten polymorphic microsatellite loci were developed from a genomic library enriched for AC and AG repeats. Levels of polymorphism were evaluated in 106 individuals from S. titanus European and American populations. An average of 16 alleles per locus was detected and the observed heterozygosity ranged from 0.141 to 0.813. Cross‐species amplification was successful in four other Cicadellidae species. These 10 microsatellites are valuable markers for population genetic and phylogeographical studies of S. titanus.     

Characterization of polymorphic microsatellite DNA markers in Banksia nivea, formerly Dryandra nivea

A genomic library was constructed and 10 novel polymorphic microsatellite markers were developed for Banksia nivea subsp. uliginosa, a woody shrub endemic to the southwest of Western Australia. Polymorphism was investigated for these markers in 30 individuals from one population located in restricted habitat at the base of the Whicher Range east of Busselton. Expected heterozygosity ranged from 0.124 to 0.898 and averaged 0.570. Observed heterozygosity ranged from 0.133 to 0.933 and averaged 0.544. The number of alleles per locus ranged from 2.0 to 16.0 and averaged 7.3. These markers also produced amplification products in three other Banksia species and will be used to assay genetic diversity in these species.     

How much effort is required to isolate nuclear microsatellites from plants?

The attributes of codominance, reproducibility and high resolution have all contributed towards the current popularity of nuclear microsatellites as genetic markers in molecular ecological studies. One of their major drawbacks, however, is the development phase required to obtain working primers for a given study species. To facilitate project planning, we have reviewed the literature to quantify the workload involved in isolating nuclear microsatellites from plants. We highlight the attrition of loci at each stage in the process, and the average effort required to obtain 10 working microsatellite primer pairs.     

Novel microsatellite DNA markers for the threatened African endemic tree species,Milicia excelsa (Moraceae), and cross‐species amplification in Milicia regia

Eleven microsatellite primer pairs were developed for the tropical African tree Milicia excelsa. Genomic DNA was enriched for dinucleotide (TC_n and TG_n) and tretranucleotide (GATA_n), and 188 random clones were sequenced from both orientations. We designed and tested 44 oligonucleotide primer pairs, which were evaluated using genomic DNA from 30 M. excelsa mature trees collected from a natural population in Benin. Eleven of the 44 markers showed good amplification and were polymorphic. The number of putative alleles for polymorphic primer pairs varied from three to seven, with expected and observed heterozygosities ranging from 0.10 to 0.64 and from 0.10 to 0.80, respectively. All 11 loci amplified the related species Milicia regia, indicating that these primers will be useful for population and ecology genetic studies in other species of the genus Milicia.     

Development and characterization of 45 novel microsatellite markers for sea cucumber (Apostichopus japonicus)

Simple sequence repeat‐enriched library screening and expressed sequence tag database mining were adopted to develop microsatellite markers for sea cucumber (Apostichopus japonicus). Eighty‐three microsatellite loci were selected for polymorphism assessment using 48 individuals. The results showed that 45 novel loci were polymorphic. The number of alleles ranged from two to 16, and the values of observed and expected heterozygosities varied from 0 to 0.9375 and from 0.1135 to 0.9674, respectively. No significant linkage disequilibrium between pairs of loci was found and 26 loci conformed to the Hardy–Weinberg equilibrium. These markers are therefore a potential tool for studies in the population structure and linkage map construction for A. japonicus.     

Characterization of polymorphic microsatellite DNA markers for Acacia saligna (Labill.) H.L.Wendl. (Mimosaceae)

A genomic library was constructed and 10 novel polymorphic microsatellite markers developed for Acacia saligna, a species complex being developed for agroforestry across southern Australia. Polymorphism was investigated for these markers in 30 individuals from two subspecies. Alleles per locus averaged 4.0 for ssp. saligna and 5.2 for ssp. lindleyi. Population specific alleles were detected for all loci. The microsatellite markers will be used for diagnostic assessment of the four main subspecies of the species complex.     

A new set of highly polymorphic microsatellites for the white and black anglerfish (Lophiidae)

A microsatellite‐enriched genomic library was obtained using individuals of the black anglerfish (Lophius budegassa) and eight polymorphic microsatellites were successfully optimized. The genetic analysis of 50 black anglerfish individuals captured in the Cantabrian coasts revealed high polymorphism with a mean of number of alleles per locus of N_a = 10.5 (5–28 alleles) and a mean expected heterozygosity of 0.740 (0.521–0.962). This microsatellite set was also functional with a sample from the white anglerfish (Lophius piscatorious) showing a high polymorphism.