Isolation and characterization of microsatellite markers from Brown Eared-pheasant (Crossoptilon mantchuricum)

The Brown Eared-pheasant (Crossoptilon mantchuricum) is an endangered species endemic to China. Here we developed eight microsatellite loci using a modified biotin-capture method. In the analyses of 28 individuals sampled, the number of alleles per locus ranged from four to nine. The expected (H _E) and observed (H _O) heterozygosities were 0.466–0.825 and 0.619–0.847, respectively. Results that eight microsatellite loci were highly polymorphic indicated that these markers are sufficiently powerful to address such questions as genetic diversity and population genetic structure of C. mantchuricum.     

Microsatellite analysis of genetic diversity in the Chinese alligator (<Emphasis Type="Italic">Alligator sinensis</Emphasis>) Changxing captive population

Chinese alligator (Alligator sinensis) is a critically endangered species endemic to China. In this study, the extent of genetic variation in the captive alligators of the Changxing Reserve Center was investigated using microsatellite markers derived from American alligators. Out of 22 loci employed, 21 were successfully amplified in the Chinese alligator. Sequence analysis showed loci in American alligators had a bigger average size than that of the Chinese alligators and the longest allele of an individual locus almost always existed in the species with longer stretch of repeat units. Eight of the 22 loci were found to be polymorphic with a total of 26 alleles present among 32 animals scored, yielding an average of 3.25 alleles per polymorphic locus. The expected heterozygosity (H _E) ranged at a moderate level from 0.4385 to 0.7163 in this population. Compared to that in the American alligators, a lower level of microsatellite diversity existed in the Changxing population as revealed by about 46% fewer alleles per locus and smaller H _E at the homologous loci. The average exclusion power and the ability to detect shared genotypes and multiple paternity were evaluated for those markers. Results suggested that when the polymorphic loci were combined, they could be sensitive markers in genetic diversity study and relatedness inference within the Chinese alligator populations. The level of genetic diversity present in the current Changxing population indicated an important resource to complement reintroductions based on the individuals from the other population. In addition, the microsatellite markers and their associated diversity characterized in this population could be utilized to further investigate the genetic status of this species.     

Microsatellite-based assessment of genetic diversity in stripe rust resistant NUWYT candidate lines

Stripe rust is one of the most devastating diseases, caused by Puccinia striiformis f. sp. tritici, affecting a huge amount of wheat crops worldwide. In this study, the genetic diversity of 16 National Uniform Wheat Yield Trial (NUWYT) candidate lines was evaluated by using 22 screened microsatellite markers. These lines were found resistant for stripe rust at adult plant stage. These wheat microsatellite markers identified a total of 38 alleles, with an average of 2.3 alleles per microsatellite locus. The number of alleles ranged from one to five alleles and the highest number of alleles were associated with B genome (25), as compared to D (11) and A (2) genomes. The allelic polymorphism index content (PIC) reflecting the gene diversity of these microsatellite markers ranged from 0.00 to 0.66, with an average of 0.27. The maximum PIC value of 0.66 was observed for xgwm 159-5B and 0.64 for xgwm 413-1B. The gene diversity ranged from 0.00 to 0.71, with an average of 0.30. The genetic similarity matrix was used to construct a dendrogram and the cluster analysis was performed by the use of unweighted pair-group method with arithmetic average algorithm. This divided the entire 16 candidate lines into three main clusters on the basis of their similarity. Our results indicate that the genetic diversity among the 16 candidate NUWYT lines was very narrow.     

Polymorphic microsatellite DNA markers for the ark shell Scapharca subcrenata (bivalve: Arcidae)

We have isolated and characterized twelve novel polymorphic microsatellite loci from Scapharca subcrenata to analyse the population structure. The number of observed alleles per locus ranged from 3 to 17. Observed heterozygosity (H _O) ranged from 0.321 to 0.929. Cross-species amplification was tested successfully in three other bivalve species. These microsatellite markers will be useful for genetic diversity studies of S. subcrenata and other Lamellibranchia species.     

Characterization of microsatellite loci developed for <Emphasis Type="Italic">Amaranthus hypochondriacus</Emphasis> and their cross-amplifications in wild species

The present study reports isolation and characterization of 12 polymorphic microsatellite markers for Amaranthus hypochondriacus. A total of 92 alleles were detected across the 20 accessions, with an average of 7.7 alleles per locus. The observed (H _O ) and expected (H _E ) heterozygosity values ranged from 0 to 0.95 and from 0.49 to 0.92, respectively. At significance threshold (P < 0.05), nine loci deviated from Hardy-Weinberg equilibrium (HWE), whereas significant linkage disequilibria (LD) were observed between five pairs of loci. The 12 loci were successfully amplified in 18 other amaranth species representing cultivated grain and vegetable species, their putative progenitors and wild species. These results demonstrate wide potential applicability of these markers for the study of intra- and inter-specific genetic diversity as well as evolutionary relationships among cultivated and wild amaranths.     

Isolation and characterization of microsatellite loci in the endangered freshwater fish <Emphasis Type="Italic">Varicorhinus alticorpus</Emphasis> (Cyprinidae)

Varicorhinus alticorpus (Cyprinidae) is an endangered freshwater fish with a scattered distribution in the southern and eastern regions of Taiwan. We described the development of nine polymorphic microsatellite loci in V. alticorpus for genetic studies. These new markers were tested in 20 individuals of the rare species. The number of alleles ranged from 4 to 13. Expected (H _E) and observed (H _O) heterozygosities ranged from 0.692 to 0.892 (averaged at 0.821) and from 0.000 to 0.350 (averaged at 0.088), respectively. All loci are significantly deviated from Hardy–Weinberg expectations due to the heterozygote deficiency. This suite of highly polymorphic microsatellites provides the first chance to undertake a conservation program for this species in Taiwan. It is essential to evaluate its genetic diversity and the fine-scale population structure. Tzen-Yuh Chiang, Teh-Wang Lee, and Feng-Jiau Lin contributed equally to this work.     

Newly developed polymorphic microsatellite markers in Job's tears (Coix lacryma‐jobi L.)

A microsatellite‐enriched library of Job's tears (Coix lacryma‐jobi L. var. Ma‐yuen Stapf) was constructed using a modified biotin–streptavidin capture method. After screening, 17 polymorphic microsatellites were used for diversity analysis among 30 Job's tears accessions. The number of alleles ranged from one to five alleles per locus with an average of 2.8 alleles. Expected heterozygosity (H_E) and polymorphism information content (PIC) ranged from 0 to 0.676 and from 0 to 0.666, respectively. The newly developed microsatellite markers are expected to be very valuable tools for evaluation of genetic diversity among large germplasm collection of Job's tears present in our Korean Gene Bank.     

Rapid isolation and characterization of microsatellites from the genome of pearl oyster (Pinctada martensi Dunker)

A new set of 34 microsatellite markers was isolated and characterized in pearl oyster Pincada martensi from a CA₁₆-enriched genomic library. Of 60 microsatellite markers screened in a wild population which contained 30 individuals, 34 markers occurred unambiguous bands, including seven monomorphic loci, 5 primer pairs (HHM844, HHM852, HHM888, HHM891, HHM899) were duplicated. These polymorphic markers showed high genetic variability represented mainly by the number of alleles, which ranged from 2 to 6, the expect heterozygosity which ranged from 0.1653 to 0.7622, and the polymorphic information content which ranged from 0.1516 to 0.7279. These markers could increase the available molecular resources that can be used to analyze genetic diversity and structure of pearl oyster.     

Isolation of microsatellite DNA and analysis on genetic diversity of endangered fish,Hucho taimen (Pallas)

Hucho taimen (Pallas) is an endangered species in China. To evaluate the species’ genetic diversity and population genetic structure, we isolated six polymorphic microsatellite markers from its genomic libraries by (ACA)₁₆ enriched, and characterized using 61 wild individuals. The number of alleles per locus ranged from two to seven, expected heterozygosity ranged from 0.3200 to 0.7410, polymorphism information content ranged from 0.3047 to 0.6896. These markers will be useful for the genetic variation assessment of taimen.     

Isolation and characterization of the first microsatellite markers for the endangered swan mussel <Emphasis Type="Italic">Anodonta cygnea</Emphasis> L. (Bivalvia: Unionoidea)

Freshwater molluscs are globally critically threatened, yet little is known about their genetic diversity. We describe the isolation and characterization of the first microsatellite markers for the European swan mussel, Anodonta cygnea. Nine loci were polymorphic in a panel of 110 individuals from seven German and Estonian populations. Number of alleles ranged from 2 to 14 and heterozygosity levels ranged from 0.021 to 0.498 for H _o and from 0.069 to 0.634 for H _e. Deficiency of heterozygous genotypes was observed in six loci. The length of the microsatellite repeat motives was positively correlated with the allele numbers in the respective loci.     

Characterization of new microsatellite loci in Pieris amamioshimensis (Ericaceae), a species nearly extinct in the wild

A set of 11 polymorphic microsatellite markers has been developed and characterized for the critically endangered species Pieris amamioshimensis. Fifty‐nine individuals of an ex‐situ population were used to identify these markers. The total number of alleles for each locus ranged from 3 to 9, with an average of 5.4. The expected heterozygosities (H_S) and observed heterozygosities (H_O) ranged from 0.47 to 0.77 and 0.22 to 0.88, respectively. In total, four loci exhibited significant deviations from Hardy–Weinberg equilibrium: two loci showed significant heterozygosity excess and the other two loci showed significant heterozygosity deficit. The polymorphism information content (0.43 ≤ PIC ≤ 0.73), the probability of exclusions (PE₁ = 0.9565, PE₂ = 0.9969 and PE₃ = 0.9999) and probabilities for identity (PI = 3.78 × 10^?9 and PI‐Sib = 2.35 × 10^?4) suggest that these markers are useful for estimating not only genetic diversity but also parentage, for the ex‐situ conservation management of populations.     

Microsatellite loci developed for black-crowned night-heron (Nycticorax nycticorax) and their cross-amplifications in Ardeidae

The present study reports isolation and characterization of 11 polymorphic microsatellite markers for Nycticorax nycticorax. A total of 82 alleles were detected with an average of 7.5 alleles per locus. The observed heterozygosity (H _O) ranged from 0.25 to 1.00, and the expected heterozygosity (H _E) ranged from 0.51 to 0.88. Analyses revealed no evidence for Linkage disequilibrium between any two loci, and only one locus was significantly deviated from HWE with the estimation of exact P values by the Markov chain method (P < 0.001). The 11 loci were successfully amplified in 11 other Ardeidae species. These results demonstrate these markers can be used for the study of intra- and inter-specific genetic diversity.     

Eleven novel microsatellite markers developed from the living fossil Ginkgo biloba (Ginkgoaceae)

Ginkgo biloba L. (Ginkgoaceae), a famous and widely cultivated plant, is often referred to as a living fossil. Here we isolated and characterized 11 polymorphic microsatellite loci from G. biloba using a modified biotin-capture method. These loci showed high levels of allelic diversity, alleles sizes ranged from 4 to 16 per locus with polymorphism information content ranging from 0.432 to 0.909 (average = 0.752). The observed and expected heterozygosities ranged from 0.208 to 0.708 (average = 0.484) and from 0.501 to 0.934 (average = 0.795), respectively. These microsatellite markers can be used as genetic markers for further studies on genetic diversity, genetic structure and the gene flow among populations of this species.     

Development of polymorphic microsatellite markers in sesame (Sesamum indicum L.)

Fifty microsatellite sequences (SSRs) were isolated from an enriched library of sesame (Sesamum indicum L.) using a modified protocol. After screening, 10 polymorphic microsatellites were used to determine their usefulness in diversity analysis among 16 sesame accessions. The number of alleles ranged from three to six alleles per locus with an average of 4.6 alleles. The fragment size varied from 150 bp to 307 bp. Expected heterozygosites (H_E) and polymorphism information contents (PICs) ranged from 0.437 to 0.858 and 0.34 to 0.80, respectively, which indicates the highly informative nature of the microsatellites reported here. These microsatellite markers will be very useful in diversity analysis among a large germplasm collection of sesame present in our Korean gene bank and also in the establishment of its core collection.     

First development and characterization of 27 novel microsatellite markers in the dobsonfly Neoneuromus ignobilis (Megaloptera: Corydalidae) at genome-scale level

The dobsonfly species Neoneuromus ignobilis Navás (Megaloptera: Corydalidae) is endemic to but widely distributed from eastern and southeastern Asia, being an important insect indicator for freshwater biomonitoring. At present, there is no report on the development of microsatellites of Megaloptera. Here, we developed 27 novel microsatellite markers of N. ignobilis from 850,920 candidate microsatellites with the stringent screening criteria considering the amplification success rate, the presence or absence of stutter peaks, the peak intensity, the polymorphism of the loci, the heterozygosity, and the number of alleles. The allele number of 27 microsatellite markers ranges from 3 to 12 with an average value of 6.19 per locus. The observed heterozygosity (H_O) and expected heterozygosity (H_E) revealed a range from 0.000 to 0.947 and 0.000 to 0.842, respectively. We constructed three panels (MP panel, most polymorphic; SS panel, most stringent strategy; ALL panel, total 27 microsatellite markers) and compared the analyses on population genetic diversity and structure. The result showed that the MP panel can significantly improve the analyses of individual assignment and genetic diversity. Accordingly, we advocate selecting the most polymorphic microsatellite marker for analyzing population genetics based on microsatellite data. The present work represents the first study on the microsatellite development of Megaloptera.

     

Polymorphic microsatellites identified by cross-species amplifications in the European Coot Fulica atra

We tested the cross-amplification of 26 microsatellites developed for passerines and an additional three developed for Gallinula species in eight European Coots from two populations. Sixteen microsatellite markers successfully amplified, of which nine were polymorphic with 2–6 alleles (mean 3.7 alleles) and an expected heterozygosity (H _e) ranging from 0.375 to 0.805 (mean H _e = 0.589). On average, we found 2.22 alleles/locus and a mean H _e of 0.440 in one nest, and 2.56 alleles/locus and a mean H _e of 0.494 in the other one. These nine polymorphic markers could be of potential use in studies of genetic variability, population structure and reproductive strategy of European Coots.     

Identification and characterization of microsatellite markers for the population genetic structure in endemic red-tailed barb,Gonoproktopterus curmuca

Gonoproktopterus curmuca is an endangered red tailed barb found in Southern part of Western Ghat, India. As a part of stock-specific, propagation assisted rehabilitation and management program, polymorphic microsatellites markers were used to study the genetic diversity and population structure of this species from the three River systems of Southern Western Ghats, such as Periyar River, the Chalakkudy River, and the Chaliyar River. From selected eight polymorphic microsatellite markers, the number of alleles per locus ranged from 2 to 8, and the average number of alleles among 3 populations ranged from 5.0 to 5.75. The mean observed (H_ob) and expected (H_ex) heterozygosity ranged from 0.5148 to 0.5360 and from 0.5996 to 0.6067, respectively. Significant deviations from Hardy–Weinberg Equilibrium expectation were found at majority of the loci (except Gcur MFW72 and Gcur MFW19) and in all three populations in which heterozygote deficits were apparent. The analysis of molecular variance indicates that the percent of variance among populations and within populations were 6.73 and 93.27, respectively. The pairwise F_ST values between populations indicate that there were significant deviations in genetic differentiations for the red-tailed barb populations from these three Rivers of the Western Ghats, India. The microsatellites methods reported a low degree of gene diversity and lack of genetic heterogeneity in the population of G. curmuca, which strongly emphasize the need of fishery management, conservation and rehabilitation of G. curmuca.     

Isolation and characterization of 18 polymorphic microsatellite loci from freshwater pearl mussel (<Emphasis Type="Italic">Cristaria plicata</Emphasis>)

Cristaria plicata was an important freshwater mussel for pearl culture in China. 18 polymorphic microsatellite markers were isolated and characterized using (CA)₁₅-enriched genomic library of C. plicata. These loci showed high levels of genetic polymorphism testing on 60 individuals sampled from Poyang Lake of Jiangxi Province, China. The number of alleles per locus ranged from 4 to 18. The expected (H _E) and observed heterozygosities (H _O) were 0.7232–0.8961 and 0.0000–1.0000, respectively. Four microsatellite loci were significantly deviated from Hardy–Weinberg equilibrium and no linkage disequilibrium was found. These microsatellite loci will be useful for assessment of genetic diversity and population structure in C. plicata.     

Genetic diversity of the Chestnut blight fungus Cryphonectria parasitica in four French populations assessed by microsatellite markers

Microsatellites are powerful markers to infer population genetic parameters. Here, 13 microsatellite loci isolated from a genomic and a cDNA library of Cryphonectria parasitica were used to characterize the genetic diversity and structure of four French populations. Twelve of these loci were polymorphic within populations, and average gene diversity (H_e) was estimated to be 0.35. There was a lower genetic diversity in a south-eastern population relative to three south-western populations. In these three populations, microsatellite genotypic diversity was higher than vegetative compatibility type diversity. A high genetic differentiation (G_ST = 0.27) suggested a low gene flow and/or founder effects of French populations which are in agreement with low dispersal of spores and different introductions of this species in southern France. This study demonstrates the significance of these microsatellite loci to assess gene flow and reproductive system in this important pathogen.     

濒危植物羊踯躅全基因组SSR标记开发与鉴定研究

该研究利用基于全基因组限制性酶切位点简化基因组测序技术（RAD seq技术）,开发濒危植物羊踯躅（Rhododendron molle G. Don）全基因组SSR标记,并对3个群体共63份羊踯躅材料进行验证鉴定,为进一步研究羊踯躅的遗传多样性和群体遗传结构以及保护利用提供技术支持。结果显示：（1）羊踯躅基因组测序获得原始数据7.653G bp,过滤后为7.513G bp;经组装发现,羊踯躅171.534 M bp的基因组分布在498 252 contigs中。（2）通过SSR检测,在11 961 SSR位点中获得了11 687对SSR分子标记,并且二核苷酸为基序的重复类型最丰富,达51.76%。（3）随机选取128对SSR标记在6个羊踯躅株系中进行PCR扩增,获得20对高多态性的SSR标记。（4）用所选的20对多态性SSR标记对3个群体共63份羊踯躅材料进行验证分析发现,这些多态性SSR标记位点的等位基因数为4~16个,期望杂合度（H_e）为0.489~0.908。 研究表明,羊踯躅的SSR丰度适中,且二核苷酸为羊踯躅中最丰富的重复序列,该实验进一步证明RAD seq技术是一种经济有效的基因测序方法,实验中开发的SSR引物将有助于进一步研究羊踯躅和其他近缘种的群体结构和多样性。