Characterization of chloroplast microsatellite loci from whole chloroplast genome of Camellia taliensis and their utilization for evaluating genetic diversity of Camellia reticulata (Theaceae)

This study characterized chloroplast microsatellite markers for Camellia reticulata, a famous ornamental and edible economic tree species only distributed in Southwestern China. Thirty-two chloroplast microsatellite markers were originally annotated in the whole chloroplast genome of Camellia taliensis, ten polymorphic microsatellite markers were tested in 96 individuals from four natural populations of C. reticulata. Alleles numbered from two to four, and average value of Shannon's Information index, Nei's gene diversity, total genetic diversity, genetic diversity within populations, gene differentiation coefficient and gene flow index were 0.408, 0.225, 0.217, 0.102, 0.530 and 0.443, respectively. Our results showed high genetic differentiation and limited gene flow among the studied populations, which may be explained by recent fragmentation of the remnant populations due to human destruction and disturbance of natural habitats of the species. The identified cpSSR markers will be useful for the future studies on population genetics, conservation biology and phylogeography of C. reticulata.     

Analysis of microsatellite markers in the genome of the plant pathogen Ceratocystis fimbriata

Ceratocystis fimbriata sensu lato represents a complex of cryptic and commonly plant pathogenic species that are morphologically similar. Species in this complex have been described using morphological characteristics, intersterility tests and phylogenetics. Microsatellite markers have been useful to study the population structure and origin of some species in the complex. In this study we sequenced the genome of C. fimbriata. This provided an opportunity to mine the genome for microsatellites, to develop new microsatellite markers, and map previously developed markers onto the genome. Over 6000 microsatellites were identified in the genome and their abundance and distribution was determined. Ceratocystis fimbriata has a medium level of microsatellite density and slightly smaller genome when compared with other fungi for which similar microsatellite analyses have been performed. This is the first report of a microsatellite analysis conducted on a genome sequence of a fungal species in the order Microascales. Forty-seven microsatellite markers have been published for population genetic studies, of which 35 could be mapped onto the C. fimbriata genome sequence. We developed an additional ten microsatellite markers within putative genes to differentiate between species in the C. fimbriata s.l. complex. These markers were used to distinguish between 12 species in the complex.     

Isolation and characterization of microsatellite markers for Achyranthes bidentata (Amaranthaceae) using next-generation sequencing platform

Achyranthes bidentata Blume (Amaranthaceae) is a perennial herb that is widely distributed in India, Java, China, and Japan. The natural resources of A. bidentata in its geo-authentic product area have rapidly declined in recent years because of the over-collection of its roots. To devise adequate conservation and management strategies for this species, its genetic diversity and population structure should be characterized. Roche 454 pyrosequencing combined with magnetic bead enrichment was used to develop microsatellite markers for A. bidentata. A total of 903 microsatellite loci were identified from 42,004 individual sequence reads. One hundred microsatellite loci were selected to test the primer amplification efficiency across 16 individuals from two A. bidentata populations. Of these tested markers, 8 yielded polymorphic amplification products, 29 yielded single alleles. For polymorphic primer pairs, the number of alleles per locus ranged from 2 to 4, with an average of 2.75. The observed and expected heterozygosities ranged from 0.353 to 0.671 and 0.250 to 0.938, respectively. The inbreeding coefficient varied from −0.692 to 0.627. This set of markers will provide useful tools for examining genetic diversity and population structure, and aid in better understanding of the conservation of A. bidentata.     

Characterization of nineteen microsatellite markers and development of multiplex PCRs for the wedge clam Donax trunculus (Mollusca: Bivalvia)

The wedge clam Donax trunculus is an Atlantic-Mediterranean warm-temperate species found from Senegal to the northern coast of France, including the Mediterranean and Black Sea. It is commercially exploited in several European countries and constitutes an important fishing resource due to its high economical value. To contribute to its conservation and management, nineteen microsatellite markers were isolated from two enriched genomic libraries. These loci were characterized in 30 clams from a single population from northwest Spain. The number of alleles per locus ranged from 2 to 17 and observed and expected heterozygosity varied from zero to 0.714 and from 0.078 to 0.950, respectively. Linkage disequilibrium was not detected and nine loci were in agreement with Hardy–Weinberg equilibrium. Fifteen polymorphic markers were arranged into three multiplex PCR sets to reduce both time and cost of microsatellite genotyping. This is the first time that polymorphic microsatellite markers have been reported for D. trunculus. These new markers provide a valuable resource for future population genetics studies and management and culture of this species.     

Microsatellite DNA analysis reveals lower than expected genetic diversity in the threatened leopard cat (<Emphasis Type="Italic">Prionailurus bengalensis</Emphasis>) in South Korea

To optimize conservation efforts, it is necessary to determine the risk of extinction by collecting reliable population information for a given species. We developed eight novel, polymorphic microsatellite markers and used these markers in conjunction with twelve existing markers to measure genetic diversity of South Korean populations of leopard cat (Prionailurus bengalensis), a species for which population size and habitat area data are unknown in the country, to assess its conservation status. The average number of alleles and the observed heterozygosity of the species were 3.8 and 0.41, respectively, and microsatellite diversity was lower than the average genetic diversity of 57 populations of 12 other felid species, and lower than that of other mammal populations occurring in South Korea, including the raccoon dog (Nyctereutes procyonoides), water deer (Hydropotes inermis), and endangered long-tailed goral (Naemorhedus caudatus). Furthermore, analysis of genetic structure in the national leopard cat population showed no clear genetic differentiation, suggesting that it is not necessary to divide the South Korean leopard cat population into multiple management units for the purposes of conservation. These results indicate that the genetic diversity of the leopard cat in South Korea is unexpectedly low, and that the risk of local extinction is, as a result, substantial. Thus, it is necessary to begin appropriate conservation efforts at a national level to conserve the leopard cat population in South Korea.     

Isolation and characterization of 18 polymorphic microsatellite markers for the “Female Ginseng” Angelica sinensis (Apiaceae) and cross-species amplification

Angelica sinensis (Apiaceae) is an endangered alpine herb that is widely used as a medicinal plant in traditional Chinese medicine (TCM). Wild populations of A. sinensis have become quite rare in China. Thus, population genetics studies of this species are urgently needed for its effective conservation and sustainable use. However, to date, no microsatellite loci have been isolated in A. sinensis. To address this issue, we isolated 18 polymorphic loci and genotyped 120 individuals collected from 6 populations. The number of alleles per locus ranged from 1.2 to 5.5, and the average was 2.4. The observed and expected heterozygosity per locus for a population varied, respectively, from 0.000 to 0.983 (averaged at 0.198) and from 0.066 to 0.661 (averaged at 0.333). Deviation from the Hardy–Weinberg equilibrium (p < 0.01) was observed for 4 to 14 loci in various populations. These microsatellite markers were cross-amplified in 10 species affinis, and 7 loci were successfully amplified in all species. These microsatellite markers are useful for genetic studies, the conservation management of A. sinensis, and identification of A. sinensis.     

Cross-species amplification and characterization of microsatellite loci in Pinus mugo Turra

Pinus mugo (dwarf mountain pine) is an important component of European mountain ecosystems. However, little is known about the present genetic structure and population differentiation of this species at the DNA level, possibly due to a lack of nuclear microsatellite markers (SSR) developed for Pinus mugo. Therefore in this study we transferred microsatellite markers originally developed for Pinus sylvestris and Pinus taeda to Pinus mugo. This cross-species amplification approach is much faster and less expensive than isolation and characterization of new microsatellite markers. The transfer rates from the source species to Pinus mugo were moderately low (26%). There were no differences in microsatellite repeat motifs between the source species and Pinus mugo. Nuclear microsatellite markers successfully transferred to Pinus mugo can be applied to various genetic studies on this species, due to the high level of their polymorphism and high value of polymorphic information content.     

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 markers for Lycium ruthenicum (Solananeae)

We developed microsatellite markers in Lycium ruthenicum, a desert plant widely distributed in northwestern China. In order to investigate its population genetic structure, genetic diversity, and its evolutionary history, we have isolated 11 novel microsatellite loci primers and characterized them in 24 individuals from 3 populations of L. ruthenicum using the combined biotin capture technique. For these microsatellites, one to seven alleles per locus were identified. The observed heterozygosities ranged from 0 to 0.958, meanwhile the expected heterozygosities ranged from 0 to 0.841. These microsatellite markers could be first useful for population level studies like genetic diversity and structure in this species.     

Identification of microsatellite loci from Epimedium koreanum and cross‐species amplification in four species of Epimedium (Berberidaceae)

Nineteen polymorphic microsatellite markers were isolated and characterized from a trinucleotide enriched partial genomic library of Epimedium koreanum. The average allele number of these microsatellites was 5.9 per locus, ranging from two to 11. The observed heterozygosity (H_O) and expected heterozygosity (H_E) at the population level were 0.00–0.90 and 0.12–0.90, respectively. In addition, the results of cross‐species amplification of this set of microsatellite markers in four closely related Epimedium species, E. brevicornum, E. sagittatum, E. pubescens and E. wushanense, revealed that these microsatellite markers were useful for population genetic structure evaluation and genotype analysis of major Epimedium species that have been used as traditional Chinese medicines.     

Development of Microsatellite Markers and Analysis of Genetic Diversity and Population Structure of Colletotrichum gloeosporioides from Ethiopia

Twenty three polymorphic microsatellite markers were developed for citrus plant pathogenic fungus, Colletotrichum gloeosporioides, and were used to analyze genetic diversity and population structure of 163 isolates from four different geographical regions of Ethiopia. These loci produced a total of 118 alleles with an average of 5.13 alleles per microsatellite marker. The polymorphic information content values ranged from 0.104 to 0.597 with an average of 0.371. The average observed heterozygosity across all loci varied from 0.046 to 0.058. The gene diversity among the loci ranged from 0.106 to 0.664. Unweighted Neighbor-joining and population structure analysis grouped these 163 isolates into three major groups. The clusters were not according to the geographic origin of the isolates. Analysis of molecular variance showed 85% of the total variation within populations and only 5% among populations. There was low genetic differentiation in the total populations (F_ST = 0.049) as evidenced by high level of gene flow estimate (N_m = 4.8 per generation) among populations. The results show that Ethiopian C. gloeosporioides populations are generally characterized by a low level of genetic diversity. The newly developed microsatellite markers were useful in analyzing the genetic diversity and population structure of the C. gloeosporioides populations. Information obtained from this study could be useful as a base to design strategies for better management of leaf and fruit spot disease of citrus in Ethiopia.     

Development of novel microsatellite markers for the grassland species Lolium multiflorum,Lolium perenne and Festuca pratensis

Twelve microsatellite markers were isolated from Lolium multiflorum. Allelic variability and cross‐species amplification were assessed on 16 individuals of each of the three grassland species L. multiflorum, Lolium perenne and Festuca pratensis. Cross‐species amplification success was 100% for L. perenne and 83% for F. pratensis. The number of alleles detected ranged from one to 14 with an average of 3.4. While three microsatellite loci were polymorphic in all three species, one marker produced species‐specific alleles in all three species. These microsatellite markers provide a valuable tool for population genetic studies within and among species of the Festuca–Lolium complex.     

Development of novel tetra- and trinucleotide microsatellite markers for giant grouper <Emphasis Type="Italic">Epinephelus lanceolatus</Emphasis> using 454 pyrosequencing

Giant grouper (Epinephelus lanceolatus) is a commercially important species, but its wild population has recently been classified as vulnerable. This species has significant potential for use in aquaculture, though a greater understanding of population genetics is necessary for selective breeding programs to minimize kinship for genetically healthy individuals. High-throughput pyrosequencing of genomic DNA was used to identify and characterize novel tetra- and trinucleotide microsatellite markers in giant grouper from Sabah, Malaysia. In total, of 62,763 sequences containing simple sequence repeats (SSRs) were obtained, and 78 SSR loci were selected to possibly contain tetra- and trinucleotide repeats. Of these loci, 16 had tetra- and 8 had trinucleotide repeats, all of which exhibited polymorphisms within easily genotyped regions. A total of 143 alleles were identified with an average of 5.94 alleles per locus, with mean observed and expected heterozygosities of 0.648 and 0.620, respectively. Among of them, 15 microsatellite markers were identified without null alleles and with Hardy–Weinberg equilibrium. These alleles showed a combined non-exclusion probability of 0.01138. The probability of individual identification (P_ID) value combined with in descending order 12 microsatellite markers was 0.00008, which strongly suggests that the use of the microsatellite markers developed in this study in various combinations would result in a high resolution method for parentage analysis and individual identification. These markers could be used to establish a broodstock management program for giant grouper and to provide a foundation for genetic studies such as population structure, parentage analysis, and kinship selection.     

Development of microsatellite markers in Qualea grandiflora (Vochysiaceae) and transferability to congeneric species,typical trees of the Brazilian savanna

This study aimed to isolate and characterize microsatellite markers for Qualea grandiflora and to test their transferability to congeneric species Qualea multiflora and Qualea parviflora. These three species are widespread in the Cerrado, the largest, richest and probably the most threatened tropical savanna in the world. We characterized ten markers in 40 individuals belonging to two populations of Q. grandiflora and eight markers in 20 individuals belonging to one population of Q. multiflora and Q. parviflora. In Q. grandiflora, considering all 40 analyzed individuals, the number of alleles per locus ranged from eight to 21, and the average was 11.60. The mean number of alleles per locus was 8.8 and 7.3 in each population. The observed and expected heterozygosities (H_o and H_e) within populations varied from 0.235 to 0.944 and from 0.225 to 0.932, respectively. In Q. multiflora the number of alleles varied from two to 11 with an average of 5.75; the H_o ranged from 0.150 to 0.950, while H_e ranged from 0.191 to 0.817. In Q. parviflora, considering the seven polymorphic loci, the number of alleles ranged from two to 13, with an average of 7.5, while H_o ranged from 0.211 to 0.944, and H_e ranged from 0.193 to 0.906. The polymorphism level of the microsatellite markers here described enable them as powerful tools for future population genetic studies in these species, helping to answer ecological and evolutionary questions.     

Development of an integrative database with 499 novel microsatellite markers for Macaca fascicularis

Background

Cynomolgus macaques (Macaca fascicularis) are a valuable resource for linkage studies of genetic disorders, but their microsatellite markers are not sufficient. In genetic studies, a prerequisite for mapping genes is development of a genome-wide set of microsatellite markers in target organisms. A whole genome sequence and its annotation also facilitate identification of markers for causative mutations. The aim of this study is to establish hundreds of microsatellite markers and to develop an integrative cynomolgus macaque genome database with a variety of datasets including marker and gene information that will be useful for further genetic analyses in this species.

Results

We investigated the level of polymorphisms in cynomolgus monkeys for 671 microsatellite markers that are covered by our established Bacterial Artificial Chromosome (BAC) clones. Four hundred and ninety-nine (74.4%) of the markers were found to be polymorphic using standard PCR analysis. The average number of alleles and average expected heterozygosity at these polymorphic loci in ten cynomolgus macaques were 8.20 and 0.75, respectively.

Conclusion

BAC clones and novel microsatellite markers were assigned to the rhesus genome sequence and linked with our cynomolgus macaque cDNA database (QFbase). Our novel microsatellite marker set and genomic database will be valuable integrative resources in analyzing genetic disorders in cynomolgus macaques.     

Development and characterization of microsatellite markers for rice leaffolder, Cnaphalocrocis medinalis (Guenée) and cross-species amplification in other Pyralididae

Rice leaffolder, Cnaphalocrocis medinalis (Guenée), is a destructive and widespread pest on rice. In this study, 20 microsatellite markers were isolated and characterized from C. medinalis partial genomic libraries using the method of fast isolation by AFLP of sequence containing repeats. Of these markers, 18 markers displayed polymorphisms. Polymorphisms were evaluated in 48 individuals from two natural populations. The number of alleles per locus ranged from 2 to 15, and the expected and observed heterozygosities ranged from 0.324 to 0.934 and from 0.304 to 0.917, respectively. Cross-species amplification was also performed to test the transferability of the 20 microsatellite markers and a moderate level of cross amplication was observed across the three species of Pyralididae (26.67 %). These microsatellite loci would facilitate the future study on population genetics and molecular genetics of rice leaffolder and would also be useful for study in Chilo suppressalis, Scirpophaga incertulas and Pyrausta nubilalis.     

Rapid development of 36 polymorphic microsatellite markers for Tetranychus truncatus by transferring from Tetranychus urticae

Tetranychus truncatus Ehara is a phytophagous spider mite that is now one of the most important pests of agricultural and economic crops in East and Southeast Asia. However, population genetics and other studies of T. truncatus have been impeded by the lack of microsatellite markers, which are expensive and time-consuming to identify. Previous studies indicated a high potential of cross-amplification of microsatellites in Tetranychus species, meaning that the microsatellite flanking sequences are sufficiently homologous among Tetranychus species that the primers for one species may work in another species. Here, we tested 205 primer pairs designed from the whole genome sequence of Tetranychus urticae Koch, a sister species of T. truncatus, for microsatellite markers in three populations of T. truncatus in China (N = 94). About half (102) of these primer pairs yielded the desired PCR products, 36 of which revealed polymorphism in T. truncatus. Each of the 36 markers harbored between 2 and 23 alleles, with a mean polymorphic information content of 0.589 (0.119–0.922 range). The mean observed and expected heterozygosity across loci and the three populations were 0.468 and 0.628, respectively. Of the 36 primer pairs, 22 also worked in Tetranychus piercei, but only a few of them worked in T. ludeni and T. phaselus. Cross-amplification is thus a cost-effective way to develop microsatellite markers, which can be of great value in population genetics studies.     

Development, characterization and transferability of microsatellite markers for the plant genus Phlox (Polemoniaceae)

In order to study diversification and microevolution in Phlox, we developed nine polymorphic microsatellite loci. In 20 individuals of Phlox pilosa from a single population, the average number of alleles per locus was 10.0 ± 5.1, and average observed and expected heterozygosities were 0.611 ± 0.234 and 0.769 ± 0.170, respectively. Most of these markers amplified successfully in 11 additional species of Phlox, representing a broad diversity of the genus, and some also amplified in more distantly related members of the Polemoniaceae. These microsatellite markers will be valuable for investigation of evolutionary processes in this important study system.     

Comparative genetic diversity of wild and hachery populations of Korean threadsail filefish Stephanolepis cirrhifer using cross-species microsatellite markers

The threadsail filefish Stephanolepis cirrhifer is a highly commercial fisheries resource in Korea that suffers intensive anthropogenic pressure across much of its range. For basic information about its current genetic status in relation to stock enhancement, the level and distribution of genetic variation between a wild and a hatchery-bred population were investigated using 10 microsatellite markers developed for Thamnaconus modestus. High levels of polymorphism were observed between the two populations. A total of 95 different alleles were found at all loci, with some alleles being unique. The allelic variability ranged from six to 13 in the wild population and from five to 13 in the hatchery one. The average observed and expected heterozygosities were estimated to be 0.72 and 0.80 in the wild sample and 0.70 and 0.79 in the hatchery one, respectively. These results showed similar genetic variability in the hatchery population, as compared with the wild population and significant genetic differentiation between the wild population and the hatchery samples (F _ST = 0.016, P < 0.05). Genetic drift in the intensive breeding practices for stock enhancement has probably promoted differentiation between populations. Significant deviations from Hardy-Weinberg equilibrium were detected in both populations. Our results indicate that further studies using species-specific microsatellite markers will be necessary for a more reliable assessment of genetic diversity of the species.     

Establishment of a microsatellite panel covering the sugi (Cryptomeria japonica) genome,and its application for localization of a male-sterile gene (ms-2)

A microsatellite (simple sequence repeat; SSR) panel for Cryptomeria japonica was established, using both newly developed and previously reported markers, to construct a frame of linkage map and facilitate localization of important genes in this species. In this study, 32 new expressed sequence tag SSRs (EST-SSRs) and 12 new genomic SSRs (gSSRs) were developed. Their average polymorphism information content (PIC) values were 0.549 and 0.776, respectively. The markers were mapped onto a high-density linkage map. The SSR panel that was established to cover the genome consisted of 46 gSSRs and 47 EST-SSRs. The number of SSR markers in each linkage group, the average map distance between loci within a linkage group, and the average PIC values in each linkage group ranged from 6 to 13, 6.77 to 19.88 and 0.475 to 0.712, respectively. The utility of the SSR panel was tested by using it to localize a male-sterile gene, ms-2. The ms-2 locus was successfully localized on the linkage group 5 using 33 SSR markers (three SSRs per linkage group) which were selected from the SSR panel based on the existence of polymorphisms and the absence of null alleles in the mapping population for ms-2. A partial linkage map surrounding the ms-2 locus was then constructed using a further 57 single nucleotide polymorphisms and three SSRs, to facilitate future development of markers tightly linked to the ms-2 locus for use in marker-assisted selection. The SSR panel covering the C. japonica genome will allow researchers to localize important genes efficiently.