Characterization of eight microsatellite loci for the sea urchin Meoma ventricosa (Spatangoida,Brissidae) through Next Generation Sequencing

Eight microsatellite loci were characterized for Meoma ventricosa (Lamarck, 1816), a burrowing sea urchin that can be afflicted by a bacterial disease causing localized mass mortality. For the analyzed population (29 individuals from St. Croix, US Virgin Islands), we observed 8.125 mean number of alleles, 0.640 mean observed heterozygosity (H_o) and 0.747 mean expected heterozygosity (H_e). Two loci showed significant deviations from Hardy–Weinberg equilibrium. Overall, the described loci were characterized by a moderately high level of polymorphism suggesting that these markers are useful for a population genetic study in the Caribbean Sea.     

Isolation and characterization of 15 polymorphic microsatellite markers for the fig-pollinating wasp,Blastophaga nipponica (Hymenoptera: Agaonidae)

We developed microsatellite markers for the fig-pollinating wasp Blastophaga nipponica Grandi using a dual-suppression-PCR technique. Twenty-one candidates of microsatellite loci were obtained, of which 15 yielded scorable patterns. The degree of polymorphism for the 15 loci was further characterized using summary statistics describing the genetic variation in 60 individuals from three natural populations in Japan. All 15 loci were polymorphic and yielded 2–27 alleles per locus. Overall observed heterozygosity (H _O) and expected heterozygosity (H _E) were 0.465 and 0.631, respectively. As expected, based on the inbreeding tendency of this species, the mean inbreeding coefficient (F _IS) was high (= 0.255). These markers will contribute to studies on the population structure of this species.     

Population genetic characterization of the endangered dung beetle Copris tripartitus (Coleoptera: Scarabaeidae) using novel microsatellite markers

The dung beetle Copris tripartitus (Coleoptera: Scarabaeidae) has long been considered an endangered insect in South Korea; the detection of recent population increases leaves its endangered status uncertain. Population genetic analysis subsequent to development of molecular markers is essential for establishing proper conservation strategies. In this study, we developed ten microsatellite markers specifically for C. tripartitus. Sixty-eight individuals of C. tripartitus collected from six South Korean localities were genotyped to validate these markers and preliminarily assess population genetic characteristics. Per-locus observed number of alleles, observed heterozygosity (H_O), and expected heterozygosity (H_E) ranged from 5–12, 0.499–0.958, and 0.54–0.743, respectively. All populations showed higher H_O than H_E, negative values of inbreeding coefficient, and, overall, no sign of recent population bottlenecks (excluding one population, Seosan). This suggests that C. tripartitus did not suffer from genetic drift and inbreeding, which are typically severe in small, isolated populations. Nevertheless, detection of only one of the two gene pools in some populations and resultant genetic subdivision into two population groups may suggest that the population size is not enough to cover both gene pools. Thus, a more extended period of protection may be required to ensure higher genetic diversity of widespread populations and achieve the long-term conservation goal.     

Genetic variation and population genetic structure of Rhizophora apiculata (Rhizophoraceae) in the greater Sunda Islands,Indonesia using microsatellite markers

Genetic variations within and among Rhizophora apiculata populations in the Greater Sunda Islands of Indonesia were studied using microsatellite markers. The study found 38 alleles on five loci in 15 populations. The observed (H _o) and expected (H _e) heterozygosity values are 0.338 and 0.378, respectively. Inbreeding effect from self-pollination might explain its heterozygote deficiency. Population genetic differentiation (F _ST = 0.381) was similar to other mangrove species. The genetic diversity of R. apiculata populations along the coastline inside the archipelago (e.g., Buleleng, Donggala, Mamuju, and Takalar) was higher than those of population along the coastline outside the archipelago, especially northern Sumatra populations (i.e., Langkat, Tapanuli Tengah, Dumai, and Padang). The isolation by distances and sea currents directions as well as their connectivity might affect the gene flow and genetic exchange. The more isolated with fewer connections by sea currents, the smaller gene flow and genetic exchange observed between populations. The higher genetic exchange, on the contrary, occurred when population location was closer to the meeting point of the sea currents. The study also showed that the patterns of sea current movement seemed to have influence genetic clustering of populations which fell into three main groups (Sunda Shelf Mangroves) and one isolated population (New Guinea Mangroves).     

跨种扩增筛选大鳄龟微卫星标记及遗传多样性分析

跨种扩增是一种能够快速、有效地获得物种微卫星标记的方法。本研究利用在近缘种中已发表的微卫星DNA引物,对大鳄龟（Macroclemys temminckii）进行跨种PCR扩增,在合成的69对引物中获得8对具有多态性的微卫星位点。对PCR扩增产物进行统计,得出观测杂合度（H_o）的范围是0.041 7~0.954 5,平均为0.384 8;期望杂合度（H_E）的范围为0.041 7~0.811 8,平均为0.479 1;多态信息含量范围为0.040 0~0.759 2,平均为0.423 2;经过卡方检验后,部分微卫星位点符合哈迪-温伯格平衡。总体来说,这些位点是研究大鳄龟遗传结构的良好分子标记。     

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.     

白条草蜥多态性微卫星位点的分离与鉴定

白条草蜥(Takydromus wolteri)是一种年产多窝卵的蜥蜴。为了对其婚配制度、同一雌性个体所产卵的窝内和窝间的父权状况、种群的遗传结构和物种的进化历史等研究内容进行探讨,本研究筛选出白条草蜥的9个具有高度多态性的微卫星位点。微卫星位点筛选自包含(AC)n和(ATAG)n重复片度的微卫星富集文库。在白条草蜥安徽滁州种群的16～32个个体中对上述位点进行检测后发现,上述座位的等位基因数目范围为12～20个,期望杂合度范围为0.894～0.955,观测杂合度范围0.565～0.938,表明这些微卫星标记具有良好的遗传多样性,它们将在白条草蜥的种群遗传结构、基因流水平、种群分化和婚配制度的研究中发挥重要作用。     

Isolation and characterization of polymorphic microsatellite markers from Australia short-finned eel (Anguilla australis Richardson)

Seventeen microsatellite DNA loci from the Australian short‐finned eel (Anguilla australis Richardson) were isolated and their amplification characteristics were described. The polymerase chain reaction primers were tested on 40 eel individuals. The primers amplified loci with relatively high numbers of alleles, ranging from five to 14 with an average of nine per locus. Mean observed heterozygosity (H_O) and expected heterozygosity (H_E) were 0.6779 and 0.7374, respectively, indicating that these markers would be useful for population studies. No loci deviated significantly from Hardy–Weinberg equilibrium (P = 0.05) and no evidence was found for genotypic disequilibrium among loci at a 5% significance level.     

Isolation and characterization of 12 microsatellite loci from Korean water deer (Hydropotes inermis argyropus) for population structure analysis in South Korea

The main goals of this study were to isolate microsatellites markers of Korean water deer (Hydropotes inermis argyropus) and understand the genetic status of the species in South Korea. Twelve new microsatellite loci were isolated and characterized to establish basic population genetic parameters for 45 H. i. argyropus specimens in South Korea. There were no significant regional or genetic structure differences between the mid-eastern and southwestern populations in South Korea according to the population genetic analyses. The number of alleles per locus ranged from two to 13 with an average of 6.08. Mean expected (H _E) and observed heterozygosity (H _O) were 0.622 and 0.533, respectively. Microsatellite variability was also not significant between the two regions (F _ST=0.012). These new markers should facilitate the future population genetics studies of Korean water deer and other closely related species.     

Development of 240 novel EST-SSRs in Eucalyptus L’Hérit

Simple sequence repeat (SSR) markers derived from expressed sequence tag (EST) resources provide great potential for comparative mapping, direct gene tagging of quantitative trait loci and functional diversity studies. Here we report on the development of 240 novel EST-SSRs for the important tree genus Eucalyptus L’Hérit. Of the 240 EST-SSR loci, 218 (90.8 %) were polymorphic among 12 individuals of E. grandis Hill ex Maiden, with the number of alleles per locus (N _a), observed heterozygosity (H _o), expected heterozygosity (H _e) and polymorphic information content (PIC) averaging at 5.0, 0.403, 0.598 and 0.529, respectively. High rates of cross-species/subgenus amplification were observed. The EST-SSRs developed herein would be a valuable addition of functional markers for genetics and breeding applications in a wide range of eucalypt species. The primer sequences for the 240 EST-SSRs have been deposited in the Probe database of GenBank (IDs Pr016588534–773).     

Development of a set of genomic microsatellite markers in tea (Camellia L.) (Camelliaceae)

Tea (Camellia L.) is the world’s most consumed health drink and is also important economically. Due to its self-incompatible and outcrossing nature, tea is composed of highly heterogeneous germplasm. It is a perennial, slow-growing crop and hence the successful release of new improved cultivars following conventional breeding methods takes years. In this context, a DNA marker-based molecular breeding approach holds great promise in accelerating genetic improvement programs in tea. Here we describe the isolation of a set of highly polymorphic genomic microsatellite markers using the enrichment approach, which may be useful for phylogenetic and marker-assisted breeding programs in tea. The enriched library comprising 3,205 clones was screened for the presence of microsatellites using a three-primer-based colony PCR method. Four hundred positive clones were selected and sequenced, to reveal 153 sequences containing simple sequence repeats. Seventy-eight primer pairs were designed from repeat-positive sequences, out of which 40 primer pairs produced successful amplifications. Twenty-two of these primer pairs, when tested on a panel of 21 diverse tea clones and accessions, were found to be highly polymorphic, resulting in 137 alleles with an average of 6.76 alleles per primer pair. The polymorphic information content (PIC), expected heterozygosity (H _e) and observed heterozygosity (H _o) of the polymorphic markers ranged from 0.1 to 0.9, 0.1–0.9 and 0.0–0.8, with average values of 0.6 ± 0.18, 0.7 ± 0.17 and 0.5 ± 0.22, respectively. These markers can be applied for various diversity analyses, mapping programs and genotyping of tea crop.     

Development of 12 microsatellite markers for Aconitum brachypodum (Ranunculaceae), a critically endangered and endemic medicinal plant

In this study, 12 microsatellite markers were developed from two microsatellite-enriched libraries (AG, AC) of Aconitum brachypodum, which were constructed using a FIASCO method. Polymorphism of each locus was assessed in 24 individuals of A. brachypodum. Number of alleles per locus (N_A) ranged from 2 to 4. The average allele number of the microsatellites was 2.58 per locus. The observed (H_O) and expected (H_E) heterozygosities ranged from 0.125 to 0.875 and 0.117 to 0.612, respectively. Polymorphic information content (PIC) ranged from 0.110 to 0.531. Among the 12 microsatellite markers, three deviated from Hardy–Weinberg equilibrium significantly. These markers will facilitate further studies on the population genetics of A. brachypodum.     

Development of microsatellite marker loci for European hazelnut (<Emphasis Type="Italic">Corylus avellana</Emphasis> L.) from ISSR fragments

Polymorphic microsatellite markers were developed for European hazelnut (Corylus avellana L.) from the sequences of inter-simple sequence repeat (ISSR) fragments and flanking regions. Twenty-five ISSR primers were used to generate fragments for cloning. Of the 520 unique sequences obtained, 41 contained long internal repeats (≥20 bp) with flanking sequences sufficient for primer design. From these, we developed 23 new polymorphic microsatellite loci. The flanking sequences were obtained for fragment ends by chromosome walking, and an additional 47 polymorphic markers were developed. Two additional polymorphic markers were developed from a GA-enriched library. The 72 new marker loci were characterized using 50 diverse hazelnut accessions. For the internal repeat loci, the number of alleles per locus ranged from 2 to 16, with a mean of 7.52. Mean values for expected heterozygosity (H_e), observed heterozygosity (H_o), and polymorphism information content (PIC) were 0.62, 0.59, and 0.58, respectively. The estimated frequency of null alleles (r) was ≥0.05 at six of the 23 loci. For the 47 marker loci developed from fragment ends, the number of alleles per locus ranged from 2 to 16, with a mean of 7.30. Mean values for H_e, H_o, and PIC were 0.62, 0.47, and 0.58, respectively. The estimated frequency of null alleles (r) was ≥0.10 at 18 of the 47 loci. Of the 70 loci developed from ISSR and flanking sequences, 50 segregated in our mapping population and were assigned to linkage groups.     

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.     

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.     

Development and characterization of microsatellite markers for genomic analysis of yarrow (Achillea millefolium L.)

Yarrow (Achillea millefoilum L.) is an important medicinal plant with different medicinal and ornamental uses. In this study, SSR markers were developed for the first time from the genome A. millefolium using the slightly modified Hamilton protocol. Three yarrow genomic libraries were constructed, enriched for microsatellite motifs AG, AC and ATG. A total of 30 primer pairs were developed of which 16 were polymorphic in 26 A. millefolium accessions. One accession from A. tenuifolia species was also included to assess the transferability of new developed SSR primers in other species. The average allele number of SSR markers was 8.5 per locus and ranged from 2 to 14. The observed heterozygosity (H _O) varied from 0 to 0.96 with an average of 0.52 and the expected heterozygosity (H _E) ranged from 0.07 to 0.49 with an average of 0.39. Among primers, Am142 showed the highest polymorphic information content (PIC) and the lowest value was obtained from Am59 primer with an average of 0.33. Three loci (AmK59, AmK344 and AmK329) deviated significantly from Hardy-Wrinberg Equlibrium (HWE) and one locus (Am344) might have null alleles. Cluster and PCoA analyses classified A. millefolium accessions according to their geographical distribution and A. tenuifolia species completely separated from A. millefoliulm genotypes.     

Isozyme analysis of genetic variability and population structure of Lactuca L. germplasm

Isozymes were used to investigate the genetic variability, population structure, and relationships of Lactuca germplasm. The isozyme systems revealed 16 putative loci of a total of 31 alleles. Out of these 16 loci, 11 were polymorphic. The average values of expected heterozygosity (H_e), observed heterozygosity (H_o), mean number of alleles per locus (A) and effective number of alleles per locus (A_e) were 0.2227, 0.266, 1.3005 and 1.369, respectively. The average fixation indices were lower than zero for most of the accessions studied, indicating an excess of heterozygotes. Genetic differentiation among accessions (F_ST) exhibited that 51.3% of the isozyme variation was recorded among accessions, and 48.7% of the genetic variation resided within accessions. The average values of total heterozygosity (H_T) and intra-accessional genetic diversity (H_S) were 0.352 and 0.171, respectively. Moreover, the inter-accessional genetic diversity (D_ST) ranged from 0 to 0.424 with an average of 0.18. Cluster analysis revealed that L. sativa cultivars were distributed throughout different Lactuca species. Thereby, isozymes results confirms the hypothesis of the polyphyletic origin of L. sativa. This high level of genetic variation proved that isozymes are efficient for polymorphism analysis of Lactuca germplasm.     

Genetic diversity and parentage assignment in Dojo loach,Misgurnus anguillicaudatus based on microsatellite markers

In this study, genetic diversity and structure of three Misgurnus anguillicaudatus populations from three different geographical locations in China (Hunan, Hubei and Henan province) were investigated using microsatellite markers. High level of genetic diversity of all three populations was revealed by expected heterozygosity (H_e), observed heterozygosity (H_o) and allele number. Significant genetic differentiations were found between all pairs of populations. The efficiency of eight microsatellite markers in parentage assignment of 540 progeny from twenty full-sib families was evaluated. Simulation based on allele frequency data demonstrated that probabilities of exclusion per locus range from 0.313 to 0.825 when no parent information is available and 0.504 to 0.904 when one parent is known. The assignment success rate based on the real data using eight markers was 96.85%. This study indicates that these M. anguillicaudatus resources are valuable genetic and breeding material for aquaculture and the microsatellite markers will be useful for investigation of genetic background and molecular marker-assisted selective breeding in this species.     

Genetic diversity and population structure of Armillaria luteo-virens (Physalacriaceae) in Qinghai-Tibet Plateau revealed by SSR markers

Armillaria luteo-virens is a type of ectomycorrhiza whose fruit body is widely used as an edible mushroom in the Qinghai-Tibetan Plateau. In total, 404 individuals of A. luteo-virens from 23 geographically separate populations were analyzed using eight pairs of SSR markers. The average number of alleles among nine populations was 28.65 with a range from 20 to 38. The mean observed (H_O) and expected (H_E) heterozygosity ranged from 0.446 to 0.501 and from 0.499 to 0.554, respectively. An analysis of molecular variance (AMOVA) showed that most genetic variation occurred within populations (83.32%). The mean value of F_ST (0.176) suggested low genetic differentiation among populations. The 23 studied populations were divided into two main clusters based on the NJ dendrogram, which was also confirmed by STRUCTURE analysis. This study may aid in not only the protection of this species but also the management of genetic resources for future artificial cultivation.