Isolation and characterization of Brachymystax lenok microsatellite loci and cross‐species amplification in Hucho spp. and Parahucho perryi

We isolated and characterized eight polymorphic microsatellite markers for Brachymystax lenok (Pallas, 1773) from genomic libraries enriched for (GATA)_n, (GACA)_n and (ATG)_n microsatellites. The number of alleles per locus ranged from two to 17. Heterozygosity ranged from 0.2 to 0.95. In addition, cross‐species amplification was successful for seven loci in Hucho hucho, eight in H. taimen and seven in Parahucho perryi.     

Characterization of microsatellite DNA markers in a critically endangered species,Mekong giant catfish,Pangasianodon gigas

Microsatellite DNA markers for a critically endangered Mekong giant catfish (Pangasianodon gigas Roberts and Vidthayanon, 1991) were developed from fin clips collected from captive fish using (GT)₁₅ probe. The number of alleles per locus ranged from two to four. The expected heterozygosities ranged from 0.13 to 0.68. Also, these primers were successfully amplified in four closely related species, Pangasius bocourti, Pangasius conchophilus, Pangasius larnaudii and Pangasius sanitwongsei with the number of alleles per locus ranged from 1 to 13, 1 to 16, 1 to 12 and 1 to 4, respectively. These markers should prove to be very useful for the evaluation of genetic diversity for this species and other related Pangasius species.     

Identification and characterization of microsatellite DNA markers developed in an endangered cyprinid of the Mekong River,the seven‐line barb (Probarbus jullieni Sauvage, 1880)

Microsatellite markers for an endangered cyprinid species of the Mekong River, the seven‐line barb (Probarbus jullieni Sauvage, 1880) were developed from the wild caught samples using (GT)₁₅ probe. The number of alleles per locus ranged from seven to 16. The expected heterozygosities ranged from 0.47 to 0.91. Also, these primers were successfully amplified in the two closely related species, P. labeamajor and P. labeaminor. These markers have proven to be very useful for the population genetic structure study in this species and other related cyprinids.     

Analysis of genetic diversity in the endangered Hucho taimen from China

Hucho taimen are listed as endangered in China. The population size has declined recently, prompting an increase in the level of listing from grade three in 2002 to grade five in 2006. We analyzed the genetic diversity of wild populations using 17 microsatellite markers to establish a scientific basis for conservation of this species. We collected tissue samples from four populations in the Heilongjiang River basin: Huma River (HM), Hutou (HT), Haiqing (HQ), and Zhuaji (ZJ). A total of 21 loci were amplified, 18 of which were polymorphic. The number of alleles per locus ranged from 2 to 9 (mean: 4.1905). There were 13 highly polymorphic loci and 5 moderately polymorphic loci. Analysis of five genetic diversity parameters (Na, Ne, Ho, He, and PIC) suggested moderate levels of diversity within the populations. The populations were ranked HT > HQ > ZJ > HM, but the differences in diversity were not statistically significant (P > 0.05). A comparison of variation among all four populations suggested Hardy–Weinberg disequilibrium at 20% of the loci. Genetic differentiation (Fst) was 0.0644 and the gene flow among populations was estimated at 3.36 individuals per generation. The majority of diversity (93.88%) occurred among individuals within a population. In contrast, relatively little (6.12%) of the genetic diversity was distributed between the populations. An analysis of genetic differentiation and genetic distance between pairs of populations revealed that both parameters were higher in comparisons of the HM population to the HT, HQ, and ZJ populations than among the three latter populations. This suggests that the HM population has a distinct genetic structure. We hypothesize that habitat degradation and excessive fishing, not low genetic diversity, has caused the decline in H. taimen populations. However, this species should be protected from further declines in genetic diversity.     

Isolation of Polymorphic RAPD-SSR Markers from Xinjiang Arctic Grayling (<Emphasis Type="Italic">Thymallus arcticus grubei</Emphasis>) and a Test of Cross-Species Amplification

A total of 18 polymorphic microsatellite loci were isolated and characterized from RAPD products in the Xinjiang Arctic Grayling (Thymallus arcticus grubei). The number of alleles (N_a) per locus varied from 2 to 10. Observed (H_o) and expected (H_e) heterozygosities ranged from 0.64 to 0.92, and from 0.63 to 0.88, respectively. Considerable differences were found among HBH, FH and FY populations in the number of alleles, effective number of alleles, number of genotypes at all of these loci. These new RAPD-SSR markers have provided a helpful tool for genetic analyses and resources conservation of T. arcticus grubei. Five additional fish species, Amur grayling (Thymallus grubii), Taimen (Hucho taimen), Sea perch (Lateolabrax japonicus), Lenok (Brachymystax lenok) and Red seam bream (Pagrosomus major) were assessed for cross-species amplification. Three of the five species showed at least one polymorphic locus. In addition, seven loci were found to be polymorphic in at least one species.     

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.     

Isolation and characterization of 13 microsatellite loci from Incarvillea mairei (Bignoniaceae), an endemic species to the Himalaya-Hengduan mountains region

Incarvillea mairei (H. Léveillé) Grierson (Bignoniaceae) is an endemic species to the Himalaya-Hengduan Mountains region. Here, we developed 13 microsatellite markers from I. mairei using a modified biotin-streptavidin capture method. Number of alleles per locus (NA) ranged from 3 to 7 with an average of 4.615. The observed (H _O) and expected (H _E) heterozygosities were from 0.050 to 0.800 and from 0.249 to 0.815, respectively. Additionally, among the 13 identified microsatellite markers, 12 of them were successfully amplified in other three congeneric species, and most of them showed polymorphic. Obtained evidences suggest that these markers provide a useful tool for further study of the population genetic structure and the breeding system in this species or/and infra-generic species.     

Analysis of molecular genetic diversity and population structure in Amaranthus germplasm using SSR markers

We assessed the molecular genetic diversity and population structure of Amaranthus species accessions using 11 simple sequence repeat markers. A total of 122 alleles were detected, and the number of alleles per marker (N_A) ranged from 6 to 21 with an average of 11.1 alleles. The frequency of major alleles per locus ranged from 0.148 to 0.695, with an average value of 0.496 per marker. The overall polymorphic information content values were 0.436–0.898, with an average value of 0.657. The observed heterozygosity (H_O) and expected heterozygosity (H_E) ranged from 0.056 to 0.876 and from 0.480 to 0.907, with average values of 0.287 and 0.698, respectively. The average H_O (0.240) was lower than the H_E and gene flow (Nm), and showed substantial genetic variability among all populations of amaranth accessions. The sample groupings did not strictly follow the geographic affiliations of the accessions. A similar pattern was obtained using model-based structure analysis without grouping by species type. Knowledge of the genetic diversity and population structure of amaranth can be used to select representative genotypes and manage Amaranthus germplasm breeding programs.     

基于SSR标记探讨三种金花茶植物的遗传多样性和遗传结构

薄叶金花茶、小花金花茶和小瓣金花茶是三种濒危金花茶植物,为了解珍稀濒危植物遗传多样性和遗传结构,该研究利用微卫星标记对他们的7个种群共184个个体进行了遗传多样性和遗传结构分析。结果表明:11个位点共检测到等位基因92个。在物种水平上,小瓣金花茶平均等位基因数(N_A)为3.9、有效等位基因数(N_E)为2.328、观测杂合度(H_o)为0.520、期望杂合度(H_e)为0.501,高于薄叶金花茶和小花金花茶。在种群水平上,有效等位基因数(N_E)在1.788～2.466之间,期望杂合度(H_e)在0.379～0.543之间;种群间遗传分化系数(FST)在0.143 7～0.453 3之间,种群间基因流(N_m)在0.301 5～1.488 9之间。AMOVA分子变异分析显示65.72%的变异存在于种群内。三种金花茶具有较低水平的遗传多样性和高水平的种群间遗传分化。STRUCTURE和PCoA种群遗传结构分析结果将取样种群分为2组,即薄叶金花茶和小花金花茶大部分个体分为一组,小瓣金花茶大部分个体分为一组。现存所有种群应根据实际情况尽快采取就地保护或迁地保护措施。     

Characterization of polymorphic microsatellite loci in the aphid species Macrosiphum euphorbiae (Hemiptera: Aphididae)

Fourteen microsatellite loci were isolated and characterized in the potato aphid, Macrosiphum euphorbiae Thomas, by screening a genomic library with the oligonucleotide probes (GA)₁₀ (GT)₁₀ and (GATA)₄. Allelic diversity was estimated in samples collected from potato fields in Tunisia. Ten loci displayed polymorphism that ranged from two to four alleles per locus and the observed heterozygosity ranged from zero to one. These markers could be used to study the population genetic structure of this polyphagous aphid species.     

Transferability and characterization of microsatellite markers from Byrsonima cydoniifolia A. Juss. (MALPIGHIACEAE) in seven related taxa from Cerrado biome reveal genetic relationships

Byrsonima Rich. is one of the largest genera of the Malpighiaceae family with 97 species occurrence in Brazil and multiple potentialities, including pharmaceutical and food industries. In this study, 17 microsatellite markers characterized in Byrsonima cydoniifolia were tested for seven related taxa, all species are native to Brazil and four are endemic. Genomic DNA was extracted from leaves tissues and 17 microsatellite markers were used to cross-amplification of microsatellite regions. Polymorphism and genetic diversity were evaluated for B. intermedia, B. verbascifolia, B. laxiflora, B. subterranea, B. umbellata, B. linearifolia. from 16 individuals and for B. viminifolia from 14 individuals. Transferred microsatellite markers panels ranged from 11 (64.8%) in B. viminifolia to 6 (35.2%) in B. umbellata. The total number of alleles per locus ranged from 5 (B. linearifolia) to 8 (B. subterranea) alleles. B. umbellata showed lower values of observed and expected heterozygosity (H_O?=?0.312; H_E?=?0.436) and B. subterranea presented the highest values (H_O?=?0.687; H_E?=?0.778). A greater number of microsatellite markers should be developed for B. umbellata. The microsatellite marker panels transferred to the species B. intermedia, B. verbascifolia, B. laxiflora, B. subterranea, B. viminifolia and B. linearifolia are very informative, with a high combined probability of exclusion of paternity (Q?≥?0.976) and the low combined probability of identity (I?≤?9.91?×?10^–6), potentially suitable for future genetic-population studies, supporting strategies for maintaining the genetic diversity and for exploration of Byrsonima species as genetic resources.

     

Twelve polymorphic expressed sequence tags‐derived markers for Plasmopara halstedii,the causal agent of sunflower downy mildew

Twelve expressed sequence tags‐derived markers were isolated from Plasmopara halstedii (Oomycetes), the causal agent of sunflower downy mildew. A total of 25 single nucleotide polymorphisms and five indels were detected by single‐strand conformation polymorphism analysis and developed for high‐throughput genotyping of 32 isolates. There was a high level of genetic diversity (H_E = 0.484). Observed heterozygosity ranged from 0 to 0.143 indicating that P. halstedii is probably a selfing species. These markers were also useful in detecting significant genetic variations among French populations (F_ST = 0.193) and between French and Russian populations (F_ST = 0.23). Cross‐amplification tests on three closely related species indicated that no loci amplified in other Oomycete species.     

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.     

Isolation and characterization of first microsatellite markers for the noble crayfish, <Emphasis Type="Italic">Astacus astacus</Emphasis>

Noble crayfish (Astacus astacus) is listed as vulnerable by the IUCN Red List of Threatened Species in Europe. However, very little is known about genetic diversity and structuring of noble crayfish populations, mainly because of the lack of informative genetic markers. We describe the isolation and characterization of the first microsatellite markers for this species, which were obtained by screening 4,000 recombinant clones. Eight loci revealed polymorphisms in a panel of 172 individuals from seven populations in Northern Europe. Number of alleles per locus ranged from two to 10 (average 4.4) and heterozygosity levels among populations varied from 0 to 0.80 for H _o and from 0 to 0.72 for H _e.     

Isolation and characterization of microsatellite loci in <Emphasis Type="Italic">Pedicularis verticillata</Emphasis> L. using PCR-based isolation of microsatellite arrays (PIMA)

Pedicularis verticillata L. is a highly valuable herb for traditional Chinese medical treatment. In this report, 11 microsatellite loci from P. verticillata were isolated. The simple sequence repeat (SSR) markers were screened in 23 samples of wild populations of P. verticillata, and eight samples from its sister P. ikomai. The number of alleles ranged from 4 to 13, and value of expected (H _E) and observed (H ₀) heterozygosity was 0.62609–0.89662 and 0–0.95652, respectively. All loci were significantly deviated from Hardy–Weinberg expectations due to the heterozygote deficiency, indicating a dramatic loss of genetic polymorphisms in the restrictedly distributed species. The markers amplified well in the two species are useful for examining genetic diversity and population genetic structure, which, in turn, can provide information for establishing conservation strategy for these endangered species.     

Isolation of polymorphic tetranucleotide microsatellite markers for the silky short‐tailed bat Carollia brevicauda

We isolated 10 polymorphic microsatellite markers for the silky short‐tailed bat, Carollia brevicauda, from genomic libraries enriched for (AAGG)_n repetitive elements. The number of alleles ranged from six to 25 per locus with the observed heterozygosity ranging from 0.29 to 0.95. These markers will be useful for analysis of questions concerning population genetic structure and models of speciation. Results of cross‐species amplification in Carollia castanea and Carollia perspicillata are also reported.     

Isolation and characterization of polymorphic tetranucleotide microsatellite loci in the chestnut-crowned babbler (Pomatostomus ruficeps)

We describe 18 microsatellite markers isolated in the cooperatively breeding chestnut‐crowned babbler (Pomatostomus ruficeps). The number of alleles ranged from seven to 16 per locus (mean N_a = 10.4 ± 0.54 SE) and the expected heterozygosity ranged from 0.732 to 0.889 (mean H_E = 0.836 ± 0.01 SE). Three of the 18 loci exhibited significant heterozygote deficiency, but the remaining 15 will be used to analyse population genetic structure and the mating system of this highly social species.     

Isolation and characterization of eight microsatellite loci from <Emphasis Type="Italic">Lycorma delicatula</Emphasis> (White) (Hemiptera: Fulgoridae) for population genetic analysis in Korea

Lycorma delicatula (White) is native to China but is becoming an important insect pest in Korea. Polymorphic DNA markers like microsatellites are widely used for characterizing dispersal patterns and capacity of invasive insect pests which can contribute to designing effective management of the species. To facilitate such population genetic studies of L. delicatula in Korea, we isolated and characterized eight microsatellite loci for L. delicatula using a hybridization-biotin enrichment method. We further used these novel microsatellite loci to determine population genetic parameters for 33 L. delicatula specimens collected from Cheonan, South Korea where outbreaks of this species were first reported in Korea. The number of alleles per locus ranged from three to ten, with an average of 6.25. The mean expected (H _E) and observed heterozygosities (H _O) were 0.575 and 0.626, respectively. The eight loci showed no deviation from Hardy–Weinberg equilibrium according to the adjusted significance threshold (P = 0.00625), and there was no linkage disequilibrium between each pair of these eight markers. Bayesian cluster analysis using the program structure revealed no evidence of genetic structuring in L. delicatula samples from Cheonan. These new microsatellite markers will be widely applicable to future ecological genetic studies of L. delicatula, including assessment of the level of gene flow and genetic connectivity among populations that are necessary for effective management and monitoring of the species.     

Genetic Diversity and Genetic Structure of an Endangered Species, Trillium tschonoskii

The genetic diversity and genetic structure of Trillium tschonoskii (Maxim) were investigated using amplified fragment length polymorphism markers. Eight primer combinations were carried out on 105 different individuals sampled from seven populations. Of the 619 discernible DNA fragments generated, 169 (27.3%) were polymorphic. The percentage of polymorphic bands within populations ranged from 4.52 to 10.50. Genetic diversity (H_E) within populations ranged from 0.0130 to 0.0379, averaging 0.0536 at the species level. Genetic differentiation among populations was detected based on Nei's genetic diversity analysis (53.03%) and analysis of molecular variance (AMOVA) (52.43%). AMOVA indicated significant genetic differentiation among populations (52.43% of the variance) and within populations (47.57% of the variance) (p < 0.0002). Gene flow was low (0.4429) among populations. Species breeding system and limited gene flow among populations are plausible reasons for the high genetic differentiation observed for this species. We propose an appropriate strategy for conserving the genetic resources of T. tschonoskii in China.