Analysis of 22 heterologous microsatellite markers for genetic variability in Indian goats

The genetic variability of 22 heterologous microsatellite markers was analyzed in two Indian goat breeds, namely Bengal and Chegu. The heterozygosity, polymorphism information content (PIC), and probability of identity of two individuals were calculated for all microsatellite loci in both the breeds. The observed number of alleles varied between 4 and 13 at the studied microsatellite loci. The evaluated microsatellite loci exhibited high mean heterozygosity of 0.69 +/- 0.11 and 0.66 +/- 0.07 in Bengal and Chegu goats, respectively. The mean PIC values of the studied loci in these breeds were 0.79 +/- 0.08 and 0.78 +/- 0.05, respectively. The probability of identity of two random individuals from different breeds, taking into account, all the 22 microsatellite loci was as low as 5.523 x 10(-40). On the basis of these results, we propose that these microsatellite markers may be used with reliability for studying genetic diversity and for identification of individuals in Indian goat breeds.     

秦川母牛群体遗传特性的微卫星标记研究

为了从DNA分子水平揭示秦川牛群体遗传多态性和群体遗传结构,寻找可用于秦川牛的微卫星标记,本研究选择了12个普通牛（Bos taurus）微卫星标记检测了90头秦川母牛各微卫星位点的遗传变异及多态性。结果表明,在秦川母牛群体中,12个微卫星位点共检测到了247个等位基因,各位点的等位基因数在13（INRA005）～33个（HEL13）之间,平均每个微卫星位点的等位基因数为21个;总有效等位基因数和平均每个位点平均有效等位基因数（Ne）分别分为142．6229和11．8852。各位点平均基因频率取样方差（V（pij））为2．6036×10^-4。12个微卫星位点平均观察杂合度（Ho）和平均期望杂合度（He）在0．7842（INRA005）～0．9775（BM315）和0．7952（BM315）～0．9446（HEL13）之间。12个位点平均多态信息含量（PIC）在0．7653（INRA005）～0．9420（HEL13）之间,平均为0．8965．12个微卫星位点均属于高度多态位点,这表明秦川母牛群体中所检测各微卫星位点具有丰富的遗传多态性,具备较大的选择潜力。12个微卫星位点的平均固定指数（F）为-0．0076,即各位点杂合子的缺陷度不高,即偏离Hardy—Weinberg平衡的程度不大。     

Isolation and characterization of 11 microsatellite loci from the black fly, Simulium negativum (Diptera: Simuliidae)

Eleven polymorphic microsatellite loci were isolated and developed from the black fly, Simulium negativum, a member of the Simulium arcticum sibling species complex. The observed heterozygosity of the 11 loci ranged from 0.03 to 0.83. The number of alleles per locus ranged from 8 to 19. Significant linkage disequilibria were encountered only for the primer pairs BF7-1 with BF7-5 and BF6-32 with BF7-16. Presumably, these microsatellite loci can be used to study genetic structure within the entire S. arcticum complex.     

Characterization of nine novel microsatellite loci for the Venus clam (Cyclina sinensis)

The Venus clam, Cyclina sinensis, is one of the most important bivalves in China marine aquaculture. Using (CA)(15)-enriched genomic libraries of this species, nine novel polymorphic microsatellite loci were isolated and characterized. The mean number of observed alleles per locus was 16 (range 8-24). The observed and expected heterozygosity ranged from 0.119 to 0.872 and from 0.626 to 0.931, respectively. Three loci had significant departure from Hardy-Weinberg equilibrium and non-significant linkage disequilibrium was found among all nine loci. These highly informative microsatellite markers should be useful for population genetic analyses of C. sinensis.     

林麝微卫星座位的分离和鉴定

利用改进的磁珠富集法从林麝(Moschus berezovskii)的基因组中分离到10个新的(AC)n重复微卫星座位,并分析其在24个林麝个体(取样于中国四川金凤山群体)中的多态性。其中,5个位点在24个林麝个体中具有4—13个等位基因,观察杂合度和期望杂合度分别是0.429—0.957和0.587—0.902,平均多态信息含量是0.730。表明这5个微卫星位点具有高度的多态性,可以用于林麝遗传多样性研究,对林麝的保护具有很重要的意义。     

Molecular genetic studies of Anopheles stephensi in Pakistan

Anopheles stephensi Liston s.l. (Diptera: Culicidae) is one of the major vectors of malaria in Pakistan, India, Iran and Afghanistan. In parts of its range this species has shown increases in both relative and absolute abundance in what is hypothesized to be a response to human-mediated environmental change resulting from extensive irrigation. We attempted to detect the molecular genetic signatures of this population instability based on three samples obtained from two villages (149/6R and 111/6R) within an irrigation zone in Punjab Province and from one village (Azakhel) outside the irrigation scheme in Northwest Frontier Province (NWFP), Pakistan, using seven microsatellite loci and 682 basepairs of the mitochondrial CO1 gene. For microsatellite loci, high levels of genetic diversity were observed within populations (mean alleles per locus 10.71-11.57; mean heterozygosity 0.703-0.733). Deviation from Hardy-Weinberg expectations was observed for only two microsatellite loci in 21 tests. No genetic differentiation was observed between populations and average pairwise F(ST) values did not differ significantly from zero for any population pair or either marker system. Tests of population expansion for both mitochondrial and microsatellite loci were inconclusive.     

Isolation and characterization of microsatellite loci in Psilopeganum sinense Hemsl (Rutaceae), an endangered herb endemic to Yangtze River valley

Twenty-nine primer pairs flanking microsatellite repeats were designed from an AC-enriched genomic library of Psilopeganum sinense and tested using 24 individuals derived from a natural population. A total of 11 microsatellite loci were found polymorphic. The number of alleles per locus varied from two to eight, with an average value of 3.7. The ranges of observed and expected heterozygosities were 0.000-0.750 and 0.365-0.800, respectively. These microsatellite loci have been directly applied to the ongoing conservation genetic studies of P. sinense.     

Identification, Inheritance, and Variation of Microsatellite Markers in the Black Scallop Mimachlamys varia

Five polymorphic microsatellite loci were identified in the black scallop Mimachlamys varia after construction of a genomic library enriched for (GT)n. To examine the transmission pattern of microsatellite alleles, several families were created and genotypes scored for three loci. The expected Mendelian ratios were found in 12 of 14 segregations examined. Unexpected segregations may be explained by a genotyping error (allelic dropout), given that when a specific allele was treated as dominant, the phenotypic ratios conformed to Mendelian expectations. The five loci were also examined in two samples from the Spanish coast. The two localities displayed similar mean values for the number of alleles per locus (7.2-8.4), allelic richness (7.2-7.9), and observed (0.389-0.484) and expected heterozygosity (0.545-0.618). Significant Hardy-Weinberg deviations were observed at three loci, with heterozygote deficiency occurring in all cases. Global multilocus θ value and allele frequencies at one locus revealed significant differentiation between the two localities.     

基于转录组测序的大熊猫多态性微卫星标记筛选

结合已公布的大熊猫Ailuropoda melanoleuca基因组和本实验室所测6只大熊猫的转录组数据,筛选多态性微卫星位点并分析其组成及特征。结果显示:共获得326个多态性微卫星位点,其中二碱基多态性微卫星最多,共228个,占69.93%;三、四、五、六碱基所占比例分别为9.51%、14.11%、5.21%、1.22%。根据分析结果中缺失率与标准差2项指标以及位点序列长度,选取20个多态性二碱基微卫星位点,用于25只大熊猫个体血液DNA进行PCR验证并做后续分析。结果表明:不同位点的等位基因数为2～8,平均等位基因数为3.70,观测杂合度、期望杂合度分别为0～1.000、0.280～0.784,平均值分别为0.472和0.532。在Bonferroni校正后,证实4个位点显著偏离哈迪-温伯格平衡,所有位点未观察到显著连锁不平衡(P>0.01)。20个位点多态信息含量(PIC)在0.246～0.734,其中具有高度多态性的位点9个(PIC>0.50),11个位点呈中度多态性(0.25相似文献   

Isolation and characterization of microsatellite loci in Ainsliaea faurieana (Asteraceae), an endemic plant species on Yakushima Island, Japan, and cross-species amplification in closely related taxa

Fourteen microsatellite loci were isolated and characterized for Ainsliaea faurieana, an endemic perennial plant species on Yakushima Island. In an analysis of 37 individuals from Yakushima Island, the observed number of alleles ranged from two to six. The expected and observed heterozygosities were 0.109-0.811 and 0.000-0.27, respectively. The inbreeding coefficient was 0.489-1.000. The interspecific applicability of these microsatellites was evaluated by analysing three closely related taxa from the Ryukyu Islands. All primer pairs for the 14 loci tested successfully amplified in all taxa.     

Polymorphic microsatellite markers isolated from the neptune whelk Neptunea arthritica

Eight polymorphic microsatellite DNA loci were isolated from the neptune whelk Neptunea arthritica, which is an important fishery resource in northern Japan. The number of alleles at the loci ranged from two to six, with observed and expected heterozygosities of 0.192-0.807 and 0.233-0.738, respectively. The observed variations suggest that these loci can be used as markers for population and kinship analyses in this species.     

Characterization of 13 polymorphic microsatellite loci in the Japanese land leech

We developed 13 polymorphic microsatellite loci of the Japanese land leech (Haemadipsa japonica; Haemadipsidea) using an Illumina MiSeq sequencing approach. A total of 42,064 nuclear DNA contigs were filtered for microsatellite motifs, among which 30,873 simple sequence repeat loci were identified. From these sequences, we selected 30 primer sets, and 13 of these loci were successfully amplified. Polymorphism of the 13 loci was tested using 16 individuals sampled from sixteen populations across Japan. The number of alleles and polymorphism information content varied from 5 to 17 and 0.335 to 0.883, respectively, and observed and expected heterozygosity values ranged from 0.143 to 0.875 and 0.349 to 0.893, respectively, indicating that these loci are polymorphic. Furthermore, we established useful multiplex PCR using these loci. The 13 microsatellite loci described in this paper are the first nuclear microsatellite markers for a land leech species.     

Novel polymorphic microsatellite markers developed in the cotton bollworm Helicoverpa armigera （Lepidoptera：Noctuidae）

A novel set of five polymorphic di- or trinucleofide microsatellite loci suitable for population genetic study were developed from an enriched genomic library for the pest insect cotton bollworm, Helicoverpa armigera, and cross-amplifiability of these and other published loci was tested in a closely related species, the tobacco budworm, H.assulta. The expected heterozygosity at these loci ranges from 0.62 to 0.91 in the cotton bollworm. The observed allele numbers varies from 4 to 12 in the limited number of individuals tested. Although a large proportion of cloned microsatellite sequences are present in multi-copy in the cotton bollworm genome, the overwhelming majority of the finalized polymorphic diallelic loci are tri-nucleotide microsatellites - an unexpected outcome, which should facilitate subsequent genotyping analysis.     

Polymorphic nuclear microsatellite loci for studies of brown crab, Cancer pagurus L

Twelve polymorphic microsatellite DNA loci were isolated from the brown crab, Cancer pagurus L., by construction of microsatellite-enriched genomic libraries. Genotyping of 40 individuals from Norfolk (UK) revealed variable levels of locus polymorphism with an average of 7.75 alleles per locus (range 2-22). The observed and expected heterozygosities per locus ranged from 0.025 to 0.868 and from 0.025 to 0.947, respectively. No evidence of linkage disequilibrium was detected between pairs of loci and genotype proportions at all loci conformed to Hardy-Weinberg equilibrium expectations. The microsatellite loci developed constitute a suite of genetic markers applicable to numerous areas of C. pagurus research.     

Mutation and evolution of microsatellite loci in Neurospora

The patterns of mutation and evolution at 13 microsatellite loci were studied in the filamentous fungal genus Neurospora. First, a detailed investigation was performed on five microsatellite loci by sequencing each microsatellite, together with its nonrepetitive flanking regions, from a set of 147 individuals from eight species of Neurospora. To elucidate the genealogical relationships among microsatellite alleles, repeat number was mapped onto trees constructed from flanking-sequence data. This approach allowed the potentially convergent microsatellite mutations to be placed in the evolutionary context of the less rapidly evolving flanking regions, revealing the complexities of the mutational processes that have generated the allelic diversity conventionally assessed in population genetic studies. In addition to changes in repeat number, frequent substitution mutations within the microsatellites were detected, as were substitutions and insertion/deletions within the flanking regions. By comparing microsatellite and flanking-sequence divergence, clear evidence of interspecific allele length homoplasy and microsatellite mutational saturation was observed, suggesting that these loci are not appropriate for inferring phylogenetic relationships among species. In contrast, little evidence of intraspecific mutational saturation was observed, confirming the utility of these loci for population-level analyses. Frequency distributions of alleles within species were generally consistent with the stepwise mutational model. By comparing variation within species at the microsatellites and the flanking-sequence, estimated microsatellite mutation rates were approximately 2500 times greater than mutation rates of flanking DNA and were consistent with estimates from yeast and fruit flies. A positive relationship between repeat number and variance in repeat number was significant across three genealogical depths, suggesting that longer microsatellite alleles are more mutable than shorter alleles. To test if the observed patterns of microsatellite variation and mutation could be generalized, an additional eight microsatellite loci were characterized and sequenced from a subset of the same Neurospora individuals.     

Isolation and characterization of 19 highly polymorphic microsatellite markers in the devil stinger, Inimicus japonicus (Synanceiidae)

Inimicus japonicus, the devil stinger, has an extensive distribution along the coast of China, Japan and the Korean Peninsula. Nineteen highly polymorphic microsatellite markers were isolated and characterized in I. japonicus. Twenty-eight individuals from a wild population were tested for polymorphism using this set of polymorphic microsatellite markers. The number of alleles per locus ranged from 4 to 14. The ranges of observed and expected heterozygosity were 0.500-0.892 and 0.521-0.910, respectively. Significant deviations from Hardy-Weinberg equilibrium were detected at two loci. To the best of our knowledge, these were the first microsatellite loci characterized from the Synanceiidae; they can be used for estimating genetic diversity, population structure studies, parentage analysis, genetic linkage map construction, germplasm classification and identification, gene identification, quantitative trait loci mapping, and marker-assisted selection in breeding of I. japonicus and other species of this family.     

Microsatellite marker development and analysis in the eastern oyster (Crassostrea virginica): confirmation of null alleles and non-Mendelian segregation ratios

Eighteen microsatellite markers were developed for the Crassostrea virginica nuclear genome, including di-, tri-, and tetranucleotide microsatellite repeat regions that included perfect, imperfect, and compound repeat sequences. A reference panel with DNA from the parents and four progeny of 10 full-sib families was used for a preliminary confirmation of polymorphism at these loci and indications of null alleles. Null alleles were discovered at three loci; in two instances, primer redesign enabled their amplification. Two to five representative alleles from each locus were sequenced to ensure that the targeted loci were amplifying. The sequence analysis revealed not only variation in the number of simple sequence repeat units, but also polymorphisms in the microsatellite flanking regions. A total of 3626 bp of combined microsatellite flanking region from the 18 loci was examined, revealing indels as well as nucleotide site substitutions. Overall, 16 indels and 146 substitutions were found with an average of 4.5% polymorphism across all loci. Eight markers were tested on the parents and 39-61 progeny from each of four families for examination of allelic inheritance patterns and genotypic ratios. Twenty-six tests of segregation ratios revealed eight significant departures from expected Mendelian ratios, three of which remained significant after correction for multiple tests. Deviations were observed in both the directions of heterozygote excess and deficiency.     

New methods to identify conserved microsatellite loci and develop primer sets of high cross-species utility - as demonstrated for birds

We have developed a new approach to create microsatellite primer sets that have high utility across a wide range of species. The success of this method was demonstrated using birds. We selected 35 avian EST microsatellite loci that had a high degree of sequence homology between the zebra finch Taeniopygia guttata and the chicken Gallus gallus and designed primer sets in which the primer bind sites were identical in both species. For 33 conserved primer sets, on average, 100% of loci amplified in each of 17 passerine species and 99% of loci in five non-passerine species. The genotyping of four individuals per species revealed that 24-76% (mean 48%) of loci were polymorphic in the passerines and 18-26% (mean 21%) in the non-passerines. When at least 17 individuals were genotyped per species for four Fringillidae finch species, 71-85% of loci were polymorphic, observed heterozygosity was above 0.50 for most loci and no locus deviated significantly from Hardy-Weinberg proportions. This new set of microsatellite markers is of higher cross-species utility than any set previously designed. The loci described are suitable for a range of applications that require polymorphic avian markers, including paternity and population studies. They will facilitate comparisons of bird genome organization, including genome mapping and studies of recombination, and allow comparisons of genetic variability between species whilst avoiding ascertainment bias. The costs and time to develop new loci can now be avoided for many applications in numerous species. Furthermore, our method can be readily used to develop microsatellite markers of high utility across other taxa.     

Fifteen polymorphic microsatellite markers for the giant spiny frog, Paa spinosa

We characterized 15 polymorphic microsatellite loci for the giant spiny frog, Paa spinosa. These loci were highly polymorphic when screened in 38 individuals from two different populations, with nine to 23 alleles per locus. The range of observed and expected heterozygosities was 0.231-0.916 and 0.296-0.944, respectively. These polymorphic loci will be useful for assessing genetic diversity, population structure, gene flow, population assignment and determining paternity in the giant spiny frog.     

Microsatellite markers for eastern hemlock (Tsuga canadensis)

We describe polymerase chain reaction primer pairs and reaction conditions for amplification of 15 microsatellite loci from eastern hemlock (Tsuga canadensis). The primers were tested on 23 individuals from a natural population in southwestern North Carolina, USA. These primers yielded an average of 5.9 alleles per locus (range of 2-14), an average observed heterozygosity of 0.45 (range 0.14-0.73), and an average polymorphic information content of 0.54 (range 0.28-0.86). In addition, eight of the primer pairs were found to amplify microsatellite loci in one or more additional species of Tsuga.