应用微卫星标DNA记对三个封闭群兔遗传分析

目的检测国内Et本大耳白兔、青紫蓝兔、新西兰白兔的遗传背景及遗传结构,为封闭群兔遗传检测方法建立和标准化提供基础资料。方法应用18个微卫星标记及荧光标记一半自动基因分型技术对三个群体95个个体进行Hardy．Weinberg检测,统计每个位点等位基因频率、杂合度、F值、遗传距离等信息。结果三个种群平均等位基因观测数为3．167、4．556、3．444,平均观测杂合度为0．444、0．5230、0．4976。18个位点平均多态信息含量（PIC）为0．410、0．549、0．470,日本大耳白兔6个位点HWE检验（P〈0．05）,显著偏离Hardy—Weinberg平衡,并在Satl3,INRCCDDV0088位点基因型完全纯和,新西兰白兔和青紫蓝兔分别有2个位点显著偏离Hardy—Weinberg平衡。三个种群遗传距离：青紫蓝兔与新西兰白兔遗传距离最近,为0．124,与日本大耳白兔遗传关系最远,为0．320;新西兰白兔与日本大耳白兔较远,为0：10。结论三个种群有各自不同遗传特征,遗传多样性较高,种群间分化明显。个别位点偏离遗传平衡,推测人工繁育过程中存在一定问题。     

High levels of gene flow and heterozygote excess characterize Rhizoctonia solani AG-1 IA (Thanatephorus cucumeris) from Texas

To date, much of the genetics of the basidiomycete Thanatephorus cucumeris (anamorph = Rhizoctonia solani) remains unknown. Here, we present a population genetics study using codominant markers to augment laboratory analyses. Seven single-copy nuclear RFLP markers were used to examine 182 isolates of Rhizoctonia solani AG-1 IA collected from six commercial rice fields in Texas. Thirty-six multilocus RFLP genotypes were identified. Population subdivision analyses indicated a high degree of gene flow/migration between the six geographic populations. Tests for Hardy-Weinberg equilibrium (HWE) among the 36 multilocus RFLP genotypes revealed that four of the seven loci did not significantly differ from HWE. Subsequent analysis demonstrated that departures from HWE at the three remaining loci were due to an excess of heterozygotes. Data presented here suggest that R. solani AG-1 IA is actively outbreeding (heterothallic). Possible explanations for heterozygote excess, which was observed at all seven RFLP loci, are discussed.     

Population genetics of the yellow fever mosquito in Trinidad: comparisons of amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP) markers

Recent development of DNA markers provides powerful tools for population genetic analyses. Amplified fragment length polymorphism (AFLP) markers result from a polymerase chain reaction (PCR)-based DNA fingerprinting technique that can detect multiple restriction fragments in a single polyacrylamide gel, and thus are potentially useful for population genetic studies. Because AFLP markers have to be analysed as dominant loci in order to estimate population genetic diversity and genetic structure parameters, one must assume that dominant (amplified) alleles are identical in state, recessive (unamplified) alleles are identical in state, AFLP fragments segregate according to Mendelian expectations and that the genotypes of an AFLP locus are in Hardy-Weinberg equilibrium (HWE). The HWE assumption is untestable for natural populations using dominant markers. Restriction fragment length polymorphism (RFLP) markers segregate as codominant alleles, and can therefore be used to test the HWE assumption that is critical for analysing AFLP data. This study examined whether the dominant AFLP markers could provide accurate estimates of genetic variability for the Aedes aegypti mosquito populations of Trinidad, West Indies, by comparing genetic structure parameters using AFLP and RFLP markers. For AFLP markers, we tested a total of five primer combinations and scored 137 putative loci. For RFLP, we examined a total of eight mapped markers that provide a broad coverage of mosquito genome. The estimated average heterozygosity with AFLP markers was similar among the populations (0.39), and the observed average heterozygosity with RFLP markers varied from 0.44 to 0.58. The average FST (standardized among-population genetic variance) estimates were 0.033 for AFLP and 0.063 for RFLP markers. The genotypes at several RFLP loci were not in HWE, suggesting that the assumption critical for analysing AFLP data was invalid for some loci of the mosquito populations in Trinidad. Therefore, the results suggest that, compared with dominant molecular markers, codominant DNA markers provide better estimates of population genetic variability, and offer more statistical power for detecting population genetic structure.     

Microsatellite variability and heterozygote excess in Elymus trachycaulus populations from British Columbia in Canada

Microsatellite markers were used to assess the genetic diversity and population structure in four populations of Elymus trachycaulus from British Columbia and one population of Elymus alaskanus from Northwest Territories. Fourteen microsatellite loci were used in this study. Our results indicated that E. trachycaulus is highly polymorphic, with an average percentage of polymorphic loci of 96.5% over the four populations. Average expected heterozygosity values (H_E or gene diversity) varied from 0.418 to 0.585 with a mean of 0.497. Most of the genetic variation was found within populations (85%) and the differentiation among populations was found to be 15% (F_st = 0.15). Interpopulation genetic distances corresponded well with the geographic distance between the population sites of origin, as well as morphological characteristics. Tests for Hardy–Weinberg equilibrium (HWE) for all loci and all populations revealed that all loci significantly differ from HWE. Subsequent analysis indicated that departure from HWE at some loci was due to an excess of heterozygotes. Possible explanations for heterozygote excess are discussed. The most likely reason for observed heterozygote excess could be due to the polyploidy nature of the species.     

Sixteen microsatellite loci for the Bufo boreas group

The four species that comprise the Bufo boreas group of toads are critically imperiled in all or portions of their geographical ranges. We present data from 16 microsatellite loci isolated from B. boreas that cross‐amplify in these four species. These markers have proven useful in the analyses of population structure and conservation genetics, and provide a powerful tool for future researchers who seek to understand the conservation genetics of these rare toad species. Polymorphism was assessed for 339 individuals from seven populations representing the four species. All loci were polymorphic (X? = 8, range of four to 19 alleles). Three loci were not in Hardy–Weinberg equilibrium (HWE) in one population, and one of these loci was out of HWE in a second population (P < 0.003 after Bonferroni correction for multiple tests). However, there were no systematic deviations from HWE across all study populations. Small populations in fragmented habitat appear to explain the existing HWE deviations.     

Powerful Haplotype-Based Hardy-Weinberg Equilibrium Tests for Tightly Linked Loci

Recently, there have been many case-control studies proposed to test for association between haplotypes and disease, which require the Hardy-Weinberg equilibrium (HWE) assumption of haplotype frequencies. As such, haplotype inference of unphased genotypes and development of haplotype-based HWE tests are crucial prior to fine mapping. The goodness-of-fit test is a frequently-used method to test for HWE for multiple tightly-linked loci. However, its degrees of freedom dramatically increase with the increase of the number of loci, which may lack the test power. Therefore, in this paper, to improve the test power for haplotype-based HWE, we first write out two likelihood functions of the observed data based on the Niu''s model (NM) and inbreeding model (IM), respectively, which can cause the departure from HWE. Then, we use two expectation-maximization algorithms and one expectation-conditional-maximization algorithm to estimate the model parameters under the HWE, IM and NM models, respectively. Finally, we propose the likelihood ratio tests LRT and LRT for haplotype-based HWE under the NM and IM models, respectively. We simulate the HWE, Niu''s, inbreeding and population stratification models to assess the validity and compare the performance of these two LRT tests. The simulation results show that both of the tests control the type I error rates well in testing for haplotype-based HWE. If the NM model is true, then LRT is more powerful. While, if the true model is the IM model, then LRT has better performance in power. Under the population stratification model, LRT is still more powerful. To this end, LRT is generally recommended. Application of the proposed methods to a rheumatoid arthritis data set further illustrates their utility for real data analysis.     

Development, characterisation, inheritance, and cross-species utility of American lobster (Homarus americanus) microsatellite and mtDNA PCR-RFLP markers.

Effective management of exploited species demands contemporary knowledge of population structure and mating patterns. Genetic markers can prove useful in providing this knowledge. Despite its commercial importance, genetic markers for American lobster (Homarus americanus) are limited. We developed 12 tetra- and 1 trinucleotide microsatellite loci for American lobster that exhibit little stuttering after PCR amplification. Gene diversity of these loci ranged from 0.516 to 0.929. A four-locus multiplex permits rapid genotyping of progeny in parentage experiments with a paternity exclusion probability over the four loci of 97.8%. We examined the loci for conformity to Hardy-Weinberg expectations (HWE) and linkage using individuals from one location and found that four loci deviated from HWE. We also tested inheritance and pairwise linkage using 48 embryos from each of two females. With the exception of two loci that were derived from the same clone and separated by 72 bp, no evidence of linkage was found. We, for the first time, demonstrate the occurrence of multiple paternity in American lobster. We also observed an apparent occurrence of dispermic androgenesis, possibly the first documentation of such an event within a species. Ten of the loci amplified in European lobster (Homarus gammarus), although two were monomorphic and one deviated significantly from HWE. We quantified mitochondrial DNA (mtDNA) sequence variation through the use of PCR amplification of two DNA fragments, followed by digestion with restriction enzymes; eight haplotypes were detected. One of the two fragments amplified in European lobster. Both sets of markers should prove useful for population discrimination purposes, and the microsatellites, in particular the four-locus multiplex, should prove highly amenable to rapidly addressing questions about mating patterns.     

从公共数据库中利用数据挖掘的方法开发15条黄金鲈EST-SSR标记

黄金鲈是美国中西部地区重要的淡水经济型鱼类。TypeⅠ型标记对于比较作图和遗传学研究有相当重要的作用, 但是黄金鲈的TypeⅠ标记数量相当少。研究通过数据挖掘的方法从公共数据库中挖掘黄金鲈的TypeⅠ型即EST-SSR标记。实验结果分析了21968条EST序列, 约14.4%的序列包含不同类型的核心重复单元。CA重复是最丰富的二碱基重复单元。从中挑选62条包含SSR的EST序列设计引物, 筛选得到15对多态的微卫星位点。群体遗传学参数显示15对EST-SSR位点的等位基因数范围为4—19(平均数为9), 观测杂合度和期望杂合度的范围分别为0.103—0.929和0.116—0.934, 有4个位点偏离HWE平衡。研究得到的EST-SSR标记适用于黄金鲈的群体遗传学和基因组作图研究。     

Correction method for null alleles in species with variable microsatellite flanking regions, a case study of the black-lipped pearl oyster Pinctada margaritifera

In bivalves, heterozygote deficiencies and departures from Hardy-Weinberg equilibrium (HWE) in microsatellite analysis are common and mainly attributed to inbreeding, genetic patchiness (Walhund effect), or null alleles. We checked for the occurrence of null alleles at 3 microsatellite loci in 3 populations of black-lipped pearl oyster, Pinctada margaritifera, using a step-by-step method to re-amplify homozygotes and null individuals with redesigned primer pair combinations. After amplification with original primer pairs, the 3 populations exhibited null alleles, absence of structure, and significant departure from HWE for all 3 loci due to heterozygote deficiencies. After 3 re-amplification steps, with modified primer sets, all loci were corrected for null alleles. Once corrected, all populations appeared at HWE, demonstrating that null alleles were responsible for the initial disequilibrium of the populations. Furthermore, analysis from corrected genotypes demonstrates significant genetic differentiation for one population from the other 2.     

Population admixture: detection by Hardy-Weinberg test and its quantitative effects on linkage-disequilibrium methods for localizing genes underlying complex traits

In association studies searching for genes underlying complex traits, the results are often inconsistent, and population admixture has been recognized qualitatively as one major potential cause. Hardy-Weinberg equilibrium (HWE) is often employed to test for population admixture; however, its power is generally unknown. Through analytical and simulation approaches, we quantify the power of the HWE test for population admixture and the effects of population admixture on increasing the type I error rate of association studies under various scenarios of population differentiation and admixture. We found that (1) the power of the HWE test for detecting population admixture is usually small; (2) population admixture seriously elevates type I error rate for detecting genes underlying complex traits, the extent of which depends on the degrees of population differentiation and admixture; (3) HWE testing for population admixture should be performed with random samples or only with controls at the candidate genes, or the test can be performed for combined samples of cases and controls at marker loci that are not linked to the disease; (4) testing HWE for population admixture generally reduces false positive association findings of genes underlying complex traits but the effect is small; and (5) with population admixture, a linkage disequilibrium method that employs cases only is more robust and yields many fewer false positive findings than conventional case-control analyses. Therefore, unless random samples are carefully selected from one homogeneous population, admixture is always a legitimate concern for positive findings in association studies except for the analyses that deliberately control population admixture.     

实验兔三个封闭群微卫星DNA多态性遗传分析

目的 对日本大耳白兔、青紫蓝兔、新西兰兔三个封闭群体开展群体遗传学分析.方法 利用10个微卫星位点,进行Hardy-Weinberg平衡(HWE)检验,统计三个种群的基因频率、观测杂合度、期望杂合度、F值和遗传距离.结果 青紫蓝品种在12L1E11位点,新西兰品种在INRACCDDV0087位点与INRACCDDV0203位点,日本大耳白兔在Sat12位点与INRACCDDV0203,P＜0.05,显著偏离HWE,多数表现为杂合子缺陷;三个群体在Sat13、So144、6L1F10、7L1F1、12L4A1、INRACCDDV0016点上均符合HWE;各位点平均等位基因数5.9,种群整体基因频率差别较大,其范围为0 -0.9060;三个种群的平均观测杂合度为0.6204,平均期望杂合度为0.6178;群体间分化系数(Fst)平均为0.0750,日本大耳白兔和青紫蓝兔遗传距离最近为0.1223,青紫蓝兔与新西兰兔遗传距离最远为0.1934.结论 三个种群的遗传结构均表现出遗传稳定性和均一性,在10个微卫星位点上呈现高度多态性,种群间遗传分化明显.     

Characterization of two New York City Jewish populations at six short tandem repeat loci

The Hasidic and non-Hasidic Jewish communities of New York City represent two subpopulations with long-documented histories of restrictive marriage patterns and a high degree of endogamy. As part of a continuing study into their genetic structure, allele frequencies were determined for the six tetrameric short tandem repeat (STR) loci: FESFPS, F13AO1, vWA, CSF1PO, TPOX, and THO1. All loci were tested for Hardy-Weinberg equilibrium (HWE) by three tests: chi-square analysis, Monte Carlo chi-square analysis. and the exact test. The non-Hasidic population failed to meet HWE at the F13A01, FESFPS, and CSF1PO loci by all three tests. The Hasidic population also failed to meet HWE at the same loci by some of the tests. Comparison of the Hasidic to the non-Hasidic population using an R x C contingency table demonstrated a similarity at only the vWA locus. Significant differences exist when comparing the two Jewish populations to a reference Caucasian population.     

大鸨东方亚种遗传多样性的微卫星分析

为了探索东方亚种数量正在减少的原因和制定科学、有效的保护措施,利用微卫星DNA对大鸨东方亚种(Otistardadybowskii)的遗传多样性进行了研究。应用3个大鸨指名亚种的微卫星位点和13个波斑鸨的微卫星位点扩增了47个个体的基因组DNA,筛选出8个具有多态性的微卫星。其中有3个微卫星的多态性较低,其余5个微卫星的多态性较高。各位点的观察杂合度为0.0435-1.0000,平均杂合度(h)为0.6595;各位点多态信息含量(PIC)为0.0416-0.8520,平均为0.5497;有效等位基因数(E)为1.04-7.46,平均为3.61。4个位点符合HardyWeinberg平衡,4个位点偏离了HardyWeinberg平衡。多方面比较发现,大鸨东方亚种遗传多样性很低,且低于指名亚种,这可能由于其种群较小、历史遗传瓶颈作用、生境破碎化、分布地域紧缩等原因造成的。     

Significant evidence for linkage disequilibrium over a 5-cM region among Afrikaners

We explore the extent of deviations from Hardy-Weinberg equilibrium (HWE) at a marker locus and linkage disequilibrium (LD) between pairs of marker loci in the Afrikaner population of South Africa. DNA samples were used for genotyping of 23 loci on six chromosomes. The samples were collected from 91 healthy unrelated Afrikaner adults. Exact tests were used to determine evidence for deviations from HWE at a single marker locus or LD between pairs of marker loci. At the 0.05 level of significance, evidence was found for deviation from HWE at only one of the 23 loci. At the same level of significance, LD was found among 8 of the 34 intrachromosomal pairs of loci. On chromosome 21, there was evidence for LD (P = 0.02) between a pair of loci with a genetic distance of 5.51 cM. On chromosome 2, there was evidence for LD between a pair of loci with a genetic distance of 5.28 cM (P = 0.002) and a pair of loci with a genetic distance of 3.68 cM (P = 0.0004). Detailed analysis of LD for one locus pair indicated that only a few of all alleles participated in the LD and that strong LD was most often positive. Our findings indicate that Afrikaans-speaking Afrikaners represent one of those special populations deemed particularly suitable for disequilibrium mapping.     

Characterization of 20 microsatellite loci by multiplex PCR in swimming crab, Portunus trituberculatus

The swimming crab, Portunus trituberculatus (Crustacea: Decapoda: Brachyura), is one of the most important fishery resources in East Asia. The aim of this work is to develop microsatellite multiplex PCR systems for 20 microsatellites which were isolated from a microsatellite-enriched genomic library and to explore the genetic diversity of P. trituberculatus captured in the coastal regions of the Yellow Sea in Korea. We prepared four multiplex PCR systems (two hexplex and tetraplex systems each) which could be used for the genotyping of 20 markers. Most markers in this system are highly informative with mean polymorphism information contents and observed heterozygosity of 0.913 and 0.930. The total number of alleles observed in this study was 724, and the mean allelic number per locus was 36.2. Significant deviations from the Hardy–Weinberg equilibrium (HWE) were observed at six loci in the Wonsando Island population, but all the loci deviated from the HWE in the Jeonjangpo population. The genetic differentiation between the two populations and the phylogenetic tree constructed by the unweighted pair group method with the arithmetic average suggested that there is no significant genetic difference between Wonsando Island and Jeonjangpo populations. The prepared multiplex PCR systems and population genetic data will be helpful to study phylogeographic analysis as well as to prepare strategies of stock management and aquaculture of P. trituberculatus.     

Isolation and characterization of 19 novel microsatellite loci in Rhododendron aureum and Rhododendron brachycarpum (Ericaceae)

Nineteen polymorphic microsatellite loci are described for Rhododendron aureum Georgi (Ericaceae), an endangered species in Korea, and the closely related Rhododendron brachycarpum. The sequences containing repeat motifs were identified using low-depth next generation sequencing and 148 microsatellite loci were examined for amplification success and the detection of polymorphisms. All 19 loci were polymorphic and the number of alleles for these markers varied from 4 to 25, with an average of 15 alleles per locus. Three R. aureum loci showed departure from Hardy–Weinberg equilibrium (HWE) and one R. brachycarpum locus deviated significantly from HWE. These microsatellite loci will be useful for investigating the genetic diversity and population structure of both species.     

Genetic diversity analysis by microsatellite markers in four captive populations of the sika deer (Cervus nippon)

The Chinese sika deer (Cervus nippon) is a rare and vulnerable animal in China for medical use. In this study, the genetic diversity and population genetic structure of 113 Chinese sika deer from 4 populations (Linyi Farm, LF; Linyi Park, LP, Yangzhou Farm, YF; Yangzhou Zoo, YZ) were investigated with 14 microsatellite loci. Eighty-three alleles were detected at the 14 loci in all populations. The expected heterozygosity ranged from 0.257 to 0.863 and the observed heterozygosity from 0.226 to 0.821. The polymorphism information content at different loci ranged from 0.217 to 0.825. The results of the HWE (Hardy–Weinberg equilibrium) tests indicated that only four loci (CEH-5, BL42, Mber70, and CEH-2) were in HWE (P > 0.01). The mean number of alleles per population ranged from 3.21 to 5.64, observed and expected heterozygosities ranged from 0.568 to 0.685, respectively. Positive inbreeding coefficient (F_IS) values were found in every population. F_ST values ranged from 0.101 in the LF to 0.155 in the YZ. The genetic identity ranged from 0.1236 to 0.1645. The genetic distance ranged from 0.4746 to 0.6025. The results of this study indicate moderate genetic variation and polymorphism across the loci. Appropriate breeding strategies should be designed for deer in captivity.     

Hardy weinberg expectations in canine breeds: implications for genetic studies

Hardy-Weinberg equilibrium (HWE) is a useful indicator of genotype frequencies within a population and whether they are based on a valid definition of alleles and a randomly mating sample. HWE assumes a stable population of adequate size without selective pressures and is used in human genetic studies as a guide to data quality by comparing observed genotype frequencies to those expected within a population. The calculation of genetic associations in case-control studies assume that the population is "in HWE." Canine breed populations deviate away from many of the criteria for HWE, and if genetic markers are not in HWE, conventional statistical analysis cannot be performed. To date, little attention has been paid as to whether genetic markers in dog breeds are distributed in compliance to HWE. In this study, 109 single-nucleotide polymorphisms (SNPs) were genotyped from 13 genes in a cohort of 894 dogs encompassing 33 breeds. Analysis of the entire cohort of dogs revealed a significant deviation away from HWE for all SNPs tested (P < 0.00001); analysis of the cohort stratified by breed and subbreed indicated that the majority of the markers complied with HWE expectation. This suggests that canine case-control association studies will be valid if performed within defined breeds.     

Microsatellite loci from red sea urchins (Strongylocentrotus franciscanus)

Strongylocentrotus franciscanus is a keystone marine herbivore in the Pacific North West of North America, that is commercially harvested for its roe in wild fisheries and is under development as an aquaculture species. We report the development of 11 novel, highly polymorphic di‐ and trinucleotide loci for use in population studies of this species. In a survey of over 200 individuals from three coastal sites in British Columbia, observed heterozygosities of the loci ranged from 0.39 to 0.85 and an average of 26 alleles per locus were resolved. Seven of the 11 microsatellite loci were in Hardy–Weinburg equiulibruim (HWE).     

Genuine Bayesian multiallelic significance test for the Hardy-Weinberg equilibrium law

Statistical tests that detect and measure deviation from the Hardy-Weinberg equilibrium (HWE) have been devised but are limited when testing for deviation at multiallelic DNA loci is attempted. Here we present the full Bayesian significance test (FBST) for the HWE. This test depends neither on asymptotic results nor on the number of possible alleles for the particular locus being evaluated. The FBST is based on the computation of an evidence index in favor of the HWE hypothesis. A great deal of forensic inference based on DNA evidence assumes that the HWE is valid for the genetic loci being used. We applied the FBST to genotypes obtained at several multiallelic short tandem repeat loci during routine parentage testing; the locus Penta E exemplifies those clearly in HWE while others such as D10S1214 and D19S253 do not appear to show this.