The Loss of Genetic Diversity in Sichuan Taimen as Revealed by DNA Fingerprinting

Species endangerment often derives from the “endangerment” of genetic diversity, thus loss of genetic diversity is an important cause of species extinction. Since historical specimens were unavailable, previous studies mainly described the genetic diversity status in the current population rather than the loss of genetic variation over time. In this study, we collected samples during1998–1999 and obtained historical specimens from 1957 to 1958. Based on the two sets of fish, we determined the changes in genetic diversity of Sichuan taimen using DNA fingerprinting. The differences in genetic parameters between the present samples and historical taimens revealed their loss of genetic variation. As a result, the existing populations have lower viability, and proper management has to be implemented to preserve genetic diversity.     

葡萄品种DNA指纹数据库的构建及遗传多样性分析

利用SSR分子标记技术对314份葡萄品种进行了DNA指纹数据库构建和遗传多样性分析,为葡萄品种鉴定、亲缘关系分析和植物品种权保护提供科学依据。结果表明:9对引物共扩增出199个等位基因,多态性位点为199个,多态性比率达100%,每个标记检测到的位点数在17~31之间,平均为22.1个;多态性信息含量(PIC)值变幅在0.793~0.886之间,平均值为0.839。本研究发现3组同名异物品种和9组疑似同物异名品种,除此之外的290份品种中,70份品种仅需1对引物即可区分开,其余品种需要引物组合来实现品种之间的区分。最少选用8对引物即可完全区分开290份葡萄品种。最终利用8对多态性SSR引物构建了314份供试材料的DNA指纹数据库,聚类分析结果表明:263份二倍体供试材料可被分为真葡萄亚属和圆叶葡萄亚属两大类,而真葡萄亚属又被分为15个亚类。51份多倍体供试材料被分为3组,聚类结果与供试材料已知的系谱来源基本吻合。     

Assessing Genetic Diversity of Brazilian Reef Fishes by Chromosomal and DNA Markers

Little is known on genetics of Brazilian coral reef fish and most of this information is limited to chromosome characterization of major representative species. The diploid chromosome number in marine fish varies from 2n= 22–26 to 2n = 240–260. Despite of this apparent diversity, most studied marine species have a diploid complement with 48 acrocentric chromosomes. This latter trend is mostly observed among Perciformes, an important major taxon of coral reef fishes. Studies in the families Pomacentridae, Pomacanthidae and Chaetodontidae, for example, have shown a common karyotype pattern entirely formed by 48 uniarmed chromosomes. However, rare numerical and structural chromosome polymorphisms and cryptic chromosome rearrangements involving heterochromatin segments and/or nucleolar organizing sites have been reported among such fishes. Although new chromosome forms can contribute to the establishment of genetically isolated populations, their role in reef fish speciation at marine realm still is an open question. More recently, genomic DNA analyses using RAPD and microsatellites, and sequencing and RFLP of mitochondrial DNA have increasingly been used in Atlantic reef fish species. Genetic homogeneity over wide geographical ranges has been reported for different fish groups, in contrast to several cases of population substructuring related to environmental constraints or evolutionary history. Amazonas outflow and upwelling on the Southeastern coast of Brazil are believed to be strong barriers to dispersal of some reef species. Moreover, it is suggested that the pattern of speciation and population structure at South Atlantic is quite distinctive from Pacific Ocean, even when comparing closely related taxa. Further genetic studies are strongly encouraged in Brazilian reef fishes in order to provide a reliable scenario of the genetic structure in this important and diverse fish group.     

Detection of Clinically Relevant Genetic Variants in Autism Spectrum Disorder by Whole-Genome Sequencing

Autism Spectrum Disorder (ASD) demonstrates high heritability and familial clustering, yet the genetic causes remain only partially understood as a result of extensive clinical and genomic heterogeneity. Whole-genome sequencing (WGS) shows promise as a tool for identifying ASD risk genes as well as unreported mutations in known loci, but an assessment of its full utility in an ASD group has not been performed. We used WGS to examine 32 families with ASD to detect de novo or rare inherited genetic variants predicted to be deleterious (loss-of-function and damaging missense mutations). Among ASD probands, we identified deleterious de novo mutations in six of 32 (19%) families and X-linked or autosomal inherited alterations in ten of 32 (31%) families (some had combinations of mutations). The proportion of families identified with such putative mutations was larger than has been previously reported; this yield was in part due to the comprehensive and uniform coverage afforded by WGS. Deleterious variants were found in four unrecognized, nine known, and eight candidate ASD risk genes. Examples include CAPRIN1 and AFF2 (both linked to FMR1, which is involved in fragile X syndrome), VIP (involved in social-cognitive deficits), and other genes such as SCN2A and KCNQ2 (linked to epilepsy), NRXN1, and CHD7, which causes ASD-associated CHARGE syndrome. Taken together, these results suggest that WGS and thorough bioinformatic analyses for de novo and rare inherited mutations will improve the detection of genetic variants likely to be associated with ASD or its accompanying clinical symptoms.     

Summarizing Specific Profiles in Illumina Sequencing from Whole-Genome Amplified DNA

Advances in both high-throughput sequencing and whole-genome amplification (WGA) protocols have allowed genomes to be sequenced from femtograms of DNA, for example from individual cells or from precious clinical and archived samples. Using the highly curated Caenorhabditis elegans genome as a reference, we have sequenced and identified errors and biases associated with Illumina library construction, library insert size, different WGA methods and genome features such as GC bias and simple repeat content. Detailed analysis of the reads from amplified libraries revealed characteristics suggesting that majority of amplified fragment ends are identical but inverted versions of each other. Read coverage in amplified libraries is correlated with both tandem and inverted repeat content, while GC content only influences sequencing in long-insert libraries. Nevertheless, single nucleotide polymorphism (SNP) calls and assembly metrics from reads in amplified libraries show comparable results with unamplified libraries. To utilize the full potential of WGA to reveal the real biological interest, this article highlights the importance of recognizing additional sources of errors from amplified sequence reads and discusses the potential implications in downstream analyses.     

栽培灯盏花DNA指纹图谱研究及遗传多样性分析

目的:采用ISSR标记构建云南省栽培灯盏花DNA指纹图谱并进行遗传多样性分析.方法:从20对候选引物中筛选出7对引物对云南10个地方栽培灯盏花进行扩增,通过扩增带型的差异构建其DNA指纹图谱,利用Popgen32、NTSYS2软件进行遗传一致性系数和遗传多样分析.结果:7对引物在10份共扩增出48条谱带,其中33条具有多态性,占68.8％,表明各居群的遗传差异较大.10个地方种质资源被聚为2个大类,野生居群被聚为一类,栽培灯盏花聚为一类,其中栽培灯盏花有两大亚类.结论:通过构建DNA指纹图谱容易地把灯盏花种植资源相互区分鉴别出来,10个地方灯盏花种质资源遗传多样性丰富,栽培灯盏花与野生具有差异,栽培种质材料之间也有差异.     

Changes in Colorectal Carcinoma Genomes under Anti-EGFR Therapy Identified by Whole-Genome Plasma DNA Sequencing

Monoclonal antibodies targeting the Epidermal Growth Factor Receptor (EGFR), such as cetuximab and panitumumab, have evolved to important therapeutic options in metastatic colorectal cancer (CRC). However, almost all patients with clinical response to anti-EGFR therapies show disease progression within a few months and little is known about mechanism and timing of resistance evolution. Here we analyzed plasma DNA from ten patients treated with anti-EGFR therapy by whole genome sequencing (plasma-Seq) and ultra-sensitive deep sequencing of genes associated with resistance to anti-EGFR treatment such as KRAS, BRAF, PIK3CA, and EGFR. Surprisingly, we observed that the development of resistance to anti-EGFR therapies was associated with acquired gains of KRAS in four patients (40%), which occurred either as novel focal amplifications (n = 3) or as high level polysomy of 12p (n = 1). In addition, we observed focal amplifications of other genes recently shown to be involved in acquired resistance to anti-EGFR therapies, such as MET (n = 2) and ERBB2 (n = 1). Overrepresentation of the EGFR gene was associated with a good initial anti-EGFR efficacy. Overall, we identified predictive biomarkers associated with anti-EGFR efficacy in seven patients (70%), which correlated well with treatment response. In contrast, ultra-sensitive deep sequencing of KRAS, BRAF, PIK3CA, and EGFR did not reveal the occurrence of novel, acquired mutations. Thus, plasma-Seq enables the identification of novel mutant clones and may therefore facilitate early adjustments of therapies that may delay or prevent disease progression.     

High-Quality Exome Sequencing of Whole-Genome Amplified Neonatal Dried Blood Spot DNA

Stored neonatal dried blood spot (DBS) samples from neonatal screening programmes are a valuable diagnostic and research resource. Combined with information from national health registries they can be used in population-based studies of genetic diseases. DNA extracted from neonatal DBSs can be amplified to obtain micrograms of an otherwise limited resource, referred to as whole-genome amplified DNA (wgaDNA). Here we investigate the robustness of exome sequencing of wgaDNA of neonatal DBS samples. We conducted three pilot studies of seven, eight and seven subjects, respectively. For each subject we analysed a neonatal DBS sample and corresponding adult whole-blood (WB) reference sample. Different DNA sample types were prepared for each of the subjects. Pilot 1: wgaDNA of 2x3.2mm neonatal DBSs (DBS_2x3.2) and raw DNA extract of the WB reference sample (WB_ref). Pilot 2: DBS_2x3.2, WB_ref and a WB_ref replica sharing DNA extract with the WB_ref sample. Pilot 3: DBS_2x3.2, WB_ref, wgaDNA of 2x1.6 mm neonatal DBSs and wgaDNA of the WB reference sample. Following sequencing and data analysis, we compared pairwise variant calls to obtain a measure of similarity—the concordance rate. Concordance rates were slightly lower when comparing DBS vs WB sample types than for any two WB sample types of the same subject before filtering of the variant calls. The overall concordance rates were dependent on the variant type, with SNPs performing best. Post-filtering, the comparisons of DBS vs WB and WB vs WB sample types yielded similar concordance rates, with values close to 100%. WgaDNA of neonatal DBS samples performs with great accuracy and efficiency in exome sequencing. The wgaDNA performed similarly to matched high-quality reference—whole-blood DNA—based on concordance rates calculated from variant calls. No differences were observed substituting 2x3.2 with 2x1.6 mm discs, allowing for additional reduction of sample material in future projects.     

Construction of Fingerprinting and Genetic Diversity of Mulberry Cultivars in China by ISSR Markers

The ISSR fingerprintings of 24 mulberry cultivars were constructed. Totally 80 bands were produced using 17 primers selected from 20 primers. Of them, 40 bands showed polymorphism. From the bands amplified, there were three independent ways to identify the mulberry varieties, such as unique ISSR markers, unique band patterns and a combination of the band patterns provided by different primers. ISSRs were very effective in differentiating the mulberry varieties. The mean genetic similarity coefficient, the mean Nei's gene diversity (h), and the mean Shannon's Information index (I) of mulberry cultivars were 0.8731, 0.1210, and 0.1942, respectively. This suggests that the genetic diversity of mulberry cultivars was low and the genetic base was narrow. Both UPGMA cluster and PCA (Principal Coordinates Analysis) analysis showed clear genetic relationships among the 24 mulberry cultivars. The major clusters were related to known pedigree relationships.     

酵母菌SRAP-PCR反应体系的优化及遗传多样性分析

从8个正向引物和11个反向引物随机组合成的88个引物对中筛选出14个引物组合,对19株酵母菌进行PCR扩增。采用正交试验设计方法,对SRAP的影响因素进行研究,从Mg2+、dNTPs、DNA浓度、引物、TaqDNA聚合酶设计5因素4水平进行优化试验,以确定PCR电泳分析的最佳条件。14个引物组合共扩增出279条带,其中92.5%呈多态性,相似系数范围为0.60~1.00。     

Genetic Diversity of Pleurotus pulmonarius Revealed by RAPD,ISSR, and SRAP Fingerprinting

Pleurotus pulmonarius is one of the most widely cultivated and popular edible fungi in the genus Pleurotus. Three molecular markers were used to analyze the genetic diversity of 15 Chinese P. pulmonarius cultivars. In total, 21 random amplified polymorphic DNA (RAPD), 20 inter-simple sequence repeat (ISSR), and 20 sequence-related amplified polymorphism (SRAP) primers or primer pairs were selected for generating data based on their clear banding profiles produced. With the use of these RAPD, ISSR, and SRAP primers or primer pairs, a total of 361 RAPD, 283 ISSR, and 131 SRAP fragments were detected, of which 287 (79.5 %) RAPD, 211 (74.6 %) ISSR, and 98 (74.8 %) SRAP fragments were polymorphic. Unweighted Pair-Group Method with Arithmetic Mean (UPGMA) trees of these three methods were structured similarly, grouping the 15 tested strains into four clades. Subsequently, visual DNA fingerprinting and cluster analysis were performed to evaluate the resolving power of the combined RAPD, ISSR, and SRAP markers in the differentiation among these strains. The results of this study demonstrated that each method above could efficiently differentiate P. pulmonarius cultivars and could thus be considered an efficient tool for surveying genetic diversity of P. pulmonarius.     

牛鞭草品种EST-SSR指纹图谱构建及遗传多样性分析

为探讨牛鞭草(Hemarthria spp.)品种间的遗传多样性,从86对EST-SSR引物中筛选出8对引物对牛鞭草属6个品种进行指纹图谱的构建及遗传多样性分析。结果表明,8对EST-SSR引物对牛鞭草属6个品种共扩增出193条清晰条带,多态性条带161条,多态性比例为83.4%。每条引物的多态信息含量(PIC)为0.480~0.695,平均为0.602。UPGMA聚类分析表明,牛鞭草属6个品种在相似系数为0.652处可分为两大类群。8对EST-SSR引物均能将6个品种完全区分开,以3对EST-SSR引物扩增的电泳图谱为基础,建立了牛鞭草属6个品种的指纹图谱标准模式图,每个品种都有唯一的指纹图谱。牛鞭草属6个品种的平均Nei’s基因多样性指数为0.333,平均Shannon信息指数为0.496,品种间的相似系数介于0.399~0.782之间。可见,牛鞭草属植物品种的遗传多样性较丰富,种间差异明显。     

中国桑树选育品种ISSR指纹图谱的构建及遗传多样性分析

利用ISSR标记构建了24个选育桑品种的指纹图谱,用3种独立的方法（特殊的标记;特异的谱带类型;不同引物提供的谱带类型组合）可以有效地鉴别桑树选育品种,证明ISSR标记在桑树品种的鉴别方面是一个有效的工具和方法。17个ISSR引物共扩增出80条带,40条带具有多态性,占50．0％。24份选育桑树品种间平均遗传相似系数、Nei’S基因多样性（gene diversity）和Shannon’S信息指数分别为0．8731,0．1210和0．1942。桑树选育品种间的遗传多样性较低,说明中国选育桑品种间遗传距离较小,亲缘关系较近,。遗传基础较狭窄。UPGMA法聚类和PCA分析都清楚地显示了24个桑树选育品种的亲缘关系,聚类结果与桑树品种的系谱基本一致。     

Horizontal Transfer of Genetic Material among Saccharomyces Yeasts

The genus Saccharomyces consists of several species divided into the sensu stricto and the sensu lato groups. The genomes of these species differ in the number and organization of nuclear chromosomes and in the size and organization of mitochondrial DNA (mtDNA). In the present experiments we examined whether these yeasts can exchange DNA and thereby create novel combinations of genetic material. Several putative haploid, heterothallic yeast strains were isolated from different Saccharomyces species. All of these strains secreted an a- or alpha-like pheromone recognized by S. cerevisiae tester strains. When interspecific crosses were performed by mass mating between these strains, hybrid zygotes were often detected. In general, the less related the two parental species were, the fewer hybrids they gave. For some crosses, viable hybrids could be obtained by selection on minimal medium and their nuclear chromosomes and mtDNA were examined. Often the frequency of viable hybrids was very low. Sometimes putative hybrids could not be propagated at all. In the case of sensu stricto yeasts, stable viable hybrids were obtained. These contained both parental sets of chromosomes but mtDNA from only one parent. In the case of sensu lato hybrids, during genetic stabilization one set of the parental chromosomes was partially or completely lost and the stable mtDNA originated from the same parent as the majority of the nuclear chromosomes. Apparently, the interspecific hybrid genome was genetically more or less stable when the genetic material originated from phylogenetically relatively closely related parents; both sets of nuclear genetic material could be transmitted and preserved in the progeny. In the case of more distantly related parents, only one parental set, and perhaps some fragments of the other one, could be found in genetically stabilized hybrid lines. The results obtained indicate that Saccharomyces yeasts have a potential to exchange genetic material. If Saccharomyces isolates could mate freely in nature, horizontal transfer of genetic material could have occurred during the evolution of modern yeast species.     

Optimal Sequencing Strategies for Surveying Molecular Genetic Diversity

Two commonly used measures of genetic diversity for intraspecies DNA sequence data are based, respectively, on the number of segregating sites, and on the average number of pairwise nucleotide differences. Expressions are derived for their variance in the presence of intragenic recombination for a panmictic population of fixed size that is at neutral equilibrium at the region sequenced. We show that, in contrast to the slow decrease in variance with increasing sample size, if the recombination rate is nonzero, the asymptotic rate of decrease of variance with increasing sequence length, for fixed sample size, is quite rapid. In particular, it is close to that which would be obtained by sequencing independent chromosome regions. The correlation between measures of diversity from linked regions is also examined. For a given total number of bases sequenced in a particular region, optimal sequencing strategies are derived. These typically involve sequencing relatively few (three to 10) long copies of the region. Under optimal strategies, the variances of the two measures are very similar for most parameter values considered. Results concerning optimal sequencing strategies will be sensitive to gross departures from the underlying assumptions, such as population bottlenecks, selective sweeps, and substantial population substructure.     

Whole-Genome Genetic Diversity in a Sample of Australians with Deep Aboriginal Ancestry

Australia was probably settled soon after modern humans left Africa, but details of this ancient migration are not well understood. Debate centers on whether the Pleistocene Sahul continent (composed of New Guinea, Australia, and Tasmania) was first settled by a single wave followed by regional divergence into Aboriginal Australian and New Guinean populations (common origin) or whether different parts of the continent were initially populated independently. Australia has been the subject of relatively few DNA studies even though understanding regional variation in genomic structure and diversity will be important if disease-association mapping methods are to be successfully evaluated and applied across populations. We report on a genome-wide investigation of Australian Aboriginal SNP diversity in a sample of participants from the Riverine region. The phylogenetic relationship of these Aboriginal Australians to a range of other global populations demonstrates a deep common origin with Papuan New Guineans and Melanesians, with little evidence of substantial later migration until the very recent arrival of European colonists. The study provides valuable and robust insights into an early and important phase of human colonization of the globe. A broader survey of Australia, including diverse geographic sample populations, will be required to fully appreciate the continent''s unique population history and consequent genetic heritage, as well as the importance of both to the understanding of health issues.     

Identification of EMS-Induced Mutations in Drosophila melanogaster by Whole-Genome Sequencing

Next-generation methods for rapid whole-genome sequencing enable the identification of single-base-pair mutations in Drosophila by comparing a chromosome bearing a new mutation to the unmutagenized sequence. To validate this approach, we sought to identify the molecular lesion responsible for a recessive EMS-induced mutation affecting egg shell morphology by using Illumina next-generation sequencing. After obtaining sufficient sequence from larvae that were homozygous for either wild-type or mutant chromosomes, we obtained high-quality reads for base pairs composing ~70% of the third chromosome of both DNA samples. We verified 103 single-base-pair changes between the two chromosomes. Nine changes were nonsynonymous mutations and two were nonsense mutations. One nonsense mutation was in a gene, encore, whose mutations produce an egg shell phenotype also observed in progeny of homozygous mutant mothers. Complementation analysis revealed that the chromosome carried a new functional allele of encore, demonstrating that one round of next-generation sequencing can identify the causative lesion for a phenotype of interest. This new method of whole-genome sequencing represents great promise for mutant mapping in flies, potentially replacing conventional methods.