分子标记技术在苹果育种中的应用

DNA分子标记是现代分子生物学发展出来的一类重要的遗传标记,已广泛应用于遗传图谱的构建、种质资源的管理和鉴定、基因的定位与克隆等。综述了分子标记在苹果育种中的应用。     

核果类果树DNA分子标记研究进展

介绍了DNA分子标记的种类及其在核果类果树亲缘关系上的鉴定、基因标记、定位与克隆、分子标记遗传图谱的构建以及分子标记辅助育种等上的应用进展,探讨了分子标记在核果类果树上的应用前景及尚需解决的问题。     

DNA分子标记在果树遗传育种研究中的应用

DNA分子标记是随着分子生物学技术的发展出现的一类重要的遗传标记,近年来发展非常迅速,已在果树遗传育种研究的各个方面得到广泛的应用。介绍了几种DNA分子标记技术的原理,综述了DNA分子标记在果树种质资源研究、分子遗传图谱构建、基因定位、分子辅助选择等方面的应用,并对其在果树上的应用前景和存在问题进行了评述。     

PCR-RAPD分子生物学技术及其在植物抗病性研究中的应用

PCR—RAPD技术是一种高效的基因组DNA多态性分析技术,能够在对生物细胞或组织中DNA遗传多样性、亲缘关系及系统进化分子标记检测的同时进行基因定位与遗传作图。本综述了PCR—RAPD技术的基本原理和应用范围,以及近年来在植物抗感病品种(品系)间亲缘远近关系分析、植物抗病性遗传基因的DNA分子标记与检测、植物抗病基因的标记和定位、植物抗病基因的分离与克隆、植物抗病育种的分子标记辅助选择与检测等植物抗病性分子机制研究方面的应用,并对该技术所存在的问题及应用前景进行了探讨。     

分子标记技术在蚕学研究中的应用

介绍了近年来DNA分子标记技术在绢丝昆虫的进化及亲缘关系分析、家蚕品种真实性鉴定、家蚕分子连锁图的构建、基因标记和定位等方面应用的重要进展 ,并展望了家蚕分子标记辅助育种的前景     

BRCA2的BRC4基元与RAD51相互作用位点的研究进展

乳腺癌易感基因2(breast cancer susceptibility gene 2,BRCA2),是人体内一种与乳腺、卵巢、胰腺等部位的肿瘤有关的抑癌基因。人的RAD51(h RAD51)是参与DNA同源重组修复过程的关键蛋白。BRCA2蛋白通过其结构中8个高度保守的BRC重复基元来调控h RAD51通过同源重组对DNA损伤进行的修复,从而阻止细胞癌变。在BRCA2的8个BRC重复基元中,BRC4与同源重组酶h RAD51的相互作用较为明显。综述了BRCA2的BRC4基元与h RAD51相互作用位点的研究进展,为了解BRCA2与RAD51相互作用的分子机理提供基础。     

生物技术用于黄瓜遗传育种

“十五”期间,生物技术在黄瓜遗传育种上的应用更加广泛,分子标记辅助育种、单倍体育种以及黄瓜基因工程改良取得了重要进展。本文综述了黄瓜基因分子标记、遗传图谱构建、基因定位、基因克隆与表达、品种DNA指纹图谱分析、分子技术鉴定病害、单倍体和三倍体培养、遗传转化体系建立及基因工程改良方面的最新进展,并讨论了存在的问题和前景。     

作物分子标记辅助选择的研究进展、影响因素及其发展策略

随着分子标记技术及其检测手段的发展,开发和应用成本的降低,分子标记辅助选择(MAS)在作物育种上的应用优势日益明显。本文综述了近年来MAS在基因聚合、基因转移和数量性状改良上的研究进展。总结了MAS的影响因素,包括标记与基因间的距离、目标性状的遗传率、群体大小、所用分子标记的数目、类型和相位等。并提出育种和定位同步进行、选择合适分子标记类型和数量、简化DNA提取方法、背景选择的逐步选择法、确定合适选择方案等MAS发展策略。     

集群分离分析法在大豆性状遗传定位中的研究进展

大豆是重要的油料作物,同时也是人类食用植物蛋白及畜牧业饲料蛋白的主要来源,在国家粮食结构和粮食安全中占有重要地位。利用简单高效的遗传定位方法,对大豆主要农艺性状进行相关基因挖掘,开发紧密连锁分子标记,有利于加快大豆的分子标记辅助选择及分子设计育种进程。集群分离分析法（BSA,bulked segregant analysis）是一种利用样本混池的建库方式对极端性状进行QTL定位的方法,因其具有“快速、准确、经济、实用”的特点,已成为当下应用较为广泛的基因定位方法。随着高通量测序技术的兴起,基于全基因组重测序的BSA方法更为广泛地应用在粮油作物、蔬菜花卉等物种中,并且成功定位出许多农艺性状相关的基因。本文简要介绍了BSA方法及流程步骤,总结了BSA在大豆农艺性状、抗逆性状以及雄性不育性状遗传定位中研究进展,并讨论了下一代测序（NGS,next-generation sequencing）背景下BSA的机遇与挑战,以及BSA在大豆分子标记辅助选择（MAS）育种中发展趋势,以期为高产优质大豆品种的选育提供重要的理论基础。     

SSR分子标记在荔枝上的研究进展

SSR作为第二代基于PCR的一种新型DNA分子遗传标记,数量丰富、多态性高、重复性好、分布于整个基因组、特异位点扩增、发生频率高、共显性遗传等,在果树育种中具有极大的应用价值和开发潜力.本文主要简述了SSR标记的原理和特点,并从分子指纹图谱构建、种质资源的遗传多样性分析、核心种质的发掘、品种鉴定、分子标记辅助育种、遗传图谱构建与基因定位等多方面介绍了近年来SSR技术在荔枝上的应用现状及前景.     

Double‐digest RAD sequencing using Ion Proton semiconductor platform (ddRADseq‐ion) with nonmodel organisms

Research in evolutionary biology involving nonmodel organisms is rapidly shifting from using traditional molecular markers such as mtDNA and microsatellites to higher throughput SNP genotyping methodologies to address questions in population genetics, phylogenetics and genetic mapping. Restriction site associated DNA sequencing (RAD sequencing or RADseq) has become an established method for SNP genotyping on Illumina sequencing platforms. Here, we developed a protocol and adapters for double‐digest RAD sequencing for Ion Torrent (Life Technologies; Ion Proton, Ion PGM) semiconductor sequencing. We sequenced thirteen genomic libraries of three different nonmodel vertebrate species on Ion Proton with PI chips: Arctic charr Salvelinus alpinus, European whitefish Coregonus lavaretus and common lizard Zootoca vivipara. This resulted in ~962 million single‐end reads overall and a mean of ~74 million reads per library. We filtered the genomic data using Stacks, a bioinformatic tool to process RAD sequencing data. On average, we obtained ~11 000 polymorphic loci per library of 6–30 individuals. We validate our new method by technical and biological replication, by reconstructing phylogenetic relationships, and using a hybrid genetic cross to track genomic variants. Finally, we discuss the differences between using the different sequencing platforms in the context of RAD sequencing, assessing possible advantages and disadvantages. We show that our protocol can be used for Ion semiconductor sequencing platforms for the rapid and cost‐effective generation of variable and reproducible genetic markers.     

New view of population genetics of zooplankton: RAD‐seq analysis reveals population structure of the North Atlantic planktonic copepod Centropages typicus

Detection of population genetic structure of zooplankton at medium‐to‐small spatial scales in the absence of physical barriers has remained challenging and controversial. The large population sizes and high rates of gene flow characteristic of zooplankton have made resolution of geographical differentiation very difficult, especially when using few genetic markers and assuming equilibrium conditions. Next‐generation sequencing now allows simultaneous sampling of hundreds to thousands of genetic markers; new analytical approaches allow studies under nonequilibrium conditions and directional migration. Samples of the North Atlantic Ocean planktonic copepod, Centropages typicus, were analysed using restriction site‐associated DNA (RAD) sequencing on a PROTON platform. Although prior studies revealed no genetic differentiation of populations across the geographical range of the species, analysis of RAD tags showed significant structure across the North Atlantic Ocean. We also compared the likelihood for models of connectivity among NW Atlantic populations under various directional flow scenarios that replicate oceanographic conditions of the sampled domain. High‐density marker sampling with RAD sequencing markedly outperformed other technical and analytical approaches in detection of population genetic structure and characterization of connectivity of this high geneflow zooplankton species.     

PMERGE: Computational filtering of paralogous sequences from RAD‐seq data

Restriction‐site associated DNA sequencing (RAD‐seq) can identify and score thousands of genetic markers from a group of samples for population‐genetics studies. One challenge of de novo RAD‐seq analysis is to distinguish paralogous sequence variants (PSVs) from true single‐nucleotide polymorphisms (SNPs) associated with orthologous loci. In the absence of a reference genome, it is difficult to differentiate true SNPs from PSVs, and their impact on downstream analysis remains unclear. Here, we introduce a network‐based approach, PMERGE that connects fragments based on their DNA sequence similarity to identify probable PSVs. Applying our method to de novo RAD‐seq data from 150 Atlantic salmon (Salmo salar) samples collected from 15 locations across the Southern Newfoundland coast allowed the identification of 87% of total PSVs identified through alignment to the Atlantic salmon genome. Removal of these paralogs altered the inferred population structure, highlighting the potential impact of filtering in RAD‐seq analysis. PMERGE is also applied to a green crab (Carcinus maenas) data set consisting of 242 samples from 11 different locations and was successfully able to identify and remove the majority of paralogous loci (62%). The PMERGE software can be run as part of the widely used Stacks analysis package.     

Identification of sex‐specific molecular markers using restriction site‐associated DNA sequencing

A major barrier to evolutionary studies of sex determination and sex chromosomes has been a lack of information on the types of sex‐determining mechanisms that occur among different species. This is particularly problematic in groups where most species lack visually heteromorphic sex chromosomes, such as fish, amphibians and reptiles, because cytogenetic analyses will fail to identify the sex chromosomes in these species. We describe the use of restriction site‐associated DNA (RAD) sequencing, or RAD‐seq, to identify sex‐specific molecular markers and subsequently determine whether a species has male or female heterogamety. To test the accuracy of this technique, we examined the lizard Anolis carolinensis. We performed RAD‐seq on seven male and ten female A. carolinensis and found one male‐specific molecular marker. Anolis carolinensis has previously been shown to possess male heterogamety and the recently published A. carolinensis genome facilitated the characterization of the sex‐specific RAD‐seq marker. We validated the male specificity of the new marker using PCR on additional individuals and also found that it is conserved in some other Anolis species. We discuss the utility of using RAD‐seq to identify sex‐determining mechanisms in other species with cryptic or homomorphic sex chromosomes and the implications for the evolution of male heterogamety in Anolis.     

RAD sequencing resolved phylogenetic relationships in European shrub willows (Salix L. subg. Chamaetia and subg. Vetrix) and revealed multiple evolution of dwarf shrubs

The large and diverse genus Salix L. is of particular interest for decades of biological research. However, despite the morphological plasticity, the reconstruction of phylogenetic relationships was so far hampered by the lack of informative molecular markers. Infrageneric classification based on morphology separates dwarf shrubs (subg. Chamaetia) and taller shrubs (subg. Vetrix), while previous phylogenetic studies placed species of these two subgenera just in one largely unresolved clade. Here we want to test the utility of genomic RAD sequencing markers for resolving relationships at different levels of divergence in Salix. Based on a sampling of 15 European species representing 13 sections of the two subgenera, we used five different RAD sequencing datasets generated by Ipyrad to conduct phylogenetic analyses. Additionally we reconstructed the evolution of growth form and analyzed the genetic composition of the whole clade. The results showed fully resolved trees in both ML and BI analysis with high statistical support. The two subgenera Chamaetia and Vetrix were recognized as nonmonophyletic, which suggests that they should be merged. Within the Vetrix/Chamaetia clade, a division into three major subclades could be observed. All species were confirmed to be monophyletic. Based on our data, arctic‐alpine dwarf shrubs evolved four times independently. The structure analysis showed five mainly uniform genetic clusters which are congruent in sister relationships observed in the phylogenies. Our study confirmed RAD sequencing as a useful genomic tool for the reconstruction of relationships on different taxonomic levels in the genus Salix.     

A resource of genome‐wide single‐nucleotide polymorphisms generated by RAD tag sequencing in the critically endangered European eel

Reduced representation genome sequencing such as restriction‐site‐associated DNA (RAD) sequencing is finding increased use to identify and genotype large numbers of single‐nucleotide polymorphisms (SNPs) in model and nonmodel species. We generated a unique resource of novel SNP markers for the European eel using the RAD sequencing approach that was simultaneously identified and scored in a genome‐wide scan of 30 individuals. Whereas genomic resources are increasingly becoming available for this species, including the recent release of a draft genome, no genome‐wide set of SNP markers was available until now. The generated SNPs were widely distributed across the eel genome, aligning to 4779 different contigs and 19 703 different scaffolds. Significant variation was identified, with an average nucleotide diversity of 0.00529 across individuals. Results varied widely across the genome, ranging from 0.00048 to 0.00737 per locus. Based on the average nucleotide diversity across all loci, long‐term effective population size was estimated to range between 132 000 and 1 320 000, which is much higher than previous estimates based on microsatellite loci. The generated SNP resource consisting of 82 425 loci and 376 918 associated SNPs provides a valuable tool for future population genetics and genomics studies and allows for targeting specific genes and particularly interesting regions of the eel genome.     

Antarctic krill population genomics: apparent panmixia,but genome complexity and large population size muddy the water

Antarctic krill (Euphausia superba; hereafter krill) are an incredibly abundant pelagic crustacean which has a wide, but patchy, distribution in the Southern Ocean. Several studies have examined the potential for population genetic structuring in krill, but DNA‐based analyses have focused on a limited number of markers and have covered only part of their circum‐Antarctic range. We used mitochondrial DNA and restriction site‐associated DNA sequencing (RAD‐seq) to investigate genetic differences between krill from five sites, including two from East Antarctica. Our mtDNA results show no discernible genetic structuring between sites separated by thousands of kilometres, which is consistent with previous studies. Using standard RAD‐seq methodology, we obtained over a billion sequences from >140 krill, and thousands of variable nucleotides were identified at hundreds of loci. However, downstream analysis found that markers with sufficient coverage were primarily from multicopy genomic regions. Careful examination of these data highlights the complexity of the RAD‐seq approach in organisms with very large genomes. To characterize the multicopy markers, we recorded sequence counts from variable nucleotide sites rather than the derived genotypes; we also examined a small number of manually curated genotypes. Although these analyses effectively fingerprinted individuals, and uncovered a minor laboratory batch effect, no population structuring was observed. Overall, our results are consistent with panmixia of krill throughout their distribution. This result may indicate ongoing gene flow. However, krill's enormous population size creates substantial panmictic inertia, so genetic differentiation may not occur on an ecologically relevant timescale even if demographically separate populations exist.     

Construction of an SSR and RAD Marker-Based Genetic Linkage Map for Carnation (<Emphasis Type="Italic">Dianthus caryophyllus</Emphasis> L.)

A high-density genetic map, an essential tool for comparative genomic studies and quantitative trait locus fine mapping, can also facilitate genome sequence assembly. The sequence-based marker technology known as restriction site-associated DNA (RAD) enables synchronous, single nucleotide polymorphism marker discovery, and genotyping using massively parallel sequencing. We constructed a high-density linkage map for carnation (Dianthus caryophyllus L.) based on simple sequence repeat (SSR) markers in combination with RAD markers developed by double-digest RAD sequencing (ddRAD-seq). A total of 2404 (285 SSR and 2119 RAD) markers could be assigned to 15 linkage groups spanning 971.5 cM, with an average marker interval of 0.4 cM. The total length of scaffolds with identified map positions was 95.6 Mb, which is equivalent to 15.4 % of the estimated genome size. The generated map is the first SSR and RAD marker-based high-density linkage map reported for carnation. The ddRAD-seq pipeline developed in this study should also help accelerate genetic and genomics analyses and molecular breeding of carnation and other non-model crops.     

Over-expression of RAD51 or RAD54 but not RAD51/4 enhances extra-chromosomal homologous recombination in the human sarcoma (HT-1080) cell line

RAD51 and RAD54, members of the RAD52 epistasis group, play key roles in homologous recombination (HR). The efficiency of homologous recombination (HR) can be increased by over-expression of either of them. A vector that allows co-expression of RAD51 and RAD54 was constructed to investigate interactions between the two proteins during extra-chromosomal HR. The efficiency of extra-chromosomal HR evaluated by GFP extra-chromosomal HR was enhanced (110-245%) in different transfected Human sarcoma (HT-1080) cell colonies. We observed that RAD51 clearly promotes extra-chromosomal HR; however, the actions of RAD54 in extra-chromosomal HR were weak. Our data suggest that RAD51 may function as a universal factor during HR, whereas RAD54 mainly functions in other types of HR (gene targeting or intra-chromosomal HR), which involves interaction with chromosomal DNA.     

Intraspecific DNA contamination distorts subtle population structure in a marine fish: Decontamination of herring samples before restriction‐site associated sequencing and its effects on population genetic statistics

Wild specimens are often collected in challenging field conditions, where samples may be contaminated with the DNA of conspecific individuals. This contamination can result in false genotype calls, which are difficult to detect, but may also cause inaccurate estimates of heterozygosity, allele frequencies and genetic differentiation. Marine broadcast spawners are especially problematic, because population genetic differentiation is low and samples are often collected in bulk and sometimes from active spawning aggregations. Here, we used contaminated and clean Pacific herring (Clupea pallasi) samples to test (a) the efficacy of bleach decontamination, (b) the effect of decontamination on RAD genotypes and (c) the consequences of contaminated samples on population genetic analyses. We collected fin tissue samples from actively spawning (and thus contaminated) wild herring and nonspawning (uncontaminated) herring. Samples were soaked for 10 min in bleach or left untreated, and extracted DNA was used to prepare DNA libraries using a restriction site‐associated DNA (RAD) approach. Our results demonstrate that intraspecific DNA contamination affects patterns of individual and population variability, causes an excess of heterozygotes and biases estimates of population structure. Bleach decontamination was effective at removing intraspecific DNA contamination and compatible with RAD sequencing, producing high‐quality sequences, reproducible genotypes and low levels of missing data. Although sperm contamination may be specific to broadcast spawners, intraspecific contamination of samples may be common and difficult to detect from high‐throughput sequencing data and can impact downstream analyses.