SNP markers: Methods of analysis, ways of development, and comparison on an example of common wheat

SNPs (single nucleotide polymorphisms), which belong to the last-generation molecular markers, occur at high frequencies in both animal and plant genomes. The development of SNP markers allows to automatize and enhance tenfolds the effectiveness of genotype analysis. This review summarizes literature data on methods of SNP polymorphism analysis. Various methods of developing SNP markers are considered, taking common wheat Triticum aestivum L. as an example. These markers are compared to other DNA markers, in order to ensure adequate choice of marker type for solving various molecular genetic problems.     

SNP分子标记及其在木本植物遗传育种的应用

木本植物因其生命周期长、基因组杂合度高、基因组较大、遗传背景不清晰等特性,制约了其研究进程。随着现代生物技术的发展,DNA分子标记技术在木本植物研究领域的应用越来越多,其中单核苷酸多态性(SNP)作为第三代分子标记技术以其高效、快速、稳定、可靠等诸多优点得到广泛应用。本文简述SNP标记的特点、开发方法、检测方法及其在木本植物遗传多样性和亲缘关系分析、品种鉴定、连锁图谱构建和辅助育种等方面的研究进展,为更好地应用SNP技术开展木本植物研究提供参考。     

Single-nucleotide polymorphism versus microsatellite markers in a combined linkage and segregation analysis of a quantitative trait

Increasingly, single-nucleotide polymorphism (SNP) markers are being used in preference to microsatellite markers. However, methods developed for microsatellites may be problematic when applied to SNP markers. We evaluated the results of using SNPs vs. microsatellites in Monte Carlo Markov chain (MCMC) oligogenic combined segregation and linkage analysis methods. These methods were developed with microsatellite markers in mind. We selected chromosome 7 from the Collaborative Study on the Genetics of Alcoholism dataset for analysis because linkage to an electrophysiological trait had been reported there. We found linkage in the same region of chromosome 7 with the Affymetrix SNP data, the Illumina SNP data, and the microsatellite marker data. The MCMC sampler appears to mix with both types of data. The sampler implemented in this MCMC oligogenic combined segregation and linkage analysis appears to handle SNP data as well as microsatellite data and it is possible that the localizations with the SNP data are better.     

植物外源基因组成分鉴定方法的研究进展

具外源基因组成分(外源染色体/染色体片段/基因)植株是目前进行基因组学研究以及作物改良的重要材料。迄今为止,已建立了基于性状观测、染色体分析、特异蛋白、DNA序列4种鉴定外源基因组成分的策略。其中,基于DNA序列的分子标记技术是当前鉴定外源基因组成分的主要手段,文中归纳了用于小麦、甘蓝等重要作物外源基因组成分的分子标记,且对简单重复序列(Simple sequence repeat, SSR)、插入缺失(Insertion-deletion,InDel)、单核苷酸多态性(Single nucleotide polymorphism,SNP)等9种标记进行了系统的比较。相比单一的鉴定方法,组合法更全面精准,文中对各组合法的应用情况进行统计和分析,提供了小麦族、芸薹族等作物的最佳鉴定组合。新一代分子标记InDel、SNP易实现高通量检测,对于外源渗入基因的精细定位展现了一定的优越性。此外,可以考虑一些新鉴定方法的加入,如微阵列比较基因组杂交(Microarray-based comparative genomic hybridization,array-CGH)、抑制差减杂交(Suppression subtractive hybridization,SSH)。     

Application of real-time PCR-based SNP detection for mapping of Net2, a causal D-genome gene for hybrid necrosis in interspecific crosses between tetraploid wheat and Aegilops tauschii

Available information on genetically assigned molecular markers is not sufficient for efficient construction of a high-density linkage map in wheat. Here, we report on application of high resolution melting (HRM) analysis using a real-time PCR apparatus to develop single nucleotide polymorphism (SNP) markers linked to a hybrid necrosis gene, Net2, located on wheat chromosome 2D. Based on genomic information on barley chromosome 2H and wheat expressed sequence tag libraries, we selected wheat cDNA sequences presumed to be located near the Net2 chromosomal region, and then found SNPs between the parental Ae. tauschii accessions of the synthetic wheat mapping population. HRM analysis of the PCR products from F(2) individuals' DNA enabled us to assign 44.4% of the SNP-representing cDNAs to chromosome 2D despite the presence of the A and B genomes. In addition, the designed SNP markers were assigned to chromosome 2D of Ae. tauschii. The order of the assigned SNP markers in synthetic hexaploid wheat was confirmed by comparison with the markers in barley and Ae. tauschii. Thus, the SNP-genotyping method based on HRM analysis is a useful tool for development of molecular markers at target loci in wheat.     

Population structure and genetic diversity of coffee progenies derived from Catuaí and Híbrido de Timor revealed by genome-wide SNP marker

The use of single nucleotide polymorphism (SNP) molecular markers has provided advances in selection methodologies used in breeding programs of different crops, reducing cost and time of cultivar release. Despite the great economic and social importance of Coffea arabica, studies with SNP markers are scarce and a small number of SNP are available for this species, when compared with other crops of agronomic importance. Thus, the objective of this study was to identify and validate SNP molecular markers for the species Coffea arabica and to introduce these markers to genetic breeding by means of an accurate analysis of the diversity and genetic structure of breeding populations of this species. After quality filtering, 11,187 SNP markers were selected from the coffee population obtained from crosses between the genotypes Catuaí and Híbrido de Timor. A great number of markers were distributed in the 11 chromosomes, within transcribed regions, and were used to estimate the genetic dissimilarity among the individuals of the breeding population. Dendrogram analysis and a Bayesian approach demonstrated the formation of two groups and the discrimination of all genotypes evaluated. The expressive number of SNP molecular markers distributed throughout C. arabica genome was efficient to discriminate all the accessions evaluated in the experiment, clustering them according to their genealogies. This work identified mixtures within the progenies. The genotyping data also provided detailed information about the parental genotypes and led to the identification of new candidate parents to be introduced to the breeding program. The study discussed population structure and its consequence in obtaining improved varieties of C. arabica.     

Application of SNPs for population genetics of nonmodel organisms: new opportunities and challenges

Recent improvements in the speed, cost and accuracy of next generation sequencing are revolutionizing the discovery of single nucleotide polymorphisms (SNPs). SNPs are increasingly being used as an addition to the molecular ecology toolkit in nonmodel organisms, but their efficient use remains challenging. Here, we discuss common issues when employing SNP markers, including the high numbers of markers typically employed, the effects of ascertainment bias and the inclusion of nonneutral loci in a marker panel. We provide a critique of considerations specifically associated with the application and population genetic analysis of SNPs in nonmodel taxa, focusing specifically on some of the most commonly applied methods.     

Development and evaluation of single-nucleotide polymorphism markers in allotetraploid rapeseed (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

Single-nucleotide polymorphisms (SNPs) and insertion–deletions (INDELs) are currently the important classes of genetic markers for major crop species. In this study, methods for developing SNP markers in rapeseed (Brassica napus L.) and their in silico mapping and use for genotyping are demonstrated. For the development of SNP and INDEL markers, 181 fragments from 121 different gene sequences spanning 86 kb were examined. A combination of different selection methods (genome-specific amplification, hetero-duplex analysis and sequence analysis) allowed the detection of 18 singular fragments that showed a total of 87 SNPs and 6 INDELs between 6 different rapeseed varieties. The average frequency of sequence polymorphism was estimated to be one SNP every 247 bp and one INDEL every 3,583 bp. Most SNPs and INDELs were found in non-coding regions. Polymorphism information content values for SNP markers ranged between 0.02 and 0.50 in a set of 86 varieties. Using comparative genetics data for B. napus and Arabidopsis thaliana, an allocation of SNP markers to linkage groups in rapeseed was achieved: a unique location was determined for seven gene sequences; two and three possible locations were found for six and four sequences, respectively. The results demonstrate the usefulness of existing genomic resources for SNP discovery in rapeseed.     

Search for and analysis of single nucleotide polymorphisms (SNPs) in rice (Oryza sativa, Oryza rufipogon) and establishment of SNP markers.

We searched for SNPs in 417 regions distributed throughout the genome of three Oryza sativa ssp. japonica cultivars, two indica cultivars, and a wild rice (O. rufipogon). We found 2800 SNPs in approximately 250,000 aligned bases for an average of one SNP every 89 bp, or one SNP every 232 bp between two randomly selected strains. Graphic representation of the frequency of SNPs along each chromosome showed uneven distribution of polymorphism-rich and -poor regions, but little obvious association with the centromere or telomere. The 94 SNPs that we found between the closely related cultivars 'Nipponbare' and 'Koshihikari' can be converted into molecular markers. Our establishment of 213 co-dominant SNP markers distributed throughout the genome illustrates the immense potential of SNPs as molecular markers not only for genome research, but also for molecular breeding of rice.     

Discovery and characterization of single nucleotide polymorphisms in coho salmon,Oncorhynchus kisutch

Molecular population genetic analyses have become an integral part of ecological investigation and population monitoring for conservation and management. Microsatellites have been the molecular marker of choice for such applications over the last several decades, but single nucleotide polymorphism (SNP) markers are rapidly expanding beyond model organisms. Coho salmon (Oncorhynchus kisutch) is native to the north Pacific Ocean and its tributaries, where it is the focus of intensive fishery and conservation activities. As it is an anadromous species, coho salmon typically migrate across multiple jurisdictional boundaries, complicating management and requiring shared data collection methods. Here, we describe the discovery and validation of a suite of novel SNPs and associated genotyping assays which can be used in the genetic analyses of this species. These assays include 91 that are polymorphic in the species and one that discriminates it from a sister species, Chinook salmon. We demonstrate the utility of these SNPs for population assignment and phylogeographic analyses, and map them against the draft trout genome. The markers constitute a large majority of all SNP markers described for coho salmon and will enable both population‐ and pedigree‐based analyses across the southern part of the species native range.     

家蚕非滞育红卵突变体Re-nd突变基因的定位克隆

【目的】家蚕Bombyx mori非滞育红卵突变体Re-nd是唯一在非滞育状态下卵色呈现鲜红色的突变品种。本研究通过基因连锁分析和定位克隆的方法确定Re-nd的突变基因所在的染色体及紧密连锁位置,为后续Re-nd的功能研究及应用奠定基础。【方法】以家蚕卵色突变体Re-nd和野生型大造进行杂交,配制基因连锁分析群体材料和定位克隆群体材料;针对家蚕全染色体进行SNP标记开发,利用BC₁代群体材料进行基因连锁分析,确定Re-nd的突变基因所在的染色体;针对定位的Re nd的突变基因所在染色体进行SNP标记开发,利用BC₁群体材料对Re-nd的突变基因进行定位克隆。【结果】基因连锁分析结果显示Re-nd的突变表型与第6号染色体上的SNP标记完全连锁;初步定位克隆结果显示Re-nd的突变基因位于SNP标记SNP7和SNP17之间,物理距离4.04 Mb;以SNP7和SNP17之间筛选出的6个SNP标记和25个重组个体进行精细定位克隆,结果显示Re-nd的突变基因所在的区域位于SNP10和SNP12两个SNP标记之间的nscaf2853上,物理距离949.3 kb左右。【结论】将Re-nd的突变基因定位于第6号染色体的2个SNP标记SNP10和SNP12之间,物理距离约949.3 kb。本研究为后续Re-nd突变基因的精细定位及功能应用研究奠定了基础。     

Identification of sex-linked SNP markers in wild populations of monomorphic birds

Single-nucleotide polymorphism (SNP) analysis is a powerful tool for population genetics, pedigree reconstruction and phenotypic trait mapping. However, the untapped potential of SNP markers to discriminate the sex of individuals in species with reduced sexual dimorphism or of individuals during immature stages remains a largely unexplored avenue. Here, we developed a novel protocol for molecular sexing of birds based on the detection of unique Z- and W-linked SNP markers. Our method is based on the identification of two unique loci, one in each sexual chromosome. Individuals are considered males when they show no calls for the W-linked SNP and are heterozygous or homozygous for the Z-linked SNP, while females exhibit both Z- and W-linked SNP calls. We validated the method in the Jackdaw (Corvus monedula). The reduced sexual dimorphism in this species makes it difficult to identify the sex of individuals in the wild. We assessed the reliability of the method using 36 individuals of known sex and found that their sex was correctly assigned in 100% of cases. The sex-linked markers also proved to be widely applicable for discriminating males and females from a sample of 927 genotyped individuals at different maturity stages, with an accuracy of 99.5%. Since SNP markers are increasingly used in quantitative genetic analyses of wild populations, the approach we propose has great potential to be integrated into broader genetic research programmes without the need for additional sexing techniques.     

Comparison of SSR and SNP Markers in Estimation of Genetic Diversity and Population Structure of Indian Rice Varieties

Simple sequence repeat (SSR) and Single Nucleotide Polymorphic (SNP), the two most robust markers for identifying rice varieties were compared for assessment of genetic diversity and population structure. Total 375 varieties of rice from various regions of India archived at the Indian National GeneBank, NBPGR, New Delhi, were analyzed using thirty six genetic markers, each of hypervariable SSR (HvSSR) and SNP which were distributed across 12 rice chromosomes. A total of 80 alleles were amplified with the SSR markers with an average of 2.22 alleles per locus whereas, 72 alleles were amplified with SNP markers. Polymorphic information content (PIC) values for HvSSR ranged from 0.04 to 0.5 with an average of 0.25. In the case of SNP markers, PIC values ranged from 0.03 to 0.37 with an average of 0.23. Genetic relatedness among the varieties was studied; utilizing an unrooted tree all the genotypes were grouped into three major clusters with both SSR and SNP markers. Analysis of molecular variance (AMOVA) indicated that maximum diversity was partitioned between and within individual level but not between populations. Principal coordinate analysis (PCoA) with SSR markers showed that genotypes were uniformly distributed across the two axes with 13.33% of cumulative variation whereas, in case of SNP markers varieties were grouped into three broad groups across two axes with 45.20% of cumulative variation. Population structure were tested using K values from 1 to 20, but there was no clear population structure, therefore Ln(PD) derived Δk was plotted against the K to determine the number of populations. In case of SSR maximum Δk was at K=5 whereas, in case of SNP maximum Δk was found at K=15, suggesting that resolution of population was higher with SNP markers, but SSR were more efficient for diversity analysis.     

EST-SNP genotyping of citrus species using high-resolution melting curve analysis

Citrus taxonomy is very complex mainly due to specific aspects of its reproductive biology. A number of studies have been performed using various molecular markers in order to evaluate the level of genetic variability in Citrus. SNP markers have been used for genetic diversity assessment using a variety of different methods. Recently, the availability of EST database and whole genome sequences has made it possible to develop more markers such as SNPs. In the present study, the high-resolution melting curve analysis (HRM) was used to detect SNPs or INDELs in Citrus genus for the first time. We aimed to develop a panel of SNPs to differentiate Citrus genotypes which can also be applied to Citrus biodiversity studies. The results showed that 21 SNP containing markers produced distinct polymorphic melting curves among the Citrus spp. investigated through HRM analysis. It was proved that HRM is an efficient, cost-effective, and accurate method for discriminating citrus SNPs as well as a method to analyze more polymorphisms in a single PCR amplicon, representing a useful tool for genetic, biodiversity, and breeding studies. SNPs developed based on Citrus sinensis EST database showed a good transferability within the Citrus genus. Moreover, HRM analysis allowed the discrimination of citrus genotypes at specific level and the resulting genetic distance analysis clustered these genotypes into three main branches. The results suggested that the panel of SNP markers could be used in a variety of applications in citrus biodiversity assessment and breeding programs using HRM analysis.     

SNP Discovery and Linkage Map Construction in Cultivated Tomato

Few intraspecific genetic linkage maps have been reported for cultivated tomato, mainly because genetic diversity within Solanum lycopersicum is much less than that between tomato species. Single nucleotide polymorphisms (SNPs), the most abundant source of genomic variation, are the most promising source of polymorphisms for the construction of linkage maps for closely related intraspecific lines. In this study, we developed SNP markers based on expressed sequence tags for the construction of intraspecific linkage maps in tomato. Out of the 5607 SNP positions detected through in silico analysis, 1536 were selected for high-throughput genotyping of two mapping populations derived from crosses between ‘Micro-Tom’ and either ‘Ailsa Craig’ or ‘M82’. A total of 1137 markers, including 793 out of the 1338 successfully genotyped SNPs, along with 344 simple sequence repeat and intronic polymorphism markers, were mapped onto two linkage maps, which covered 1467.8 and 1422.7 cM, respectively. The SNP markers developed were then screened against cultivated tomato lines in order to estimate the transferability of these SNPs to other breeding materials. The molecular markers and linkage maps represent a milestone in the genomics and genetics, and are the first step toward molecular breeding of cultivated tomato. Information on the DNA markers, linkage maps, and SNP genotypes for these tomato lines is available at http://www.kazusa.or.jp/tomato/.     

Comparing single-nucleotide polymorphism marker-based and microsatellite marker-based linkage analyses

We compared linkage analysis results for an alcoholism trait, ALDX1 (DSM-III-R and Feigner criteria) using a nonparametric linkage analysis method, which takes into account allele sharing among several affected persons, for both microsatellite and single-nucleotide polymorphism (SNP) markers (Affymetrix and Illumina) in the Collaborative Study on the Genetics of Alcoholism (COGA) dataset provided to participants at the Genetic Analysis Workshop 14 (GAW14). The two sets of linkage results from the dense Affymetrix SNP markers and less densely spaced Illumina SNP markers are very similar. The linkage analysis results from microsatellite and SNP markers are generally similar, but the match is not perfect. Strong linkage peaks were found on chromosome 7 in three sets of linkage analyses using both SNP and microsatellite marker data. We also observed that for SNP markers, using the given genetic map and using the map by converting 1 megabase pair (1 Mb) to 1 centimorgan (cM), did not change the linkage results. We recommend the use of the 1 Mb-to-1 cM converted map in a first round of linkage analysis with SNP markers in which map integration is an issue.     

Comparative Analysis of Disease-Linked Single Nucleotide Polymorphic Markers from Brassica rapa for Their Applicability to Brassica oleracea

Numerous studies using single nucleotide polymorphisms (SNPs) have been conducted in humans, and other animals, and in major crops, including rice, soybean, and Chinese cabbage. However, the number of SNP studies in cabbage is limited. In this present study, we evaluated whether 7,645 SNPs previously identified as molecular markers linked to disease resistance in the Brassica rapa genome could be applied to B. oleracea. In a BLAST analysis using the SNP sequences of B. rapa and B. oleracea genomic sequence data registered in the NCBI database, 256 genes for which SNPs had been identified in B. rapa were found in B. oleracea. These genes were classified into three functional groups: molecular function (64 genes), biological process (96 genes), and cellular component (96 genes). A total of 693 SNP markers, including 145 SNP markers [BRH—developed from the B. rapa genome for high-resolution melt (HRM) analysis], 425 SNP markers (BRP—based on the B. rapa genome that could be applied to B. oleracea), and 123 new SNP markers (BRS—derived from BRP and designed for HRM analysis), were investigated for their ability to amplify sequences from cabbage genomic DNA. In total, 425 of the SNP markers (BRP-based on B. rapa genome), selected from 7,645 SNPs, were successfully applied to B. oleracea. Using PCR, 108 of 145 BRH (74.5%), 415 of 425 BRP (97.6%), and 118 of 123 BRS (95.9%) showed amplification, suggesting that it is possible to apply SNP markers developed based on the B. rapa genome to B. oleracea. These results provide valuable information that can be utilized in cabbage genetics and breeding programs using molecular markers derived from other Brassica species.     

Identification and characterization of SSR,SNP and InDel molecular markers from RNA-Seq data of guar (<Emphasis Type="Italic">Cyamopsis tetragonoloba</Emphasis>, L. Taub.) roots

Background

Guar [Cyamopsis tetragonoloba, L. Taub.] is an important industrial crop because of the commercial applications of the galactomannan gum contained in its seeds. Plant breeding programmes based on marker-assisted selection require a rich resource of molecular markers. As limited numbers of such markers are available for guar, molecular breeding programmes have not been undertaken for the genetic improvement of this important crop. Hence, the present work was done to enrich the molecular markers resource of guar by identifying high quality SSR, SNP and InDel markers from the RNA-Seq data of the roots of two guar varieties.

Results

We carried out RNA-Seq analysis of the roots of two guar varieties, namely, RGC-1066 and M-83. A total of 102,479 unigenes with an average length of 1016 bp were assembled from about 30 million high quality pair-end reads generated by an Illumina HiSeq 2500 platform. The assembled unigenes had 86.55% complete and 97.71% partially conserved eukaryotic genes (CEGs). The functional annotation of assembled unigenes using BLASTX against six databases showed that the guar unigenes were most similar to Glycine max. We could assign GO terms to 45,200 unigenes using the UniProt database. The screening of 102,479 unigenes with MISA and SAMtools version 1.4 softwares resulted in the identification of 25,040 high-confidence molecular markers which consisted of 18,792 SSRs, 5999 SNPs and 249 InDels. These markers tagged most of the genes involved in root development, stress tolerance and other general metabolic activities. Each of the 25,040 molecular markers was characterized, particularly with respect to its position in the unigene. For 71% of the molecular markers, we could determine the names, products and functions of the unigenes. About 80% of the markers, from a random sample of molecular markers, showed PCR amplification.

Conclusions

We have identified and characterized 25,040 high confidence SSR, SNP and InDel molecular markers in guar. It is expected that these markers will be useful in molecular breeding programmes and will also be helpful in studying molecular mechanisms of root development, stress tolerance and gum synthesis in guar.

     

A pipeline for high throughput detection and mapping of SNPs from EST databases

Single nucleotide polymorphisms (SNPs) represent the most abundant type of genetic variation that can be used as molecular markers. The SNPs that are hidden in sequence databases can be unlocked using bioinformatic tools. For efficient application of these SNPs, the sequence set should be error-free as much as possible, targeting single loci and suitable for the SNP scoring platform of choice. We have developed a pipeline to effectively mine SNPs from public EST databases with or without quality information using QualitySNP software, select reliable SNP and prepare the loci for analysis on the Illumina GoldenGate genotyping platform. The applicability of the pipeline was demonstrated using publicly available potato EST data, genotyping individuals from two diploid mapping populations and subsequently mapping the SNP markers (putative genes) in both populations. Over 7000 reliable SNPs were identified that met the criteria for genotyping on the GoldenGate platform. Of the 384 SNPs on the SNP array approximately 12% dropped out. For the two potato mapping populations 165 and 185 SNPs segregating SNP loci could be mapped on the respective genetic maps, illustrating the effectiveness of our pipeline for SNP selection and validation.     

梁山慈竹体细胞突变体No.30的SLAF-seq特异分子标记分析

梁山慈竹体细胞突变体No.30具有纤维素含量高、木质素含量高、纤维细胞长度长、纤维细胞长宽比大等特点,但由于梁山慈竹主要进行无性繁殖,不易获得纯合植株,突变体No.30的分子水平鉴定一直无法进行,严重阻碍了该突变体的推广利用。对梁山慈竹体细胞突变体No.30进行特异分子标记分析,并初步确定其性状变异来源。本研究利用SLAF-seq技术分别对突变体No.30以及同地区野生型梁山慈竹群体（CK）的基因组进行了简化基因组测序。经过了测序、建库、多态位点开发、特异多态性位点筛选等过程,最后对特异多态性位点筛选结果进行注释分析。结果发现突变体No.30的GC含量、纯合InDel数低于野生型梁山慈竹,而检测到的InDel总数以及杂合InDel数却高于野生群体。对SLAF-seq得到的InDel、SNP位点进行特异性筛选,获得了突变体No.30中稳定存在的特异InDel/SNP标签,包括特异C-T转换9381个、特异A-G转换9472个、特异InDel 329个。通过对这些特异SLAF标签进行注释,发现有6个特异SNP标签和1个特异InDel与纤维素合成相关,3个特异SNP标签与木质素合成相关,以及4个特异SNP标签和1个特异InDel与纤维细胞形态建成有关。这些结果表明突变体No.30在体细胞培养的过程中在基因组水平上发生了突变,并初步确定了其性状变异来源。但这些特异SLAF标签与突变体No.30在纤维素含量和木质素含量,以及纤维细胞发育等性状方面的相关性仍需进一步验证。