Discovery of polymorphisms in starch-related genes in rice germplasm by amplification of pooled DNA and deeply parallel sequencing

High-throughput sequencing of pooled DNA was applied to polymorphism discovery in candidate genes involved in starch synthesis. This approach employed semi- to long-range PCR (LR-PCR) followed by next-generation sequencing technology. A total of 17 rice starch synthesis genes encoding seven classes of enzymes, including ADP-glucose pyrophosphorylase (AGPase), granule starch synthase (GBSS), soluble starch synthase (SS), starch branching enzyme (BE), starch debranching enzyme (DBE) and starch phosphorylase (SPHOL) and phosphate translocator (GPT1) from 233 genotypes were PCR amplified using semi- to long-range PCR. The amplification products were equimolarly pooled and sequenced using massively parallel sequencing technology (MPS). By detecting single nucleotide polymorphism (SNP)/Indels in both coding and noncoding areas of the genes, we identified genetic differences and characterized the SNP/Indel variation and distribution patterns among individual starch candidate genes. Approximately, 60.9 million reads were generated, of which 54.8 million (90%) mapped to the reference sequences. The average coverage rate ranged from 12,708 to 38,300 times for SSIIa and SSIIIb, respectively. SNPs and single/multiple-base Indels were analysed in a total assembled length of 116,403 bp. In total, 501 SNPs and 113 Indels were detected across the 17 starch-related loci. The ratio of synonymous to nonsynonymous SNPs (Ka/Ks) test indicated GBSSI and isoamylase 1 (ISA1) as the least diversified (most purified) and conservative genes as the studied populations have been through cycles of selection. This report demonstrates a useful strategy for screening germplasm by MPS to discover variants in a specific target group of genes.     

香菇三个功能基因部分序列的单核苷酸多态性分析

以15个香菇栽培品种为材料,对尿嘧啶核苷酸-胞嘧啶核苷酸激酶基因(UMP-CMP kinase gene,uck1)、分裂原活化蛋白激酶基因(mitogen-activated protein kinase gene,mapk)和外切β-1,3葡聚糖酶基因(exo-β-1,3-glucanase-encoding gene,exg1)进行了部分序列的单核苷酸多态性(single nucleotide polymorphism,SNP)分析。结果表明,测序中出现的双峰,是菌丝双核体细胞中两细胞核之间的差异造成的。在采用uck1、mapk和exg1的3,126bp中,共发现48处多态性位点,发生频率为1/65bp,其中36个属于转换、12个为颠换。从群体发生频率上,38个属于超过10%的常见SNP,10个属于罕见SNP。不同基因的SNP发生频率不同,uck1、mapk和exg1的SNP发生频率分别为1.40%、0.82%和2.41%。外显子区SNP数量高于内含子,3个基因在外显子区域分布28个,内含子分布20个。外显子的28个SNP位点中,11个为错义突变,17个为同义突变。错义突变引起了编码氨基酸的改变。对SNP位点的聚类分析表明,15个栽培品种间存在的多态性位点在1–36之间,15个品种的SNP类型不同。uck1,mapk,exg1的SNP可用于香菇栽培品种的鉴别。     

‘Satellite DNA transcripts have diverse biological roles in Drosophila'

Recent years have seen considerable progress in applying single nucleotide polymorphisms (SNPs) to population genetics studies. However, relatively few have attempted to use them to study the genetic differentiation of wild bird populations and none have examined possible differences of exonic and intronic SNPs in these studies. Here, using 144 SNPs, we examined population genetic differentiation in the saker falcon (Falco cherrug) across Eurasia. The position of each SNP was verified using the recently sequenced saker genome with 108 SNPs positioned within the introns of 10 fragments and 36 SNPs in the exons of six genes, comprising MHC, MC1R and four others. In contrast to intronic SNPs, both Bayesian clustering and principal component analyses using exonic SNPs consistently revealed two genetic clusters, within which the least admixed individuals were found in Europe/central Asia and Qinghai (China), respectively. Pairwise D analysis for exonic SNPs showed that the two populations were significantly differentiated and between the two clusters the frequencies of five SNP markers were inferred to be influenced by selection. Central Eurasian populations clustered in as intermediate between the two main groups, consistent with their geographic position. But the westernmost populations of central Europe showed evidence of demographic isolation. Our work highlights the importance of functional exonic SNPs for studying population genetic pattern in a widespread avian species.     

Sequence diversity, haplotype analysis, association mapping and functional marker development in the waxy and starch synthase IIa genes for grain-yield-related traits in hexaploid wheat (Triticum aestivum L.)

Development of high-yielding cereal crops could meet increasing global demands for food, feed and bio-fuels. Wheat is one of the world??s most important cereal crops. The biosynthesis of starch is the major determinant of yield in wheat. Two starch biosynthesis genes, the waxy (Wx) genes and the starch synthase IIa (SSIIa) genes, were amplified and sequenced in 92 diverse wheat genotypes using genome-specific primers. Nucleotide diversity, haplotype analysis and association mapping were performed. The first exon (5??-UTR) and the first intron of the three homoeologous Wx genes were isolated using expressed sequence tag sequences. The Wx genes contained 12 exons separated by 11 introns. SNP (single nucleotide polymorphism) frequency ranged from 1 SNP/3,648?bp for Wx-D1 to 1 SNP/135?bp for SSIIa-A1, with an average of 1 SNP/230?bp. The average SNP frequencies in exon and intron regions were 1 SNP/322?bp and 1 SNP/228?bp, respectively. Thirty, 23 and 5 SNPs were identified and formed five, six and five haplotypes for SSIIa-A1, SSIIa-B1 and SSIIa-D1, respectively. However, no association was found between these SNPs and seven yield-related traits. Twenty-two, 15 and 1 SNPs were detected and formed nine, five and two haplotypes for Wx-A1, Wx-B1 and Wx-D1, respectively. Three unique nucleotides C+A+T at SNP5, SNP6 and SNP12 formed Wx-B1-H3, which was significantly associated with increased grain weight, thousand kernel weight, and total starch content in three spring wheat genotypes and five winter wheat genotypes. Cost-effective and co-dominant SNP markers were developed using temperature-switch (TS)-PCR and are being used for marker-assisted selection of doubled haploid lines with enhanced grain yield and starch content in winter wheat breeding programs.     

Gene-associated single nucleotide polymorphism discovery in perennial ryegrass (Lolium perenne L.)

Molecular genetic marker development in perennial ryegrass has largely been dependent on anonymous sequence variation. The availability of a large-scale EST resource permits the development of functionally-associated genetic markers based on SNP variation in candidate genes. Genic SNP loci and associated haplotypes are suitable for implementation in molecular breeding of outbreeding forage species. Strategies for in vitro SNP discovery through amplicon cloning and sequencing have been designed and implemented. Putative SNPs were identified within and between the parents of the F₁(NA₆ × AU₆) genetic mapping family and were validated among progeny individuals. Proof-of-concept for the process was obtained using the drought tolerance-associated LpASRa2 gene. SNP haplotype structures were determined and correlated with predicted amino acid changes. Gene-length LD was evaluated across diverse germplasm collections. A survey of SNP variation across 100 candidate genes revealed a high frequency of SNP incidence (c. 1 per 54 bp), with similar proportions in exons and introns. A proportion (c. 50%) of the validated genic SNPs were assigned to the F₁(NA₆ × AU₆) genetic map, showing high levels of coincidence with previously mapped RFLP loci. The perennial ryegrass SNP resource will enable genetic map integration, detailed LD studies and selection of superior allele content during varietal development.     

Polymorphism identification within 50 equine gene-specific sequence tagged sites

The continued discovery of polymorphisms in the equine genome will be important for future studies using genomic screens and fine mapping for the identification of disease genes. Segments of 50 equine genes were examined for variability in 10 different horse breeds using a pool-and-sequence method. We identified 11 single nucleotide polymorphisms (SNPs) in 9380 bp of sequenced exon, and 25 SNPs, six microsatellites, and one insertion/deletion in 16961 bp of sequenced intron. Of all genes studied 52% contained at least one polymorphism, and polymorphisms were found at an overall rate of 1/613 bp. Several of the putative SNPs were tested and verified by restriction enzyme analysis using natural restriction sites or ones created by primer mutagenesis. The lowest allele frequency for a SNP detected in pooled samples was 10%. Three of the SNPs verified in the diverse horse pool were further tested in six breed-specific horse pools and were found to be reasonably variable within breeds. The pool-and-sequence method allows identification of polymorphisms in horse populations and will be a valuable tool for future disease gene and comparative mapping in horses.     

SNP haplotypes of the <Emphasis Type="Italic">BADH1</Emphasis> gene and their association with aroma in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Betaine aldehyde dehydrogenase (BADH) is a key enzyme involved in the synthesis of glycinebetaine—a powerful osmoprotectant against salt and drought stress in a large number of species. Rice is not known to accumulate glycinebetaine but it has two functional genes coding for the BADH enzyme. A non-functional allele of the BADH2 gene located on chromosome 8 is a major factor associated with rice aroma. However, similar information is not available regarding the BADH1 gene located on chromosome 4 despite the similar biochemical function of the two genes. Here we report on the discovery and validation of SNPs in the BADH1 gene by re-sequencing of diverse rice varieties differing in aroma and salt tolerance. There were 17 SNPs in introns with an average density of one per 171 bp, but only three SNPs in exons at a density of one per 505 bp. Each of the three exonic SNPs led to changes in amino acids with functional significance. Multiplex SNP assays were used for genotyping of 127 diverse rice varieties and landraces. In total 15 SNP haplotypes were identified but only four of these, corresponding to two protein haplotypes, were common, representing more than 85% of the cultivars. Determination of population structure using 54 random SNPs classified the varieties into two groups broadly corresponding to indica and japonica cultivar groups, aromatic varieties clustering with the japonica group. There was no association between salt tolerance and the common BADH1 haplotypes, but aromatic varieties showed specific association with a BADH1 protein haplotype (PH2) having lysine₁₄₄ to asparagine₁₄₄ and lysine₃₄₅ to glutamine₃₄₅ substitutions. Protein modeling and ligand docking studies show that these two substitutions lead to reduction in the substrate binding capacity of the BADH1 enzyme towards gamma-aminobutyraldehyde (GABald), which is a precursor of the major aroma compound 2-acetyl-1-pyrroline (2-AP). This association requires further validation in segregating populations for potential utilization in the rice breeding programs.     

Single-nucleotide polymorphism frequency in a set of selected lines of bread wheat (Triticum aestivum L.).

Information on single-nucleotide polymorphisms (SNPs) in hexaploid bread wheat is still scarce. The goal of this study was to detect SNPs in wheat and examine their frequency. Twenty-six bread wheat lines from different origins worldwide were used. Specific PCR-products were obtained from 21 genes and directly sequenced. SNPs were discovered from the alignment of these sequences. The overall sequence polymorphism observed in this sample appears to be low; 64 single-base polymorphisms were detected in approximately 21.5 kb (i.e., 1 SNP every 335 bp). The level of polymorphism is highly variable among the different genes studied. Fifty percent of the genes studied contained no sequence polymorphism, whereas most SNPs detected were located in only 2 genes. As expected, taking into account a synthetic line created with a wild Triticum tauschii parent increases the level of polymorphism (101 SNPs; 1 SNP every 212 bp). The detected SNPs are available at http://urgi.versailles.inra.fr/GnpSNP">http://urgi.versailles.inra.fr/GnpSNP. Data on linkage disequilibrium (LD) are still preliminary. They showed a significant level of LD in the 2 most polymorphic genes. To conclude, the genome size of hexaploid wheat and its low level of polymorphism complicate SNP discovery in this species.     

Discovery of SNPs in soybean genotypes frequently used as the parents of mapping populations in the United States and Korea

Single nucleotide polymorphisms (SNPs) including insertion/deletions (indels) serve as useful and informative genetic markers. The availability of high-throughput and inexpensive SNP typing systems has increased interest in the development of SNP markers. After fragments of genes were amplified with primers derived from 110 soybean GenBank ESTs, sequencing data of PCR products from 15 soybean genotypes from Korea and the United States were analyzed by SeqScape software to find SNPs. Among 35 gene fragments with at least one SNP among the 15 genotypes, SNPs occurred at a frequency of 1 per 2,038 bp in 16,302 bp of coding sequence and 1 per 191 bp in 16,960 bp of noncoding regions. This corresponds to a nucleotide diversity (theta) of 0.00017 and 0.00186, respectively. Of the 97 SNPs discovered, 78 or 80.4% were present in the six North American soybean mapping parents. The addition of "Hwaeomputkong," which originated from Japan, increased the number to 92, or 94.8% of the total number of SNPs present among the 15 genotypes. Thus, Hwaeomputkong and the six North American mapping parents provide a diverse set of soybean genotypes that can be successfully used for SNP discovery in coding DNA and closely associated introns and untranslated regions.     

Patterns of divergence among conifer ESTs and polymorphism in Pinus sylvestris identify putative selective sweeps

Finding genes that are under positive selection is a difficult task, especially in non-model organisms. Here, we have analyzed expressed sequence tag (EST) data from 4 species (Pinus pinaster, Pinus taeda, Picea glauca, and Pseudotsuga menziesii) to investigate selection patterns during their evolution and to identify genes likely to be under positive selection. To confirm selection, population samples of these genes have been sequenced in Pinus sylvestris, a species that was not included in the EST data set. The estimates of branch-specific Ka/Ks (nonsynonymous/synonymous substitution rates) across all genes in the EST data set were similar or smaller than estimates from other higher plant species. There was no evidence for the traditional indication of positive selection, Ka/Ks above 1. However, several lines of evidence based on polymorphism patterns suggest that genes with high Ka/Ks (0.20-0.52) in the EST data set are in fact more affected by positive selection in P. sylvestris than genes with low Ka/Ks (0.01-0.04). The high Ka/Ks genes have a lower level of polymorphism and more negative Tajima's D than the low Ka/Ks genes. Further, in the high Ka/Ks group, the Hudson-Kreitman-Aguade test is significant. This suggests that the EST data set is a good starting point for finding genes under positive selection in conifers and that even moderate Ka/Ks values could be indicative of selection. A group of 5 genes with high Ka/Ks collectively show evidence for positive selection within P. sylvestris.     

The development of genome-wide single nucleotide polymorphisms in blue wildebeest using the DArTseq platform

Blue wildebeest (Connochaetes taurinus taurinus) are economically important antelope that are widely utilised in the South African wildlife industry. However, very few genomic resources are available for blue wildebeest that can assist in breeding management and facilitate research. This study aimed to develop a set of genome-wide single nucleotide polymorphism (SNP) markers for blue wildebeest. The DArTseq genotyping platform, commonly used in polyploid plant species, was selected for SNP discovery. A limited number of published articles have described the use of the DArTseq platform in animals and, therefore, this study also provided a unique opportunity to assess the performance of the DArTseq platform in an animal species. A total of 20,563 SNPs, each located within a 69 bp sequence, were generated. The developed SNP markers had a high average scoring reproducibility (>99%) and a low percentage missing data (~9.21%) compared to other reduced representation sequencing approaches that have been used in animal studies. Furthermore, the number of candidate SNPs per nucleotide position decreased towards the 3′ end of sequence reads, and the ratio of transitions (Ts) to transversions (Tv) remained similar for each read position. These observations indicate that there was no read position bias, such as the identification of false SNPs due to low sequencing quality, towards the tail-end of sequencing reads. The DArTseq platform was also successful in identifying a large number of informative SNPs with desirable polymorphism parameters such as a high minor allele frequency (MAF). The Bos taurus genome was used for the in silico mapping of the marker sequences and a total of 6020 (29.28%) sequences were successfully mapped against the bovine genome. The marker sequences mapped to all of the bovine chromosomes establishing the genome-wide distribution of the SNPs. Moreover, the high observed Ts:Tv ratio (2.84:1) indicate that the DArTseq platform targeted gene-rich regions of the blue wildebeest genome. Finally, functional annotation of the marker sequences revealed a wide range of different putative functions indicating that these SNP markers can be useful in functional gene studies. The DArTseq platform, therefore, represents a high-throughput, robust and cost-effective genotyping platform, which may find adoption in several other African antelope and animal species.     

DigiPINS: a database for vertebrate exonic single nucleotide polymorphisms and its application to cancer association studies

Single nucleotide polymorphisms (SNPs), which are the most abundant form of genetic variations in numerous organisms, have emerged as important tools for the study of complex genetic traits and deciphering of genome evolution. High-throughput genome sequencing projects worldwide provide an unprecedented opportunity for whole-genome SNP analysis in a variety of species. To facilitate SNP discovery in vertebrates, we have developed a web-based, user-friendly, and fully automated application, DigiPINS, for genome-wide identification of exonic SNPs from EST data. Currently, the database can be used to the mining of exonic SNPs in six complete genomes (Homo sapiens, Mus musculus, Rattus norvegicus, Canis familiaris, Gallus gallus and Danio rerio). In addition to providing information on sequence conservation, DigiPINS allows compilation of comprehensive sets of polymorphisms within cancer candidate genes or identification of novel cancer markers, making it potentially useful for cancer association studies. The DigiPINS server is available via the internet at http://pbil.univ-lyon1.fr/gem/DigiPINS/query_DigiPINS.php.     

Evidence for purifying selection against synonymous mutations in mammalian exonic splicing enhancers

Silent sites in mammals have classically been assumed to be free from selective pressures. Consequently, the synonymous substitution rate (Ks) is often used as a proxy for the mutation rate. Although accumulating evidence demonstrates that the assumption is not valid, the mechanism by which selection acts remain unclear. Recent work has revealed that the presence of exonic splicing enhancers (ESEs) in coding sequence might influence synonymous evolution. ESEs are predominantly located near intron-exon junctions, which may explain the reduced single-nucleotide polymorphism (SNP) density in these regions. Here we show that synonymous sites in putative ESEs evolve more slowly than the remaining exonic sequence. Differential mutabilities of ESEs do not appear to explain this difference. We observe that substitution frequency at fourfold synonymous sites decreases as one approaches the ends of exons, consistent with the existing SNP data. This gradient is at least in part explained by ESEs being more abundant near junctions. Between-gene variation in Ks is hence partly explained by the proportion of the gene that acts as an ESE. Given the relative abundance of ESEs and the reduced rates of synonymous divergence within them, we estimate that constraints on synonymous evolution within ESEs causes the true mutation rate to be underestimated by not more than approximately 8%. We also find that Ks outside of ESEs is much lower in alternatively spliced exons than in constitutive exons, implying that other causes of selection on synonymous mutations exist. Additionally, selection on ESEs appears to affect nonsynonymous sites and may explain why amino acid usage near intron-exon junctions is nonrandom.     

High resolution melting analysis of almond SNPs derived from ESTs

High resolution melting curve (HRM) is a recent advance for the detection of SNPs. The technique measures temperature induced strand separation of short PCR amplicons, and is able to detect variation as small as one base difference between samples. It has been applied to the analysis and scan of mutations in the genes causing human diseases. In plant species, the use of this approach is limited. We applied HRM analysis to almond SNP discovery and genotyping based on the predicted SNP information derived from the almond and peach EST database. Putative SNPs were screened from almond and peach EST contigs by HRM analysis against 25 almond cultivars. All 4 classes of SNPs, INDELs and microsatellites were discriminated, and the HRM profiles of 17 amplicons were established. The PCR amplicons containing single, double and multiple SNPs produced distinctive HRM profiles. Additionally, different genotypes of INDEL and microsatellite variations were also characterised by HRM analysis. By sequencing the PCR products, 100 SNPs were validated/revealed in the HRM amplicons and their flanking regions. The results showed that the average frequency of SNPs was 1:114 bp in the genic regions, and transition to transversion ratio was 1.16:1. Rare allele frequencies of the SNPs varied from 0.02 to 0.5, and the polymorphic information contents of the SNPs were from 0.04 to 0.53 at an average of 0.31. HRM has been demonstrated to be a fast, low cost, and efficient approach for SNP discovery and genotyping, in particular, for species without much genomic information such as almond.     

Natural selection of protein structural and functional properties: a single nucleotide polymorphism perspective

Background

The rates of molecular evolution for protein-coding genes depend on the stringency of functional or structural constraints. The Ka/Ks ratio has been commonly used as an indicator of selective constraints and is typically calculated from interspecies alignments. Recent accumulation of single nucleotide polymorphism (SNP) data has enabled the derivation of Ka/Ks ratios for polymorphism (SNP A/S ratios).

Results

Using data from the dbSNP database, we conducted the first large-scale survey of SNP A/S ratios for different structural and functional properties. We confirmed that the SNP A/S ratio is largely correlated with Ka/Ks for divergence. We observed stronger selective constraints for proteins that have high mRNA expression levels or broad expression patterns, have no paralogs, arose earlier in evolution, have natively disordered regions, are located in cytoplasm and nucleus, or are related to human diseases. On the residue level, we found higher degrees of variation for residues that are exposed to solvent, are in a loop conformation, natively disordered regions or low complexity regions, or are in the signal peptides of secreted proteins. Our analysis also revealed that histones and protein kinases are among the protein families that are under the strongest selective constraints, whereas olfactory and taste receptors are among the most variable groups.

Conclusion

Our study suggests that the SNP A/S ratio is a robust measure for selective constraints. The correlations between SNP A/S ratios and other variables provide valuable insights into the natural selection of various structural or functional properties, particularly for human-specific genes and constraints within the human lineage.     

Re-sequencing of DNA from a diverse panel of cattle reveals a high level of polymorphism in both intron and exon

In order to assess the extent of DNA sequence variation in cattle, introns and exons from both the leptin and Amyloid Precursor Protein (APP) genes have been sequenced in a panel of DNAs derived from 22 diverse animals. Direct DNA sequencing of PCR products was used; thus, 44 chromosomes were studied. Polymorphisms were identified by manual scanning of sequence chromatograms and computerized sequence analysis. Twenty Single Nucleotide Polymorphisms (SNPs) were detected in 1788 bp sequenced from the leptin gene, giving a frequency of 1 SNP per 89 bp. Twenty-four SNPs were detected in a 458-bp fragment of the APP gene; 23 of the polymorphisms were contained in a 302-bp intron 16 fragment. This equates to an SNP frequency of 1 per 13 bp for the intron. We can thus conclude that this portion of the bovine APP gene constitutes a hypermutable region. Nucleotide sequence diversity values of 0.019 and 0.0026 were obtained for APP and leptin respectively. Received: 22 April 1999 / Accepted: 25 July 1999