Genome-wide single nucleotide polymorphism discovery and validation in adzuki bean

Adzuki bean, also known as red bean (Vigna angularis), with 2n = 22 chromosomes, is an important legume crop in East Asian countries, including China, Japan, and Korea. For single nucleotide polymorphism (SNP) discovery, we used Vigna accessions, V. angularis IT213134 and its wild relative V. nakashimae IT178530, because of the lack of DNA sequence polymorphism in the cultivated species. Short read sequences of IT213134 and IT178530 of approximately 37 billion and 35 billion bp were produced using the Illumina HiSeq 2000 system to a sequencing depth of 61.5× and 57.7×, respectively. After de novo assembly was carried out with trimmed HiSeq reads from IT213134, 98,441 contigs of various sizes were produced with N50 of 13,755 bp. Using Burrows–Wheeler Aligner software, trimmed short reads of V. nakashimae IT178530 were successfully mapped to IT213134 contigs. All sequence variations at the whole-genome level were examined between the two Vigna species. Of the 1,565,699 SNPs, 59.4 % were transitions and 40.6 % were transversions. A total of 213,758 SNPs, consisting of 122,327 non-synonymous and 91,431 synonymous SNPs, were identified in coding sequences. For SNP validation, 96 SNPs in the genic region were chosen from among IT213134 contigs longer than 10 kb. Of these 96 SNPs, 88 were confirmed by Sanger sequencing of 10 adzuki bean genotypes from various geographic origins as well as IT213134 and its wild relative IT178530. These genome-wide SNP markers will enrich the existing Vigna resources and, specifically, could be of value for constructing a genetic map and evaluating the genetic diversity of adzuki bean.     

Transcriptome sequencing to produce SNP-based genetic maps of onion

We used the Roche-454 platform to sequence from normalized cDNA libraries from each of two inbred lines of onion (OH1 and 5225). From approximately 1.6 million reads from each inbred, 27,065 and 33,254 cDNA contigs were assembled from OH1 and 5225, respectively. In total, 3,364 well supported single nucleotide polymorphisms (SNPs) on 1,716 cDNA contigs were identified between these two inbreds. One SNP on each of 1,256 contigs was randomly selected for genotyping. OH1 and 5225 were crossed and 182 gynogenic haploids extracted from hybrid plants were used for SNP mapping. A total of 597 SNPs segregated in the OH1 × 5225 haploid family and a genetic map of ten linkage groups (LOD ≥8) was constructed. Three hundred and thirty-nine of the newly identified SNPs were also mapped using a previously developed segregating family from BYG15-23 × AC43, and 223 common SNPs were used to join the two maps. Because these new SNPs are in expressed regions of the genome and commonly occur among onion germplasms, they will be useful for genetic mapping, gene tagging, marker-aided selection, quality control of seed lots, and fingerprinting of cultivars.     

Use of an expressed sequence tag-based method for single nucleotide polymorphism identification and discrimination of Citrus species and cultivars

Sweet orange [Citrus sinensis (L.) Osbeck] represents the most important Citrus species, followed by clementine (C. clementina Hort. ex Tan.). Citrus species and genotypes are difficult to recognize as they have a moderate level of diversity due to nucellar selection, vegetative propagation and origin by single spontaneous mutation. Despite the large number of available sequences and the existence of a draft assembly of sweet orange and clementine, there are currently no single nucleotide polymorphism (SNP) databases for Citrus species. For this purpose, the QualitySNP software was used to discover SNPs in 19 Citrus species starting from 540,000 expressed sequence tags (ESTs) assembled in 52,000 contigs. The vast majority of ESTs, contigs and SNPs were found in C. clementina and C. sinensis: 4,400 out of 16,000 contigs (27 %) of C. clementina and 4,100 out of 17,000 contigs (24 %) of C. sinensis contained putative SNPs. A total of 3,634 sequences were associated with enzymes belonging to 121 metabolic KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways, among which the secondary metabolite pathway was the most represented. A total of 163 SNPs from 52 contigs and genes of specific functional categories were validated and 81 polymorphic sites were found. Thirty-seven selected SNPs, validated by Sanger sequencing, confirmed that polymorphisms were mainly between species, while poor within-species variability was discovered. This work provides a collection of 15,879 putative SNP markers that could be exploited by the Citrus community. Furthermore, the validated SNPs associated with specific genes could be used for functional genetic studies in germplasm diversity analysis, mapping and breeding.     

Development of single nucleotide polymorphism markers in the large and complex rubber tree genome using next-generation sequence data

The development of single nucleotide polymorphism (SNP) markers provides the opportunity to improve many areas of plant breeding and population genetics. Unfortunately, for species such as the rubber tree (Hevea brasiliensis), the use of next-generation sequencing for genomic SNP discovery is very difficult because of the large genome size and the abundance of repeated sequences. Access to a set of validated SNP markers is a significant advantage for rubber researchers who wish to apply SNPs in scientific research. Here, we performed genomic sequencing of H. brasiliensis and generated 10,993,648 short reads, which were assembled into 10,071 contigs (N50 = 3078) by a de novo assembly strategy. A total of 2446 contigs presented no hits in the current H. brasiliensis genome assembly and may therefore be considered novel genomic sequences of rubber tree. A total of 143 putative polymorphic positions were selected, gene annotations were available for 58.7 % of the markers, and all of the sequences could be anchored to the released H. brasiliensis genome. These SNPs were validated in eight genotypes of H. brasiliensis and 15 F1 plants from a mapping population, resulting in 30 (20.9 %) positions correctly classified. The analysis revealed key candidate genes responsible for defence mechanisms and provided markers for further genetic improvement of Hevea in breeding programmes.     

Characterisation of single nucleotide polymorphisms in sugarcane ESTs

Commercial sugarcane cultivars (Saccharum spp. hybrids) are both polyploid and aneuploid with chromosome numbers in excess of 100; these chromosomes can be assigned to 8 homology groups. To determine the utility of single nucleotide polymorphisms (SNPs) as a means of improving our understanding of the complex sugarcane genome, we developed markers to a suite of SNPs identified in a list of sugarcane ESTs. Analysis of 69 EST contigs showed a median of 9 SNPs per EST and an average of 1 SNP per 50 bp of coding sequence. The quantitative presence of each base at 58 SNP loci within 19 contiguous sequence sets was accurately and reliably determined for 9 sugarcane genotypes, including both commercial cultivars and ancestral species, through the use of quantitative light emission technology in pyrophosphate sequencing. Across the 9 genotypes tested, 47 SNP loci were polymorphic and 11 monomorphic. Base frequency at individual SNP loci was found to vary approximately twofold between Australian sugarcane cultivars and more widely between cultivars and wild species. Base quantity was shown to segregate as expected in the IJ76-514 × Q165 sugarcane mapping population, indicating that SNPs that occur on one or two sugarcane chromosomes have the potential to be mapped. The use of SNP base frequencies from five of the developed markers was able to clearly distinguish all genotypes in the population. The use of SNP base frequencies from a further six markers within an EST contig was able to help establish the likely copy number of the locus in two genotypes tested. This is the first instance of a technology that has been able to provide an insight into the copy number of a specific gene locus in hybrid sugarcane. The identification of specific and numerous haplotypes/alleles present in a genotype by pyrophosphate sequencing or alternative techniques ultimately will provide the basis for identifying associations between specific alleles and phenotype and between allele dosage and phenotype in sugarcane.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Transcriptome-based single nucleotide polymorphism markers for genome mapping in Japanese pear (Pyrus pyrifolia Nakai)

The development of single nucleotide polymorphism (SNP) markers in Japanese pear (Pyrus pyrifolia Nakai) offers the opportunity to use DNA markers for marker-assisted selection in breeding programs because of their high abundance, codominant inheritance, and potential for automated high-throughput analysis. We developed a 1,536-SNP bead array without a reference genome sequence from more than 44,000 base changes on the basis of a large-scale expressed sequence tag (EST) analysis combined with 454 genome sequencing data of Japanese pear ‘Housui’. Among the 1,536 SNPs on the array, 756 SNPs were genotyped, and 609 SNP loci were mapped to linkage groups on a genetic linkage map of ‘Housui’, based on progeny of an interspecific cross between European pear (Pyrus communis L.) ‘Bartlett’ and ‘Housui’. The newly constructed genetic linkage map consists of 951 loci, comprising 609 new SNPs, 110 pear genomic simple sequence repeats (SSRs), 25 pear EST–SSRs, 127 apple SSRs, 61 pear SNPs identified by the “potential intron polymorphism” method, and 19 other loci. The map covers 22 linkage groups spanning 1341.9 cM with an average distance of 1.41 cM between markers and is anchored to reference genetic linkage maps of European pears and apples. A total of 514 contigs containing mapped SNP loci showed significant similarity to known proteins by functional annotation analysis.     

Using cDNA and genomic sequences as tools to develop SNP strategies in cassava (Manihot esculenta Crantz)

Single nucleotide polymorphisms (SNP) are the most abundant type of DNA polymorphism found in animal and plant genomes. They provide an important new source of molecular markers that are useful in genetic mapping, map-based positional cloning, quantitative trait locus mapping and the assessment of genetic distances between individuals. Very little is known on the frequency of SNPs in cassava. We have exploited the recently-developed collection of cassava expressed sequence tags (ESTs) to detect SNPs in the five cultivars of cassava used to generate the sequences. The frequency of intra-cultivar and inter-cultivar SNPs after analysis of 111 contigs was one polymorphism per 905 and one per 1,032 bp, respectively; totaling 1 each 509 bp. We have obtained further information on the frequency of SNPs in six cassava cultivars by analysis of 33 amplicons obtained from 3 EST and BAC end sequences. Overall, about 11 kb of DNA sequence was obtained for each cultivar. A total of 186 SNPs (136 and 50 from ESTs and BAC ends, respectively) were identified. Among these, 146 were intra-cultivar polymorphisms, while 80 were inter-cultivar polymorphisms. Thus the total frequency of SNPs was one per 62 bp. This information will help to develop new strategies for EST mapping as well as their association with phenotypic characteristics.     

Genome-Wide Discovery of DNA Polymorphisms in Mei (<Emphasis Type="Italic">Prunus mume</Emphasis> Sieb. et Zucc.), an Ornamental Woody Plant,with Contrasting Tree Architecture and their Functional Relevance for Weeping Trait

Next-generation sequencing technologies provide opportunities to ascertain the genetic basis of phenotypic differences, even in the closely related cultivars via detection of large amount of DNA polymorphisms. In this study, we performed whole-genome re-sequencing of two mei cultivars with contrasting tree architecture. 75.87 million 100 bp pair-end reads were generated, with 92 % coverage of the genome. Re-sequencing data of two former upright mei cultivars were applied for detecting DNA polymorphisms, since we were more interested in variations conferring weeping trait. Applying stringent parameters, 157,317 mutual single nucleotide polymorphisms (SNPs) and 15,064 mutual insertions-deletions (InDels) were detected and found unevenly distributed within and among the mei chromosomes, which lead to the discovery of 220 high-density, 463 low-density SNP regions together with 80 high-density InDel regions. Additionally, 322 large-effect SNPs and 433 large-effect InDels were detected, and 10.09 % of the SNPs were observed in coding regions. 5.25 % SNPs in coding regions resulted in non-synonymous changes. Ninety SNPs were chosen randomly for validation using high-resolution melt analysis. 93.3 % of the candidate SNPs contained the predicted SNPs. Pfam analysis was further conducted to better understand SNP effects on gene functions. DNA polymorphisms of two known QTL loci conferring weeping trait and their functional effect were also analyzed thoroughly. This study highlights promising functional markers for molecular breeding and a whole-genome genetic basis of weeping trait in mei.     

Local de novo assembly of RAD paired-end contigs using short sequencing reads

Despite the power of massively parallel sequencing platforms, a drawback is the short length of the sequence reads produced. We demonstrate that short reads can be locally assembled into longer contigs using paired-end sequencing of restriction-site associated DNA (RAD-PE) fragments. We use this RAD-PE contig approach to identify single nucleotide polymorphisms (SNPs) and determine haplotype structure in threespine stickleback and to sequence E. coli and stickleback genomic DNA with overlapping contigs of several hundred nucleotides. We also demonstrate that adding a circularization step allows the local assembly of contigs up to 5 kilobases (kb) in length. The ease of assembly and accuracy of the individual contigs produced from each RAD site sequence suggests RAD-PE sequencing is a useful way to convert genome-wide short reads into individually-assembled sequences hundreds or thousands of nucleotides long.     

In silico single nucleotide polymorphism discovery and application to marker-assisted selection in soybean

The general approach to discovering single nucleotide polymorphisms (SNPs) requires locus-specific PCR amplification. To enhance the efficiency of SNP discovery in soybean, we used in silico analysis prior to re-sequencing as it is both rapid and inexpensive. In silico analysis was performed to detect putative SNPs in expressed sequence tag (EST) contigs assembled using publicly available ESTs from 18 different soybean genotypes. SNP validation by direct sequencing of six soybean cultivars and a wild soybean genotype was performed with PCR primers designed from EST contigs aligned with at least 5 out of 18 soybean genotypes. The efficiency of SNP discovery among the confirmation genotypes was 81.2%. Furthermore, the efficiency of SNP discovery between Pureunkong and Jinpumkong 2 genotypes was 47.4%, a great improvement on our previous finding based on direct sequencing (22.3%). Using SNPs between Pureunkong and Jinpumkong 2 in EST contigs, which were linked to target traits, we were able to genotype 90 recombinant inbred lines by high-resolution melting (HRM) analysis. These SNPs were mapped onto the expected locations near quantitative trait loci for water-logging tolerance and seed pectin concentration. Thus, our protocol for HRM analysis can be applied successfully not only to genetic diversity studies, but also to marker-assisted selection (MAS). Our study suggests that a combination of in silico analysis and HRM can reduce the cost and labor involved in developing SNP markers and genotyping SNPs. The markers developed in this study can also easily be applied to MAS if the markers are associated with the target traits.     

A high-throughput apple SNP genotyping platform using the GoldenGate™ assay

EST data generated from 14 apple genotypes were downloaded from NCBI and mapped against a reference EST assembly to identify Single Nucleotide Polymorphisms (SNPs). Mapping of these SNPs was undertaken using 90% of sequence similarity and minimum coverage of four reads at each SNP position. In total, 37,807 SNPs were identified with an average of one SNP every 187 bp from a total of 6888 unique EST contigs. Identified SNPs were checked for flanking sequences of ≥ 60 bp along both sides of SNP alleles for reliable design of a custom high-throughput genotyping assay. A total of 12,299 SNPs, representing 6525 contigs, fit the selected criterion of ≥ 60 bp sequences flanking a SNP position. Of these, 1411 SNPs were validated using four apple genotypes. Based on genotyping assays, it was estimated that 60% of SNPs were valid SNPs, while 26% of SNPs might be derived from paralogous regions.     

Association of the FADS2 Gene with ω-6 and ω-3 PUFA Concentration in the Egg Yolk of Japanese Quail

This study focused on the association of polymorphisms of the FADS2 gene with fatty acid profiles in egg yolk of eight Japanese quail lines selected for high and low ω-6:ω-3 PUFA ratio (h2 = 0.36–0.38). For the identification of polymorphisms within the FADS2 gene 1350 bp of cDNA sequence were obtained encoding 404 amino acids. Five synonymous SNPs were found by comparative sequencing of animals of the high and low lines. These SNPs were genotyped by single base extension on 160 Japanese quail. The association analysis, comprising analysis of variance and family based association test (FBAT), revealed significant effects of SNP3 and SNP4 genotypes on the egg yolk fatty acid profiles, especially the ω-6 and ω-3 PUFAs (P < 0.05). No effects of the other SNPs were found—indicating that these are not in linkage disequilibrium with the causal polymorphism. The results of this study promote FADS2 as a functional candidate gene for traits related to ω-6 and ω-3 PUFA concentration in the egg yolk.     

Automated SNP Detection in Expressed Sequence Tags: Statistical Considerations and Application to Maritime Pine Sequences

We developed an automated pipeline for the detection of single nucleotide polymorphisms (SNPs) in expressed sequence tag (EST) data sets, by combining three DNA sequence analysis programs: Phred, Phrap and PolyBayes. This application requires access to the individual electrophoregram traces. First, a reference set of 65 SNPs was obtained from the sequencing of 30 gametes in 13 maritime pine (Pinus pinaster Ait.) gene fragments (6671 bp), resulting in a frequency of 1 SNP every 102.6 bp. Second, parameters of the three programs were optimized in order to retrieve as many true SNPs, while keeping the rate of false positive as low as possible. Overall, the efficiency of detection of true SNPs was 83.1%. However, this rate varied largely as a function of the rare SNP allele frequency: down to 41% for rare SNP alleles (frequency < 10%), up to 98% for allele frequencies above 10%. Third, the detection method was applied to the 18498 assembled maritime pine (Pinus pinaster Ait.) ESTs, allowing to identify a total of 1400 candidate SNPs, in contigs containing between 4 and 20 sequence reads. These genetic resources, described for the first time in a forest tree species, were made available at http://www.pierroton.inra/genetics/Pinesnps. We also derived an analytical expression for the SNP detection probability as a function of the SNP allele frequency, the number of haploid genomes used to generate the EST sequence database, and the sample size of the contigs considered for SNP detection. The frequency of the SNP allele was shown to be the main factor influencing the probability of SNP detection.     

金针菇基因组测序与组装策略分析

采用二代和三代测序技术分别对金针菇单核体菌株“6-3”进行测序,应用4种组装策略进行基因组的de novo组装,对比组装效果。基因组组装的参数方面,仅使用二代测序组装的效果最差,长度大于10kb的Contig全长只有24.6Mb,Contig N50只有23kb,组装率只有59.27%。采用三代组装二代校正的组装策略效果最好,长度大于10kb的Contig全长为38.3Mb,Contig N50为2.8Mb,组装率高达92.16%。保守单拷贝基因拼接效果方面,4种组装策略获得基因组序列与BUSCO数据库里的担子菌的保守单拷贝基因比对,基因完整性均大于94%。在组装准确性方面,经过PCR扩增、Sanger测序验证,三代组装二代校正的基因组序列完整并且连续,同时序列上碱基的SNP、InDel数量最少。综上所述,三代组装二代校正得到的基因组序列具有Contig N50值大、组装率高、碱基准确性高的特点,是食用菌基因组测序较为理想的方案。     

High-throughput genotyping in citrus accessions using an SNP genotyping array

We developed a 384 multiplexed SNP array, named CitSGA-1, for the genotyping of Citrus cultivars, and evaluated the performance and reliability of the genotyping. SNPs were surveyed by direct sequence comparison of the sequence tagged site (STS) fragment amplified from genomic DNA of cultivars representing the genetic diversity of citrus breeding in Japan. Among 1497 SNPs candidates, 384 SNPs for a high-throughput genotyping array were selected based on physical parameters of Illumina’s bead array criteria. The assay using CitSGA-1 was applied to a hybrid population of 88 progeny and 103 citrus accessions for breeding in Japan, which resulted in 73,726 SNP calls. A total of 351 SNPs (91 %) could call different genotypes among the DNA samples, resulting in a success rate for the assay comparable to previously reported rates for other plant species. To confirm the reliability of SNP genotype calls, parentage analysis was applied, and it indicated that the number of reliable SNPs and corresponding STSs were 276 and 213, respectively. The multiplexed SNP genotyping array reported here will be useful for the efficient construction of linkage map, for the detection of markers for marker-assisted breeding, and for the identification of cultivars.     

Complete chloroplast genome of cultivated flowering cherry, <Emphasis Type="Italic">Prunus</Emphasis> ×<Emphasis Type="Italic">yedoensis</Emphasis> ‘Somei-yoshino’ in comparison with wild <Emphasis Type="Italic">Prunus yedoensis</Emphasis> Matsum. (Rosaceae)

Prunus ×yedoensis Matsum. ‘Somei-yoshino’ is the most common and widespread cultivar of the ornamental flowering cherries. We hereby report its complete chloroplast (cp) genome sequences generated by whole-genome next-generation sequencing approach. The cp genome size was 157,792 bp in length consisting of four regions; large single-copy region (85,914 bp), small single-copy region (19,120 bp), and a pair of inverted repeat regions (26,379 bp). The genome contained a total of 131 genes, including 86 coding genes, 8 rRNA genes, and 37 tRNA genes. A total of 92 simple sequence repeats (SSRs) were detected within the cp genome. Its molecular features were compared with the complete cp genome of wild P. yedoensis, which occurs rarely in natural habitats of Mt. Halla in Jeju Island, Korea, displaying nearly indistinguishable morphology as P. ×yedoensis ‘Somei-yoshino’. Although both cp genomes were structured highly alike, the sequence variations between them were revealed in several single-nucleotide polymorphisms (SNPs). Using additional individuals of wild and cultivated flowering cherries, PCR amplification confirmed that those SNPs were phylogenetically informative, providing distinction between wild and cultivated flowering cherries. In future study, the SNPs and SSRs reported in this study could be used to identify wild individuals from morphologically identical cultivars of flowering cherries and also to conserve the genetic diversity of wild flowering cherries in Jeju Island.     

High-throughput SNP discovery and genotyping in durum wheat (<Emphasis Type="Italic">Triticum durum</Emphasis> Desf.)

We describe the application of complexity reduction of polymorphic sequences (CRoPS^®) technology for the discovery of SNP markers in tetraploid durum wheat (Triticum durum Desf.). A next-generation sequencing experiment was carried out on reduced representation libraries obtained from four durum cultivars. SNP validation and minor allele frequency (MAF) estimate were carried out on a panel of 12 cultivars, and the feasibility of genotyping these SNPs in segregating populations was tested using the Illumina Golden Gate (GG) technology. A total of 2,659 SNPs were identified on 1,206 consensus sequences. Among the 768 SNPs that were chosen irrespective of their genomic repetitiveness level and assayed on the Illumina BeadExpress genotyping system, 275 (35.8%) SNPs matched the expected genotypes observed in the SNP discovery phase. MAF data indicated that the overall SNP informativeness was high: a total of 196 (71.3%) SNPs had MAF >0.2, of which 76 (27.6%) showed MAF >0.4. Of these SNPs, 157 were mapped in one of two mapping populations (Meridiano × Claudio and Colosseo × Lloyd) and integrated into a common genetic map. Despite the relatively low genotyping efficiency of the GG assay, the validated CRoPS-derived SNPs showed valuable features for genomics and breeding applications such as a uniform distribution across the wheat genome, a prevailing single-locus codominant nature and a high polymorphism. Here, we report a new set of 275 highly robust genome-wide Triticum SNPs that are readily available for breeding purposes.     

Ewing Sarcoma: influence of TP53 Arg72Pro and MDM2 T309G SNPs

The Ewing Sarcoma is an important tumor of bone and soft tissue. The SNPs Arg72Pro of TP53 and T309G of MDM2 have been associated with many cancer types and have been differently distributed among populations worldwide. Based on a case–control design, this study aimed to assess the role of these SNPs in 24 Ewing Sarcoma patients, compared to 91 control individuals. DNA samples were extracted from blood and genotyped for both SNPs by PCR–RFLP and confirmed by DNA sequencing. The results showed an association between the G allele of the T309G and Ewing Sarcoma (P = 0.02). Comparing to the TT carriers, the risk of G allele carriers was 3.35 (95 % CI = 1.22–9.21) with P = 0.02. At the genotypic level, an association of the TT genotype with the control group (P = 0.03) was found. Comparing to the TT genotype, the risk of TG and GG was 2.97 (95 % CI = 1.03–8.58) with P = 0.04 and 5.00 (95 % CI = 1.23–20.34) with P = 0.02, respectively. No associations regarding the Arg72Pro SNP were found. Considering that the T309G has been associated with several types of cancer, including sarcomas, our results indicate that this SNP may also be important to Ewing Sarcoma predisposition.