Genome-wide single nucleotide polymorphism and Insertion-Deletion discovery through next-generation sequencing of reduced representation libraries in common bean

Single nucleotide polymorphisms (SNPs) and insertions-deletions (InDels) are valuable molecular markers for genomics and genetics studies and molecular breeding. The advent of next-generation sequencing techniques has enabled researchers to approach high-throughput and cost-effective SNP and InDel discovery on a genomic scale. In this report, 36 common bean genotypes grown in Canada were used to construct reduced representation libraries for next-generation sequencing. Using 76 million sequence reads generated by the Illumina HiSeq 2000 Sequencing System, we identified a total of 43,698 putative SNPs and 1,267 putative InDels. Of the SNPs, 43,504 were bi-allelic and 194 were tri-allelic, and the InDels comprised 574 insertions and 693 deletions. The putative bi-allelic SNPs were distributed across all 11 chromosomes with the highest number of SNPs observed in chromosome 2 (4,788), and the lowest in chromosome 10 (2,941). With the aid of the recent release of the first chromosome-scale version of Phaseolus vulgaris, 24,907 bi-allelic SNPs, 79 tri-allelic SNPs, 315 insertions, and 377 deletions were located in 8,758, 77, 273, and 364 genes, respectively. Among these 24,907 bi-allelic SNPs, 7,168 nonsynonymous bi-allelic SNPs were identified within 36 common bean genotypes that were located in 4,303 genes. A total of 113 putative SNPs were randomly chosen for validation using high-resolution melt analysis. Of the 113 candidate SNPs, 105 (92.9 %) contained the predicted SNPs.     

Genome-wide DNA polymorphisms in elite indica rice inbreds discovered by whole-genome sequencing

Advances in next-generation sequencing technologies have aided discovery of millions of genome-wide DNA polymorphisms, single nucleotide polymorphisms (SNPs) and insertions-deletions (InDels), which are an invaluable resource for marker-assisted breeding. Whole-genome resequencing of six elite indica rice inbreds (three cytoplasmic male sterile and three restorer lines) resulted in the generation of 338?million 75-bp paired-end reads, which provided 85.4% coverage of the Nipponbare genome. A total of 2?819?086 nonredundant DNA polymorphisms including 2?495?052 SNPs, 160?478 insertions and 163?556 deletions were discovered between the inbreds and Nipponbare, providing an average of 6.8 SNPs/kb across the genome. Distribution of SNPs and InDels in the chromosome was nonrandom with SNP-rich and SNP-poor regions being evident across the genome. A contiguous 4.3-Mb region on chromosome 5 with extremely low SNP density was identified. Overall, 83?262 nonsynonymous SNPs spanning 16?379 genes and 3620 nonsynonymous InDels in 2625 genes have been discovered which provide valuable insights into the basis underlying performance of the inbreds and the hybrids between these inbred combinations. SNPs and InDels discovered from this diverse set of indica rice inbreds not only enrich SNP resources for molecular breeding but also enable the study of genome-wide variations on hybrid performance.     

Kazusa mammalian cDNA resources: towards functional characterization of KIAA gene products.

The Kazusa cDNA project pioneered an extensive sequencing project of human cDNAs in their entirety and focused sequencing efforts particularly on large cDNAs encoding large proteins. More than 2000 human genes, referred to as 'KIAA' genes, were initially identified through this cDNA project. Since many KIAA genes still remain functionally uncharacterized, our current focus is to determine their biological functions in vivo. In this review, we describe the current status of the Kazusa mammalian cDNA resources and the future direction of the functional characterization of KIAA genes.     

Identification of genome-wide variations among three elite restorer lines for hybrid-rice

Rice restorer lines play an important role in three-line hybrid rice production. Previous research based on molecular tagging has suggested that the restorer lines used widely today have narrow genetic backgrounds. However, patterns of genetic variation at a genome-wide scale in these restorer lines remain largely unknown. The present study performed re-sequencing and genome-wide variation analysis of three important representative restorer lines, namely, IR24, MH63, and SH527, using the Solexa sequencing technology. With the genomic sequence of the Indica cultivar 9311 as the reference, the following genetic features were identified: 267,383 single-nucleotide polymorphisms (SNPs), 52,847 insertion/deletion polymorphisms (InDels), and 3,286 structural variations (SVs) in the genome of IR24; 288,764 SNPs, 59,658 InDels, and 3,226 SVs in MH63; and 259,862 SNPs, 55,500 InDels, and 3,127 SVs in SH527. Variations between samples were also determined by comparative analysis of authentic collections of SNPs, InDels, and SVs, and were functionally annotated. Furthermore, variations in several important genes were also surveyed by alignment analysis in these lines. Our results suggest that genetic variations among these lines, although far lower than those reported in the landrace population, are greater than expected, indicating a complicated genetic basis for the phenotypic diversity of the restorer lines. Identification of genome-wide variation and pattern analysis among the restorer lines will facilitate future genetic studies and the molecular improvement of hybrid rice.     

Genomewide discovery of DNA polymorphisms in rice cultivars with contrasting drought and salinity stress response and their functional relevance

Next‐generation sequencing technologies provide opportunities to understand the genetic basis of phenotypic differences, such as abiotic stress response, even in the closely related cultivars via identification of large number of DNA polymorphisms. We performed whole‐genome resequencing of three rice cultivars with contrasting responses to drought and salinity stress (sensitive IR64, drought‐tolerant Nagina 22 and salinity‐tolerant Pokkali). More than 356 million 90‐bp paired‐end reads were generated, which provided about 85% coverage of the rice genome. Applying stringent parameters, we identified a total of 1 784 583 nonredundant single‐nucleotide polymorphisms (SNPs) and 154 275 InDels between reference (Nipponbare) and the three resequenced cultivars. We detected 401 683 and 662 509 SNPs between IR64 and Pokkali, and IR64 and N22 cultivars, respectively. The distribution of DNA polymorphisms was found to be uneven across and within the rice chromosomes. One‐fourth of the SNPs and InDels were detected in genic regions, and about 3.5% of the total SNPs resulted in nonsynonymous changes. Large‐effect SNPs and InDels, which affect the integrity of the encoded protein, were also identified. Further, we identified DNA polymorphisms present in the differentially expressed genes within the known quantitative trait loci. Among these, a total of 548 SNPs in 232 genes, located in the conserved functional domains, were identified. The data presented in this study provide functional markers and promising target genes for salinity and drought tolerance and present a valuable resource for high‐throughput genotyping and molecular breeding for abiotic stress traits in rice.     

Genome analysis of the domestic dog (Korean Jindo) by massively parallel sequencing

Although pioneering sequencing projects have shed light on the boxer and poodle genomes, a number of challenges need to be met before the sequencing and annotation of the dog genome can be considered complete. Here, we present the DNA sequence of the Jindo dog genome, sequenced to 45-fold average coverage using Illumina massively parallel sequencing technology. A comparison of the sequence to the reference boxer genome led to the identification of 4 675 437 single nucleotide polymorphisms (SNPs, including 3 346 058 novel SNPs), 71 642 indels and 8131 structural variations. Of these, 339 non-synonymous SNPs and 3 indels are located within coding sequences (CDS). In particular, 3 non-synonymous SNPs and a 26-bp deletion occur in the TCOF1 locus, implying that the difference observed in cranial facial morphology between Jindo and boxer dogs might be influenced by those variations. Through the annotation of the Jindo olfactory receptor gene family, we found 2 unique olfactory receptor genes and 236 olfactory receptor genes harbouring non-synonymous homozygous SNPs that are likely to affect smelling capability. In addition, we determined the DNA sequence of the Jindo dog mitochondrial genome and identified Jindo dog-specific mtDNA genotypes. This Jindo genome data upgrade our understanding of dog genomic architecture and will be a very valuable resource for investigating not only dog genetics and genomics but also human and dog disease genetics and comparative genomics.     

LRP5 sequence and polymorphisms in the baboon

Background  LRP5 is known to have an important relationship with bone density and a variety of other biological processes. Mapping to human chromosome 11q13.2, LRP5 shows considerable evolutionary conservation. Orthologs of this gene exist in many species, although comparison of human LRP5 with other non-human primates has not been performed until now.
Methods  We reported the complementary DNA (cDNA) sequence and deduced amino acid sequence for baboon LRP5 , and compared the baboon and human sequences. cDNA sequences for 21 baboons were examined to identify single-nucleotide polymorphisms (SNPs).
Results  Sequences of coding regions in human and baboon LRP5 showed 97– 99% homology. Twenty-five SNPs were identified in the coding region of baboon LRP5 .
Conclusion  The observed degree of coding sequence homology in LRP5 led us to expect that the baboon may serve as a useful model for future research into the role(s) of this gene in primate metabolic diseases.     

Comprehensive Characterization of Human Genome Variation by High Coverage Whole-Genome Sequencing of Forty Four Caucasians

Whole genome sequencing studies are essential to obtain a comprehensive understanding of the vast pattern of human genomic variations. Here we report the results of a high-coverage whole genome sequencing study for 44 unrelated healthy Caucasian adults, each sequenced to over 50-fold coverage (averaging 65.8×). We identified approximately 11 million single nucleotide polymorphisms (SNPs), 2.8 million short insertions and deletions, and over 500,000 block substitutions. We showed that, although previous studies, including the 1000 Genomes Project Phase 1 study, have catalogued the vast majority of common SNPs, many of the low-frequency and rare variants remain undiscovered. For instance, approximately 1.4 million SNPs and 1.3 million short indels that we found were novel to both the dbSNP and the 1000 Genomes Project Phase 1 data sets, and the majority of which (∼96%) have a minor allele frequency less than 5%. On average, each individual genome carried ∼3.3 million SNPs and ∼492,000 indels/block substitutions, including approximately 179 variants that were predicted to cause loss of function of the gene products. Moreover, each individual genome carried an average of 44 such loss-of-function variants in a homozygous state, which would completely “knock out” the corresponding genes. Across all the 44 genomes, a total of 182 genes were “knocked-out” in at least one individual genome, among which 46 genes were “knocked out” in over 30% of our samples, suggesting that a number of genes are commonly “knocked-out” in general populations. Gene ontology analysis suggested that these commonly “knocked-out” genes are enriched in biological process related to antigen processing and immune response. Our results contribute towards a comprehensive characterization of human genomic variation, especially for less-common and rare variants, and provide an invaluable resource for future genetic studies of human variation and diseases.     

Sequence polymorphisms in porcine homologs of murine coat colour‐related genes

Herein, we report the variability among 57 porcine homologs of murine coat colour‐related genes. We identified single nucleotide polymorphisms (SNPs) and insertions/deletions (InDels) within 44 expressed gene sequences by aligning eight pig complementary DNA (cDNA) samples. The sequence alignment revealed a total of 485 SNPs and 15 InDels. The polymorphisms were then validated by performing matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) with reference DNA samples obtained from 384 porcine individuals. Of the 384 individuals, three parents of the experimental F₂ family were included to detect polymorphisms between them for linkage mapping. We also genotyped previously reported polymorphisms of 12 genes, and one SNP each in three genes that were detected by performing a BLAST search of the Trace database. A total of 211 SNPs and three InDels were successfully genotyped from our porcine DNA panel. We detected SNPs in 33 of the 44 genes among the parents of an experimental F₂ family and then constructed a linkage map of the 33 genes for this family. The linkage assignment of each gene to the porcine chromosomes was consistent with the location of the BAC clone in the porcine genome and the corresponding gene sequence. We confirmed complete substitutions of EDNRB and MLPH in the Jinhua and Clawn miniature breeds, respectively. Furthermore, we identified polymorphic alleles exclusive to each pig group: 13 for Jinhua, two for Duroc, three for Meishan, four for the Japanese wild boar, one for the Clawn miniature pig and four for the Potbelly pig.     

Molecular cloning, characterization, and genomic localization of a human potassium channel gene.

Potassium (K+) channels are critical for a variety of cell functions, including modulation of action potentials, determination of resting membrane potential, and development of memory and learning. In addition to their role in regulating myocyte excitability, cardiac K+ channels control heart rate and coronary vascular tone and are implicated in the development of arrhythmias. We report here the cloning and sequencing of a k+ channel gene, KCNA1, derived from a human cardiac cDNA library and the chromosomal localization of the corresponding genomic clone. Oligonucleotides based on a delayed rectifier K+ channel gene were used in PCR reactions with human genomic DNA to amplify the S4-S6 regions of several different K+ channel genes. These sequences were used to isolate clones from a human cardiac cDNA library. We sequenced one of these clones, HCK1. HCK1 contains putative S2-S6 domains and shares approximately 70% sequence homology with previously isolated Shaker homologues. HCK1 was used to screen human cosmid libraries and a genomic clone was isolated. By sequencing the genomic clones, a putative S1 domain and translation initiation sequences were identified. Genomic mapping using human-rodent somatic cell panels and in situ hybridization with human metaphase chromosomes have localized KCNA1 to the distal short arm of human chromosome 12. This work is an important step in the study of human cardiac K+ channel structure and function and will be of use in the study of human inherited disease.     

Expressed sequence tags for the chicken genome from a normalized 10-day-old White Leghorn whole embryo cDNA library: 1. DNA sequence characterization and linkage analysis

Expressed sequence tags (ESTs) provide a rapid and reliable method for gene discovery as well as a resource for the large-scale analysis of gene expression of known and unknown genes. Here we describe a normalized cDNA library developed from a 10-day-old White Leghorn chicken whole embryo. The utility of the library was evaluated by partial sequencing of 99 randomly selected insert-containing clones and the analysis of EST-targeted genomic regions for single nucleotide polymorphisms (SNPs) in the East Lansing chicken reference DNA mapping panel. Using stringent match criteria of percent identity of 80 or higher across a length of 50 or more bases, 46 ESTs matched database sequences including previously reported Gallus gallus genes. Thirty-seven of the 50 primer pairs developed from 50 unique ESTs amplified a single fragment. The size of the 37 amplicons ranged from 276 to 693 bp for a total of 17,508 and an average of 473. About 70% of the SNPs detected were either G-->A or C-->T transition. The number of SNPs detected within the amplicons from EST-targeted genomic regions ranged from 0 to 4 for a total of 65 and a frequency of about 1 every 470 bases. About 35% of the amplicons contained only 1 SNP, while 19% had 4 SNPs. Using the SNPs that were informative in the East Lansing reference panel, 17 ESTs were mapped on the East Lansing chicken genetic map. The ESTs described, as well as the nucleotide variants identified within the EST-targeted genomic regions, represent significant resources for genome analysis in the chicken.     

A Genomic Resource for the Development,Improvement, and Exploitation of Sorghum for Bioenergy

With high productivity and stress tolerance, numerous grass genera of the Andropogoneae have emerged as candidates for bioenergy production. To optimize these candidates, research examining the genetic architecture of yield, carbon partitioning, and composition is required to advance breeding objectives. Significant progress has been made developing genetic and genomic resources for Andropogoneae, and advances in comparative and computational genomics have enabled research examining the genetic basis of photosynthesis, carbon partitioning, composition, and sink strength. To provide a pivotal resource aimed at developing a comparative understanding of key bioenergy traits in the Andropogoneae, we have established and characterized an association panel of 390 racially, geographically, and phenotypically diverse Sorghum bicolor accessions with 232,303 genetic markers. Sorghum bicolor was selected because of its genomic simplicity, phenotypic diversity, significant genomic tools, and its agricultural productivity and resilience. We have demonstrated the value of sorghum as a functional model for candidate gene discovery for bioenergy Andropogoneae by performing genome-wide association analysis for two contrasting phenotypes representing key components of structural and non-structural carbohydrates. We identified potential genes, including a cellulase enzyme and a vacuolar transporter, associated with increased non-structural carbohydrates that could lead to bioenergy sorghum improvement. Although our analysis identified genes with potentially clear functions, other candidates did not have assigned functions, suggesting novel molecular mechanisms for carbon partitioning traits. These results, combined with our characterization of phenotypic and genetic diversity and the public accessibility of each accession and genomic data, demonstrate the value of this resource and provide a foundation for future improvement of sorghum and related grasses for bioenergy production.     

Integration of genetic and genomic methods for identification of genes and gene variants encoding QTLs in the nonhuman primate

We have developed an integrated approach, using genetic and genomic methods, in conjunction with resources from the Southwest National Primate Research Center (SNPRC) baboon colony, for the identification of genes and their functional variants that encode quantitative trait loci (QTL). In addition, we use comparative genomic methods to overcome the paucity of baboon specific reagents and to augment translation of our findings in a nonhuman primate (NHP) to the human population. We are using the baboon as a model to study the genetics of cardiovascular disease (CVD). A key step for understanding gene–environment interactions in cardiovascular disease is the identification of genes and gene variants that influence CVD phenotypes. We have developed a sequential methodology that takes advantage of the SNPRC pedigreed baboon colony, the annotated human genome, and current genomic and bioinformatic tools. The process of functional polymorphism identification for genes encoding QTLs involves comparison of expression profiles for genes and predicted genes in the genomic region of the QTL for individuals discordant for the phenotypic trait mapping to the QTL. After comparison, genes of interest are prioritized, and functional polymorphisms are identified in candidate genes by genotyping and quantitative trait nucleotide analysis. This approach reduces the time and labor necessary to prioritize and identify genes and their polymorphisms influencing variation in a quantitative trait compared with traditional positional cloning methods.     

Development of PCR-based SNP markers for rice blast resistance genes at the<Emphasis Type="Italic"> Piz</Emphasis> locus

We assessed the utility of single-nucleotide polymorphisms (SNPs) and small insertion/deletion polymorphisms (InDels) as DNA markers in genetic analysis and breeding of rice. Toward this end, we surveyed SNPs and InDels in the chromosomal region containing the Piz and Piz-t rice blast resistance genes and developed PCR-based markers for typing the SNPs. Analysis of sequences from a blast-susceptible Japanese cultivar and two cultivars each containing one of these genes revealed that SNPs are abundant in the Piz and Piz-t regions (on average, one SNP every 248 bp), but the number of InDels was much lower. The dense distribution of SNPs facilitated the generation of SNP markers in the vicinity of the genes. For typing these SNPs, we used a modified allele-specific PCR method. Of the 49 candidate allele-specific markers, 33 unambiguously and reproducibly discriminated between the two alleles. We used the markers for mapping the Piz and Piz-t genes and evaluating the size of DNA segments introgressed from the Piz donor cultivar in Japanese near-isogenic lines containing Piz. Our findings suggest that, because of its ability to generate numerous markers within a target region and its simplicity in assaying genotypes, SNP genotyping with allele-specific PCR is a valuable tool for gene mapping, map-based cloning, and marker-assisted selection in crops, especially rice.Communicated by D.J. Mackill     

Identification of novel SNPs in glioblastoma using targeted resequencing

High-throughput sequencing opens avenues to find genetic variations that may be indicative of an increased risk for certain diseases. Linking these genomic data to other "omics" approaches bears the potential to deepen our understanding of pathogenic processes at the molecular level. To detect novel single nucleotide polymorphisms (SNPs) for glioblastoma multiforme (GBM), we used a combination of specific target selection and next generation sequencing (NGS). We generated a microarray covering the exonic regions of 132 GBM associated genes to enrich target sequences in two GBM tissues and corresponding leukocytes of the patients. Enriched target genes were sequenced with Illumina and the resulting reads were mapped to the human genome. With this approach we identified over 6000 SNPs, including over 1300 SNPs located in the targeted genes. Integrating the genome-wide association study (GWAS) catalog and known disease associated SNPs, we found that several of the detected SNPs were previously associated with smoking behavior, body mass index, breast cancer and high-grade glioma. Particularly, the breast cancer associated allele of rs660118 SNP in the gene SART1 showed a near doubled frequency in glioblastoma patients, as verified in an independent control cohort by Sanger sequencing. In addition, we identified SNPs in 20 of 21 GBM associated antigens providing further evidence that genetic variations are significantly associated with the immunogenicity of antigens.