Nucleotide diversity and linkage disequilibrium in cold-hardiness- and wood quality-related candidate genes in Douglas fir

Nuclear sequence variation and linkage disequilibrium (LD) were studied in 15 cold-hardiness- and 3 wood quality-related candidate genes in Douglas fir [Pseudotsuga menziesii (Mirb.) Franco]. This set of genes was selected on the basis of its function in other plants and collocation with cold-hardiness-related quantitative trait loci (QTL). The single-nucleotide polymorphism (SNP) discovery panel represented 24 different trees from six regions in Washington and Oregon plus parents of a segregating population used in the QTL study. The frequency of SNPs was one SNP per 46 bp across coding and noncoding regions on average. Haplotype and nucleotide diversities were also moderately high with H(d) = 0.827 +/- 0.043 and pi = 0.00655 +/- 0.00082 on average, respectively. The nonsynonymous (replacement) nucleotide substitutions were almost five times less frequent than synonymous ones and substitutions in noncoding regions. LD decayed relatively slowly but steadily within genes. Haploblock analysis was used to define haplotype tag SNPs (htSNPs). These data will help to select SNPs for association mapping, which is already in progress.     

Single-nucleotide polymorphisms detected in expressed sequence tags of melon (Cucumis melo L.).

A search was performed for single-nucleotide polymorphisms (SNP) and short insertions-deletions (indels) in 34 melon (Cucumis melo L.) expressed sequence tag (EST) fragments between two distantly related melon genotypes, a group Inodorus 'Piel de sapo' market class breeding line T111 and the Korean accession PI 161375. In total, we studied 15 kb of melon sequence. The average frequency of SNPs between the two genotypes was one every 441 bp. One indel was also found every 1666 bp. Seventy-five percent of the polymorphisms were located in introns and the 3'untranslated regions. On average, there were 1.26 SNPs plus indels per amplicon. We explored three different SNP detection systems to position five of the SNPs in a melon genetic map. Three of the SNPs were mapped using cleaved amplified polymorphic sequence (CAPS) markers, one SNP was mapped using the single primer extension reaction with fluorescent-labelled dideoxynucleotides, and one indel was mapped using polyacrilamide gel electrophoresis separation. The discovery of SNPs based on ESTs and a suitable system for SNP detection has broad potential utility in melon genome mapping.     

Single nucleotide polymorphism discovery in common bean

Single nucleotide polymorphisms (SNPs) were discovered in common bean (Phaseolus vulgaris L.) via resequencing of sequence-tagged sites (STSs) developed by PCR primers previously designed to soybean shotgun and bacterial artificial chromosome (BAC) end sequences, and by primers designed to common bean genes and microsatellite flanking regions. DNA fragments harboring SNPs were identified in single amplicons from six contrasting P. vulgaris genotypes of the Andean (Jalo EEP 558, G 19833, and AND 277) and Mesoamerican (BAT 93, DOR 364, and Rudá) gene pools. These genotypes are the parents of three common bean recombinant inbred line mapping populations. From an initial set of 1,880 PCR primer pairs tested, 265 robust STSs were obtained, which could be sequenced in each one of the six common bean genotypes. In the resulting 131,120?bp of aligned sequence, a total of 677 SNPs were identified, including 555 single-base changes (295 transitions and 260 transversions) and 122 small nucleotide insertions/deletions (indels). The frequency of SNPs was 5.16 SNPs/kb and the mean nucleotide diversity, expressed as Halushka??s theta, was 0.00226. This work represents one of the first efforts aimed at detecting SNPs in P. vulgaris. The SNPs identified should be an important resource for common bean geneticists and breeders for quantitative trait locus discovery, marker-assisted selection, and map-based cloning. These SNPS will be also useful for diversity analysis and microsynteny studies among legume species.     

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.     

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.     

Estimation of DNA sequence diversity in bovine cytokine genes

DNA sequence variation provides the fundamental material for improving livestock through selection. In cattle, single nucleotide polymorphisms and small insertions/deletions (collectively referred to here as SNPs) have been identified in cytokine genes and scored in a reference population to determine linkage map positions. The aim of the present study was twofold: first, to estimate the SNP frequency in a reference population of beef cattle, and second, to determine cytokine haplotypes in a group of sires from commercial populations. Forty-five SNP markers in DNA segments from nine cytokine gene loci were analyzed in 26 reference parents. Comparison of all 52 haploid genomes at each PCR amplicon locus revealed an average of one SNP per 143 bp of sequence, whereas comparison of any two chromosomes identified heterozygous sites, on average, every 443 bp. The combination of these 45 SNP genotypes was sufficient to uniquely identify each of the 26 animals. The average number of haplotype alleles (4.4) per PCR amplicon (688 bp) and the percentage heterozygosity among founding parents (50%) were similar to those for microsatellite markers in the same population. For 49 sires from seven common breeds of beef cattle, SNP genotypes (1225 total) were obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) at three amplicon loci. All three of the amplicon haplotypes were correctly deduced for each sire without the use of parent or progeny genotypes. The latter allows a wide range of genetic studies in commercial populations of cattle where genotypic information from relatives may not be available. Received: 16 June 2000 / Accepted: 23 August 2000     

BARCSoySNP23: a panel of 23 selected SNPs for soybean cultivar identification

This report describes a set of 23 informative SNPs (BARCSoySNP23) distributed on 19 of the 20 soybean linkage groups that can be used for soybean cultivar identification. Selection of the SNPs to include in this set was made based upon the information provided by each SNP for distinguishing a diverse set of soybean genotypes as well as the linkage map position of each SNP. The genotypes included the ancestors of North American cultivars, modern North American cultivars and a group of Korean cultivars. The procedure used to identify this subset of highly informative SNP markers resulted in a significant increase in the power of identification versus any other randomly selected set of equal number. This conclusion was supported by a simulation which indicated that the 23-SNP panel can uniquely distinguish 2,200 soybean cultivars, whereas sets of randomly selected 23-SNP panels allowed the unique identification of only about 50 cultivars. The 23-SNP panel can efficiently distinguish each of the genotypes within four maturity group sets of additional cultivars/lines that have identical classical pigmentation and morphological traits. Comparatively, the 13 trinucleotide SSR set published earlier (BARCSoySSR13) has more power on a per locus basis because of the multi-allelic nature of SSRs. However, the assay of bi-allelic SNP loci can be multi-plexed using non-gel based techniques allowing for rapid determination of the SNP alleles present in soybean genotypes, thereby compensating for their relatively low information content. Both BARCSoySNP23 and BARCSoySSR13 were highly congruent relative to identifying genotypes and for estimating population genetic differences.     

Single nucleotide polymorphisms in Cryptomeria japonica: their discovery and validation for genome mapping and diversity studies

In order to develop a large set of single-nucleotide polymorphisms (SNPs) in Cryptomeria japonica, for a wide range of applications, we adopted a systematic EST (expressed sequence tags) re-sequencing approach. We examined a group of four genotypes comprising parents of a mapping population as well as representatives of two main lines from natural populations. We sequenced 5,170 gene fragments, representing analysis of over 1.3?Mb of DNA sequences in C. japonica. This analysis leads to the discovery of 13,413 SNPs in 3,744 amplicons, with an average of one SNP for every 101.0?bp (one SNP for every 78.3?bp in introns and for every 106.7?bp in exon regions). Nucleotide diversity in C. japonica (???=?0.0045) was found to be similar to values recorded in highly polymorphic forest tree species such as pine. We also validated the use of the SNPs as molecular markers for genetic diversity studies using the high throughput SNP genotyping platform GoldenGate. From 1,536 candidate SNP sites tested, 1,164 (75.8?%) were confirmed to be polymorphic. We anticipate that the genome-wide SNP markers reported here will be useful for evaluating the species?? range-wide genetic structure and in marker-assisted selection used as part of the C. japonica tree improvement program.     

Development of SNP markers and haplotype analysis of the candidate gene for rhg1, which confers resistance to soybean cyst nematode in soybean

Soybean cyst nematode (SCN; Heterodera glycines Ichinohe) is one of the most destructive pests in the cultivation of soybean (Glycine max (L.) Merr.) worldwide. Markers based on the SCN resistance gene will enable efficient marker-assisted selection (MAS). We sequenced the candidate gene rhg1 in six resistant and two susceptible soybean genotypes and identified 37 SNPs (single nucleotide polymorphisms) among the sequences, of which 11 were in the coding region. Seven of these 11 SNPs led to changes in the amino acid sequence of the gene. The amino acid sequence we obtained differs from the previously published one by a stretch of 26–27 amino acids. Six codominant allele-specific SNP markers based on agarose gel detection were developed and tested in 70 genotypes, among which occurred only nine different haplotypes. Two neutrality tests (Tajima’s D and Fu and Li’s F) were significant for the six SNP loci in the 70 genotypes, which is consistent with intensive directional selection. A strong LD pattern was detected among five SNPs except 2868T > C. Two SNPs (689C > A and 757C > T) formed one haplotype (689C-757C) that was perfectly associated with SCN resistance. The new allele-specific PCR markers located in the alleged sequence of the rhg1 candidate gene, combined with the microsatellite marker BACR-Satt309, will significantly improve the efficiency of MAS during the development of SCN-resistant cultivars.     

A first insight into peach [Prunus persica (L.) Batsch] SNP variability

Three factors may have reduced the diversity at both individual gene and whole genome levels in cultivated peach: its self-compatible mating system, the narrow genetic basis of most commercial cultivars, and the recent strong selection towards agronomically interesting traits. Previous diversity analyses with markers such as simple sequence repeats (SSRs) have revealed low levels of genetic variability. Here, we sequenced 23 genome-wide distributed DNA fragments in 47 occidental peach varieties, also observing reduced variability levels. On average, there was one single nucleotide polymorphism (SNP) every 598 bp and one indel every 4,189 bp. As expected, variability was higher in non-coding than in coding regions (one SNP every 390 non-coding bp versus one in 1,850 bp in coding DNA). In general, SNPs were observed at relatively high frequency, mean minor allele frequency?=?0.225, meaning that a large proportion of the SNPs discovered by sequencing similar germplasm will be useful for other purposes, such as association mapping. The average heterozygosity of the varieties was 0.28, with a low correlation between SSR and SNP heterozygosity. The whole sequence of two candidate genes, a pectate lyase 1 candidate for fruit firmness (CGPAA2668) and a sucrose synthase 1 candidate for sugar content (CGPPB6189), in the 47 varieties revealed that they both may have suffered a process of balancing selection.     

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.     

Development and Evaluation of SoySNP50K,a High-Density Genotyping Array for Soybean

The objective of this research was to identify single nucleotide polymorphisms (SNPs) and to develop an Illumina Infinium BeadChip that contained over 50,000 SNPs from soybean (Glycine max L. Merr.). A total of 498,921,777 reads 35–45bp in length were obtained from DNA sequence analysis of reduced representation libraries from several soybean accessions which included six cultivated and two wild soybean (G. soja Sieb. et Zucc.) genotypes. These reads were mapped to the soybean whole genome sequence and 209,903 SNPs were identified. After applying several filters, a total of 146,161 of the 209,903 SNPs were determined to be ideal candidates for Illumina Infinium II BeadChip design. To equalize the distance between selected SNPs, increase assay success rate, and minimize the number of SNPs with low minor allele frequency, an iteration algorithm based on a selection index was developed and used to select 60,800 SNPs for Infinium BeadChip design. Of the 60,800 SNPs, 50,701 were targeted to euchromatic regions and 10,000 to heterochromatic regions of the 20 soybean chromosomes. In addition, 99 SNPs were targeted to unanchored sequence scaffolds. Of the 60,800 SNPs, a total of 52,041 passed Illumina’s manufacturing phase to produce the SoySNP50K iSelect BeadChip. Validation of the SoySNP50K chip with 96 landrace genotypes, 96 elite cultivars and 96 wild soybean accessions showed that 47,337 SNPs were polymorphic and generated successful SNP allele calls. In addition, 40,841 of the 47,337 SNPs (86%) had minor allele frequencies ≥10% among the landraces, elite cultivars and the wild soybean accessions. A total of 620 and 42 candidate regions which may be associated with domestication and recent selection were identified, respectively. The SoySNP50K iSelect SNP beadchip will be a powerful tool for characterizing soybean genetic diversity and linkage disequilibrium, and for constructing high resolution linkage maps to improve the soybean whole genome sequence assembly.     

High-throughput genotyping with the GoldenGate assay in the complex genome of soybean

Large numbers of single nucleotide polymorphism (SNP) markers are now available for a number of crop species. However, the high-throughput methods for multiplexing SNP assays are untested in complex genomes, such as soybean, that have a high proportion of paralogous genes. The Illumina GoldenGate assay is capable of multiplexing from 96 to 1,536 SNPs in a single reaction over a 3-day period. We tested the GoldenGate assay in soybean to determine the success rate of converting verified SNPs into working assays. A custom 384-SNP GoldenGate assay was designed using SNPs that had been discovered through the resequencing of five diverse accessions that are the parents of three recombinant inbred line (RIL) mapping populations. The 384 SNPs that were selected for this custom assay were predicted to segregate in one or more of the RIL mapping populations. Allelic data were successfully generated for 89% of the SNP loci (342 of the 384) when it was used in the three RIL mapping populations, indicating that the complex nature of the soybean genome had little impact on conversion of the discovered SNPs into usable assays. In addition, 80% of the 342 mapped SNPs had a minor allele frequency >10% when this assay was used on a diverse sample of Asian landrace germplasm accessions. The high success rate of the GoldenGate assay makes this a useful technique for quickly creating high density genetic maps in species where SNP markers are rapidly becoming available. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the USDA and does not imply approval of a product to the exclusion of others that may be suitable.     

GWAS of agronomic traits in soybean collection included in breeding pool in Kazakhstan

Background

In recent years soybean is becoming one of the most important oilseed crops in Kazakhstan. Only within the last ten years (2006–2016), the area under soybean is expanded from 45 thousand hectares (ha) in 2006 to 120 thousand ha in 2016. The general trend of soybean expansion is from south-eastern to eastern and northern regions of the country, where average temperatures are lower and growing seasons are shorter. These new soybean growing territories were poorly examined in terms of general effects on productivity level among the diverse sample of soybean accessions. In this study, phenotypic data were collected in three separate regions of Kazakhstan and entire soybean sample was genotyped for identification of marker-trait associations (MTA).

Results

In this study, the collection of 113 accessions representing five different regions of the World was planted in 2015–2016 in northern, eastern, and south-eastern regions of Kazakhstan. It was observed that North American accessions showed the highest yield in four out of six trials especially in Northern Kazakhstan in both years. The entire sample was genotyped with 6 K SNP Illumina array. 4442 SNPs found to be polymorphic and were used for whole genome genotyping purposes. Obtained SNP markers data and field data were used for GWAS (genome-wide association study). 30 SNPs appear to be very significant in 42 MTAs in six studied environments.

Conclusions

The study confirms the efficiency of GWAS for the identification of molecular markers which tag important agronomic traits. Overall thirty SNP markers associated with time to flowering and maturation, plant height, number of fertile nodes, seeds per plant and yield were identified. Physical locations of 32 identified out of 42 total MTAs coincide well with positions of known analogous QTLs. This result indicates importance of revealed MTAs for soybean growing regions in Kazakhstan. Obtained results would serve as required prerequisite for forming and realization of specific breeding programs towards effective adaptation and increased productivity of soybean in three different regions of Kazakhstan.

     

SNP marker diversity in common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

Single nucleotide polymorphism (SNP) markers have become a genetic technology of choice because of their automation and high precision of allele calls. In this study, our goal was to develop 94 SNPs and test them across well-chosen common bean (Phaseolus vulgaris L.) germplasm. We validated and accessed SNP diversity at 84 gene-based and 10 non-genic loci using KASPar technology in a panel of 70 genotypes that have been used as parents of mapping populations and have been previously evaluated for SSRs. SNPs exhibited high levels of genetic diversity, an excess of middle frequency polymorphism, and a within-genepool mismatch distribution as expected for populations affected by sudden demographic expansions after domestication bottlenecks. This set of markers was useful for distinguishing Andean and Mesoamerican genotypes but less useful for distinguishing within each gene pool. In summary, slightly greater polymorphism and race structure was found within the Andean gene pool than within the Mesoamerican gene pool but polymorphism rate between genotypes was consistent with genepool and race identity. Our survey results represent a baseline for the choice of SNP markers for future applications because gene-associated SNPs could themselves be causative SNPs for traits. Finally, we discuss that the ideal genetic marker combination with which to carry out diversity, mapping and association studies in common bean should consider a mix of both SNP and SSR markers.     

DNA sequence polymorphism of the Rhg4 candidate gene conferring resistance to soybean cyst nematode in Chinese domesticated and wild soybeans

Rhg4 is one of the major resistant genes conferring resistance to soybean cyst nematode races 1, 3 and 4. In order to better understand its sequence diversity among different Chinese soybean populations and the impact of human activities on it, we designed 5 primer sets based on its sequence deposited in Genbank (Genbank accession No. AF506518) to obtain the Rhg4 sequence from 104 Chinese cultivated and wild soybean genotypes, and then analyzed the DNA sequence polymorphism in different Chinese soybean populations. The alignment of Rhg4 sequence included 5,216 nucleotide base pairs. A total of 67 single nucleotide polymorphisms (SNPs) including 59 single base changes and 8 DNA insertion-deletions (InDels) were identified with a SNP frequency of 1/78. Except for a 14-base InDel, there were 29 SNPs in coding regions, and among them, 13 were non-synonymous (9 in functional domains with 1 in a leucine-rich repeats region, 2 in a transmembrane region and 6 in a Ser/Thr kinase domain). The probability of substitution at each site was not the same, there were two hot spots, one was in the 5'-untranslated region between positions 124 and 804, and the other was in the region between positions 2520 and 3733. Sequence diversity analysis among 104 soybean genotypes showed π?=?0.00102 and θ?=?0.00218 for Rhg4. A domestication bottleneck was found because of lower sequence diversity and 58% unique SNPs loss in landraces compared with Glycine soja. Intensive selection increased the sequence diversity of cultivars, which had higher diversity and more unique SNPs than landraces. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11032-012-9703-1) contains supplementary material, which is available to authorized users.