Single nucleotide polymorphism mapping and alignment of recombinant chromosome substitution lines in barley

Single nucleotide polymorphism (SNP) genotyping is useful for assessing genetic variation in germplasm collections, genetic map development and detection of alien chromosome substitutions. In this study, a diversity analysis using 1,301 SNPs on a set of 37 barley accessions was conducted. This analysis showed a high polymorphism rate between the malting barley cultivar 'Haruna Nijo' and the food barley cultivar 'Akashinriki'. Haruna Nijo and Akashinriki are donors of the barley expressed sequence tag (EST) collections. A doubled haploid (DH) population derived from the cross between Haruna Nijo and Akashinriki was genotyped with 1,448 SNPs. Of these 1,448 SNPs, 734 were polymorphic and distributed on barley linkage groups (chromosomes) as follows: 1H (86), 2H (125), 3H (120), 4H (100), 5H (127), 6H (88) and 7H (88). By using cMAP, we integrated the SNP markers across high-density maps. The SNPs were also used to genotype 98 BC(3)F(4) recombinant chromosome substitution lines (RCSLs) developed from the same cross (Haruna Nijo/Akashinriki). These data were used to create graphical genotypes for each line and thus estimate the location, extent and total number of introgressions from Akashinriki in the Haruna Nijo background. The 35 selected RCSLs sample most of the Akashinriki food barley genome, with only a few missing segments. These resources bring new alleles into the malting barley gene pool from food barley.     

Fine genetic characterization of elite maize germplasm using high-throughput SNP genotyping

To investigate the genetic structure of Chinese maize germplasm, the MaizeSNP50 BeadChip with 56,110 single nucleotide polymorphisms (SNPs) was used to genotype a collection of 367 inbred lines widely used in maize breeding of China. A total of 41,819 informative SNPs with minor allele number of more than 0.05 were used to estimate the genetic diversity, relatedness, and linkage disequilibrium (LD) decay. Totally 1,015 SNPs evenly distributed in the genome were selected randomly to evaluate the population structure of these accessions. Results showed that two main groups could be determined i.e., the introduced germplasm and the local germplasm. Further, five subgroups corresponding to different heterotic groups, that is, Reid Yellow Dent (Reid), Lancaster Sure Crop (Lancaster), P group (P), Tang Sipingtou (TSPT), and Tem-tropic I group (Tem-tropic I), were determined. The genetic diversity of within subgroups was highest in the Tem-Tropic I and lowest in the P. Most lines in this panel showed limited relatedness with each other. Comparisons of gene diversity showed that there existed some conservative genetic regions in specific subgroups across the ten chromosomes, i.e., seven in the Lancaster, seven in the Reid, six in the TSPT, five in the P, and two in the Tem-Tropical I. In addition, the results also revealed that there existed fifteen conservative regions transmitted from Huangzaosi, an important foundation parent, to its descendants. These are important for further studies since the outcomes may provide clues to understand why Huangzaosi could become a foundation parent in Chinese maize breeding. For the panel of 367 elite lines, average LD distance was 391 kb and varied among different chromosomes as well as in different genomic regions of one chromosome. This analysis uncovered a high natural genetic diversity in the elite maize inbred set, suggesting that the panel can be used in association study, esp. for temperate regions.     

Hierarchical high-throughput SNP genotyping of the human Y chromosome using MALDI-TOF mass spectrometry

We have established the use of a primer extension/mass spectrometry method (the PinPoint assay) for high-throughput SNP genotyping of the human Y chromosome. 118 markers were used to define 116 haplogroups and typing was organised in a hierarchical fashion. Twenty multiplex PCR/primer extension reactions were set up and each sample could be assigned to a haplogroup with only two to five of these multiplex analyses. A single aliquot of one enzyme was found to be sufficient for both PCR and primer extension. We observed 100% accuracy in blind validation tests. The technique thus provides a reliable, cost-effective and automated method for Y genotyping, and the advantages of using a hierarchical strategy can be applied to any DNA segment lacking recombination.     

Prospects for using multimarker dihaploid lines for mapping the barley genome

Model population of spring barley Oregon Wolfe Pack dihaploid lines, derived from F1 of cross between dominant marker stock and recessive line, was studied on presence of morphological markers. For detection of DNA variability RAPD-analysis of these genotypes was carried out. Polymorphic fragment correlating with morphological trait was detected using primer P89.     

Development and high-throughput genotyping of substitution lines carring the chromosome segments of indica 9311 in the background of japonica Nipponbare

Chromosome segment substitution lines (CSSLs) are useful for the precise mapping of quartitative trait loci (QTLs) and dissection of the genetic basis of complex traits.In this study,two whole-genome sequenced rice cultivars,the japonica Nipponbare and indica 9311 were used as recipient and dtonor,respectively.A population with 57 CSSLs was developed after crossing and back-crossing assisted by mo lecular rnarkers,and genotypes were identified using a high-throughput resequencing strategy,Detailed graphical genotypes of 38 lines were constructed based on resequencing data.These CSSLs had a total of 95 substituted segments derived from indica 9311,with an average of about 2.5 segments pet CSSL and eight segments per chromosome,and covered about 87.4％ of the rice whole genome.A multiple linear regression QTL analysis mapped four QTLs for 1000-grain weight.The largest-effect QTL was located in a region on chromosome 5 that contained a cloned major QTL GW5/qSW5 for grain size in rice.These CSSLs with a background of Nipponbare may provide powerful tools for future whole-genome discovery and functional study of essential genes/QTLs in rice,and offer ideal materials and foundations for japonica breeding.     

Sequence-tagged-site-facilitated PCR for barley genome mapping

Summary Speed, efficiency, and safety considerations have led many genome mapping projects to evaluate polymerase chain reaction (PCR) sequence amplification as an alternative to Southern blot analysis. However, the availability of informative primer sequences can be a limiting factor in PCR-based mapping. An alternative to random amplified polymorphism detection (RAPD) is the sequence-tagged-site (STS) approach. If informative primer sequences could be derived from known sequences, then current maps, which are based on both known function and anonymous clones, might be easily converted to maps utilizing PCR technology. In this paper, four pairs of primer sequences were obtained from published sequences, and four pairs were obtained by sequencing portions of DNA clones from genomic clones derived from a random genomic library used in the North American Barley Genome Mapping Project (NABGMP). These primers were used to screen for polymorphisms in the progeny of a winter x spring and a spring x spring barley cross. Two types of polymorphisms were distinguished using these primer sets: (1) insertion/deletion events that could be read directly from agarose gels, and (2) point mutation events. The latter were identified using polyacrylamide-gel electrophoresis of PCR products following digestion with restriction endonucleases (four-base cutters). To determine whether the PCR-based polymorphisms were allelic to polymorphisms identified by the clones from which the primer sequences derived, chromosomal assignments and (when possible) co-segregation analysis was performed.     

Genotools SNP manager: a new software for automated high-throughput MALDI-TOF mass spectrometry SNP genotyping

Analysis of single nucleotide polymorphisms (SNPs) is a rapidly growing field of research that provides insights into the most common type of differences between individual genomes. The resulting information has a strong impact in the fields of pharmacogenomics, drug development, forensic medicine, and diagnostics of specific disease markers. The technique of matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) has been shown to be a highly suitable tool for the analysis of DNA. It supplies a very versatile method for addressing a high-throughput SNP genotyping approach. Here, we present the Bruker genotools SNP MANAGER, a new software tool suitable for highly automated MALDI-TOF MS SNP genotyping. The genotools SNP MANAGER administers the sample preparation data, calculates masses of allele-specific primer extension products, performs genotyping analysis, and displays the results. In the current study, we have used the genotools SNP MANAGER to perform an automated duplex SNP analysis of two biallelic markers from the promoter of the gene encoding the inflammatory mediator interleukin-6.     

Development and assessment of simple PCR markers for SNP genotyping in barley

Simple molecular marker assays underpin routine plant breeding and research activities in many laboratories worldwide. With the rapid growth of single nucleotide polymorphism (SNP) resources for many important crop plants, the availability of routine, low-tech marker assays for genotyping SNPs is of increased importance. In this study, we demonstrate that temperature-switch PCR (TSP) supports the rapid development of robust, allele-specific PCR markers for codominant SNP genotyping on agarose gel. A total of 87 TSP markers for assessing gene diversity in barley were developed and used to investigate the efficacy for marker development, assay reliably and genotyping accuracy. The TSP markers described provide good coverage of the barley genome, are simple to use, easy to interpret and score, and are amenable to assay automation. They provide a resource of informative SNP markers for assessing genetic relationships among individuals, populations and gene pools of cultivated barley (Hordeum vulgare L.) and its wild relative H. spontaneum K. Koch. TSP markers provide opportunities to use available SNP resources for marker-assisted breeding and plant genetic research, and to generate information that can be integrated with SNP data from different sources and studies. TSP markers are expected to provide similar advantages for any animal or plant species. M. J. Hayden and T. Tabone contributed equally to this work.     

Physiological and yield responses of recombinant chromosome substitution lines of barley to terminal drought in a Mediterranean‐type environment

Physiological traits and productivity of the recombinant chromosome substitution lines (RCSLs) of barley, developed through the cross of Hordeum vulgare ssp. vulgare cv. Harrington and the wild ancestor Hordeum vulgare ssp. spontaneum, were measured in plants growing in microplots (with and without irrigation) and in field conditions in two Mediterranean‐type environments, Cauquenes (rainfed) and Santa Rosa (irrigated). The objectives were to assess the degree of phenotypic variability in response to terminal drought stress and to test whether the introgression of the wild ancestor into cv. Harrington can increase the terminal drought tolerance of RCSLs of barley. Days from emergence to anthesis and from anthesis to maturity of the 80 RCSLs were reduced in only 2–4 days under water stress, in microplots. Specific leaf area (SLA) and stomatal conductance (gs) of 80 RCSLs and cv. Harrington decreased greatly under water stress in plants growing in microplots and field conditions (in 2004/05 growing season). No G × E interaction was detected except for SLA in the microplot experiment. The principal component analysis provided a clear distinction between RCSLs. Along the first principal component, it was possible to identify 24 RCSLs which represent the whole range of grain yield (GY), gs and SLA observed in the 80 RCSLs. The selected 24 RCSLs were evaluated in field conditions at Cauquenes and Santa Rosa, during two growing seasons (2007/08 and 2008/09). The gs and carbon isotope discrimination in grains (Δ¹³C) were significantly (P < 0.001) lower in the rainfed condition (Cauquenes), but the water‐soluble carbohydrates (WSC) in stems at anthesis and maturity was significantly (P < 0.001) higher than in well‐irrigated condition (Santa Rosa). Grain yield was reduced by 63% under drought conditions. Differences between RCSLs in gs, WSC and GY were significant (P < 0.001) in 2007/08. The stress tolerance index (STI) was highly (P < 0.01) correlated with GY in all environments (rainfed and irrigated conditions and the two growing seasons). The relationship between STI and Δ¹³C under rainfed condition allowed identifying drought tolerant and susceptible RCSLs; the former were high yielding lines under rainfed and irrigated conditions (and higher STI values), but with similar GY to cv. Harrington, but presented higher grain Δ¹³C values than cv. Harrington. The drought susceptible lines presented lower GY, STI and Δ¹³C values than cv. Harrington. These results suggest that H. spontaneum has contributed alleles that increase terminal drought tolerance to some of the RCSLs.     

Development of an integrated 200K SNP genotyping array and application for genetic mapping,genome assembly improvement and genome wide association studies in pear (Pyrus)

Pear (Pyrus; 2n = 34), the third most important temperate fruit crop, has great nutritional and economic value. Despite the availability of many genomic resources in pear, it is challenging to genotype novel germplasm resources and breeding progeny in a timely and cost‐effective manner. Genotyping arrays can provide fast, efficient and high‐throughput genetic characterization of diverse germplasm, genetic mapping and breeding populations. We present here 200K AXIOM^® PyrSNP, a large‐scale single nucleotide polymorphism (SNP) genotyping array to facilitate genotyping of Pyrus species. A diverse panel of 113 re‐sequenced pear genotypes was used to discover SNPs to promote increased adoption of the array. A set of 188 diverse accessions and an F₁ population of 98 individuals from ‘Cuiguan’ × ‘Starkrimson’ was genotyped with the array to assess its effectiveness. A large majority of SNPs (166 335 or 83%) are of high quality. The high density and uniform distribution of the array SNPs facilitated prediction of centromeric regions on 17 pear chromosomes, and significantly improved the genome assembly from 75.5% to 81.4% based on genetic mapping. Identification of a gene associated with flowering time and candidate genes linked to size of fruit core via genome wide association studies showed the usefulness of the array in pear genetic research. The newly developed high‐density SNP array presents an important tool for rapid and high‐throughput genotyping in pear for genetic map construction, QTL identification and genomic selection.     

A low-cost open-source SNP genotyping platform for association mapping applications

Association mapping aimed at identifying DNA polymorphisms that contribute to variation in complex traits entails genotyping a large number of single-nucleotide polymorphisms (SNPs) in a very large panel of individuals. Few technologies, however, provide inexpensive high-throughput genotyping. Here, we present an efficient approach developed specifically for genotyping large fixed panels of diploid individuals. The cost-effective, open-source nature of our methodology may make it particularly attractive to those working in nonmodel systems.     

An alternative to radiation hybrid mapping for large-scale genome analysis in barley

The presence of a monosomic gametocidal chromosome (GC) in a barley chromosome addition line of common wheat generates structural aberrations in the barley chromosome as well as in the wheat chromosomes of gametes lacking the GC. A collection of structurally aberrant barley chromosomes is analogous to a panel of radiation hybrid (RH) mapping and is valuable for high-throughput physical mapping. We developed 90 common wheat lines (GC lines) containing aberrant barley 7H chromosomes induced by a gametocidal chromosome, 2C. DNAs isolated from these GC lines provided a panel of 7H chromosomal fragments in a wheat genetic background, comparable with RH mapping panels in mammals. We used this 7H GC panel and the methodology for RH mapping to physically map PCR-based barley markers, SSRs and AFLPs, onto chromosome 7H, relying on polymorphism between the 7H chromosome and the wheat genome. We call this method GC mapping. This study describes a novel adaptation and combination of methods of inducing chromosomal rearrangements to produce physical maps of markers. The advantages of the presented method are similar to RH mapping in that non-polymorphic markers can be used and the mapping panels can be relatively easily obtained. In addition, mapping results are cumulative when using the same mapping set with new markers. The GC lines will be available from the National Bioresources Project-KOMUGI (). Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

QTL mapping of protein content in rice using single chromosome segment substitution lines

Protein content (PC) is an important component of rice nutritional quality. In order to better understand the genetic basis of this trait and increase related breeding efficiency, 21 single chromosome segment substitution (SCSS) lines grown in four sites over two growing seasons (regarded as eight environments) were used to associate PC with particular chromosome segments. Segments from 15 chromosomes were found to contain quantitative trait loci (QTLs) for PC in at least one environment. These included segments from chromosome 3 and 8, in which QTLs for PC had not previously been identified. The segment of chromosome 8 in CSSL-48 had the largest positive effect across all environments. The interaction between substitution and environment was highly significant. Some substitutions had large effects in one environment, but no effect in another (i.e. CSSL-08 and CSSL-17), while some substitutions significantly increased PC in one environment but decreased it in another (i.e. CSSL-41 and CSSL-43). By biplot and clustering analysis, the eight environments were grouped into two contrasting environment types, that is, Hainan and Jiangsu. The segment of chromosome 8 in CSSL-48 had PC-enhancing QTLs in both of the environment types. The segments in CSSL-34 had QTLs which increase PC in the Jiangsu environment but have no effect in the Hainan environment. For enhancing PC, CSSL-48 could be explored in breeding for wide adaptation across all environments, while CSSL-12, CSSL-14, CSSL-17, CSSL-41 and CSSL-43, and that in CSSL-34 could be explored in breeding for specific adaptation to the Hainan and Jiangsu environments, respectively. Near isogenic lines are under development to validate the QTLs with large effects in a range of genetic backgrounds relevant to Jiangsu and Hainan breeding programs. Secondary mapping populations are also being developed for further localising the responsible QTLs in CSSL-14, CSSL-34 and CSSL-48.     

Estimating autozygosity from high-throughput information: effects of SNP density and genotyping errors

Background

Runs of homozygosity are long, uninterrupted stretches of homozygous genotypes that enable reliable estimation of levels of inbreeding (i.e., autozygosity) based on high-throughput, chip-based single nucleotide polymorphism (SNP) genotypes. While the theoretical definition of runs of homozygosity is straightforward, their empirical identification depends on the type of SNP chip used to obtain the data and on a number of factors, including the number of heterozygous calls allowed to account for genotyping errors. We analyzed how SNP chip density and genotyping errors affect estimates of autozygosity based on runs of homozygosity in three cattle populations, using genotype data from an SNP chip with 777 972 SNPs and a 50 k chip.

Results

Data from the 50 k chip led to overestimation of the number of runs of homozygosity that are shorter than 4 Mb, since the analysis could not identify heterozygous SNPs that were present on the denser chip. Conversely, data from the denser chip led to underestimation of the number of runs of homozygosity that were longer than 8 Mb, unless the presence of a small number of heterozygous SNP genotypes was allowed within a run of homozygosity.

Conclusions

We have shown that SNP chip density and genotyping errors introduce patterns of bias in the estimation of autozygosity based on runs of homozygosity. SNP chips with 50 000 to 60 000 markers are frequently available for livestock species and their information leads to a conservative prediction of autozygosity from runs of homozygosity longer than 4 Mb. Not allowing heterozygous SNP genotypes to be present in a homozygosity run, as has been advocated for human populations, is not adequate for livestock populations because they have much higher levels of autozygosity and therefore longer runs of homozygosity. When allowing a small number of heterozygous calls, current software does not differentiate between situations where these calls are adjacent and therefore indicative of an actual break of the run versus those where they are scattered across the length of the homozygous segment. Simple graphical tests that are used in this paper are a current, yet tedious solution.     

Construction of a genetic map based on high-throughput SNP genotyping and genetic mapping of a TuMV resistance locus in Brassica rapa

Brassica rapa is a member of the Brassicaceae family and includes vegetables and oil crops that are cultivated worldwide. The introduction of durable resistance against turnip mosaic virus (TuMV) into agronomically important cultivars has been a significant challenge for genetic and horticultural breeding studies of B. rapa. Based on our previous genome-wide analysis of DNA polymorphisms between the TuMV-resistant doubled haploid (DH) line VC40 and the TuMV-susceptible DH line SR5, we constructed a core genetic map of the VCS-13M DH population, which is composed of 83 individuals derived from microspore cultures of a F₁ cross between VC40 and SR5, by analyzing the segregation of 314 sequence-characterized genetic markers. The genetic markers correspond to 221 SNPs and 31 InDels of genes as well as 62 SSRs, covering 1,115.9 cM with an average distance of 3.6 cM between the adjacent marker loci. The alignment and orientation of the constructed map showed good agreement with the draft genome sequence of Chiifu, thus providing an efficient strategy to map genic sequences. Using the genetic map, a novel dominant TuMV resistance locus (TuMV-R) in the VCS-13M DH population was identified as a 0.34 Mb region in the short arm of chromosome A6 in which four CC–NBS–LRR resistance genes and two pathogenesis-related-1 genes reside. The genetic map developed in this study can play an important role in the genetic study of TuMV resistance and the molecular breeding of B. rapa.     

An adapter ligation-mediated PCR method for high-throughput mapping of T-DNA inserts in the Arabidopsis genome

Agrobacterium transfer DNA (T-DNA) is an effective plant mutagen that has been used to create sequence-indexed T-DNA insertion lines in Arabidopsis thaliana as a tool to study gene function. Creating T-DNA insertion lines requires a dependable method for locating the site of insertion in the genome. In this protocol, we describe an adapter ligation-mediated PCR method that we have used to screen a mutant library and identify over 150,000 T-DNA insertional mutants; the method can also be applied to map individual mutants. The procedure consists of three steps: a restriction enzyme-mediated ligation of an adapter to the genomic DNA; a PCR amplification of the T-DNA/genomic DNA junction with primers specific to the adapter and T-DNA; and sequencing of the T-DNA/genomic junction to enable mapping to the reference genome. In most cases, the sequenced genomic region extends to the T-DNA border, enabling the exact location of the insert to be identified. The entire process takes 2 weeks to complete.