High-quality Single Nucleotide Polymorphism Markers Research Articles

Multi-locus genome-wide association study reveal genomic regions underlying root system architecture traits in Ethiopian sorghum germplasm.

The identification of genomic regions underlying the root system architecture (RSA) is vital for improving crop abiotic stress tolerance. To improve sorghum (Sorghum bicolor L. Moench) for environmental stress tolerance, information on genetic variability and genomic regions linked to RSA traits is paramount. The aim of this study was, therefore, to investigate common quantitative trait nucleotides (QTNs) via multiple methodologies and identify genomic regions linked to RSA traits in a panel of 274 Ethiopian sorghum accessions. Multi-locus genome-wide association study was conducted using 265,944 high-quality single nucleotide polymorphism markers. Considering the QTN detected by at least three different methods, a total of 17 reliable QTNs were found to be significantly associated with root angle, number, length, and dry weight. Four QTNs were detected on chromosome SBI-05, followed by SBI-01 and SBI-02 with three QTNs each. Among the 17 QTNs, 11 are colocated with previously identified root traits quantitative trait loci and the remaining six are genome regions with novel genes. A total of 118 genes are colocated with these up- and down-streams of the QTNs. Moreover, five QTNs were found intragenic. These QTNs are S5_8994835 (number of nodal roots), S10_55702393 (number of nodal roots), S1_56872999 (nodal root angle), S9_1212069 (nodal root angle), and S5_5667192 (root dry weight) intragenic regions of Sobic.005G073101, Sobic.010G198000, Sobic.001G273000, Sobic.009G013600, and Sobic.005G054700, respectively. Particularly, Sobic.005G073101, Sobic.010G198000, and Sobic.009G013600 were found responsible for the plant growth hormone-induced RSA. These genes may regulate root development in the seedling stage. Further analysis on these genes might be important to explore the genetic structure of RSA of sorghum.

Identification of Advantaged Genes for Lodging Resistance-Related Traits in the Temperate geng Group (Oryza sativa L.) Using a Genome-Wide Association Study

With the endless growth of the population and development of the economy and living standards, a high yield with good grain quality has become the prime objective of breeding. However, lodging is a major factor leading to a significant decline in rice (Oryza sativa L.) production and quality. We conducted genome-wide association analysis to detect quantitative trait loci (QTL)-related lodging resistance, using 395 temperate geng accessions. A total of 50 QTL affecting the six traits were detected, using 1,438,279 high-quality single nucleotide polymorphism markers. Five important QTL clusters were detected affecting the lodging resistance-related traits. The ten candidate genes were detected by performing gene differential expression analysis and haplotype analysis. Among them, LOC_Os07g48570 (OsDof-23) for qLA7.2, qSA7.2 and qPR7.3, LOC_Os08g29110 (wp2) for qLA8 and qSA8, and LOC_Os11g36440 (MHZ5) for qPR11 and qBR11.1 were considered the most likely candidate genes based on functional annotations. The results may facilitate the breeding of rice varieties resistant to lodging, to overcome the adverse effects of climate change.

Genetic diversity and population structure of pigeonpea (Cajanus cajan [L.] Millspaugh) landraces grown in Benin revealed by Genotyping-By-Sequencing.

Genetic diversity studies provide important details on target trait availability and its variability, for the success of breeding programs. In this study, GBS approach was used to reveal a new structuration of genetic diversity and population structure of pigeonpea in Benin. We used a total of 688 high-quality Single Nucleotide Polymorphism markers for a total of 44 pigeonpea genotypes. The distribution of SNP markers on the 11 chromosomes ranged from 14 on chromosome 5 to 133 on chromosome 2. The Polymorphism Information Content and gene diversity values were 0.30 and 0.34 respectively. The analysis of population structure revealed four clear subpopulations. The Weighted Neighbor Joining tree agreed with structure analyses by grouping the 44 genotypes into four clusters. The PCoA revealed that genotypes from subpopulations 1, 2 and 3 intermixed among themselves. The Analysis of Molecular Variance showed 7% of the total variation among genotypes while the rest of variation (93%) was within genotypes from subpopulations indicating a high gene exchange (Nm = 7.13) and low genetic differentiation (PhiPT = 0.07) between subpopulations. Subpopulation 2 presented the highest mean values of number of different alleles (Na = 1.57), number of loci with private alleles (Pa = 0.11) and the percentage of polymorphic loci (P = 57.12%). We discuss our findings and demonstrate how the genetic diversity and the population structure of this specie can be used through the Genome Wide Association Studies and Marker-Assisted Selection to enhance genetic gain in pigeonpea breeding programs in Benin.

Chemical and genetic variation in feral Cannabis sativa populations across the Nebraska climate gradient

Cannabis sativa is a versatile crop that can be cultivated for fiber, seed, or phytochemicals. To take advantage of this versatility and the potential of Cannabis as a feedstock for the bioeconomy, genomics-enabled breeding programs must be strengthened and expanded. This work contributes to the foundation for such by investigating the phytochemistry and genomics of feral Cannabis populations collected from seventeen counties across the climate gradient of Nebraska. Flower tissue from male and female plants (28 total) was studied using (i) gas chromatography-mass spectrometry to assess cannabinoid profiles and (ii) RNA sequencing to determine transcript abundances. Both male and female flower tissues produced cannabinoids, and, though the compounds were more abundant in female flower tissue, the primary cannabinoid in both was usually cannabidiol. The expression of genes that mediate early steps on the cannabinoid biosynthetic pathway were upregulated in female relative to male flowers, suggesting that female versus male flower tissue cannabinoid abundance may be controlled at least in part at the transcriptional level. DNA sequencing was used to place feral Cannabis plants from Nebraska into a previously described genomic context, revealing that all the plants studied here are much more similar to previously characterized hemp-type Cannabis plants than to drug-type Cannabis plants, at least at the genetic level. This work provides foundational phytochemical knowledge and a large set of high-quality single nucleotide polymorphism markers for future studies of feral Nebraska Cannabis.

Genetic Bases of Flow- and Sink-Related Traits in Rice Revealed by Genome-Wide Association Study

Rice yield is determined by source, sink, and flow and the coordination of these factors. However, the genetic base of the sink-flow is still unknown. We conducted on genome-wide association to detect quantitative trait loci (QTL) related to sink size (the number of rachis branches and spikelet number in rachis branches) and flow vascular bundle (large and small vascular bundles number in panicle neck and second internode) using 440 germplasm resources. The accessions exhibited marked variation in all traits and demonstrated complex phenotypic correlations. A total of 138 QTL affecting the 8 traits were detected using 3,188,500 high-quality single nucleotide polymorphism markers. Sixteen QTL clusters simultaneously affected flow and sink traits, which might explain the genetic base of significant correlations between flow and sink traits. The nine candidate genes in two consistent chromosomal regions simultaneously affecting multiple vascular bundle and sink size traits by performing gene-based association analysis and haplotype analysis. Among them, D2 (LOC_Os01g10040) and Gn1a (LOC_Os01g10110) for qPLVN1.1, qSLVN1.1, and qPRN1.2 and OsPIN5b (LOC_Os08g41720) for qPLVN8, qSLVN8, qSSVN8, and qSTSN8.2 were considered the most likely candidate genes based on functional annotations. The results provide useful information for improving rice yield potential via balancing sink–flow relationships by marker-assisted selection.

Single nucleotide polymorphisms reveal genetic diversity in New Mexican chile peppers (Capsicum spp.)

BackgroundChile peppers (Capsicum spp.) are among the most important horticultural crops in the world due to their number of uses. They are considered a major cultural and economic crop in the state of New Mexico in the United States. Evaluating genetic diversity in current New Mexican germplasm would facilitate genetic improvement for different traits. This study assessed genetic diversity, population structure, and linkage disequilibrium (LD) among 165 chile pepper genotypes using single nucleotide polymorphism (SNP) markers derived from genotyping-by-sequencing (GBS).ResultsA GBS approach identified 66,750 high-quality SNP markers with known map positions distributed across the 12 chromosomes of Capsicum. Principal components analysis revealed four distinct clusters based on species. Neighbor-joining phylogenetic analysis among New Mexico State University (NMSU) chile pepper cultivars showed two main clusters, where the C. annuum genotypes grouped together based on fruit or pod type. A Bayesian clustering approach for the Capsicum population inferred K = 2 as the optimal number of clusters, where the C. chinense and C. frutescens grouped in a single cluster. Analysis of molecular variance revealed majority of variation to be between the Capsicum species (76.08 %). Extensive LD decay (~ 5.59 Mb) across the whole Capsicum population was observed, demonstrating that a lower number of markers would be required for implementing genome wide association studies for different traits in New Mexican type chile peppers. Tajima’s D values demonstrated positive selection, population bottleneck, and balancing selection for the New Mexico Capsicum population. Genetic diversity for the New Mexican chile peppers was relatively low, indicating the need to introduce new alleles in the breeding program to broaden the genetic base of current germplasm.ConclusionsGenetic diversity among New Mexican chile peppers was evaluated using GBS-derived SNP markers and genetic relatedness on the species level was observed. Introducing novel alleles from other breeding programs or from wild species could help increase diversity in current germplasm. We present valuable information for future association mapping and genomic selection for different traits for New Mexican chile peppers for genetic improvement through marker-assisted breeding.

Identify QTLs and candidate genes underlying source-, sink-, and grain yield-related traits in rice by integrated analysis of bi-parental and natural populations.

The source-sink relationship determines the ultimate grain yield of rice. In this study, we used a set of reciprocal introgression lines (ILs) derived from Xuishui09 × IR2061 to map quantitative trait loci (QTLs) that were associated with sink-, source-, and grain yield-related traits. A total of 95 QTLs influencing eight measured traits were identified using 6181 high-quality single nucleotide polymorphism markers. Nine background-independent QTLs were consistently detected in seven chromosomal regions in different genetic backgrounds. Seven QTLs clusters simultaneously affected sink-, source-, and grain yield-related traits, probably due to the genetic basis of significant correlations of grain yield with source and sink traits. We selected 15 candidate genes in the four QTLs consistently identified in the two populations by performing gene-based association and haplotype analyses using 2288 accessions from the 3K project. Among these, LOC_Os03g48970 for qTSN3b, LOC_Os06g04710 for qFLL6a, and LOC_Os07g32510 for qTGW7 were considered as the most likely candidate genes based on functional annotations. These results provide a basis for further study of candidate genes and for the development of high-yield rice varieties by balancing source–sink relationships using marker-assisted selection.

Genome-wide association study of turnip mosaic virus resistance in non-heading Chinese cabbage.

A genome-wide association study (GWAS) using 83 diverse non-heading Chinese cabbage (NHCC) accessions identified 42,526 high-quality single nucleotide polymorphism markers associated with turnip mosaic virus (TuMV) resistance. Seventeen associated loci were identified, along with the related genes that were differentially expressed between resistant and susceptible varieties, suggesting that they may be candidate genes for TuMV tolerance. Nine mutant genes of Arabidopsis were selected for inoculation with TuMV-GFP (green fluorescence protein) to further confirm the disease resistance of these genes. Quantitative polymerase chain reaction (qPCR) analysis showed that the virus content in the Arabidopsis mutants with the homologous genes of cell wall-associated proteins, pectin methyl-esterase (PME), transcription factors (TFs), resistance gene (R), VAN3/SFC protein and F-box gene were significantly higher than that in the mutants with the homologous genes of methylation and J protein. Our results provide the basis of further study of the potential function of these candidate TuMV resistance genes and demonstrate that the described diverse NHCC can be efficiently used for GWAS of various quantitative traits. Taken together, the findings of this study will be useful to improve TuMV resistance in NHCC breeding and to discover new genes related to TuMV resistance.

Genetic Bases of the Stomata-Related Traits Revealed by a Genome-Wide Association Analysis in Rice (Oryza sativa L.).

Stomatal density (D) and size (S) are an important adaptive mechanism for abiotic stress tolerance and photosynthesis capacity in rice. However, the genetic base of rice stomata-related traits still remains unclear. We identified quantitative trait loci (QTLs) associated with D and S on abaxial and adaxial leaf surfaces using genome-wide association analysis with 451 diverse accessions in two environments. D and S showed significant differences between indica (xian) and japonica (geng) accessions and significantly negative phenotypic correlations. A total of 64 QTLs influencing eight stomata-related traits were identified using 2,936,762 high-quality single nucleotide polymorphism markers. Twelve QTLs were consistently detected for the same traits in nine chromosomal regions in both environments. In addition, 12 QTL clusters were simultaneously detected for the same stomata-related traits on abaxial and adaxial leaf surfaces in the same environment, probably explaining the genetic bases of significant correlations of the stomata-related traits. We screened 64 candidate genes for the nine consistent QTL regions using haplotype analysis. Among them, LOC_Os01g66120 for qDada1, OsSPCH2 (LOC_Os02g15760) for qDada2.1 and qDaba2.1, LOC_Os02g34320 for qSada2.2, OsFLP (LOC_Os07g43420) or LOC_Os07g43530 for qSaba7.1, and LOC_Os07g41200 for qWada7 and qWaba7 were considered as the most likely candidate genes based on functional annotations. The results systematically dissected the genetic base of stomata-related traits and provide useful information for improving rice yield potential via increasing abiotic stress tolerance and photosynthesis capacity under stressed and non-stressed conditions through deploying the favorable alleles underlying stomata-related traits by marker-assisted selection.

Genome-wide association mapping reveals loci for shelf life and developmental rate of lettuce.

Two major QTL, one for shelf life that corresponds to qSL4 and one, qDEV7, for developmental rate, were identified. Associated markers will be useful in breeding for improved fresh-cut lettuce. Fresh-cut lettuce in packaged salad can have short shelf life, and visible deterioration may start within a week after processing. Yield and developmental rate are an important aspect of lettuce production. Genetic diversity and genome-wide association studies (GWAS) were performed on 493 accessions with the genotypic data of 4615 high-quality single nucleotide polymorphism markers. Population structure (Q), principal component (PC), and phylogenetic analyses displayed genetic relationships associated with lettuce types and geographic distribution. Data for shelf life, yield, developmental rate, and their stability indices were used for statistical analysis, and GWAS was performed by general and mixed linear models. The genetic relationship among the individuals was incorporated into the models using kinship matrix, PC, and Q. Broad-sense heritability (H2) across environments was 0.43 for shelf life, 0.36 for yield, and 0.60 for developmental rate. There was a negative correlation between yield and developmental rate. Significant marker-trait association (SMTA) was detected for shelf life on chromosome 4. The most significant quantitative trait locus (QTL, qSL4, P = 2.23E-17) explained 24% of the total phenotypic variation (R2). The major QTL for developmental rate was detected on chromosome 7 (qDEV7, P = 2.43E-16, R2 = 17%), while additional QTLs with smaller effect were found in all chromosomes. No SMTA was detected for yield. The study identified lettuce accessions with extended and stable shelf life, stable yield, and desirable developmental rate. Molecular markers closely linked to traits can be applied for selection of preferable genotypes and for identification of genes associated with these traits.

Genomic-wide association study for white spot resistance in a tropical maize germplasm

White spot is an important maize disease, prevalent in most high production tropical regions with a severe negative impact in grain yield. Breeding for white spot resistance is the most effective mechanism to control the disease. The objectives of this research include identifying and mapping chromosomal regions and putative genes associated to the maize response to white spot. We performed an association mapping with phenotypic data collected in two environments located at South region of Brazil, and by using a set of 355,972 high-quality single nucleotide polymorphism markers in a panel composed by 183 tropical field corn and popcorn inbred lines. Nine SNPs were significantly associated with white spot, five of them co-localized on previously reported quantitative trait loci regions, while remaining regions are novel. The total phenotypic variance explained (r2) by the nine SNPs associated with white spot severity was 64.3%, while the variance explained by each SNP ranged from 0.72 to 25.54%. The identified putative gene models are promising candidates associated with white spot resistance. Once these SNPs were validated, they can be useful for selecting superior genotypes.

Genetic mapping of anthocyanin accumulation-related genes in pepper fruits using a combination of SLAF-seq and BSA.

Anthocyanins have significant functions in stress tolerance in pepper (Capsicum annuum L.) and also benefit human health. Nevertheless, the key structural genes and regulatory genes involved in anthocyanin accumulation in pepper fruits are still not well understood and fine mapped. For the present study, 383 F2 plants from a cross between the green-fruited C. annuum line Z5 and the purple-fruited line Z6 was developed. Two separate bulked DNA pools were constructed with DNAs extracted from either 37 plants with high anthocyanin content or from 18 plants with no anthocyanin. A combination of specific-locus amplified fragment sequencing (SLAF-seq) and bulked segregant analysis (BSA) was used to identify candidates for regions associated with anthocyanin accumulation. We identified a total of 127,004 high-quality single nucleotide polymorphism (SNP) markers, and detected 1674 high-quality SNP markers associated with anthocyanin accumulation. Three candidate anthocyanin-associated regions including the intervals from 12.48 to 20.00 Mb, from 54.67 to 56.59 Mb, and from 192.17 to 196.82 Mb were identified within a 14.10-Mb interval on chromosome 10 containing 126 candidate genes. Based on their annotations, we identified 12 candidate genes that are predicted to be associated with anthocyanin expression. The present results provide an efficient strategy for genetic mapping of and valuable insights into the genetic mechanisms of anthocyanin accumulation in pepper fruit, and allow us to clone and functionally analyze the genes that influence anthocyanin accumulation in this species.

A High-Density Genetic Map of an Allohexaploid Brassica Doubled Haploid Population Reveals Quantitative Trait Loci for Pollen Viability and Fertility.

A doubled haploid (DH) mapping population was obtained from microspore culture of an allohexaploid F1 from the cross between two recently-synthesized allohexaploid Brassica lines. We used single nucleotide polymorphism (SNP) genetic variation based on restriction-site associated DNA (RAD) sequencing to construct a high density genetic linkage map of the population. RAD libraries were constructed from the genomic DNA of both parents and 146 DH progenies. A total of 2.87 G reads with an average sequencing depth of 2.59 × were obtained in the parents and of 1.41 × in the progeny. A total of 290,422 SNPs were identified from clustering of RAD reads, from which we developed 7,950 high quality SNP markers that segregated normally (1:1) in the population. The linkage map contained all 27 chromosomes from the parental A, B and C genomes with a total genetic distance of 5725.19 cM and an average of 0.75 cM between adjacent markers. Genetic distance on non-integrated linkage groups was 1534.23 cM, or 21% of total genetic distance. Out of 146 DH progenies, 91 had a complete set of 27 chromosomes as expected of a hexaploid species, and 21 out of 27 chromosomes showed high collinearity between the physical and linkage maps. The loss of chromosome(s) or chromosome segment(s) in the DH population was associated with a reduction in pollen viability. Twenty-five additive QTL were associated with pollen viability and fertility-related traits (seed number, seed yield, pod length, plant height, 1000-seed weight). In addition, 44 intra-genomic and 18 inter-genomic epistatic QTL pairs were detected for 4 phenotypic traits. This provides confidence that the DH population may be selected for improved pollen viability and fertility in a future allohexaploid Brassica species.

Candidate genes for first flower node identified in pepper using combined SLAF-seq and BSA.

First flower node (FFN) is an important trait for evaluating fruit earliness in pepper (Capsicum annuum L.), but the genetic mechanisms that control FFN are still poorly understood. In the present study, we developed 249 F2 plants derived from an intraspecific cross between the inbred pepper lines Z4 and Z5. Thirty plants with the highest FFN and 30 plants with the lowest FFN were chosen and their DNAs were pooled according to phenotype to construct two bulked DNA pools. Specific-locus amplified fragment sequencing (SLAF-seq) was combined with bulked segregant analysis (BSA) to identify candidate regions related to FFN. According to our genetic analysis, the FFN trait is quantitatively inherited. A total of 106,848 high-quality single nucleotide polymorphism (SNP) markers were obtained, and 393 high-quality SNP markers associated with FFN were detected. Ten candidate regions within an interval of 3.98 Mb on chromosome 12 harboring 23 candidate genes were identified as closely correlated with FFN. Five genes (CA12g15130, CA12g15160, CA12g15370, CA12g15360, and CA12g15390) are predicted based on their annotations to be associated with expression of the FFN trait. The present study demonstrates an efficient genetic mapping strategy and lays a good foundation for molecular marker-assisted breeding using SNP markers linked to FFN and for cloning and functional analysis of the key genes controlling FFN.

QTL Mapping for Fiber Quality and Yield Traits Based on Introgression Lines Derived from Gossypium hirsutum × G. tomentosum.

The tetraploid species Gossypium hirsutum is cultivated widely throughout the world with high yield and moderate fiber quality, but its genetic basis is narrow. A set of 107 introgression lines (ILs) was developed with an interspecific cross using G. hirsutum acc. 4105 as the recurrent parent and G. tomentosum as the donor parent. A specific locus amplified fragment sequencing (SLAF-seq) strategy was used to obtain high-throughput single nucleotide polymorphism (SNP) markers. In total, 3157 high-quality SNP markers were obtained and further used for identification of quantitative trait loci (QTLs) for fiber quality and yield traits evaluated in multiple environments. In total, 74 QTLs were detected that were associated with five fiber quality traits (30 QTLs) and eight yield traits (44 QTLs), with 2.02–30.15% of the phenotypic variance explained (PVE), and 69 markers were found to be associated with these thirteen traits. Eleven chromosomes in the A sub-genome (At) harbored 47 QTLs, and nine chromosomes in the D sub-genome (Dt) harbored 27 QTLs. More than half (44 QTLs = 59.45%) showed positive additive effects for fiber and yield traits. Five QTL clusters were identified, with three in the At, comprised of thirteen QTLs, and two in the Dt comprised of seven QTLs. The ILs developed in this study and the identified QTLs will facilitate further molecular breeding for improvement of Upland cotton in terms of higher yield with enhanced fiber quality.

Identification of Major Quantitative Trait Loci for Seed Oil Content in Soybeans by Combining Linkage and Genome-Wide Association Mapping.

Soybean oil is the most widely produced vegetable oil in the world and its content in soybean seed is an important quality trait in breeding programs. More than 100 quantitative trait loci (QTLs) for soybean oil content have been identified. However, most of them are genotype specific and/or environment sensitive. Here, we used both a linkage and association mapping methodology to dissect the genetic basis of seed oil content of Chinese soybean cultivars in various environments in the Jiang-Huai River Valley. One recombinant inbred line (RIL) population (NJMN-RIL), with 104 lines developed from a cross between M8108 and NN1138-2, was planted in five environments to investigate phenotypic data, and a new genetic map with 2,062 specific-locus amplified fragment markers was constructed to map oil content QTLs. A derived F2 population between MN-5 (a line of NJMN-RIL) and NN1138-2 was also developed to confirm one major QTL. A soybean breeding germplasm population (279 lines) was established to perform a genome-wide association study (GWAS) using 59,845 high-quality single nucleotide polymorphism markers. In the NJMN-RIL population, 8 QTLs were found that explained a range of phenotypic variance from 6.3 to 26.3% in certain planting environments. Among them, qOil-5-1, qOil-10-1, and qOil-14-1 were detected in different environments, and qOil-5-1 was further confirmed using the secondary F2 population. Three loci located on chromosomes 5 and 20 were detected in a 2-year long GWAS, and one locus that overlapped with qOil-5-1 was found repeatedly and treated as the same locus. qOil-5-1 was further localized to a linkage disequilibrium block region of approximately 440 kb. These results will not only increase our understanding of the genetic control of seed oil content in soybean, but will also be helpful in marker-assisted selection for breeding high seed oil content soybean and gene cloning to elucidate the mechanisms of seed oil content.

Whole-genome single-nucleotide polymorphism (SNP) marker discovery and association analysis with the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) content in Larimichthys crocea.

Whole-genome single-nucleotide polymorphism (SNP) markers are valuable genetic resources for the association and conservation studies. Genome-wide SNP development in many teleost species are still challenging because of the genome complexity and the cost of re-sequencing. Genotyping-By-Sequencing (GBS) provided an efficient reduced representative method to squeeze cost for SNP detection; however, most of recent GBS applications were reported on plant organisms. In this work, we used an EcoRI-NlaIII based GBS protocol to teleost large yellow croaker, an important commercial fish in China and East-Asia, and reported the first whole-genome SNP development for the species. 69,845 high quality SNP markers that evenly distributed along genome were detected in at least 80% of 500 individuals. Nearly 95% randomly selected genotypes were successfully validated by Sequenom MassARRAY assay. The association studies with the muscle eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) content discovered 39 significant SNP markers, contributing as high up to ∼63% genetic variance that explained by all markers. Functional genes that involved in fat digestion and absorption pathway were identified, such as APOB, CRAT and OSBPL10. Notably, PPT2 Gene, previously identified in the association study of the plasma n-3 and n-6 polyunsaturated fatty acid level in human, was re-discovered in large yellow croaker. Our study verified that EcoRI-NlaIII based GBS could produce quality SNP markers in a cost-efficient manner in teleost genome. The developed SNP markers and the EPA and DHA associated SNP loci provided invaluable resources for the population structure, conservation genetics and genomic selection of large yellow croaker and other fish organisms.

SNP-based high density genetic map and mapping of btwd1 dwarfing gene in barley.

A high-density linkage map is a valuable tool for functional genomics and breeding. A newly developed sequence-based marker technology, restriction site associated DNA (RAD) sequencing, has been proven to be powerful for the rapid discovery and genotyping of genome-wide single nucleotide polymorphism (SNP) markers and for the high-density genetic map construction. The objective of this research was to construct a high-density genetic map of barley using RAD sequencing. 1894 high-quality SNP markers were developed and mapped onto all seven chromosomes together with 68 SSR markers. These 1962 markers constituted a total genetic length of 1375.8 cM and an average of 0.7 cM between adjacent loci. The number of markers within each linkage group ranged from 209 to 396. The new recessive dwarfing gene btwd1 in Huaai 11 was mapped onto the high density linkage maps. The result showed that the btwd1 is positioned between SNP marks 7HL_6335336 and 7_249275418 with a genetic distance of 0.9 cM and 0.7 cM on chromosome 7H, respectively. The SNP-based high-density genetic map developed and the dwarfing gene btwd1 mapped in this study provide critical information for position cloning of the btwd1 gene and molecular breeding of barley.

Construction of a high-density genetic map for grape using next generation restriction-site associated DNA sequencing

BackgroundGenetic mapping and QTL detection are powerful methodologies in plant improvement and breeding. Construction of a high-density and high-quality genetic map would be of great benefit in the production of superior grapes to meet human demand. High throughput and low cost of the recently developed next generation sequencing (NGS) technology have resulted in its wide application in genome research. Sequencing restriction-site associated DNA (RAD) might be an efficient strategy to simplify genotyping. Combining NGS with RAD has proven to be powerful for single nucleotide polymorphism (SNP) marker development.ResultsAn F1 population of 100 individual plants was developed. In-silico digestion-site prediction was used to select an appropriate restriction enzyme for construction of a RAD sequencing library. Next generation RAD sequencing was applied to genotype the F1 population and its parents. Applying a cluster strategy for SNP modulation, a total of 1,814 high-quality SNP markers were developed: 1,121 of these were mapped to the female genetic map, 759 to the male map, and 1,646 to the integrated map. A comparison of the genetic maps to the published Vitis vinifera genome revealed both conservation and variations.ConclusionsThe applicability of next generation RAD sequencing for genotyping a grape F1 population was demonstrated, leading to the successful development of a genetic map with high density and quality using our designed SNP markers. Detailed analysis revealed that this newly developed genetic map can be used for a variety of genome investigations, such as QTL detection, sequence assembly and genome comparison.

Development of highly polymorphic SNP markers from the complexity reduced portion of maize [Zea mays L.] genome for use in marker-assisted breeding

The duplicated and the highly repetitive nature of the maize genome has historically impeded the development of true single nucleotide polymorphism (SNP) markers in this crop. Recent advances in genome complexity reduction methods coupled with sequencing-by-synthesis technologies permit the implementation of efficient genome-wide SNP discovery in maize. In this study, we have applied Complexity Reduction of Polymorphic Sequences technology (Keygene N.V., Wageningen, The Netherlands) for the identification of informative SNPs between two genetically distinct maize inbred lines of North and South American origins. This approach resulted in the discovery of 1,123 putative SNPs representing low and single copy loci. In silico and experimental (Illumina GoldenGate (GG) assay) validation of putative SNPs resulted in mapping of 604 markers, out of which 188 SNPs represented 43 haplotype blocks distributed across all ten chromosomes. We have determined and clearly stated a specific combination of stringent criteria (>0.3 minor allele frequency, >0.8 GenTrainScore and >0.5 Chi_test100 score) necessary for the identification of highly polymorphic and genetically stable SNP markers. Due to these criteria, we identified a subset of 120 high-quality SNP markers to leverage in GG assay-based marker-assisted selection projects. A total of 32 high-quality SNPs represented 21 haplotypes out of 43 identified in this study. The information on the selection criteria of highly polymorphic SNPs in a complex genome such as maize and the public availability of these SNP assays will be of great value for the maize molecular genetics and breeding community.