Analysis of genetic mapping in a waxy/dent maize RIL population using SSR and SNP markers

A maize genetic linkage map was generated using SSR and SNP markers in a F_7:8 recombinant inbred line (RIL) population derived from a cross of waxy corn (KW7) and dent corn (Mo17). A total of 465 markers, including 459 SSR and 6 SNP markers, were assigned to 10 linkage groups which spanned 2,656.5 cM with an average genetic distance between markers of 5.7 cM, and the number of loci per linkage group ranged from 39 to 55. The SSR (85.4%) and SNP (83.3%) markers showed Mendelian segregation ratios in the RIL population at a 5% significance threshold. In linkage analysis of six SNP loci associated with kernel starch synthesis genes (ae1, bt2, sh1, sh2, su1, and wx1), all six loci were successfully mapped and are closely linked with SSR markers in chromosomes 3 (sh2), 4 (su1 and bt2), 5 (ae1), and 9 (sh1 and wx1). The SSR markers linked with genes in starch synthesis may be utilized in marker assisted breeding programs. The resulting genetic map will be useful in dissection of quantitative traits and the identification of superior QTLs from the waxy hybrid corn. Additionally, these data support further genetic analysis and development of maize breeding programs.     

A genetic linkage map based on AFLP and SSR markers and mapping of QTL for dry-matter content in sweetpotato

We developed a genetic linkage map of sweetpotato using amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers and a mapping population consisting of 202 individuals derived from a broad cross between Xushu 18 and Xu 781, and mapped quantitative trait loci (QTL) for the storage root dry-matter content. The linkage map for Xushu 18 included 90 linkage groups with 2077 markers (1936 AFLP and 141 SSR) and covered 8,184.5 cM with an average marker distance of 3.9 cM, and the map for Xu 781 contained 90 linkage groups with 1954 markers (1824 AFLP and 130 SSR) and covered 8,151.7 cM with an average marker distance of 4.2 cM. The maps described herein have the best coverage of the sweetpotato genome and the highest marker density reported to date. These are the first maps developed that have 90 complete linkage groups, which is in agreement with the actual number of chromosomes. Duplex and triplex markers were used to detect the homologous groups, and 13 and 14 homologous groups were identified in Xushu 18 and Xu 781 maps, respectively. Interval mapping was performed first and, subsequently, a multiple QTL model was used to refine the position and magnitude of the QTL. A total of 27 QTL for dry-matter content were mapped, explaining 9.0–45.1 % of the variation; 77.8 % of the QTL had a positive effect on the variation. This work represents an important step forward in genomics and marker-assisted breeding of sweetpotato.     

A genetic linkage map of Nicotiana plumbaginifolia/ Nicotiana longiflora based on RFLP and RAPD markers

We have constructed a genetic linkage map for Nicotiana plumbaginifolia/Nicotiana longiflora (2n= 2x=20), based on the segregation of 69 RFLP and 102 RAPD loci in 99 F₂ plants from the cross N. plumbaginifolia×N. longiflora. The map consists of nine major linkage groups, each containing more than nine marker loci, and spans 1062 cM. Twenty of the RFLP markers were mapped previously to Nicotiana sylvestris (2n=2x=24) chromosomes using monosomic alien addition lines. Taxonomically, N. plumbaginifolia and N. sylvestris belong to the same section, namely the Alatae; however, cytogenetic evidence indicates that they are not closely related. Comparison of the distribution of markers common to both maps suggests that genome reorganization has occurred during the evolution of these two species. Evidence is also presented that genome reorganization may be accompanied by gain and loss of specific classes of DNA sequences in their genomes.     

Characterization of genetic diversity and population structure in wheat using array based SNP markers

Genetic diversity is crucial for successful adaptation and sustained improvement in crops. India is bestowed with diverse agro-climatic conditions which makes it rich in wheat germplasm adapted to various niches. Germplasm repository consists of local landraces, trait specific genetic stocks including introgressions from wild relatives, exotic collections, released varieties, and improved germplasm. Characterization of genetic diversity is done using morpho-physiological characters as well as by analyzing variations at DNA level. However, there are not many reports on array based high throughput SNP markers having characteristics of genome wide coverage employed in Indian spring wheat germplasm. Amongst wheat SNP arrays, 35K Axiom Wheat Breeder’s Array has the highest SNP polymorphism efficiency suitable for genetic mapping and genetic diversity characterization. Therefore, genotyping was done using 35K in 483 wheat genotypes resulting in 14,650 quality filtered SNPs, that were distributed across the B (~?50%), A (~?39%), and D (~?10%) genomes. The total genetic distance coverage was 4477.85 cM with 3.27 SNP/cM and 0.49 cM/SNP as average marker density and average inter-marker distance, respectively. The PIC ranged from 0.09 to 0.38 with an average of 0.29 across genomes. Population structure and Principal Coordinate Analysis resulted in two subpopulations (SP1 and SP2). The analysis of molecular variance revealed the genetic variation of 2% among and 98% within subpopulations indicating high gene flow between SP1 and SP2. The subpopulation SP2 showed high level of genetic diversity based on genetic diversity indices viz. Shannon’s information index (I)?=?0.648, expected heterozygosity (He)?=?0.456 and unbiased expected heterozygosity (uHe)?=?0.456. To the best of our knowledge, this study is the first to include the largest set of Indian wheat genotypes studied exclusively for genetic diversity. These findings may serve as a potential source for the identification of uncharacterized QTL/gene using genome wide association studies and marker assisted selection in wheat breeding programs.

     

Linkage map construction using InDel and SSR markers and QTL analysis of heading traits in Brassica oleracea var. capitata L.

Cabbage heading traits are important quantitative traits that greatly affect both quality and yield of cabbage. However, the genetic control of these traits remains unclear. To detect quantitative trait loci (QTLs) associated with heading traits, a double haploid (DH) population with 196 lines was created from a cabbage hybrid 01–20 × 96–100. A genetic map with insertion–deletion and simple sequence repeat markers was constructed based on the DH population, with a total length of 934.06 cM and average interval length of 2.3 cM between adjacent markers. Field experiments in three seasons were carried out to evaluate the heading traits, including head mature period (Hm), head weight (Hw), core length (Cl), head vertical diameter (Hvd), and the ratio of Cl to Hvd (Cl/Hvd). Using the map and the trait data, 13 reliable QTLs in total were identified and 5 were found in more than one season based on the adjusted means of three seasons. Major QTLs were identified for Hm (R ² = 40.4, LOD = 14.84), Hw (R ² = 28.6, LOD = 9.83), Cl (R ² = 38.8, LOD = 15.73), Hvd (R ² = 19.2, LOD = 9.26), and Cl/Hvd (R ² = 38.8, LOD = 12.75). The most significant QTLs were Hm3.1, Cl3.1, and Cl/Hvd3.1, which were detected in three seasons with the maximum contribution rate of almost 40 %. Six active regions that harbored more than one QTL were identified on five chromosomes, and one of them contained major QTLs associated with five traits. The QTLs obtained in this study should be useful for marker-assisted selection in cabbage breeding and for understanding the genetic control of these traits.     

High-resolution mapping of loci conferring resistance to sugarcane mosaic virus in maize using RFLP, SSR, and AFLP markers

Sugarcane mosaic virus (SCMV) is one of the most important virus diseases of maize in Europe. Genetic analysis on backcross five (BC5) progeny derived from the cross FAP1360A (resistant) × F7 (susceptible) confirmed that at least two dominant genes, Scm1 and Scm2, are required for resistance to SCMV in the progeny of this cross. With the aid of RFLP and SSR marker analyses, Scm1 was mapped in the region of 8.7 cM – between the nucleolus organizer region (nor) and RFLP marker bnl6.29 on the short arm of chromosome 6, while Scm2 was mapped to an interval of 26.8 cM flanked by the RFLP markers umc92 and umc102 near the centromere region of chromosome 3. Both chromosome regions were further enriched for AFLP markers by successful application of a bulked segregant analysis to this oligogenic trait. A total of 23 linked AFLP markers were identified, clustered in chromosome regions adjacent to either Scm1 or Scm2. Seven AFLP markers linked to Scm1 resided within the nor-bnl6.29 interval, and one of them, E3M8-1, showed no recombination with Scm1. Three AFLP markers linked to Scm2 are located between umc92 and umc102. Received: 13 October 1998 / Accepted: 18 January 1999     

Identification of sex-linked SNP markers using RAD sequencing suggests ZW/ZZ sex determination in Pistacia vera L.

Background

Pistachio (Pistacia vera L.) is a dioecious species that has a long juvenility period. Therefore, development of marker-assisted selection (MAS) techniques would greatly facilitate pistachio cultivar-breeding programs. The sex determination mechanism is presently unknown in pistachio. The generation of sex-linked markers is likely to reduce time, labor, and costs associated with breeding programs, and will help to clarify the sex determination system in pistachio.

Results

Restriction site-associated DNA (RAD) markers were used to identify sex-linked markers and to elucidate the sex determination system in pistachio. Eight male and eight female F₁ progenies from a Pistacia vera L. Siirt × Bağyolu cross, along with the parents, were subjected to RAD sequencing in two lanes of a Hi-Seq 2000 sequencing platform. This generated 449 million reads, comprising approximately 37.7 Gb of sequences. There were 33,757 polymorphic single nucleotide polymorphism (SNP) loci between the parents. Thirty-eight of these, from 28 RAD reads, were detected as putative sex-associated loci in pistachio. Validation was performed by SNaPshot analysis in 42 mature F₁ progenies and in 124 cultivars and genotypes in a germplasm collection. Eight loci could distinguish sex with 100% accuracy in pistachio. To ascertain cost-effective application of markers in a breeding program, high-resolution melting (HRM) analysis was performed; four markers were found to perfectly separate sexes in pistachio. Because of the female heterogamety in all candidate SNP loci, we report for the first time that pistachio has a ZZ/ZW sex determination system. As the reported female-to-male segregation ratio is 1:1 in all known segregating populations and there is no previous report of super-female genotypes or female heteromorphic chromosomes in pistachio, it appears that the WW genotype is not viable.

Conclusion

Sex-linked SNP markers were identified and validated in a large germplasm and proved their suitability for MAS in pistachio. HRM analysis successfully validated the sex-linked markers for MAS. For the first time in dioecious pistachio, a female heterogamety ZW/ZZ sex determination system is suggested.     

Bridging the genotyping gap: using genotyping by sequencing (GBS) to add high-density SNP markers and new value to traditional bi-parental mapping and breeding populations

Genotyping by sequencing (GBS) is the latest application of next-generation sequencing protocols for the purposes of discovering and genotyping SNPs in a variety of crop species and populations. Unlike other high-density genotyping technologies which have mainly been applied to general interest “reference” genomes, the low cost of GBS makes it an attractive means of saturating mapping and breeding populations with a high density of SNP markers. One barrier to the widespread use of GBS has been the difficulty of the bioinformatics analysis as the approach is accompanied by a high number of erroneous SNP calls which are not easily diagnosed or corrected. In this study, we use a 384-plex GBS protocol to add 30,984 markers to an indica (IR64) × japonica (Azucena) mapping population consisting of 176 recombinant inbred lines of rice (Oryza sativa) and we release our imputation and error correction pipeline to address initial GBS data sparsity and error, and streamline the process of adding SNPs to RIL populations. Using the final imputed and corrected dataset of 30,984 markers, we were able to map recombination hot and cold spots and regions of segregation distortion across the genome with a high degree of accuracy, thus identifying regions of the genome containing putative sterility loci. We mapped QTL for leaf width and aluminum tolerance, and were able to identify additional QTL for both phenotypes when using the full set of 30,984 SNPs that were not identified using a subset of only 1,464 SNPs, including a previously unreported QTL for aluminum tolerance located directly within a recombination hotspot on chromosome 1. These results suggest that adding a high density of SNP markers to a mapping or breeding population through GBS has a great value for numerous applications in rice breeding and genetics research.     

Construction of a high-density genetic map based on large-scale markers developed by specific length amplified fragment sequencing (SLAF-seq) and its application to QTL analysis for isoflavone content in Glycine max

Background

Quantitative trait locus (QTL) mapping is an efficient approach to discover the genetic architecture underlying complex quantitative traits. However, the low density of molecular markers in genetic maps has limited the efficiency and accuracy of QTL mapping. In this study, specific length amplified fragment sequencing (SLAF-seq), a new high-throughput strategy for large-scale SNP discovery and genotyping based on next generation sequencing (NGS), was employed to construct a high-density soybean genetic map using recombinant inbred lines (RILs, Luheidou2 × Nanhuizao, F_5:8). With this map, the consistent QTLs for isoflavone content across various environments were identified.

Results

In total, 23 Gb of data containing 87,604,858 pair-end reads were obtained. The average coverage for each SLAF marker was 11.20-fold for the female parent, 12.51-fold for the male parent, and an average of 3.98-fold for individual RILs. Among the 116,216 high-quality SLAFs obtained, 9,948 were polymorphic. The final map consisted of 5,785 SLAFs on 20 linkage groups (LGs) and spanned 2,255.18 cM in genome size with an average distance of 0.43 cM between adjacent markers. Comparative genomic analysis revealed a relatively high collinearity of 20 LGs with the soybean reference genome. Based on this map, 41 QTLs were identified that contributed to the isoflavone content. The high efficiency and accuracy of this map were evidenced by the discovery of genes encoding isoflavone biosynthetic enzymes within these loci. Moreover, 11 of these 41 QTLs (including six novel loci) were associated with isoflavone content across multiple environments. One of them, qIF20-2, contributed to a majority of isoflavone components across various environments and explained a high amount of phenotypic variance (8.7% - 35.3%). This represents a novel major QTL underlying isoflavone content across various environments in soybean.

Conclusions

Herein, we reported a high-density genetic map for soybean. This map exhibited high resolution and accuracy. It will facilitate the identification of genes and QTLs underlying essential agronomic traits in soybean. The novel major QTL for isoflavone content is useful not only for further study on the genetic basis of isoflavone accumulation, but also for marker-assisted selection (MAS) in soybean breeding in the future.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1086) contains supplementary material, which is available to authorized users.     

Construction of an ultra-high density consensus genetic map,and enhancement of the physical map from genome sequencing in <Emphasis Type="Italic">Lupinus angustifolius</Emphasis>

Key message

An ultra-high density genetic map containing 34,574 sequence-defined markers was developed in Lupinus angustifolius. Markers closely linked to nine genes of agronomic traits were identified. A physical map was improved to cover 560.5 Mb genome sequence.

Abstract

Lupin (Lupinus angustifolius L.) is a recently domesticated legume grain crop. In this study, we applied the restriction-site associated DNA sequencing (RADseq) method to genotype an F₉ recombinant inbred line population derived from a wild type × domesticated cultivar (W × D) cross. A high density linkage map was developed based on the W × D population. By integrating sequence-defined DNA markers reported in previous mapping studies, we established an ultra-high density consensus genetic map, which contains 34,574 markers consisting of 3508 loci covering 2399 cM on 20 linkage groups. The largest gap in the entire consensus map was 4.73 cM. The high density W × D map and the consensus map were used to develop an improved physical map, which covered 560.5 Mb of genome sequence data. The ultra-high density consensus linkage map, the improved physical map and the markers linked to genes of breeding interest reported in this study provide a common tool for genome sequence assembly, structural genomics, comparative genomics, functional genomics, QTL mapping, and molecular plant breeding in lupin.

     

A high-resolution map of the vicinity of the R1 locus on chromosome V of potato based on RFLP and AFLP markers

The R1 allele confers on potato a race-specific resistance to Phytophthora infestans. The corresponding genetic locus maps on chromosome V in a region in which several other resistance genes are also located. As part of a strategy for cloning R1, a high-resolution genetic map was constructed for the segment of chromosome V that is bordered by the RFLP loci GP21 and GP179 and includes the R1 locus. Bulked segregant analysis and markers based on amplified fragment length polymorphisms (AFLP markers) were used to select molecular markers closely linked to R1. Twenty-nine of approximately 3200 informative AFLP loci displayed linkage to the R1 locus. Based on the genotypic analysis of 461 gametes, eight loci mapped within the GP21–GP179 interval. Two of those could not be seperated from R1 by recombination. For genotyping large numbers of plants with respect to the flanking markers GP21 and GP179 PCR based assays were also developed which allowed marker-assisted selection of plants with genotypes Rr and rr and of recombinant plants.     

Identification of QTL for live weight and growth rate using DNA markers on chromosome 3 in an F2 population of Japanese quail

The Japanese quail (Coturnix japonica) is an important agricultural species and is an animal model for genetic researches. This study was conducted to identify quantitative trait loci (QTL) affecting live weight and growth rate on chromosome 3 in quail. Two strains of Japanese quail including wild and white were crossed reciprocally and F₁ generation was created. The birds from F₂ generation were measured for growth traits and all of 472 birds (8 pairs from the parental strains, 34 F₁ birds and 422 F₂ birds) were genotyped for microsatellite markers on chromosome 3. The results indicated chromosome wide significant QTL for hatching weight (P < 0.01) and weight at 1, 2, 3 and 4 weeks of age, average daily gain from hatch to 1, 1–2 and 3–4 weeks of age and Kleiber ratio (P < 0.05), an indirect criterion of feed efficiency. The highest QTL additive and imprinting effects (2.72 and 0.79 % of the trait variation in the F₂ population, respectively) were related to hatching weight. The identified QTL for this trait (at 7 cM relative to the centromeric region of the chromosome) had significant interaction with sex and hatch (P < 0.01). The dominance effect of QTL was significant (P < 0.05) for bodyweight at one week of age accounting for 1.69 % of the trait variation in the F₂ population.     

High-density SNP linkage map construction and QTL mapping for flavonoid-related traits in a tea plant (<Emphasis Type="Italic">Camellia sinensis</Emphasis>) using 2b-RAD sequencing

Background

Flavonoids are important components that confer upon tea plants a unique flavour and health functions. However, the traditional breeding method for selecting a cultivar with a high or unique flavonoid content is time consuming and labour intensive. High-density genetic map construction associated with quantitative trait locus (QTL) mapping provides an effective way to facilitate trait improvement in plant breeding. In this study, an F1 population (LJ43×BHZ) was genotyped using 2b-restriction site-associated DNA (2b-RAD) sequencing to obtain massive single nucleotide polymorphism (SNP) markers to construct a high-density genetic map for a tea plant. Furthermore, QTLs related to flavonoids were identified using our new genetic map.

Results

A total of 13,446 polymorphic SNP markers were developed using 2b-RAD sequencing, and 4,463 of these markers were available for constructing the genetic linkage map. A 1,678.52-cM high-density map at an average interval of 0.40 cM with 4,217 markers, including 427 frameset simple sequence repeats (SSRs) and 3,800 novel SNPs, mapped into 15 linkage groups was successfully constructed. After QTL analysis, a total of 27 QTLs related to flavonoids or caffeine content (CAF) were mapped to 8 different linkage groups, LG01, LG03, LG06, LG08, LG10, LG11, LG12, and LG13, with an LOD from 3.14 to 39.54, constituting 7.5% to 42.8% of the phenotypic variation.

Conclusions

To our knowledge, the highest density genetic map ever reported was constructed since the largest mapping population of tea plants was adopted in present study. Moreover, novel QTLs related to flavonoids and CAF were identified based on the new high-density genetic map. In addition, two markers were located in candidate genes that may be involved in flavonoid metabolism. The present study provides valuable information for gene discovery, marker-assisted selection breeding and map-based cloning for functional genes that are related to flavonoid content in tea plants.

     

Effect of population size and unbalanced data sets on QTL detection using genome-wide association mapping in barley breeding germplasm

Over the past two decades many quantitative trait loci (QTL) have been detected; however, very few have been incorporated into breeding programs. The recent development of genome-wide association studies (GWAS) in plants provides the opportunity to detect QTL in germplasm collections such as unstructured populations from breeding programs. The overall goal of the barley Coordinated Agricultural Project was to conduct GWAS with the intent to couple QTL detection and breeding. The basic idea is that breeding programs generate a vast amount of phenotypic data and combined with cheap genotyping it should be possible to use GWAS to detect QTL that would be immediately accessible and used by breeding programs. There are several constraints to using breeding program-derived phenotype data for conducting GWAS namely: limited population size and unbalanced data sets. We chose the highly heritable trait heading date to study these two variables. We examined 766 spring barley breeding lines (panel #1) grown in balanced trials and a subset of 384 spring barley breeding lines (panel #2) grown in balanced and unbalanced trials. In panel #1, we detected three major QTL for heading date that have been detected in previous bi-parental mapping studies. Simulation studies showed that population sizes greater than 384 individuals are required to consistently detect QTL. We also showed that unbalanced data sets from panel #2 can be used to detect the three major QTL. However, unbalanced data sets resulted in an increase in the false-positive rate. Interestingly, one-step analysis performed better than two-step analysis in reducing the false-positive rate. The results of this work show that it is possible to use phenotypic data from breeding programs to detect QTL, but that careful consideration of population size and experimental design are required.     

Genome-wide association analysis to identify SNP markers affecting teat numbers in an F2 intercross population between Landrace and Korean native pigs

Most reproductive traits have low heritability and are greatly affected by environmental factors. Teat number and litter size are traits related to the reproduction ability of pigs. To identify quantitative trait loci (QTLs) for teat number traits, a genome-wide association study (GWAS) was conducted using an F₂ intercross between Landrace and Korean native pigs. Genotype analysis was performed using the porcine SNP 60 K beadchip. The GWAS was performed using a mixed-effects model and linear regression approach. When a genome-wide threshold was determined using the Bonferroni method (P = 1.61 × 10^?6), 38 single nucleotide polymorphism (SNP) markers in pig chromosome 7 (SSC7) were significantly associated with three teat number traits (total teat number, left teat number, and right teat number). Among these, SNPs in 5 genes (HDDC3, LOC100156276, LOC100155863, ANPEP, SCAMP2) were selected for further study based primarily on their statistical significance. A significant association was detected in SCAMP2 g.25280 G>A for total teat number (P = 2.0 × 10^?12), HDDC3 g.1319 G>A SNP for left teat number (P = 2.3 × 10^?7), and SCAMP2 g.14198 G>A for right teat number (P = 4.7 × 10^?12). These results provide valuable information about the selective breeding for desirable teat numbers in pigs.     

Identification of QTLs for eight agronomically important traits using an ultra-high-density map based on SNPs generated from high-throughput sequencing in sorghum under contrasting photoperiods

The productivity of sorghum is mainly determined by agronomically important traits. The genetic bases of these traits have historically been dissected and analysed through quantitative trait locus (QTL) mapping based on linkage maps with low-throughput molecular markers, which is one of the factors that hinder precise and complete information about the numbers and locations of the genes or QTLs controlling the traits. In this study, an ultra-high-density linkage map based on high-quality single nucleotide polymorphisms (SNPs) generated from low-coverage sequences (~0.07 genome sequence) in a sorghum recombinant inbred line (RIL) population was constructed through new sequencing technology. This map consisted of 3418 bin markers and spanned 1591.4 cM of genome size with an average distance of 0.5 cM between adjacent bins. QTL analysis was performed and a total of 57 major QTLs were detected for eight agronomically important traits under two contrasting photoperiods. The phenotypic variation explained by individual QTLs varied from 3.40% to 33.82%. The high accuracy and quality of this map was evidenced by the finding that genes underlying two cloned QTLs, Dw3 for plant height (chromosome 7) and Ma1 for flowering time (chromosome 6), were localized to the correct genomic regions. The close associations between two genomic regions on chromosomes 6 and 7 with multiple traits suggested the existence of pleiotropy or tight linkage. Several major QTLs for heading date, plant height, numbers of nodes, stem diameter, panicle neck length, and flag leaf width were detected consistently under both photoperiods, providing useful information for understanding the genetic mechanisms of the agronomically important traits responsible for the change of photoperiod.     

A genetic linkage map of Picea abies Karst., based on RAPD markers,as a tool in population genetics

Norway spruce (Picea abies Karst.) is a most important species among European forest trees for both economical and ecological reasons. However, this species has suffered from a lack of information on the genetic side due to the scarcity of linkage data. In this study we have used a population of 72 megagametophytes from a single tree in a natural Italian stand to produce a genetic linkage map by means of RAPD markers. Ninety-six random decamers used as primers yielded 185 polymorphic loci showing Mendelian inheritance. Analysis of the segregation by multipoint analysis allowed us to define 17 major linkage groups covering a total distance of 3584 cM, with an average spacing between markers of 22 cM. Possible uses of a genetic linkage map with respect to population ecology and genetics are discussed.