Construction Of High-density Linkage Map Research Articles

A high-density linkage map construction in guava (Psidium guajava L.) using genotyping by sequencing and identification of QTLs for leaf, peel, and pulp color in an intervarietal mapping population.

Psidium guajava L. is an important fruit crop in the tropical and subtropical regions of the world. The advanced breeding methods are not employed for important commercial traits like peel and pulp color, seed hardiness, fruit size, etc., due to the scarcity of genome-wide molecular markers and high-density linkage maps. In this study, we employed single-nucleotide polymorphism (SNP) markers and identified quantitative trait loci (QTL) regions that are associated with color traits of leaf, peel, and pulp in the guava intervarietal mapping population. The mapping population was developed from the contrasting genotypes of fruit and leaf color. Variations in color among the segregating hybrids were recorded both visually and using a Color reader. A high-density linkage map of guava was constructed using the SNP markers from genotyping by sequencing (GBS) of 150 hybrid individuals of the cross 'Arka Poorna' (green) x 'Purple Local' (purple). The integrated linkage map consisted of 1426 SNPs mapped on 11 linkage groups (LG), spanning a total distance of around 730 cM with an average of 129.6 markers per LG. Through QTL analysis for color traits, a minor QTL region was identified for visually scored leaf color and peel color on LG1, whereas a major QTL was detected for pulp color in LG4. The Hunter color values (L* and, a*) also had major QTLs with overlapping marker intervals for leaf and peel colors, establishing the association of SNP markers to the trait. The QTLs harbored genes and transcription factors involved in lycopene and anthocyanin pigment biosynthesis. This is the first report of a high-density linkage map based on SNP markers in guava and QTL mapping for color characters in leaf, fruit peel and pulp. The genotyping information generated in this study can aid in genetic engineering and marker-assisted breeding in guava.

SNP-based high-density linkage map construction and QTL mapping of black spot disease resistance in Chinese sand pear.

Black spot disease (PBS) caused by Alternaria alternata is an economic disease of pear (Pyrus pyrifolia Nakai). Developing cultivars with durable PBS resistance traits is an important research objective for improving pear germplasm. The Deshengxiang is a popular pear variety in China and resistant to PBS. This study aimed to detect quantitative trait loci (QTL) associated with PBS resistance trait in pear and determine closely linked molecular markers by specific locus amplified fragment sequencing (SLAF-seq). F1 population resulting from a cross between "Deshengxiang" (female) and "Guiguan," a susceptible (male) variety, was developed and evaluated in 2016 and 2017. SLAF technology was used to discover SNPs in the F1 individuals and subsequently a high-density genetic linkage map for PBS resistance was constructed which contained 17,604 SNP markers. Based on the linkage map, the markers were distributed into 17 linkage groups, spanning 1548.48cM, with a mean marker distance of 0.09cM, representing the densest genetic map of the genus Pyrus. QTL analysis of PBS resistance identified a locus strongly related to PBS resistance at 77.68 ~ 112.99cM on linkage group 15, which was further narrowed down to 93.79 ~ 112.99cM. Two markers, Marker94293 and Marker94206, located at 97.47 and 102.93cM, were closely associated with PBS resistance, with a Δ (SNP index) value of 0.46. Co-localization of QTL interval, bioinformatics analysis, and functional annotation revealed PBS putative candidate genes. Overall, the high-density pear linkage map is a suitable reference for mapping PBS resistance trait, QTL, and genes identified in this study contribute information that could be useful for PBS improvement in pear.

High-density linkage map construction in an autotetraploid blueberry population and detection of quantitative trait loci for anthocyanin content.

Highbush blueberry (Vaccinium corymbosum, 2n = 4x = 48) is the most cultivated type of blueberry, both in New Zealand and overseas. Its perceived nutritional value is conferred by phytonutrients, particularly anthocyanins. Identifying the genetic mechanisms that control the biosynthesis of these metabolites would enable faster development of cultivars with improved fruit qualities. Here, we used recently released tools for genetic mapping in autotetraploids to build a high-density linkage map in highbush blueberry and to detect quantitative trait loci (QTLs) for fruit anthocyanin content. Genotyping was performed by target sequencing, with ∼18,000 single nucleotide polymorphism (SNP) markers being mapped into 12 phased linkage groups (LGs). Fruits were harvested when ripe for two seasons and analyzed with high-performance liquid chromatography-mass spectrometry (HPLC-MS): 25 different anthocyanin compounds were identified and quantified. Two major QTLs that were stable across years were discovered, one on LG2 and one on LG4, and the underlying candidate genes were identified. Interestingly, the presence of anthocyanins containing acylated sugars appeared to be under strong genetic control. Information gained in this study will enable the design of molecular markers for marker-assisted selection and will help build a better understanding of the genetic control of anthocyanin biosynthesis in this crop.

High-density genetic mapping identified a major locus for environmental sex expression in pumpkin (Cucurbita moschata Duch.)

Long-day length and high temperature inhibit sex expression in pumpkin (Cucurbita moschata Duch.), and therefore directly impact the production potential. In this study, female flowering patterns in photoperiod-insensitive (PPIS) and photoperiod-sensitive (PPS) germplasms differed significantly in a moderately long day and high temperature environment. However, both germplasms exhibited a similar response in short day with either low temperature or high temperature environment. Photoperiod sensitivity led to this difference in sex expression between the germplasms. For the traits of 1st female flowering node (FFFN) and number of female flowers (NFF), high-density linkage map construction and quantitative trait locus (QTL) mapping were performed using SLAF-seq technology and 162 F2 individuals generated from PPIS and PPS. In total, 4 655 SLAFs were selected and mapped on 20 linkage groups (LGs). The total map length was 2 502.01 cM with an average interval distance of 0.75 cM. Major QTLs for both FFFN and NFF were detected on LG6 with intervals of 7.89 and 17.67 cM and PVE values of 30.5% and 22.9%, respectively. Further analyses of the major locus for FFFN revealed 73 protein-coding genes. Among them, 4 were related to sex expression, photoperiod flowering, and hormone response. An InDel (insertion and deletion) marker partially correlated with FFFN of the F2 population was also developed. Our study identified the QTL for the sex expression response to environmental factors using the high-density linkage map. The identified candidate genes and markers will provide useful information about the molecular interaction between the environment and sex expression and for marker-assisted selection of pumpkin environment-insensitive resources.

Construction of high-density linkage maps and QTL mapping for growth-related traits in F1 hybrid Yunlong grouper (Epinephelus moara♀ × E. lanceolatus♂)

Owing to the increasing market demand and emerging grouper aquaculture industry, close attention has been paid to genetic breeding for growth traits in grouper. The construction of linkage maps for quantitative trait loci (QTL) analyses is an essential step for marker-assisted selection (MAS) programs. In this study, we report the first high-density linkage map of Yunlong grouper (Epinephelus moara♀ × E. lanceolatus♂) constructed by sequencing 118 F1 progenies in a full-sib family using restriction site associated DNA (RAD) technology. For the female and male maps, 12,307 and 12,340 single nucleotide polymorphism (SNP) markers were anchored onto 24 linkage groups, spanning 1909.37 and 1933.61 cM, respectively, with an average marker interval of 0.16 cM. QTL mapping revealed 39 significant QTLs for three growth traits. Candidate genes participating in ubiquitin modification, cell adhesion, G-protein coupled receptors, cell proliferation, and calcium transport were identified from these QTLs, including itgb1, col1a1, pdgfc, bmpr1a, pcdhgc5, and cacna1b. The results of this study provide a foundation for genomic and genetic studies and further analyses of the heterosis of some growth traits, thereby facilitating MAS in Yunlong grouper.

Mapping of QTL for agronomic traits using high-density SNPs with an RIL population in maize.

Genome wide association studies (GWAS) have been widely used to identify QTLs underlying quantitative traits in humans and animals, and they have also become a popular method of mapping QTLs in many crops, including maize. Advances in high-throughput genotyping technologies enable construction of high-density linkage maps using SNP markers. High-density genetic mapping must precede to find molecular markers associated with a particular trait. The objectives of this study were to (1) construct a high-density linkage map using SNP markers and (2) detect the QTLs for grain yield and quality related traits of the Mo17/KW7 RIL population. In this study, two parental lines, Mo17 (normal maize inbred line) and KW7 (waxy inbred line) and 80 F7:8 lines in the Mo17/KW7 RIL population were genotyped using the MaizeSNP50 BeadChip, an Illumina BeadChip array of 56,110 maize SNPs. Marker integration and detection of QTLs was performed using the inclusive composite interval mapping (ICIM) method within the QTL IciMapping software. This study was genotyped using the Illumina MaizeSNP50 BeadChip for maize Mo17/KW7 recombinant inbred line (RIL) population. The 2904 SNP markers were distributed along all 10 maize chromosomes. The total length of the linkage map was 3553.7cm, with an average interval of 1.22cm between SNPs. A total of 18 QTLs controlling eight traits were detected in the Mo17/KW7 RIL population. Three QTLs for plant height (PH) were detected on chromosomes 4 and 8 and showed from 16.01% (qPH8) to 19.85% (qPH4a) of phenotypic variance. Five QTLs related to ear height (EH) were identified on chromosomes 3, 4, and 6 and accounted for 3.79% (qEH6) to 27.57% (qEH4b) of phenotypic variance. Five QTLs related to water content (WC) on chromosomes 1, 4, 8, and 9 accounted for 9.55% (qWC8b) to 23.30% (qWC4) of phenotypic variance. One QTL (qAC9) relating to amylose content (AC) on chromosome 9 showed 82.10% of phenotypic variance. The high-density linkage map and putative QTLs of the maize RIL population detected in this study can be effectively utilized in waxy and normal maize breeding programs to facilitate the selection process through marker-assisted selection (MAS) breeding programs.

Breeding Aspects of Selected Ornamental Bulbous Crops

This article provides an overview of the origin, genetic diversity and methods and trends in breeding of selected ornamental geophytes (Lilium, Tulipa, Narcissus and Hippeastrum). The role of interspecific hybridisation and polyploidisation in assortment development is reviewed. A great variety of cultivars with traits of interest have been generated over the last century by using classical breeding. Geophyte breeders have been interested in a diversity of traits, including resistance to diseases, flower colour and shape, long lasting flowering and a long vase life. Shortening the long breeding process of many geophytes by reducing the juvenile phase and using in vitro techniques are reviewed. Currently, the breeding process has been enhanced by using modern molecular cytogenetic techniques. Genomic in situ hybridisation is frequently used, among other techniques, for genome differentiation in interspecific hybrids, and for assessment of the extent of intergenomic recombination in backcross progenies. Furthermore, several molecular marker techniques are used for verification of hybrid status, identification of genetic diversity, confirmation of the genetic fidelity of in vitro propagated plants and construction of high-density linkage maps. Recently, a myriad of new plant breeding technologies, such as cisgenetics and genome editing technologies have been used to improve the traits of ornamental geophytes, an endeavour that is discussed here. Breeding trends, cultivar novelties as well a new cultivars registered by international authorities during the last five years are presented in detail.

High-density linkage map construction and identification of loci regulating fruit quality traits in blueberry

Fruit quality traits play a significant role in consumer preferences and consumption in blueberry (Vaccinium corymbosum L). The objectives of this study were to construct a high-density linkage map and to identify the underlying genetic basis of fruit quality traits in blueberry. A total of 287 F1 individuals derived from a cross between two southern highbush blueberry cultivars, ‘Reveille’ and ‘Arlen’, were phenotyped over three years (2016–2018) for fruit quality-related traits, including titratable acidity, pH, total soluble solids, and fruit weight. A high-density linkage map was constructed using 17k single nucleotide polymorphisms markers. The linkage map spanned a total of 1397 cM with an average inter-loci distance of 0.08 cM. The quantitative trait loci interval mapping based on the hidden Markov model identified 18 loci for fruit quality traits, including seven loci for fruit weight, three loci for titratable acidity, five loci for pH, and three loci for total soluble solids. Ten of these loci were detected in more than one year. These loci explained phenotypic variance ranging from 7 to 28% for titratable acidity and total soluble solid, and 8–13% for pH. However, the loci identified for fruit weight did not explain more than 10% of the phenotypic variance. We also reported the association between fruit quality traits and metabolites detected by Proton nuclear magnetic resonance analysis directly responsible for these fruit quality traits. Organic acids, citric acid, and quinic acid were significantly (P < 0.05) and positively correlated with titratable acidity. Sugar molecules showed a strong and positive correlation with total soluble solids. Overall, the study dissected the genetic basis of fruit quality traits and established an association between these fruit quality traits and metabolites.

High-Density Linkage Map Construction and QTL Identification in a Diploid Blueberry Mapping Population.

Genotyping by sequencing approaches have been widely applied in major crops and are now being used in horticultural crops like berries and fruit trees. As the original and largest producer of cultivated blueberry, the United States maintains the most diverse blueberry germplasm resources comprised of many species of different ploidy levels. We previously constructed an interspecific mapping population of diploid blueberry by crossing the parent F1#10 (Vaccinium darrowii Fla4B × diploid V. corymbosum W85–20) with the parent W85–23 (diploid V. corymbosum). Employing the Capture-Seq technology developed by RAPiD Genomics, with an emphasis on probes designed in predicted gene regions, 117 F1 progeny, the two parents, and two grandparents of this population were sequenced, yielding 131.7 Gbp clean sequenced reads. A total of 160,535 single nucleotide polymorphisms (SNPs), referenced to 4,522 blueberry genome sequence scaffolds, were identified and subjected to a parent-dependent sliding window approach to further genotype the population. Recombination breakpoints were determined and marker bins were deduced to construct a high density linkage map. Twelve blueberry linkage groups (LGs) consisting of 17,486 SNP markers were obtained, spanning a total genetic distance of 1,539.4 cM. Among 18 horticultural traits phenotyped in this population, quantitative trait loci (QTLs) that were significant over at least 2 years were identified for chilling requirement, cold hardiness, and fruit quality traits of color, scar size, and firmness. Interestingly, in 1 year, a QTL associated with timing of early bloom, full bloom, petal fall, and early green fruit was identified in the same region harboring the major QTL for chilling requirement. In summary, we report here the first high density bin map of a diploid blueberry mapping population and the identification of several horticulturally important QTLs.

Transcriptome based high-throughput SSRs and SNPs discovery in the medicinal plant Lagenaria siceraria

<i>Lagenaria siceraria</i> (Molina) Standley has unique biological characteristics with high nutritional and medicinal values. It is an important pharmaceutical plant with various biologically active ingredients. Genetic improvement and deeper genomic studies require a rich resource of molecular markers. The application of next-generation sequencing technology, especially for transcriptome profiling, has greatly facilitated high throughput single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) discovery. In this study, we sequenced the transcriptome of three major cultivars of <i>L. siceraria</i> and obtained 64.88 GB of clean data. The assembled high-quality reads were clustered into 89,347 unigenes, which were annotated by non-redundant protein database, Swiss-Port, Eukaryotic Ortholog Groups, Kyoto Encyclopedia of Genes and Genomes, and gene ontology databases. A total of 8,891 SSR and 35,873 SNP markers were predicted from unigenes by MISA and SAM tools, respectively. Characterization of the predicted markers in <i>L. siceraria</i> showed that the SSR and SNP densities were 60 and 243 markers per Mb of genome, respectively, and the estimated ratio of transition to transversion of SNP was 2.016. These markers will be very useful for genetic studies in <i>L. siceraria</i>, especially for the high-density linkage map construction and genome-wide association studies. Further genomic studies based on these results will facilitate the identification of novel genes or alleles of pharmaceutical importance.

Development and Evaluation of a High-Throughput Single-Nucleotide Polymorphism Array for Large Yellow Croaker (Larimichthys crocea).

High-density single-nucleotide polymorphism (SNP) genotyping array is an essential tool for genetic analyses of animals and plants. Large yellow croaker (Larimichthys crocea) is one of the most commercially important marine fish species in China. Although plenty of SNPs have been identified in large yellow croaker, no high-throughput genotyping array is available. In this study, a high-throughput SNP array named NingXin-I with 600K SNPs was developed and evaluated. A set of 82 large yellow croakers were collected from different locations of China and re-sequenced. A total of 9.34M SNPs were identified by mapping sequence reads to the large yellow croaker reference genome. About 1.98M candidate SNPs were selected for further analyses by using criteria such as SNP quality score and conversion performance in the final array. Finally, 579.5K SNPs evenly distributed across the large yellow croaker genome with an average spacing of 1.19 kb were proceeded to array production. The performance of NingXin-I array was evaluated in 96 large yellow croaker individuals from five populations, and 83.38% SNPs on the array were polymorphic sites. A further test of the NingXin-I array in five closely related species in Sciaenidae identified 26.68–56.23% polymorphic SNP rate across species. A phylogenetic tree inferred by using the genotype data generated by NingXin-I confirmed the phylogenetic distance of the species in Sciaenidae. The performance of NingXin-I in large yellow croaker and the other species in Sciaenidae suggested high accuracy and broad application. The NingXin-I array should be valuable for quantitative genetic studies, such as genome-wide association studies (GWASs), high-density linkage map construction, haplotype analysis, and genome-based selection.

Ordering of high-density markers by the k-Optimal algorithm for the traveling-salesman problem

Construction of accurate and high-density linkage maps is a key research area of genetics. We investigated the efficiency of genetic map construction (MAP) using modifications of the k-Optimal (k-Opt) algorithm for solving the traveling-salesman problem (TSP). For TSP, different initial routes resulted in different optimal solutions. The most optimal solution could be found only by use of as many initial routes as possible. But for MAP, a large number of initial routes resulted in one optimal order. k-Opt using open route length gave a slightly higher proportion of correct orders than the method of adding one virtual marker and using closed route length. Recombination frequency (REC) and logarithm of odds (LOD) score gave similar proportions of correct order, higher than that given by genetic distance. Both missing markers and genotyping error reduced ordering accuracy, but the best order was still achieved with high probability by comparison of the optimal orders from multiple initial routes. Computation time increased rapidly with marker number, and 2-Opt took much less time than 3-Opt. The 2-Opt algorithm was compared with ordering methods used in two other software packages. The best method was 2-Opt using open route length as the criterion to identify the optimal order and using REC or LOD as the measure of distance between markers. We describe a unified software interface for using k-Opt in high-density linkage map construction for a wide range of genetic populations.

RAD-seq-Based High-Density Linkage Map Construction and QTL Mapping of Biomass-Related Traits in Sorghum using the Japanese Landrace Takakibi NOG

Sorghum [Sorghum bicolor (L.) Moench] grown locally by Japanese farmers is generically termed Takakibi, although its genetic diversity compared with geographically distant varieties or even within Takakibi lines remains unclear. To explore the genomic diversity and genetic traits controlling biomass and other physiological traits in Takakibi, we focused on a landrace, NOG, in this study. Admixture analysis of 460 sorghum accessions revealed that NOG belonged to the subgroup that represented Asian sorghums, and it was only distantly related to American/African accessions including BTx623. In an attempt to dissect major traits related to biomass, we generated a recombinant inbred line (RIL) from a cross between BTx623 and NOG, and we constructed a high-density linkage map based on 3,710 single-nucleotide polymorphisms obtained by restriction-site-associated DNA sequencing of 213 RIL individuals. Consequently, 13 fine quantitative trait loci (QTLs) were detected on chromosomes 2, 3, 6, 7, 8 and 9, which included five QTLs for days to heading, three for plant height (PH) and total shoot fresh weight and two for Brix. Furthermore, we identified two dominant loci for PH as being identical to the previously reported dw1 and dw3. Together, these results corroborate the diversified genome of Japanese Takakibi, while the RIL population and high-density linkage map generated in this study will be useful for dissecting other important traits in sorghum.

High-density genetic linkage-map construction of hawthorn and QTL mapping for important fruit traits.

Few reports exist on QTL mapping of the important economic traits of hawthorn. We hybridized the cultivars ‘Shandongdamianqiu’ (female parent) and ‘Xinbinruanzi’ (male parent), and 130 F1 individuals and the two parents were used for RAD-seq, SNP development, and high-density linkage map construction. Three genetic maps were obtained, one for each of the parents and an integrated one. In these three maps, 17 linkage groups were constructed. The female and male parent maps contained 2657 and 4088 SNP markers, respectively, and had genetic distances of 2689.65 and 2558.41 cM, respectively, whereas the integrated map was 2470.02 cM, and contained 6,384 SNP markers. QTL mapping based on six agronomic traits, namely fruit transverse diameter, vertical diameter, single fruit weight, pericarp brittleness, pericarp puncture hardness, and average sarcocarp firmness were conducted, and 25 QTLs were detected in seven linkage groups. Explained phenotypic variation rate ranged from 17.7% to 35%. This genetic map contains the largest number of molecular markers ever obtained from hawthorn and will provide an important future reference for fine QTL mapping of economic traits and molecular assisted selection of hawthorn.

Effect of marker segregation distortion on high density linkage map construction and QTL mapping in Soybean (Glycine max L.)

Marker segregation distortion is a natural phenomenon. Severely distorted markers are usually excluded in the construction of linkage maps. We investigated the effect of marker segregation distortion on linkage map construction and quantitative trait locus (QTL) mapping. A total of 519 recombinant inbred lines of soybean from orthogonal and reciprocal crosses between LSZZH and NN493-1 were genotyped by specific length amplified fragment markers and seed linoleic acid content was measured in three environments. As a result, twenty linkage groups were constructed with 11,846 markers, including 1513 (12.77%) significantly distorted markers, on 20 chromosomes, and the map length was 2475.86 cM with an average marker-interval of 0.21 cM. The inclusion of distorted markers in the analysis was shown to not only improve the grouping of the markers from the same chromosomes, and the consistency of linkage maps with genome, but also increase genome coverage by markers. Combining genotypic data from both orthogonal and reciprocal crosses decreased the proportion of distorted markers and then improved the quality of linkage maps. Validation of the linkage maps was confirmed by the high collinearity between positions of markers in the soybean reference genome and in linkage maps and by the high consistency of 24 QTL regions in this study compared with the previously reported QTLs and lipid metabolism related genes. Additionally, linkage maps that include distorted markers could add more information to the outputs from QTL mapping. These results provide important information for linkage mapping, gene cloning and marker-assisted selection in soybean.

High-density linkage map construction and mapping QTL for yield and yield components in autotetraploid alfalfa using RAD-seq

BackgroundAlfalfa (Medicago sativa L.) is an important forage crop grown worldwide. Alfalfa is called the “queen of forage crops” due to its high forage yield and nutritional characteristics. The aim of this study was to undertake quantitative trait loci (QTL) mapping of yield and yield-related traits in an F1 population of two alfalfa varieties that differ in their yield and yield-related traits.ResultsWe constructed a high-density linkage map using single nucleotide polymorphism (SNP) markers generated by restriction-site associated DNA sequencing (RAD-seq). The linkage map contains 4346 SNP and 119 simple sequence repeat (SSR) markers, with 32 linkage groups for each parent. The average marker distances were 3.00 and 1.32 cM, with coverages of 3455 cM and 4381 cM for paternal and maternal linkage maps, respectively. Using these maps and phenotypic data, we identified a total of 21 QTL for yield and yield components, including five for yield, five for plant height, five for branch number, and six for shoot diameter. Among them, six QTL were co-located for more than one trait. Five QTL explained more than 10% of the phenotypic variation.ConclusionsWe used RAD-seq to construct a linkage map for alfalfa that greatly enhanced marker density compared to previous studies. This high-density linkage map of alfalfa is a useful reference for mapping yield-related traits. Identified yield-related loci could be used to validate their usefulness in developing markers for maker-assisted selection in breeding populations to improve yield potential in alfalfa.

Construction of the First SNP-Based Linkage Map Using Genotyping-by-Sequencing and Mapping of the Male-Sterility Gene in Leaf Chicory.

We report the first high-density linkage map construction through genotyping-by-sequencing (GBS) in leaf chicory (Cichorium intybus subsp. intybus var. foliosum, 2n = 2x = 18) and the SNP-based fine mapping of the linkage group region carrying a recessive gene responsible for male-sterility (ms1). An experimental BC1 population, segregating for the male sterility trait, was specifically generated and 198 progeny plants were preliminary screened through a multiplexed SSR genotyping analysis for the identification of microsatellite markers linked to the ms1 locus. Two backbone SSR markers belonging to linkage group 4 of the available Cichorium consensus map were found genetically associated to the ms1 gene at 5.8 and 12.1 cM apart. A GBS strategy was then used to produce a high-density SNP-based linkage map, containing 727 genomic loci organized into 9 linkage groups and spanning a total length of 1,413 cM. 13 SNPs proved to be tightly linked to the ms1 locus based on a subset of 44 progeny plants analyzed. The map position of these markers was further validated by sequence-specific PCR experiments using an additional set of 64 progeny plants, enabling to verify that four of them fully co-segregated with male-sterility. A mesosynteny analysis revealed that 10 genomic DNA sequences encompassing the 13 selected SNPs of chicory mapped in a peripheral region of chromosome 5 of lettuce (Lactuca sativa L.) spanning about 18 Mbp. Since a MYB103-like gene, encoding for a transcription factor involved in callose dissolution of tetrads and exine development of microspores, was found located in the same chromosomal region, this orthologous was chosen as candidate for male-sterility. The amplification and sequencing of its CDS using accessions with contrasting phenotypes/genotypes (i.e., 4 male sterile mutants, ms1ms1, and 4 male fertile inbreds, Ms1Ms1) enabled to detect an INDEL of 4 nucleotides in its second exon, responsible for an anticipated stop codon in the male sterile mutants. This polymorphism was subsequently validated through allele-specific PCR assays and found to fully co-segregate with male-sterility, using 64 progeny plants of the same mapping BC1 population. Overall, our molecular data could be practically exploited for genotyping plant materials and for marker-assisted breeding schemes in leaf chicory.

High density linkage map construction and QTL mapping for runner production in allo-octoploid strawberry Fragaria\u2009\xd7\u2009ananassa based on ddRAD-seq derived SNPs

Recent advances in high-throughput genome sequencing technologies are now making the genetic dissection of the complex genome of cultivated strawberry easier. We sequenced Maehyang (short-day cultivar) × Albion (day-neutral cultivar) crossing populations using double digest restriction-associated DNA (ddRAD) sequencing technique that yielded 978,968 reads, 80.2% of which were aligned to strawberry genome allowing the identification of 13,181 high quality single nucleotide polymorphisms (SNPs). Total 3051 SNPs showed Mendelian segregation in F1, of which 1268 were successfully mapped to 46 linkage groups (LG) spanning a total of 2581.57 cM with an average interval genetic distance of 2.22 cM. The LGs were assigned to the 28 chromosomes of Fragaria × ananassa as determined by positioning the sequence tags on F. vesca genome. In addition, seven QTLs namely, qRU-5D, qRU-3D1, qRU-1D2, qRU-4D, qRU-4C, qRU-5C and qRU-2D2 were identified for runner production with LOD value ranging from 3.5–7.24 that explained 22–38% of phenotypic variation. The key candidate genes having putative roles in meristem differentiation for runnering and flowering within these QTL regions were identified. These will enhance our understanding of the vegetative vs sexual reproductive behavior in strawberry and will aid in setting breeding targets for developing perpetual flowering and profuse runnering cultivar.

High-resolution genetic mapping of a novel brown planthopper resistance locus, Bph34 in Oryza sativa L. X Oryza nivara (Sharma & Shastry) derived interspecific F2 population.

A BPH-resistant locus designated as Bph34 identified in Oryza nivara acc. IRGC104646 on long arm of chromosome 4 using high-resolution mapping with 50K SNP chip. BPH resistance contributed by locus showed dominant inheritance in F2 and F3. The Bph34 locus is 91kb in size and contains 11 candidate genes. In addition to SNP markers, SSR markers, RM16994 and RM17007 co-segregated with the BPH resistance. These two SSR markers can facilitate marker-assisted transfer of the Bph34 locus into elite rice cultivars in all labs. Brown planthopper (BPH, Nilaparvata lugen Stål) is one of the most destructive insects of rice (Oryza sativa L.) causing significant yield losses annually. Exploiting host plant resistance to BPH and incorporating resistant genes in susceptible commercial cultivars is economical and environmentally friendly approach to manage this pest. Here, we report high-resolution mapping of a novel genetic locus for resistance to BPH, designated as Bph34 on long arm of rice chromosome 4. The locus was mapped using an interspecific F2 population derived from a cross between susceptible indica cultivar PR122 and BPH-resistant wild species, O. nivara acc. IRGC104646. Inheritance studies performed using F2 and F2:3 populations revealed the presence of single dominant gene. Construction of high-density linkage map using 50K SNP chip (OsSNPnks) followed by QTL mapping identified single major locus at 28.8 LOD score between SNP markers, AX-95952039 and AX-95921548. The major locus contributing resistance to BPH designated as Bph34 and explained 68.3% of total phenotypic variance. The Bph34 locus is 91Kb in size on Nipponbare reference genome-IRGSP-1.0 and contains 11 candidate genes. In addition to associated SNP markers, two SSR markers, RM16994 and RM17007, also co-segregated with the Bph34 which can be used efficiently for markers assisted transfer into elite rice cultivars across the labs.

An efficient approach for the development of genome-specific markers in allohexaploid wheat (Triticum aestivum L.) and its application in the construction of high-density linkage maps of the D genome.

In common wheat, the development of genotyping platforms has been hampered by the large size of the genome, its highly repetitive elements and its allohexaploid nature. However, recent advances in sequencing technology provide opportunities to resolve these difficulties. Using next-generation sequencing and gene-targeting sequence capture, 12,551 nucleotide polymorphisms were detected in the common wheat varieties ‘Hatsumochi’ and ‘Kitahonami’ and were assigned to chromosome arms using International Wheat Genome Sequencing Consortium survey sequences. Because the number of markers for D genome chromosomes in commercially available wheat single nucleotide polymorphism arrays is insufficient, we developed markers using a genome-specific amplicon sequencing strategy. Approximately 80% of the designed primers successfully amplified D genome-specific products, suggesting that by concentrating on a specific subgenome, we were able to design successful markers as efficiently as could be done in a diploid species. The newly developed markers were uniformly distributed across the D genome and greatly extended the total coverage. Polymorphisms were surveyed in six varieties, and 31,542 polymorphic sites and 5,986 potential marker sites were detected in the D genome. The marker development and genotyping strategies are cost effective, robust and flexible and may enhance multi-sample studies in the post-genomic era in wheat.