Genotyping-by-sequencing Approach Research Articles

Cutting-Edge of Genotyping by Sequencing (GBS) for Improving Drought and Salinity Stress Tolerance in Watermelon (Citrullus lanatus L.): A Review

AbstractGenotyping by sequencing (GBS) is a very helpful approach and one of the most useful techniques for examining and analysing the genetic variation of various lines and varieties. GBS technology was used to identify single nucleotide polymorphisms (SNPs) and assess genetic variation in several watermelon accessions. GBS application in watermelon breeding programs has recently become a popular technique among many breeders. Watermelon (Citrullus lanatus L.) is a warm-season crop that is widely cultivated for its delicious fruit. And it is one of the most economically significant crops in the world. However, watermelon cultivation is frequently hampered by abiotic stressors such as drought and salinity. Recently, there has been a growing body of research on the mechanisms that allow watermelon to tolerate these stresses and improve crop yield. Generally, cucurbits are beneficial to human health, they provide necessary minerals, fibre, and nutrient components. Therefore, this review demonstrates the cutting edge of using GBS technology to identify genetic design of several features in watermelon to improve abiotic stresses (drought and saline). The application of the GBS technique has provided a distinct advantage in watermelon breeding studies. Based on GBS approach, many new candidate genes in watermelon lines control a variety of traits including saline and drought tolerance, fruit rind color, disease tolerance, nutrient components, size, and fruit shape were discovered. Modern breeding techniques are being used to develop economically viable vegetable crops that will meet customer preferences and needs. Further research is needed to enhance watermelon production.

Analysis of Genetic Diversity and Development of A Core Collection in Walnut (Juglans regia L.) Germplasm Native to China via Genotyping-by-Sequencing

Popular knowledge of the population structure and genetic diversity of a plant species is essential for designing improvement strategies. The genotyping-by-sequencing (GBS) approach has been used to simplify complex genomes and has become a popular high-throughput molecular tool for selecting and breeding many crop plants, including those with large genomes. This study aimed to construct a core collection of walnut (Juglans regia) germplasm using the GBS approach. A diversity panel of 87 walnut initial genotypes, including 25 landraces, 12 cultivars, and 50 seedling populations, mostly native to the Gansu Province of China, was subjected to GBS. A total of 110,497 high-quality SNPs were identified and used for determining distinct clusters and an optimum number of subpopulations. Structure analysis divided the genotypes into three distinct groups, which coincided with their collection site and year, suggesting a certain degree of separation in the geographical origin and pedigree among the three groups. To maximize germplasm utilization, the genotypes were posteriorly grouped according to the subgroups obtained through GBS analysis. To minimize subsample redundancy, the core collection was designed using a set of 6540 SNPs distributed across all 16 chromosomes. Finally, a core collection comprising nine walnut genotypes (10% of the entire genotype set), including five cultivars, three seedling populations, and one landrace, was assembled. Genetic structure analysis indicated that the core collection has an uneven distribution in the landrace collection, which could be related to environmental conditions, and the genotypes of the landrace collection are similar. Overall, the results of this study and the establishment of the core collection will facilitate the improvement of walnut in future breeding programs.

Novel candidate loci for morpho-agronomic and seed quality traits detected by targeted genotyping-by-sequencing in common bean.

Phaseolus vulgaris L., known as common bean, is one of the most important grain legumes cultivated around the world for its immature pods and dry seeds, which are rich in protein and micronutrients. Common bean offers a cheap food and protein sources to ameliorate food shortage and malnutrition around the world. However, the genetic basis of most important traits in common bean remains unknown. This study aimed at identifying QTL and candidate gene models underlying twenty-six agronomically important traits in common bean. For this, we assembled and phenotyped a diversity panel of 200P. vulgaris genotypes in the greenhouse, comprising determinate bushy, determinate climbing and indeterminate climbing beans. The panel included dry beans and snap beans from different breeding programmes, elite lines and landraces from around the world with a major focus on accessions of African, European and South American origin. The panel was genotyped using a cost-conscious targeted genotyping-by-sequencing (GBS) platform to take advantage of highly polymorphic SNPs detected in previous studies and in diverse germplasm. The detected single nucleotide polymorphisms (SNPs) were applied in marker-trait analysis and revealed sixty-two quantitative trait loci (QTL) significantly associated with sixteen traits. Gene model identification via a similarity-based approach implicated major candidate gene models underlying the QTL associated with ten traits including, flowering, yield, seed quality, pod and seed characteristics. Our study revealed six QTL for pod shattering including three new QTL potentially useful for breeding. However, the panel was evaluated in a single greenhouse environment and the findings should be corroborated by evaluations across different field environments. Some of the detected QTL and a number of candidate gene models only elucidate the understanding of the genetic nature of these traits and provide the basis for further studies. Finally, the study showed the possibility of using a limited number of SNPs in performing marker-trait association in common bean by applying a highly scalable targeted GBS approach. This targeted GBS approach is a cost-efficient strategy for assessment of the genetic basis of complex traits and can enable geneticists and breeders to identify novel loci and targets for marker-assisted breeding more efficiently.

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.

Genotyping-by-Sequencing Technology in Plant Taxonomy and Phylogeny.

Genotyping-by-sequencing (GBS) is a method to discover and genotype simultaneous genome-wide high-throughput single nucleotide polymorphisms(SNPs). GBS is based on reducing genome complexity with restriction enzymes. Here we describe a method developed by Elshire et al. for constructing simplified GBS libraries and recent bioinformatic approaches developed to analyze the large volume of polymorphism data generated by this method. GBS approach is suitable for population studies, taxonomic and phylogenic studies, germplasm characterization, and breeding and trait mapping for a wide range of organisms, including plants with complex genomes.

Genome-wide analyses reveal footprints of divergent selection and popping-related traits in CIMMYT's maize inbred lines.

Popcorn (Zea mays L. var. Everta) is the most ancient type of cultivated maize. However, there is little known about the genetics of popping-related traits based on genotyping-by-sequencing (GBS) technology. Here, we characterized the phenotypic variation for seven popping-related traits in maize kernels among 526 CIMMYT inbred lines (CMLs). In total, 155 083 high-quality single nucleotide polymorphism (SNP) markers were identified by a GBS approach. Several trait-associated loci were detected by genome-wide association study for color, popping expansion volume, shape, pericarp, flotation index, floury/vitreous, and protein content, explaining a majority of the observed phenotypic variance, and these were validated by a diverse panel comprising 764 tropical landrace accessions. Sixty two of the identified loci were recognized to have undergone selection. On average, there was a 55.27% frequency for alleles that promote popping in CMLs. Our work not only pinpoints previously unknown loci for popping-related traits, but also reveals that many of these loci have undergone selection. Beyond establishing a new benchmark for the genetics of popcorn, our study provides a foundation for gene discovery and breeding. It also presents evidence to investigate the role of a gradual loss of popping ability as a by-product of diversification of culinary uses throughout the evolution of teosinte-to-modern maize.

A high-density SNP-based linkage map using genotyping-by-sequencing and its utilization for improved genome assembly of chickpea (Cicer arietinum L.).

Genotyping-by-sequencing (GBS) allows rapid identification of markers for use in development of linkage maps, which expedite efficient breeding programs. In the present study, we have utilized GBS approach to identify and genotype single-nucleotide polymorphism (SNP) markers in an inter-specific RIL population of Cicer arietinum L. X C. reticulatum. A total of 141,639 raw SNPs were identified using the TASSEL-GBS pipeline. After stringent filtering, 8208 candidate SNPs were identified of which ~ 37% were localized in the intragenic regions followed by genic regions (~ 30%) and intergenic regions (~ 27%). We then utilized 6920 stringent selected SNPs from present study and 6714 SNPs and microsatellite markers available from previous studies for construction of linkage map. The resulting high-density linkage map comprising of eight linkage groups contained 13,590 markers which spanned 1299.14 cM of map length with an average marker density of 0.095 cM. Further, the derived linkage map was used to improve the available assembly of desi chickpea genome by anchoring 443 previously unplaced scaffolds onto eight linkage groups. The present efforts have refined anchoring of the desi chickpea genome assembly to 55.57% of the ~ 520 Mb of assembled desi genome. To the best of our knowledge, the linkage map generated in the present study represents one of the most dense linkage map developed for the crop till date. It will serve as a valuable resource for fine mapping and positional cloning of important quantitative trait loci (QTLs) associated with agronomical traits and also for anchoring and ordering of future genome sequence assemblies.

Large scale SNP unearthing and genetic architecture analysis in sea-captured and cultured populations of Cynoglossus semilaevis

Knowledge on population structure and genetic diversity is a focal point for association mapping studies and genomic selection. Genotyping by sequencing (GBS) represents an innovative method for large scale SNP detection and genotyping of genetic resources. Here we used the GBS approach for the genome-wide identification of SNPs in a collection of Cynoglossus semilaevis and for the assessment of the level of genetic diversity in C. semilaevis genotypes. GBS analysis generated a total of 55.12 Gb high-quality sequence data, with an average of 0.63 Gb per sample. The total number of SNP markers was 563, 109. In order to explore the genetic diversity of C. semilaevis and to select a minimal core set representing most of the total genetic variation with minimum redundancy, C. semilaevis sequences were analyzed using high quality SNPs. Based on hierarchical clustering, it was possible to divide the collection into 2 clusters. The marine fishing populations were clustered and clearly separated from the cultured populations, and the cultured populations from Hebei was also distinct from the other two local populations. These analyses showed that genotypes were clustered based on species-related features. Differential significant SNPs were also captured and validated by GBS and SNaPshot, with linkage disequilibrium and haplotype analysis, seven SNPs have been confirmed to have obvious differentiation in two populations, which may be used as the characteristic evaluation sites of sea-captured and cultured Cynoglossus semilaevis populations. And SNP markers and information on population structure developed in this study will undoubtedly support genome-wide association mapping studies and marker-assisted selection programs. These differential SNPs could be also employed as the characteristic evaluation sites of sea-captured and cultured Cynoglossus semilaevis populations in future.

Linkage Disequilibrium, Effective Population Size and Genomic Inbreeding Rates in American Mink Using Genotyping-by-Sequencing Data.

Knowledge of linkage disequilibrium (LD) patterns is necessary to determine the minimum density of markers required for genomic studies and to infer historical changes as well as inbreeding events in the populations. In this study, we used genotyping-by-sequencing (GBS) approach to detect single nucleotide polymorphisms (SNPs) across American mink genome and further to estimate LD, effective population size (Ne), and inbreeding rates based on excess of homozygosity (FHOM) and runs of homozygosity (ROH). A GBS assay was constructed based on the sequencing of ApeKI-digested libraries from 285 American mink using Illumina HiSeq Sequencer. Data of 13,321 SNPs located on 46 scaffolds was used to perform LD analysis. The average LD (r2 ± SD) between adjacent SNPs was 0.30 ± 0.35 over all scaffolds with an average distance of 51 kb between markers. The average r2 < 0.2 was observed at inter-marker distances of >40 kb, suggesting that at least 60,000 informative SNPs would be required for genomic selection in American mink. The Ne was estimated to be 116 at five generations ago. In addition, the most rapid decline of population size was observed between 100 and 200 generations ago. Our results showed that short extensions of homozygous genotypes (500 kb to 1 Mb) were abundant across the genome and accounted for 33% of all ROH identified. The average inbreeding coefficient based on ROH longer than 1 Mb was 0.132 ± 0.042. The estimations of FHOM ranged from −0.44 to 0.34 among different samples with an average of 0.15 over all individuals. This study provided useful insights to determine the density of SNP panel providing enough statistical power and accuracy in genomic studies of American mink. Moreover, these results confirmed that GBS approach can be considered as a useful tool for genomic studies in American mink.

Darwin's Fancy Revised: An Updated Understanding of the Genomic Constitution of Pigeon Breeds.

Through its long history of artificial selection, the rock pigeon (Columba livia Gmelin 1789) was forged into a large number of domestic breeds. The incredible amount of phenotypic diversity exhibited in these breeds has long held the fascination of scholars, particularly those interested in biological inheritance and evolution. However, exploiting them as a model system is challenging, as unlike with many other domestic species, few reliable records exist about the origins of, and relationships between, each of the breeds. Therefore, in order to broaden our understanding of the complex evolutionary relationships among pigeon breeds, we generated genome-wide data by performing the genotyping-by-sequencing (GBS) method on close to 200 domestic individuals representing over 60 breeds. We analyzed these GBS data alongside previously published whole-genome sequencing data, and this combined analysis allowed us to conduct the most extensive phylogenetic analysis of the group, including two feral pigeons and one outgroup. We improve previous phylogenies, find considerable population structure across the different breeds, and identify unreported interbreed admixture events. Despite the reduced number of loci relative to whole-genome sequencing, we demonstrate that GBS data provide sufficient analytical power to investigate intertwined evolutionary relationships, such as those that are characteristic of animal domestic breeds. Thus, we argue that future studies should consider sequencing methods akin to the GBS approach as an optimal cost-effective approach for addressing complex phylogenies.

Genome-wide SNP discovery through genotyping by sequencing, population structure, and linkage disequilibrium in Brazilian peach breeding germplasm

Genotyping by sequencing (GBS) is a flexible and cost-effective strategy for genome-wide SNP discovery and high-throughput genotyping. Here, we employed the GBS approach to explore population structure, genetic variability, and patterns of linkage disequilibrium (LD) among 220 peach genotypes (Prunus persica), representative of the Brazilian breeding germplasm. This selected panel is mainly composed by locally adapted peaches developed in mild winter and high relative humidity conditions, and represents a worldwide reference of peach germplasm for low-chilling areas. A total of 93,353 SNP markers were discovered, and after filtering, 18,373 high-quality SNPs were used in analyses. Thirty-four percent of selected SNPs were located in genic regions and about 70% of these in the coding sequence. Principal coordinate analysis (PCoA) and Bayesian clustering (fastSTRUCTURE) successfully detected population genetic structure in our germplasm panel, supporting the identification of three main distinct subpopulations. The distribution of the genotypes within subpopulations reflected their fruit-related traits: melting and non-melting flesh cultivars. LD patterns revealed a medium LD level in peach, with the extent of LD highly dependent on the subpopulation and genome regions. The use of GBS-SNPs to perform genome-wide association studies (GWAS) was also exploited by using phenotypic information from five fruit quality traits. The observed significant SNP-trait associations were always in the regions previously predicted by linkage and association mapping analysis, providing valuable insights to incorporate the use of marker-assisted selection in the breeding program.

Resolving relationships in an exceedingly young Neotropical orchid lineage using Genotyping-by-sequencing data

Poor morphological and molecular differentiation in recently diversified lineages is a widespread phenomenon in plants. Phylogenetic relationships within such species complexes are often difficult to resolve because of the low variability in traditional molecular loci. Furthermore, biological phenomena responsible for topological incongruence such as Incomplete Lineage Sorting (ILS) and hybridisation complicate the resolution of phylogenetic relationships among closely related taxa. In this study, we employ a Genotyping-by-sequencing (GBS) approach to disentangle evolutionary relationships within a species complex belonging to the Neotropical orchid genus Cycnoches. This complex includes seven taxa distributed through Central America and the Colombian Chocó, and is nested within a clade estimated to have first diversified in the early Quaternary. Previous phylogenies inferred from few loci failed to provide support for internal relationships within the complex. Our Neighbour-net and coalescent-based analyses inferred from ca. 13,000 GBS loci obtained from 31 individuals belonging to six of the seven traditionally accepted Cycnoches taxa provided a robust phylogeny for this group. The genus Cycnoches includes three main clades that are further supported by morphological traits and geographic distributions. Similarly, a topology reconstructed through maximum likelihood (ML) inference of concatenated GBS loci produced results that are comparable with those reconstructed through coalescence and network-based methods. Our comparative phylogenetic informativeness analyses suggest that the low support evident in the ML phylogeny might be attributed to the abundance of uninformative GBS loci, which can account for up to 50% of the total number of loci recovered. The phylogenomic framework provided here, as well as morphological evidence and geographical patterns, suggest that the six entities previously thought to be different species or subspecies might actually represent only three distinct segregates. We further discuss the limited phylogenetic informativeness found in our GBS approach and its utility to disentangle relationships within recent and rapidly evolving species complexes. Our study is the first to demonstrate the utility of GBS data to reconstruct relationships within young (~2 Ma) Neotropical plant clades, opening new avenues for studies of species complexes that populate the species-rich orchid family.

Screening populations for copy number variation using genotyping-by-sequencing: a proof of concept using soybean fast neutron mutants

BackgroundThe effective use of mutant populations for reverse genetic screens relies on the population-wide characterization of the induced mutations. Genome- and population-wide characterization of the mutations found in fast neutron populations has been hindered, however, by the wide range of mutations generated and the lack of affordable technologies to detect DNA sequence changes. In this study, we therefore aimed to test whether genotyping-by-sequencing (GBS) technology could be used to characterize copy number variation (CNV) induced by fast neutrons in a soybean mutant population.ResultsWe called CNVs from GBS data in 79 soybean mutants and assessed the sensitivity and precision of this approach by validating our results against array comparative genomic hybridization (aCGH) data for 19 of these mutants as well as targeted PCR and ddPCR assays for a representative subset of the smallest events detected by GBS. Our GBS pipeline detected 55 of the 96 events found by aCGH, with approximate detection thresholds of 60 kb, 500 kb and 1 Mb for homozygous deletions, hemizygous deletions and duplications, respectively. Among the whole set of 79 mutants, the GBS data revealed 105 homozygous deletions, 32 hemizygous deletions and 19 duplications. This included several extremely large events, exhibiting maximum sizes of ~ 11.2 Mb for a homozygous deletion, ~ 11.6 Mb for a hemizygous deletion, and ~ 50 Mb for a duplication.ConclusionsThis study provides a proof of concept that GBS can be used as an affordable high-throughput method for assessing CNVs in fast neutron mutants. The modularity of this GBS approach allows combining as many different libraries or sequencing runs as is necessary for reaching the goals of a particular study. This method should enable the low-cost genome-wide characterization of hundreds to thousands of individuals in fast neutron mutant populations or any population with large genomic deletions and duplications.

Genetic diversity of kiwifruit (Actinidia spp.), including Korean native A. arguta, using single nucleotide polymorphisms derived from genotyping-by-sequencing

Genotyping-by-sequencing (GBS) was used to investigate the genetic diversity and population structure of kiwifruits (Actinidia spp.). Using single nucleotide polymorphisms detected by GBS, phylogenetic tree and population structure were constructed for 89 kiwifruit accessions including Korean native A. arguta. The kiwifruit accessions were clearly divided into two groups in the phylogenetic tree. These groups were characterized by the presence or absence of hairs on pericarp. In the population structure analysis, the peak of delta K was detected at K = 5, suggesting that the 89 kiwifruit accessions were divided into five clusters. Each cluster represented A. chinensis, A. deliciosa, A. eriantha with wild accessions, female A. arguta, and male A. arguta. The result of the population structure supported the genetic background of each accession. We also performed genetic diversity analysis of A. arguta accessions. Consequently, A. arguta accessions were characterized by sex and 13 A. arguta accessions, occupying 33.3% of the total collection, were selected as a core set for use as germplasm to develop disease and cold stress resistant cultivars in future kiwifruit breeding programs. These results suggest that GBS approach is suitable for genetic diversity analysis of kiwifruits. Moreover, our results could be applied in kiwifruit breeding program to develop disease and cold stress resistant cultivars using Korean native A. arguta. Particularly, the developed Korean native A. arguta core set will be used as breeding materials for crop improvement strategies.

Ancestry informative alleles captured with reduced representation library sequencing in Theobroma cacao.

As the source of chocolate, cacao has become one of the most important crops in the world. The identification of molecular markers to understand the demographic history, genetic diversity and population structure plays a pivotal role in cacao breeding programs. Here, we report the use of a modified genotyping-by-sequencing (GBS) approach for large-scale single nucleotide polymorphism (SNP) discovery and allele ancestry mapping. We identified 12,357 bi-allelic SNPs after filtering, of which, 7,009 variants were ancestry informative. The GBS approach proved to be rapid, cost-effective, and highly informative for ancestry assignment in this species.

Genotyping-by-sequencing performance in selected livestock species

Application of next generation sequencing for large scale genotyping in livestock is limited by high costs and challenging data analysis process. However, available restriction enzyme-based enrichment techniques like e.g. genotyping-by-sequencing (GBS) are promising tools allowing reduction of financial outlies by a high sample multiplexing and narrowing down the sequenced genome areas to the randomly distributed read tags. In this study, we tested the performance of standard, PstI endonuclease-adapted GBS protocol for population genetics in cattle, horse and sheep with application of different, including low-depth sequencing setups. It was found that the detected SNPs display desirable polymorphism parameters and are evenly scattered across the whole genome including gene coding regions. It was also shown that the SNPs can be successfully applied in population genetics, revealing the genetic differentiation of the studied breeds. The GBS approach represents a cost-effective alternative to existing genotyping methods which may find adoption in various research applications.

The Terpene Synthase Gene Family of Carrot (Daucus carota L.): Identification of QTLs and Candidate Genes Associated with Terpenoid Volatile Compounds.

Terpenes are an important group of secondary metabolites in carrots influencing taste and flavor, and some of them might also play a role as bioactive substances with an impact on human physiology and health. Understanding the genetic and molecular basis of terpene synthases (TPS) involved in the biosynthesis of volatile terpenoids will provide insights for improving breeding strategies aimed at quality traits and for developing specific carrot chemotypes possibly useful for pharmaceutical applications. Hence, a combination of terpene metabolite profiling, genotyping-by-sequencing (GBS), and genome-wide association study (GWAS) was used in this work to get insights into the genetic control of terpene biosynthesis in carrots and to identify several TPS candidate genes that might be involved in the production of specific monoterpenes. In a panel of 85 carrot cultivars and accessions, metabolite profiling was used to identify 31 terpenoid volatile organic compounds (VOCs) in carrot leaves and roots, and a GBS approach was used to provide dense genome-wide marker coverage (>168,000 SNPs). Based on this data, a total of 30 quantitative trait loci (QTLs) was identified for 15 terpenoid volatiles. Most QTLs were detected for the monoterpene compounds ocimene, sabinene, β-pinene, borneol and bornyl acetate. We identified four genomic regions on three different carrot chromosomes by GWAS which are both associated with high significance (LOD ≥ 5.91) to distinct monoterpenes and to TPS candidate genes, which have been identified by homology-based gene prediction utilizing RNA-seq data. In total, 65 TPS candidate gene models in carrot were identified and assigned to known plant TPS subfamilies with the exception of TPS-d and TPS-h. TPS-b was identified as largest subfamily with 32 TPS candidate genes.

Genotyping by sequencing reveals the interspecific C. maxima / C. reticulata admixture along the genomes of modern citrus varieties of mandarins, tangors, tangelos, orangelos and grapefruits.

The mandarin horticultural group is an important component of world citrus production for the fresh fruit market. This group formerly classified as C. reticulata is highly polymorphic and recent molecular studies have suggested that numerous cultivated mandarins were introgressed by C. maxima (the pummelos). C. maxima and C. reticulata are also the ancestors of sweet and sour oranges, grapefruit, and therefore of all the “small citrus” modern varieties (mandarins, tangors, tangelos) derived from sexual hybridization between these horticultural groups. Recently, NGS technologies have greatly modified how plant evolution and genomic structure are analyzed, moving from phylogenetics to phylogenomics. The objective of this work was to develop a workflow for phylogenomic inference from Genotyping By Sequencing (GBS) data and to analyze the interspecific admixture along the nine citrus chromosomes for horticultural groups and recent varieties resulting from the combination of the C. reticulata and C. maxima gene pools. A GBS library was established from 55 citrus varieties, using the ApekI restriction enzyme and selective PCR to improve the read depth. Diagnostic polymorphisms (DPs) of C. reticulata/C. maxima differentiation were identified and used to decipher the phylogenomic structure of the 55 varieties. The GBS approach was powerful and revealed 30,289 SNPs and 8,794 Indels with 12.6% of missing data. 11,133 DPs were selected covering the nine chromosomes with a higher density in genic regions. GBS combined with the detection of DPs was powerful for deciphering the “phylogenomic karyotypes” of cultivars derived from admixture of the two ancestral species after a limited number of interspecific recombinations. All the mandarins, mandarin hybrids, tangelos and tangors analyzed displayed introgression of C. maxima in different parts of the genome. C. reticulata/C. maxima admixture should be a major component of the high phenotypic variability of this germplasm opening up the way for association studies based on phylogenomics.

Approaches in Characterizing Genetic Structure and Mapping in a Rice Multiparental Population.

Multi-parent Advanced Generation Intercross (MAGIC) populations are fast becoming mainstream tools for research and breeding, along with the technology and tools for analysis. This paper demonstrates the analysis of a rice MAGIC population from data filtering to imputation and processing of genetic data to characterizing genomic structure, and finally quantitative trait loci (QTL) mapping. In this study, 1316 S6:8 indica MAGIC (MI) lines and the eight founders were sequenced using Genotyping by Sequencing (GBS). As the GBS approach often includes missing data, the first step was to impute the missing SNPs. The observable number of recombinations in the population was then explored. Based on this case study, a general outline of procedures for a MAGIC analysis workflow is provided, as well as for QTL mapping of agronomic traits and biotic and abiotic stress, using the results from both association and interval mapping approaches. QTL for agronomic traits (yield, flowering time, and plant height), physical (grain length and grain width) and cooking properties (amylose content) of the rice grain, abiotic stress (submergence tolerance), and biotic stress (brown spot disease) were mapped. Through presenting this extensive analysis in the MI population in rice, we highlight important considerations when choosing analytical approaches. The methods and results reported in this paper will provide a guide to future genetic analysis methods applied to multi-parent populations.

Genome-Wide Single Nucleotide Polymorphism Discovery and the Construction of a High-Density Genetic Map for Melon (Cucumis melo L.) Using Genotyping-by-Sequencing.

Although genotyping-by-sequencing (GBS) enables the efficient and low-cost generation of large numbers of markers, the utility of resultant genotypes are limited, because they are enormously error-prone and contain high proportions of missing data. In this study, we generated single nucleotide polymorphism (SNP) markers for 109 recombinant inbred lines of melon (Cucumis melo L.) using the GBS approach and ordered them according to their physical position on the draft double haploid line DHL92 genome. Next, by investigating associations between these SNPs, we discovered that some segments on the physical map conflict with linkage relationships. Therefore, to filter out error-prone loci, 4,110 SNPs in which we have a high degree of confidence were selected as anchors to test independence with respect to unselected markers, and the resultant dataset was then analyzed using the Full-Sib Family Haplotype (FSFHap) algorithm in the software TASSEL 5.2. On the basis of this analysis, 22,933 loci that have an average rate of missing data of 0.281% were used to construct a genetic map, which spans 1,088.3 cM across 12 chromosomes and has a maximum spacing of 6.0 cM. Use of this high-quality linkage map enabled the identification of several quantitative trait loci (QTL) known to control traits in fruit and validated our approach. This study highlights the utility of GBS markers for the identification of trait-associated QTLs in melon and facilitates further investigation of genome structure.