Genotyping-by-sequencing Research Articles

Genomic insights into the NPGS intermediate wheatgrass germplasm collection

AbstractThe National Plant Germplasm System (NPGS) is a vital resource for genetic diversity, yet utilization of this resource requires a thorough understanding of the germplasm and genetic diversity. Intermediate wheatgrass (IWG, Thinopyrum intermedium) is a perennial grass species that has been improved for forage production through breeding utilizing the NPGS collection and has also been targeted for domestication as a perennial grain crop. To better characterize the IWG collection, we combined previously published forage data with new agronomic and genomic data. A total of 331 NPGS accessions were genomically profiled with genotyping‐by‐sequencing (GBS) and a genome‐wide association study (GWAS) was used to evaluate trait architecture. Along with the GWAS, in silico bulk samples were profiled by recoding GBS data to conduct association mapping through allele counting with extreme‐phenotype (XP)‐GWAS. Genomic analysis revealed two subpopulations, which were defined as European and Asian groups, and are differentiated around the Black Sea region. Phenotypic observations for forage and agronomic traits differed between the two groups (p < 0.05), even though greater than 70% of the genetic variance was partitioned within individual accessions. Finally, XP‐GWAS revealed 303 marker–trait associations for five agronomic and four forage traits. These results suggest that genetic diversity within the NPGS collection should lead to genetic gains for both forage and grain breeding as well as opportunities for breeding programs to enhance genetic diversity. More broadly, the methods we applied could be applicable to low‐resourced species, leveraging existing, and new data, to strengthen genetic characterization and breeding efficiency.

Identification of Causal Gene-Specific SNP Markers for the Development of Gynoecious Hybrids in Cucumber (Cucumis sativa L.) Using the PathoLogic Algorithm

Although the genome sequence of cucumber is publicly available, only a limited number of functional markers are in store for developing gynoecious hybrids using Indian genotypes. This study reported novel SNPs and InDels in the exonic regions of genes involved in gynoecy using two parents and their hybrid with genotyping-by-sequencing (GBS) by generating 3.547 Gb of raw data. Using NSDC reference genome GCA_000004075.2, a total of 40,143, 181,008 and 43,612 SNPs were identified, among which 514 were polymorphic between male and female parents but monomorphic between the male parent and the hybrid (confirming hybridity). We further identified that, out of those 514 SNPs, 74 were within the exonic regions of the sex-specific genes. The most interesting functional marker in this study was SNP 2,13,85,488, identified in the gene CsaV3_6G037780 G3I-38214 on chromosome 6, encoding 1-aminocyclopropane-1-carboxylate oxidase 1 (ACS1), which plays a key role in female flower production, as indicated in CuCyc with the PathoLogic algorithm. The InDel analysis also identified a variation inside the gene CsaV3_6G304050 G3I-37940, encoding histone lysine N-methyl transferase, involved in flowering and female gametophyte development. Thus, this study has identified gynoecy-specific functional markers; upon further validation, these markers will accelerate the evolution of gynoecious hybrids in India and global cucumber breeding programs.

Assessment of the Genetic Diversity and Population Structure of the Peruvian Andean Legume, Tarwi (Lupinus mutabilis), with High Quality SNPs

Lupinus mutabilis Sweet (Fabaceae), “tarwi” or “chocho”, is an important grain legume in the Andean region. In Peru, studies on tarwi have mainly focused on morphological features; however, they have not been molecularly characterized. Currently, it is possible to explore the genetic parameters of plants with reliable and modern methods such as genotyping by sequencing (GBS). Here, for the first time, we used single nucleotide polymorphism (SNP) markers to infer the genetic diversity and population structure of 89 accessions of tarwi from nine Andean regions of Peru. A total of 5922 SNPs distributed along all chromosomes of tarwi were identified. STRUCTURE analysis revealed that this crop is grouped into two clusters. A dendrogram was generated using the UPGMA clustering algorithm and, like the principal coordinate analysis (PCoA), it showed two groups that correspond to the geographic origin of the tarwi samples. AMOVA showed a reduced variation between clusters (7.59%) and indicated that variability within populations is 92.41%. Population divergence (Fst) between clusters 1 and 2 revealed low genetic difference (0.019). We also detected a negative Fis for both populations, demonstrating that, like other Lupinus species, tarwi also depends on cross-pollination. SNP markers were powerful and effective for the genotyping process in this germplasm. We hope that this information is the beginning of the path towards a modern genetic improvement and conservation strategies of this important Andean legume.

A genetic linkage map and improved genome assembly of the termite symbiont Termitomyces cryptogamus

BackgroundThe termite-fungus symbiosis is an ancient stable mutualism of two partners that reproduce and disperse independently. With the founding of each termite colony the symbiotic association must be re-established with a new fungus partner. Complementarity in the ability to break down plant substrate may help to stabilize this symbiosis despite horizontal symbiont transmission. An alternative, non-exclusive, hypothesis is that a reduced rate of evolution may contribute to stabilize the symbiosis, the so-called Red King Effect.MethodsTo explore this concept, we produced the first linkage map of a species of Termitomyces, using genotyping by sequencing (GBS) of 88 homokaryotic offspring. We constructed a highly contiguous genome assembly using PacBio data and a de-novo evidence-based annotation. This improved genome assembly and linkage map allowed for examination of the recombination landscape and its potential effect on the mutualistic lifestyle.ResultsOur linkage map resulted in a genome-wide recombination rate of 22 cM/Mb, lower than that of other related fungi. However, the total map length of 1370 cM was similar to that of other related fungi.ConclusionsThe apparently decreased rate of recombination is primarily due to genome expansion of islands of gene-poor repetitive sequences. This study highlights the importance of inclusion of genomic context in cross-species comparisons of recombination rate.

Novel QTL for chilling tolerance at germination and early seedling stages in sorghum.

Sorghum (Sorghum bicolor L.) a drought tolerant staple crop for half a billion people in Africa and Asia, an important source of animal feed throughout the world and a biofuel feedstock of growing importanceorghum's originated from tropical regions rendering the crop to be cold sensitive. Low temperature stresses such as chilling and frost greatly affect the agronomic performance of sorghum and limit its geographical distribution, posing a major problem in temperate environments when sorghum is planted early. Understanding the genetic basis of wide adaptability and of sorghum would facilitate molecular breeding programs and studies of other C4 crops. The objective of this study is to conduct quantitative trait loci analysis using genotying by sequencing for early seed germination and seedling cold tolerance in two sorghum recombinant inbred lines populations. To accomplish that, we used two populations of recombinant inbred lines (RIL) developed from crosses between cold-tolerant (CT19, ICSV700) and cold-sensitive (TX430, M81E) parents. The derived RIL populations were evaluated for single nucleotide polymorphism (SNP) using genotype-by-sequencing (GBS) in the field and under controlled environments for their response to chilling stress. Linkage maps were constructed with 464 and 875 SNPs for the CT19 X TX430 (C1) and ICSV700 X M81 E (C2) populations respectively. Using quantitative trait loci (QTL) mapping, we identified QTL conferring tolerance to chilling tolerance at the seedling stage. A total of 16 and 39 total QTL were identified in the C1 and C2 populations, respectively. Two major QTL were identified in the C1 population, and three major QTL were mapped in the C2 population. Comparisons between the two populations and with previously identified QTL show a high degree of similarity in QTL locations. Given the amount of co-localization of QTL across traits and the direction of allelic effect supports that these regions have a pleiotropic effect. These QTL regions were also identified to be highly enriched for genes encoding chilling stress and hormonal response genes. This identified QTL can be useful in developing tools for molecular breeding of sorghums with improved low-temperature germinability.

Population Structure, Genetic Diversity and Candidate Genes for the Adaptation to Environmental Stress in Picea koraiensis.

Picea koraiensis is major silvicultural and timber species in northeast China, and its distribution area is an important transition zone for genus spruce migration. The degree of intraspecific differentiation of P. koraiensis is high, but population structure and differentiation mechanisms are not clear. In this study, 523,761 single nucleotide polymorphisms (SNPs) were identified in 113 individuals from 9 populations of P. koraiensis by genotyping-by-sequencing (GBS). Population genomic analysis showed that P. koraiensis was divided into three geoclimatic regions: Great Khingan Mountains climatic region, Lesser Khingan Mountains climatic region, and Changbai Mountain climatic region. Mengkeshan (MKS) population on the northern edge of the distribution area and Wuyiling (WYL) population located in the mining area are two highly differentiated groups. Selective sweep analysis showed that MKS and WYL populations had 645 and 1126 selected genes, respectively. Genes selected in the MKS population were associated with flowering and photomorphogenesis, cellular response to water deficit, and glycerophospholipid metabolism; genes selected in the WYL population were associated with metal ion transport, biosynthesis of macromolecules, and DNA repair. Climatic factors and heavy metal stress drives divergence in MKS and WYL populations, respectively. Our findings provide insights into adaptive divergence mechanisms in Picea and will contribute to molecular breeding studies.

Molecular Markers and Their Applications in Marker-Assisted Selection (MAS) in Bread Wheat (Triticum aestivum L.)

As one of the essential cereal crops, wheat provides 20% of the calories and proteins consumed by humans. Due to population expansion, dietary shift and climate change, it is challenging for wheat breeders to develop new varieties for meeting wheat production requirements. Marker-assisted selection (MAS) has distinct advantages over conventional selection in plant breeding, such as being time-saving, cost-effective and goal-oriented. This review makes attempts to give a description of different molecular markers: sequence tagged site (STS), simple sequence repeat (SSR), genotyping by sequencing (GBS), single nucleotide polymorphism (SNP) arrays, exome capture, Kompetitive Allele Specific PCR (KASP), cleaved amplified polymorphic sequence (CAPS), semi-thermal asymmetric reverse PCR (STARP) and genotyping by target sequencing (GBTS). We also summarize some quantitative trait loci (QTL)/genes as well as their linked markers, which are potentially useful in MAS. This paper provides updated information on some markers linked to critical traits and their potential applications in wheat breeding programs.

Genomic and evolutionary relationships among wild and cultivated blueberry species

BackgroundBlueberries (Vaccinium section Cyanococcus) are an economically important fruit crop in the United States. Understanding genetic structure and relationships in blueberries is essential to advance the genetic improvement of horticulturally important traits. In the present study, we investigated the genomic and evolutionary relationships in 195 blueberry accessions from five species (comprising 33 V. corymbosum, 14 V. boreale, 81 V. darrowii, 29 V. myrsinites, and 38 V. tenellum) using single nucleotide polymorphisms (SNPs) mined from genotyping-by-sequencing (GBS) data.ResultsGBS generated ~ 751 million raw reads, of which 79.7% were mapped to the reference genome V. corymbosum cv. Draper v1.0. After filtering (read depth > 3, minor allele frequency > 0.05, and call rate > 0.9), 60,518 SNPs were identified and used in further analyses. The 195 blueberry accessions formed three major clusters on the principal component (PC) analysis plot, in which the first two PCs accounted for 29.2% of the total genetic variance. Nucleotide diversity (π) was highest for V. tenellum and V. boreale (0.023 each), and lowest for V. darrowii (0.012). Using TreeMix analysis, we identified four migration events and deciphered gene flow among the selected species. In addition, we detected a strong V. boreale lineage in cultivated blueberry species. Pairwise SweeD analysis identified a wide sweep (encompassing 32 genes) as a strong signature of domestication on the scaffold VaccDscaff 12. From this region, five genes encoded topoisomerases, six genes encoded CAP-gly domain linker (which regulates the dynamics of the microtubule cytoskeleton), and three genes coded for GSL8 (involved in the synthesis of the cell wall component callose). One of the genes, augustus_masked-VaccDscaff12-processed-gene-172.10, is a homolog of ArabidopsisAT2G25010 and encodes the protein MAINTENANCE OF MERISTEMS-like involved in root and shoot growth. Additional genomic stratification by admixture analysis identified genetic lineages and species boundaries in blueberry accessions. The results from this study indicate that V. boreale is a genetically distant outgroup, while V. darrowii, V. myrsinites, and V. tenellum are closely related.ConclusionOur study provides new insights into the evolution and genetic architecture of cultivated blueberries.

Genome-wide association study for the primary feather color trait in a native Chinese duck.

Background: To reveal candidate genes and the molecular genetic mechanism underlying primary feather color trait in ducks, a genome-wide association study (GWAS) for the primary feather color trait was performed based on the genotyping-by-sequencing (GBS) technology for a native Chinese female duck, Longyan Shan-ma ducks. Methods: Blood genomic DNA from 314 female Longyan Shan-ma duck were genotyped using GBS technology. A GWAS for the primary feather color trait with genome variations was performed using an univariate linear mixed model based on all SNPs in autosomes. Results: Seven genome-wide significant single nucleotide polymorphisms (SNPs, Bonferroni-adjusted p-value <8.03 × 10-7) within the introns of the genes STARD9, ZNF106, SLC7A5, and BANP genes were associated with the primary feather color trait. Twenty-two genome-wide suggestive SNPs (Bonferroni-adjusted p-value <1.61 × 10-5) of 17 genes (besides ZNF106 and SLC7A5) were also identified. Seven SNPs were located at one 0.22Mb region (38.65-38.87Mb) on chromosome 5, and six SNPs were located at one 0.31Mb region (19.53-19.84Mb) on chromosome 11. The functions of STARD9, SLC7A5, BANP, LOC101798015, and IPMK were involved pigmentation and follicle development, especially, STARD9 upregulated expression in black feather (haplotype-CCCC) bulb tissue compared with in pockmarked feather (haplotype-TGTT) bulb tissue, implicating these genes as candidate genes for primary feather color trait. Conclusion: The preliminarily findings suggested candidate genes and regions, and the genetic basis of primary feather color trait in a female duck.

Identification of a Small Translocation from 6R Possessing Stripe Rust Resistance to Wheat.

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici Eriks. & E. Henn, is the most devastating fungal disease of bread wheat. Here, a wheat-rye multiple disomic substitution line, SLU126 4R (4D), 5R (5D), and 6R (7D), possessing resistance against 25 races of P. striiformis f. sp. tritici, was used and crossed with Chinese Spring ph1b to induce homeologous recombination to produce introgressions with a reduced rye chromosome segment. Seedling assays confirmed that the stripe rust resistance from SLU126 was retained over multiple generations. Through genotyping-by-sequencing (GBS) platforms and aligning the putative GBS-single-nucleotide polymorphism (SNPs) to the full-length annotated rye nucleotide-binding leucine-rich repeat (NLR) genes in the parental lines (CS ph1b, SLU126, CSA, and SLU820), we identified the physical position of 26, 13, and 9 NLR genes on chromosomes 6R, 4R, and 5R, respectively. The physical positions of 25 NLR genes on chromosome 6R were identified from 568,460,437 bp to 879,958,268 bp in the 6RL chromosome segment. Based on these NLR positions on the 6RL chromosome segment, the three linked SNPs (868,123,650 to 873,285,112 bp) were validated through kompetitive allele-specific PCR (KASP) assays in SLU126 and resistance plants in the family 29-N3-5. Using these KASP markers, we identified a small piece of the rye translocation (i.e., as a possible 6DS.6DL.6RL.6DL) containing the stripe resistance gene, temporary designated YrSLU, within the 6RL segment. This new stripe rust resistance gene provides an additional asset for wheat improvement to mitigate yield losses caused by stripe rust.

Genomic location of Gb1, a unique gene conferring wheat resistance to greenbug biotype F

Greenbug (Schizaphis graminum, Rondani) is a serious insect pest in many wheat growing regions and has been infesting cereal crops in the USA for over a century. Continuous occurrence of new greenbug biotypes makes it essential to explore all greenbug resistant sources available to manage this pest. Gb1, a recessive greenbug resistance gene in DS28A, confers resistance to several economically important greenbug biotypes and is the only gene found to be resistant to greenbug biotype F. A set of 174 F2:3 lines from the cross DS28A × Custer was evaluated for resistance to greenbug biotype F in 2020 and 2022. Selective genotyping of the corresponding F2 population using single nucleotide polymorphism (SNP) markers generated by genotyping-by-sequencing (GBS) led to the identification of a candidate genomic region for Gb1. Thus, SSR markers previously mapped in this region were used to genotype the entire F2 population, and kompetitive allele specific PCR (KASP) markers were also developed from SNPs in the target region. Gb1 was placed in the terminal region of the short arm of chromosome 1A, and its location was confirmed in a second population derived from the cross DS28A × PI 697274. The combined data analysis from the two mapping populations delimited Gb1 to a < 1 Mb interval between 13,328,200 and 14,241,426 bp on 1AS.

De Novo SNP Discovery and Genotyping of Masson Pine (Pinus massoniana Lamb.) via Genotyping-by-Sequencing

Masson pine (Pinus massoniana Lamb.) is an important tree species in China, but its genomic research has been hindered due to a large genome size. Genotyping-by-sequencing (GBS) has been a powerful approach to revolutionize the field of genomic research by facilitating the discovery of thousands of single nucleotide polymorphisms (SNPs) and genotyping in non-model organisms, at relatively low cost. Here, we performed de novo SNP discovery and genotyping in 299 trees via the genotyping-by-sequencing (GBS) approach. The effort produced 9.33 × 109 sequence reads, 265,525 SNP-associated contigs, and 6,739,240 raw SNPs. Further filtering and validation of the SNP-associated contigs for reliable SNPs were performed using blasting against the Pinus tabuliformis reference genome, functional annotation, technical replicates, and custom parameter settings for the optimization. The 159,372 SNP-associated contigs were aligned and validated for SNP prediction, in which 60,038 contigs were searched with hits in the NCBI nr database. We further improved the SNP discovery and genotyping with multiple technical replicates and custom parameter settings filtering. It was found that the use of blasting, annotation, technical replicates, and specific parameter settings removed many unreliable SNPs and identified 20,055 more precise and reliable SNPs from the 10,712 filtered contigs. We further demonstrated the informativeness of the identified SNPs in the inference of some genetic diversity and structure. These findings should be useful to stimulate genomic research and genomics-assisted breeding of Masson pine.

Genome-wide assessment of genetic diversity and association mapping for salt tolerance traits in okra (Abelmoschus esculentus L. Moench) using genotyping-by-sequencing

Okra is a vegetable that is widely grown worldwide. However, there are few reports on genetic diversity analysis and genome-wide association studies (GWASs) based on large-scale molecular markers in okra. In this study, 353,001 SNP markers were developed based on a genotyping by sequencing (GBS) strategy to analyze the population structure, genetic diversity and GWAS of 180 okra germplasms from all over the world. Both phylogenetic tree and population structure analyses classified all accessions into three categories, and the classification of most accessions in the two methods were the same. Some okra accessions from warm and cool areas were classified into one category, and some from hot areas were classified into one category. The Shannon‒Wiener index (I), Nei's genetic diversity index (Hei), minor allele frequency (MAF) and polymorphic information content (PIC) of the 180 okra accessions were 0.5071, 0.3366, 0.2580 and 0.2691, respectively. The B subgroup had the highest level of genetic diversity. A total of 180 okra seedlings were treated with 120 mM (T1) and 200 mM (T2) NaCl solutions, and salt tolerance-related traits were analyzed by GWAS. Seventy-seven SNP loci were identified to be associated with four salt tolerance traits. c3341.graph_c0 was aligned in multiple SNP loci, which may be an important unigene regulating the salt tolerance of okra seedlings. To our knowledge, this was the first study to use large-scale SNP markers to carry out population genetic analysis and GWAS in okra.

Genome wide association analysis for grain micronutrients and anti-nutritional traits in mungbean [Vigna radiata (L.) R. Wilczek] using SNP markers.

Mungbean is an important food grain legume for human nutrition and nutritional food due to its nutrient-dense seed, liked palatability, and high digestibility. However, anti-nutritional factors pose a significant risk to improving nutritional quality for bio-fortification. In the present study, genetic architecture of grain micronutrients (grain iron and zinc concentration) and anti-nutritional factors (grain phytic acid and tannin content) in association mapping panel of 145 diverse mungbean were evaluated. Based on all four parameters genotypes PUSA 1333 and IPM 02-19 were observed as desired genotypes as they had high grain iron and zinc concentration but low grain phytic acid and tannin content. The next generation sequencing (NGS)-based genotyping by sequencing (GBS) identified 14,447 genome-wide SNPs in a diverse selected panel of 127 mungbean genotypes. Population admixture analysis revealed the presence of four different ancestries among the genotypes and LD decay of ∼57.6 kb kb physical distance was noted in mungbean chromosomes. Association mapping analysis revealed that a total of 20 significant SNPs were shared by both GLM and Blink models associated with grain micronutrient and anti-nutritional factor traits, with Blink model identifying 35 putative SNPs. Further, this study identified the 185 putative candidate genes. Including potential candidate genes Vradi07g30190, Vradi01g09630, and Vradi09g05450 were found to be associated with grain iron concentration, Vradi10g04830 with grain zinc concentration, Vradi08g09870 and Vradi01g11110 with grain phytic acid content and Vradi04g11580 and Vradi06g15090 with grain tannin content. Moreover, two genes Vradi07g15310 and Vradi09g05480 showed significant variation in protein structure between native and mutated versions. The identified SNPs and candidate genes are potential powerful tools to provide the essential information for genetic studies and marker-assisted breeding program for nutritional improvement in mungbean.

Genome-wide association analysis and validation with KASP markers for nut and shell traits in almond (Prunus dulcis [Mill.] D.A.Webb)

Almond shell hardness is an economically important quality trait that protects the integrity of the kernel. It is directly related with shelling or crack-out percent. In the U.S. and Australian markets, soft to semi-hard shells varieties have been used in the industry, while in the European market, hard shells are dominantly sold and distributed. The origin of a hard shell is from wild almond species. Soft shells in the USA and Australia have been selected for a higher crack-out percent or larger kernel and ease of processing. Here, we examined breeding populations totaling 264 trees that were genotyped with genotyping-by-sequencing (GBS) where shell hardness was measured with a texture analyzer, and shell weight, in-shell weights and shell thickness were measured and crack-out percent was calculated. In a genome-wide association, seven genetic loci were found to be associated with shell hardness, crack-out percent, shell thickness and shell weight. Of these seven, one was shared between multiple traits. Kompetitive allele-specific PCR (KASP) assays were developed and tested in unrelated breeding lines from Australia, Spain, France and Italy. One KASP assay was found to be highly predictive of shell hardness in this diverse panel and can be applied for use in marker-assisted breeding.

Genotyping by sequencing for estimating relative abundances of diatom taxa in mock communities

BackgroundDiatoms are present in all waters and are highly sensitive to pollution gradients. Therefore, they are ideal bioindicators for water quality assessment. Current indices used in these applications are based on identifying diatom species and counting their abundances using traditional light microscopy. Several molecular techniques have been developed to help automate different steps of this process, but obtaining reliable estimates of diatom community composition and species abundance remains challenging.ResultsHere, we evaluated a recently developed quantification method based on Genotyping by Sequencing (GBS) for the first time in diatoms to estimate the relative abundances within a species complex. For this purpose, a reference database comprised of thousands of genomic DNA clusters was generated from cultures of Nitzschia palea. The sequencing reads from calibration and mock samples were mapped against this database for parallel quantification. We sequenced 25 mock diatom communities containing up to five taxa per sample in different abundances. Taxon abundances in these communities were also quantified by a diatom expert using manual counting of cells on light microscopic slides. The relative abundances of strains across mock samples were over- or under-estimated by the manual counting method, and a majority of mock samples had stronger correlations using GBS. Moreover, one previously recognized putative hybrid had the largest number of false positive detections demonstrating the limitation of the manual counting method when morphologically similar and/or phylogenetically close taxa are analyzed.ConclusionsOur results suggest that GBS is a reliable method to estimate the relative abundances of the N. palea taxa analyzed in this study and outperformed traditional light microscopy in terms of accuracy. GBS provides increased taxonomic resolution compared to currently available quantitative molecular approaches, and it is more scalable in the number of species that can be analyzed in a single run. Hence, this is a significant step forward in developing automated, high-throughput molecular methods specifically designed for the quantification of [diatom] communities for freshwater quality assessments.

Efficiency of genotyping by sequencing in inferring genomic relatedness and molecular insights into fat tail selection in Tunisian sheep.

In developing countries, the use of simple and cost-efficient molecular technology is crucial for genetic characterization of local animal resources and better development of conservation strategies. The genotyping by sequencing (GBS) technique, also called restriction enzyme- reduced representational sequencing, is an efficient, cost-effective method for simultaneous discovery and genotyping of many markers. In the present study, we applied a two-enzyme GBS protocol (PstI/MspI) to discover and genotype SNP markers among 197 Tunisian sheep samples. A total of 100 333 bi-allelic SNPs were discovered and genotyped with an SNP call rate of 0.69 and mean sample depth 3.33. The genomic relatedness between 183 samples grouped the samples perfectly to their populations and pointed out a high genetic relatedness of inbred subpopulation reflecting the current adopted reproductive strategies. The genome-wide association study contrasting fat vs. thin-tailed breeds detected 41 significant variants including a peak positioned on OAR20. We identified FOXC1, GMDS, VEGFA, OXCT1, VRTN and BMP2 as the most promising for sheep tail-type trait. The GBS data have been useful to assess the population structure and improve our understanding of the genomic architecture of distinctive characteristics shaped by selection pressure in local sheep breeds. This study successfully investigates a cost-efficient method to discover genotypes, assign populations and understand insights into sheep adaptation to arid area. GBS could be of potential utility in livestock species in developing/emerging countries.

QTL localisation of seed-related traits in Tibetan hulless barley based on a high-density single-nucleotide polymorphism genetic map

The effective use of high-quality and high-yielding germplasm resources is of great importance for the development of hulless barley varieties. Therefore, the search for such resources has long been a goal of the breeding community. In this study, a genotyping-by-sequencing (GBS) analysis was performed on an F2 (Nierumuzha × Kunlun 10) population. A high-density genetic map of hulless barley was constructed, which contained 1 475 efficient single-nucleotide polymorphism markers with 7 052 bin markers. The total length of the seven chromosomes was 1 139.4 cM, with an average marker distance of 0.59 cM. Based on this high-density linkage map, a total of 54 quantitative trait loci (QTLs) related to the seed traits were detected, including seed colour (SC), thousand kernel weight (TKW), seed average area (SAA), seed perimeter (SP), seed length (SL), seed width (SW), seed length/width (SLW), seed diameter (SD), and seed circular degree (SCD). These QTLs explained 3.32–35.73% (mean = 11.45%) of the phenotypic interpretation, including 24 main QTLs and 30 epistatic QTLs. A total of 24 candidate genes were identified within the QTL region, including one SC-associated transcription factor (ANT1), two TSW-related genes, a transcription factor (NAC021), a gene associated with the non-homologous end joining (NHEJ) pathway (ku70), three SAA-associated genes (LOL2, NAC021, TSK), two SL-associated genes (MADS21, MADS4), six SW-associated genes (FIP1, NAC021, DREB 1A, HVA22A, CYP78A6, SAUR71), five LW-related genes (NAM-B2, CRY1, LHY, CYP710A1, WRKY72), two SP-related genes (SKIP11, TCP18), two SD-related genes (NAC021, SKIP8), and three SCD-related genes (MYB1R1, RAX3, NAC100). These genes are involved in the regulation of the cell development, material transport, signal transduction, and plant morphogenesis and play an important role in the regulation of agronomic traits in hulless barley. The high-density genetic mapping and QTL identification of the seed traits in hulless barley provide a valuable genetic resource and the basis for further molecular marker-assisted selection and genomic studies.

Genetic diversity and local adaption of alfalfa populations (Medicago sativa L.) under long-term grazing

Genomic information on alfalfa adaptation to long-term grazing is useful for alfalfa genetic improvement. In this study, 14 alfalfa populations were collected from long-term grazing sites (> 25 years) across four soil zones in western Canada. Alfalfa cultivars released between 1926 and 1980 were used to compare degree of genetic variation of the 14 populations. Six agro-morphological and three nutritive value traits were evaluated from 2018 to 2020. The genotyping-by-sequencing (GBS) data of the alfalfa populations and environmental data were used for genotype-environment association (GEA). Both STRUCTURE and UPGMA based on 19,853 SNPs showed that the 14 alfalfa populations from long-term grazing sites had varying levels of parentages from alfalfa sub-species Medicago sativa and M. falcata. The linear regression of STRUCTURE membership probability on phenotypic data indicated genetic variations of forage dry matter yield, spring vigor and plant height were low, but genetic variations of regrowth, fall plant height, days to flower and crude protein were still high for the 14 alfalfa populations from long-term grazing sites. The GEA identified 31 SNPs associated with 13 candidate genes that were mainly associated with six environmental factors of. Candidate genes underlying environmental factors were associated with a variety of proteins, which were involved in plant responses to abiotic stresses, i.e., drought, cold and salinity-alkali stresses.

QTL mapping and BSA-seq map a major QTL for the node of the first fruiting branch in cotton.

Understanding the genetic basis of the node of the first fruiting branch (NFFB) improves early-maturity cotton breeding. Here we report QTL mapping on 200 F2 plants and derivative F2:3 and F2:4 populations by genotyping by sequencing (GBS). BC1F2 population was constructed by backcrossing one F2:4 line with the maternal parent JF914 and used for BSA-seq for further QTL mapping. A total of 1,305,642 SNPs were developed between the parents by GBS, and 2,907,790 SNPs were detected by BSA-seq. A high-density genetic map was constructed containing 11,488 SNPs and spanning 4,202.12 cM in length. A total of 13 QTL were mapped in the 3 tested populations. JF914 conferred favorable alleles for 11 QTL, and JF173 conferred favorable alleles for the other 2 QTL. Two stable QTL were repeatedly mapped in F2:3 and F2:4, including qNFFB-D3-1 and qNFFB-D6-1. Only qNFFB-D3-1 contributed more than 10% of the phenotypic variation. This QTL covered about 24.7 Mb (17,130,008-41,839,226 bp) on chromosome D3. Two regions on D3 (41,779,195-41,836,120 bp, 41,836,768-41,872,287 bp) were found by BSA-seq and covered about 92.4 Kb. This 92.4 Kb region overlapped with the stable QTL qNFFB-D3-1 and contained 8 annotated genes. By qRT-PCR, Ghir_D03G012430 showed a lower expression level from the 1- to 2-leaf stage and a higher expression level from the 3- to 6-leaf stage in the buds of JF173 than that of JF914. Ghir_D03G012390 reached the highest level at the 3- and 5-leaf stages in the buds of JF173 and JF914, respectively. As JF173 has lower NFFB and more early maturity than JF914, these two genes might be important in cell division and differentiation during NFFB formation in the seedling stage. The results of this study will facilitate a better understanding of the genetic basis of NFFB and benefit cotton molecular breeding for improving earliness traits.