Identify Candidate Genomic Regions Research Articles

Whole-Genome Resequencing Reveals Signatures of Adaptive Evolution in Acanthopagrus latus and Rhabdosargus sarba.

Acanthopagrus latus and Rhabdosargus sarba are economically important marine species along the coast of China, with similar external morphological characteristics and living habits, with wide distribution and strong adaptability. To investigate the molecular mechanisms underlying the adaptive evolution of these two species, we conducted whole-genome resequencing of 10 individuals of both species from the coastal waters of Wuyu Island, Fujian, China, using high-throughput sequencing technology. We obtained SNP, InDel, CNV, and SV variation information and annotated these variations, constructing a genomic variation database for both species. By comparing the resequencing data with reference genomes, we identified 9,829,511 SNP loci in the population of A. latus and 34,051,056 SNP loci in the population of R. sarba. Using whole-genome SNP data, we employed Fst and ROD methods to identify candidate genomic regions under selection. Functional annotation and enrichment analysis using GO and KEGG databases revealed potential adaptive evolution in R. sarba associated with immune response, feeding, growth and development, and locomotion, while A. latus showed potential adaptive evolution associated with immune response, nervous system, growth and development, and metabolism.

Genome scans for selection signatures identify candidate virulence genes for adaptation of the soybean cyst nematode to host resistance.

Plant pathogens are constantly under selection pressure for host resistance adaptation. Soybean cyst nematode (SCN, Heterodera glycines) is a major pest of soybean primarily managed through resistant cultivars; however, SCN populations have evolved virulence in response to selection pressures driven by repeated monoculture of the same genetic resistance. Resistance to SCN is mediated by multiple epistatic interactions between Rhg (for resistance to H. glycines) genes. However, the identity of SCN virulence genes that confer the ability to overcome resistance remains unknown. To identify candidate genomic regions showing signatures of selection for increased virulence, we conducted whole genome resequencing of pooled individuals (Pool-Seq) from two pairs of SCN populations adapted on soybeans with Peking-type (rhg1-a, rhg2, and Rhg4) resistance. Population differentiation and principal component analysis-based approaches identified approximately 0.72-0.79 million SNPs, the frequency of which showed potential selection signatures across multiple genomic regions. Chromosomes 3 and 6 between population pairs showed the greatest density of outlier SNPs with high population differentiation. Conducting multiple outlier detection tests to identify overlapping SNPs resulted in a total of 966 significantly differentiated SNPs, of which 285 exon SNPs were mapped to 97 genes. Of these, six genes encoded members of known stylet-secreted effector protein families potentially involved in host defence modulation including venom-allergen-like, annexin, glutathione synthetase, SPRYSEC, chitinase, and CLE effector proteins. Further functional analysis of identified candidate genes will provide new insights into the genetic mechanisms by which SCN overcomes soybean resistance and inform the development of molecular markers for rapidly screening the virulence profile of an SCN-infested field.

Genomic characterization of dryland adaptation in endangered Anxi cattle in China.

Local species exhibit distinctive indigenous characteristics while showing unique productive and phenotypic traits. However, the advent of commercialization has posed a substantial threat to the survival of indigenous species. Anxi cattle, an endangered native breed in China, have evolved unique growth and reproductive characteristics in extreme desert and semidesert ecosystems. In this study, we conducted a genomic comparison of 10 Anxi cattle genomes with those of five other global populations/breeds to assess genetic diversity and identify candidate genomic regions in Anxi cattle. Population structure and genetic diversity analyses revealed that Anxi cattle are part of the East Asian cattle clade, exhibiting higher genetic diversity than commercial breeds. Through selective sweep analysis, we identified specific genetic variations linked to the environmental adaptability of Anxi cattle. Notably, we identified several candidate genes, including CERS3 involved in regulating skin permeability and antimicrobial functions, RBFOX2 associated with cardiac development, SLC16A7 participated in the regulation of pancreatic endocrine function, and SPATA3 related to reproduction. Our findings revealed the distinctive genomic features of Anxi cattle in dryland environments, provided invaluable insights for further research and breed preservation, and had important significance for enriching the domestic cattle breeding gene bank.

Simultaneously mapping loci related to two plant architecture traits by phenotypic recombination BSA/BSR in peanut (Arachis hypogaea L.).

We developed a new method phenotypic recombination BSA/BSR (PR-BSA/BSR), which could simultaneously identify the candidate genomic regions associated with two traits in a segregating population. Bulked segregant analysis sequencing (BSA-seq) has been widely used for identifying the genomic regions affecting a certain trait. In this study, we developed a modified BSA/bulked segregant RNA-sequencing (BSR-seq) method, which we named phenotypic recombination BSA/BSR (PR-BSA/BSR), to simultaneously identify candidate genomic regions associated with two traits in a segregating population. Lateral branch angle (LBA) and flower-branch pattern (FBP) are two important traits associated with the peanut plant architecture because they affect the planting density and light use efficiency. We generated an F6 population (with two segregating traits) derived from a cross between the inbred lines Pingdu9616 (erect and sequential; ES-type) and Florunner (spreading and alternating; SA-type). The selection of bulks with extreme phenotypes was a key step in this study. Specifically, 30 individuals with recombinant phenotypes [i.e., spreading and sequential (SS-type) and erect and alternating (EA-type)] were selected to generate two bulks. The transcriptomes of individuals were sequenced and then the loci related to LBA and FBP were simultaneously detected via a ΔSNP-index strategy, which involved the direction of positive and negative peaks in the ∆SNP-index plot. The LBA-related locus was mapped to a 6.82Mb region (101,743,223-108,564,267bp) on chromosome 15, whereas the FBP-related locus was mapped to a 2.16Mb region (117,682,534-119,846,824bp) on chromosome 12. Furthermore, the marker-based classical QTL mapping method was used to analyze the PF-F6 population, which confirmed our PR-BSA/BSR results. Therefore, the PR-BSA/BSR method produces accurate and reliable data.

Parent-offspring genotyped trios unravelling genomic regions with gametic and genotypic epistatic transmission bias on the cattle genome.

Several biological mechanisms affecting the sperm and ova fertility and viability at developmental stages of the reproductive cycle resulted in observable transmission ratio distortion (i.e., deviation from Mendelian expectations). Gene-by-gene interactions (or epistasis) could also potentially cause specific transmission ratio distortion patterns at different loci as unfavorable allelic combinations are under-represented, exhibiting deviation from Mendelian proportions. Here, we aimed to detect pairs of loci with epistatic transmission ratio distortion using 283,817 parent-offspring genotyped trios (sire-dam-offspring) of Holstein cattle. Allelic and genotypic parameterization for epistatic transmission ratio distortion were developed and implemented to scan the whole genome. Different epistatic transmission ratio distortion patterns were observed. Using genotypic models, 7, 19 and 6 pairs of genomic regions were found with decisive evidence with additive-by-additive, additive-by-dominance/dominance-by-additive and dominance-by-dominance effects, respectively. Using the allelic transmission ratio distortion model, more insight was gained in understanding the penetrance of single-locus distortions, revealing 17 pairs of SNPs. Scanning for the depletion of individuals carrying pairs of homozygous genotypes for unlinked loci, revealed 56 pairs of SNPs with recessive epistatic transmission ratio distortion patterns. The maximum number of expected homozygous offspring, with none of them observed, was 23. Finally, in this study, we identified candidate genomic regions harboring epistatic interactions with potential biological implications in economically important traits, such as reproduction.

Outlier analyses and genome-wide association study identify glgC and ERD6-like 4 as candidate genes for foliar water-soluble carbohydrate accumulation in Trifolium repens.

Increasing water-soluble carbohydrate (WSC) content in white clover is important for improving nutritional quality and reducing environmental impacts from pastoral agriculture. Elucidation of genes responsible for foliar WSC variation would enhance genetic improvement by enabling molecular breeding approaches. The aim of the present study was to identify single nucleotide polymorphisms (SNPs) associated with variation in foliar WSC in white clover. A set of 935 white clover individuals, randomly sampled from five breeding pools selectively bred for divergent (low or high) WSC content, were assessed with 14,743 genotyping-by-sequencing SNPs, using three outlier detection methods: PCAdapt, BayeScan and KGD-FST. These analyses identified 33 SNPs as discriminating between high and low WSC populations and putatively under selection. One SNP was located in the intron of ERD6-like 4, a gene coding for a sugar transporter located on the vacuole membrane. A genome-wide association study using a subset of 605 white clover individuals and 5,757 SNPs, identified a further 12 SNPs, one of which was associated with a starch biosynthesis gene, glucose-1-phosphate adenylyltransferase, glgC. Our results provide insight into genomic regions underlying WSC accumulation in white clover, identify candidate genomic regions for further functional validation studies, and reveal valuable information for marker-assisted or genomic selection in white clover.

Bonamia-free flat oyster (Ostrea edulis L.) seed for restoration projects: non-destructive screening of broodstock, hatchery production and test for Bonamia-tolerance

Native (flat) oyster (Ostrea edulis) beds, once a major component of the North Sea, largely disappeared from the region in the late 19th century. Flat oyster restoration is taking place at a number of locations in the North Sea. When flat oyster beds are restored in areas where O. edulis is functionally extinct it is advised to treat these as disease-free areas. Adult oysters were collected in the Dutch Delta area which is infected with the pathogenic parasite Bonamia ostreae. The aim of this research was to obtain Bonamia-free seed from parents collected in a Bonamia-infected area. In addition, the oysters were analysed to identify candidate genomic regions related to bonamiosis tolerance and exposed to Bonamia in the field to assess survival compared to a naïve control group. With the aid of a non-destructive screening method, Bonamia-free broodstock were selected. These oysters produced Bonamia-free larvae and seed. For comparison, broodstock oysters were collected in the Dutch Wadden Sea, an area free of Bonamia. These oysters also produced Bonamia-free larvae and seed. To study if the Delta area oysters had developed a degree of resistance to the disease, while the naïve Wadden Sea oysters had not, seed of both groups was challenged in Lake Grevelingen where Bonamia occurs. Survival of the pre-selected Bonamia-free oysters was significantly higher than the naïve group. Samples of seed were analysed for association of candidate genetic markers related to bonamiosis tolerance. A higher percentage of individuals with tolerance-associated marker genotypes was found in the screened group compared to the naïve one. However, mortality of the naïve group could not be related to Bonamia presence. Further challenge tests are needed before firm conclusions regarding the genetic markers can be made. The results show that hatchery production of Bonamia-free and potentially Bonamia-tolerant flat oysters is possible.

163 Genome-wide association study to identify candidate genomic regions for cow fertility in Retinta cattle breed

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

Elucidation of the Flavor Aspects and Flavor-Associated Genomic Regions in Bottle Gourd (Lagenaria siceraria) by Metabolomic Analysis and QTL-seq.

Bottle gourd (Lagenaria siceraria) is a commercially important cucurbitaceous vegetable with health-promoting properties whose collections and cultivars differ considerably in their flavor aspects. However, the metabolomic profile related to flavor has not yet been elucidated. In the present study, a comprehensive metabolite analysis revealed the metabolite profile of the strong-flavor collection “J120” and weak-flavor collection “G32”. The major differentially expressed metabolites included carboxylic acids, their derivatives, and organooxygen compounds, which are involved in amino acid biosynthesis and metabolism. QTL-seq was used to identify candidate genomic regions controlling flavor in a MAGIC population comprising 377 elite lines. Three significant genomic regions were identified, and candidate genes likely associated with flavor were screened. Our study provides useful information for understanding the metabolic causes of flavor variation among bottle gourd collections and cultivars. Furthermore, the identified candidate genomic regions may facilitate rational breeding programs to improve bottle gourd quality.

Speciation with gene flow between two Neotropical sympatric species (Pitcairnia spp.: Bromeliaceae).

The study of mechanisms that generate new species is considered fundamental for broad areas of ecology and evolution. Speciation is a continuous process in which reproductive isolation is established, and it is of fundamental importance to understand the origins of the adaptations that contribute to this process. Hybrid zones are considered natural laboratories for the study of speciation and represent ideal systems for such studies. Here, we investigated genomic differentiation between hybridizing Neotropical species Pitcairnia staminea (G. Lodd.) and P. albiflos (Herb.). Using thousands of SNPs genotyped through RAD‐seq, we estimate effective population sizes, interspecific gene flow, as well as time of divergence between these two sister species and identify candidate genomic regions for positive selection that may be related to reproductive isolation. We selected different scenarios of speciation and tested them by using approximate Bayesian computation (ABC); we found evidence of divergence with gradual reduction in gene flow between these species over time, compatible with the hypothesis of speciation with gene flow between these Pitcairnia species. The parameter estimates obtained through ABC suggested that the effective population size of P. albiflos was around three times larger than that of P. staminea. Our divergence date estimates showed that these two species diverged during the Pliocene (4.7 Mya; CI = 1.3–8.5 Mya), which has likely allowed this species to accumulate genome‐wide differences. We also detected a total of 17 of 4165 loci which showed signatures of selection with high genetic differentiation (F ST > 0.85), 12 of these loci were annotated in de novo assembled transcriptomes of both species, and 4 candidate genes were identified to be putatively involved in reproductive isolation. These four candidate genes were previously associated with the function of pollen development, pollen tube germination and orientation, abiotic stress, and flower scent in plants, suggesting an interplay between pre‐ and postpollination barriers in the evolution of reproductive isolation between such species.

Genetic characterization and curation of diploid A-genome wheat species.

A-genome diploid wheats represent the earliest domesticated and cultivated wheat species in the Fertile Crescent and include the donor of the wheat A sub-genome. The A-genome species encompass the cultivated einkorn (Triticum monococcum L. subsp. monococcum), wild einkorn (T. monococcum L. subsp. aegilopoides (Link) Thell.), and Triticum urartu. We evaluated the collection of 930 accessions in the Wheat Genetics Resource Center (WGRC) using genotyping by sequencing and identified 13,860 curated single-nucleotide polymorphisms. Genomic analysis detected misclassified and genetically identical (>99%) accessions, with most of the identical accessions originating from the same or nearby locations. About 56% (n = 520) of the WGRC A-genome species collections were genetically identical, supporting the need for genomic characterization for effective curation and maintenance of these collections. Population structure analysis confirmed the morphology-based classifications of the accessions and reflected the species geographic distributions. We also showed that T. urartu is the closest A-genome diploid to the A-subgenome in common wheat (Triticum aestivum L.) through phylogenetic analysis. Population analysis within the wild einkorn group showed three genetically distinct clusters, which corresponded with wild einkorn races α, β, and γ described previously. The T. monococcum genome-wide FST scan identified candidate genomic regions harboring a domestication selection signature at the Non-brittle rachis 1 (Btr1) locus on the short arm of chromosome 3Am at ∼70 Mb. We established an A-genome core set (79 accessions) based on allelic diversity, geographical distribution, and available phenotypic data. The individual species core set maintained at least 79% of allelic variants in the A-genome collection and constituted a valuable genetic resource to improve wheat and domesticated einkorn in breeding programs.

Genome-wide association study using haplotype libraries and repeated-measures model to identify candidate genomic regions for stillbirth in Holstein cattle

Reduced fertility is one of the main causes of economic losses on dairy farms, resulting in economic losses estimated at $938 per stillbirth case in Holstein herds. The identification of genomic regions associated with stillbirth could help to develop better management and breeding strategies aimed to reduce the frequency of undesirable gestation outcomes. Here, 10,570 cows and 50,541 birth records were used to perform a haplotype-based GWAS. A total of 41 significantly associated pseudo-SNPs (haplotypes within haplotype blocks converted to a binary classification) were identified after Bonferroni adjustment for multiple tests. A total of 117 positional candidate genes were annotated within or close (in a 200-kb interval) to significant pseudo-SNPs (haplotype blocks). The guilt-by-association functional prioritization identified 31 potential functional candidate genes for reproductive performance out of the 117 positional candidate genes annotated. These genes play crucial roles in biological processes associated with pregnancy persistence, fetus development, immune response, among others. These results helped us to better understand the genetic basis of stillbirth in dairy cattle and may be useful for the prediction of stillbirth in Holstein cattle, helping to reduce the related economic losses caused by this phenotype.

Single-step genome-wide association study uncovers known and novel candidate genomic regions for endocrine and classical fertility traits in Swedish Red and Holstein dairy cows

• In this study, the novel endocrine and classical fertility traits of Swedish dairy herds were evaluated using in-line milk progesterone profiles in ssGWAS approach. • The genomic regions associated with the endocrine and classical traits were identified. • A total of 20 QTLs, of which 18 of them are novel, were detected. The novel QTL regions embedded candidate genes ( ATG7, GPI, GJA1, PPP3CA, ECT2, ARHGAP20, PHLDB1, CACNA1D, CCNE1, CDH13, PLD1, FBN2, KIF3A, FGF12, KCNMB2, MAN1A1, KCNN2, SMAD6, MAPK8IP1, PHF21A, LPXN, MMRN1, KCNIP4, NID2, PCDHGA8, GRIA1, PCDHGB4, PHLDB2, STXBP5L, PTPRR, SRGAP1, SNX27, SPTA1, S100A10, TBC1D20 and ITCH ) that are associated with endocrine and classical fertility traits. • The candidate QTL regions provide insights into the genetic basis of endocrine and classical fertility in SR and Holstein dairy breeds. In a study aiming to identify candidate genomic regions associated with endocrine and classical fertility traits in Swedish Red (SR) and Holstein cows, data on 3955 lactations in 1164 SR and 1672 Holstein cows were examined. The dataset comprised milk progesterone (P4) levels ( n = 341,212) in 14 Swedish herds, automatically collected and analyzed in-line using the DeLaval Herd Navigator™. Endocrine traits studied were: days from calving to commencement of luteal activity (C-LA), first luteal phase length (LPL), length of first inter-luteal interval, length of first inter-ovulatory interval (IOI), luteal activity during the first 60 DIM, and proportion of samples with luteal activity during the first 60 DIM. Classical fertility traits based on insemination data were also investigated, such as days from calving to last insemination and calving interval. A total of 180 SR and 312 Holstein cows were genotyped with a low-density SNP chip and imputed to 50 K. Single-step genome-wide association (ssGWAS) was used to explore candidate genomic regions associated with fertility traits. A mixed linear single-trait animal model was fitted, considering season and parity as fixed effects and animal and permanent environment as random effects. The results revealed 990 and 415 SNPs above the threshold (-log ( p -value) ≥4) for SR and Holstein cows, respectively. The breeds shared only eight SNPs significantly associated with fertility traits. Annotation analysis revealed 281 SNPs located in 241 genes. Functional enrichment analysis using DAVID tools reduced the number to 80 genes, which were mediated in various biological processes and KEGG pathways in multiple functions, including folliculogenesis, embryogenesis, uterine growth and development, immune response, and ovarian cysts. Of the 80 genes, 67 were associated with fertility traits in SR cows and 13 in Holstein. Most genes were associated with LPL and IOI in SR cows, but in Holstein the only association with an endocrine trait was with C-LA. Twenty QTL regions that embedded 40 genes were associated with fertility traits in both breeds. All the QTLs detected, except at BTA2 and BTA19 are novel QTL regions that were not reported previously. These novel QTL regions embedded the candidate genes that include ARHGAP20, PHLDB1, CACNA1D, ATG7, CCNE1, GPI, CDH13, ECT2, PLD1, FBN2, KIF3A, FGF12, KCNMB2, GJA1, MAN1A1, KCNN2, SMAD6, MAPK8IP1, PHF21A, LPXN, MMRN1, KCNIP4, NID2, PCDHGA8, GRIA1, PCDHGB4, PHLDB2, STXBP5L, PPP3CA, PTPRR, SRGAP1, SNX27, SPTA1, S100A10, TBC1D20 and ITCH. The candidate regions may help to improve genetic progress in female fertility if used in selection decisions. A challenge for future research is to determine why different regions seem relevant for different traits and breeds, and the practical implications for genomic selection.

Sat-BSA: an NGS-based method using local de novo assembly of long reads for rapid identification of genomic structural variations associated with agronomic traits.

Advances in next generation sequencing (NGS)-based methodologies have accelerated the identifications of simple genetic variants such as point mutations and small insertions/deletions (InDels). Structural variants (SVs) including large InDels and rearrangements provide vital sources of genetic diversity for plant breeding. However, their analysis remains a challenge due to their complex nature. Consequently, novel NGS-based approaches are needed to rapidly and accurately identify SVs. Here, we present an NGS-based bulked-segregant analysis (BSA) technique called Sat-BSA (SVs associated with traits) for identifying SVs controlling traits of interest in crops. Sat-BSA targets allele frequencies at all SNP positions to first identify candidate genomic regions associated with a trait, which is then reconstructed by long reads-based local de novo assembly. Finally, the association between SVs, RNA-seq-based gene expression patterns and trait is evaluated for multiple cultivars to narrow down the candidate genes. We applied Sat-BSA to segregating F2 progeny obtained from crosses between turnip cultivars with different tuber colors and successfully isolated two genes harboring SVs that are responsible for tuber phenotypes. The current study demonstrates the utility of Sat-BSA for the identification of SVs associated with traits of interest in species with large and heterozygous genomes.

Genetic analysis of wheat grains using digital imaging and their relationship to enhance grain weight

ABSTRACT: Phenomic characterization through digital imaging (DI) can capture the three dimensional variation in wheat grain size and shape using different image orientations. Digital imaging may help identifying genomic regions controlling grain morphology using association mapping with simple sequence repeats (SSRs) markers. Accordingly, seed shape phenotypic data of a core collection of 55 wheat genotypes, previously characterized for osmotic and drought tolerance, were produced using computer based Smart grain software. Measured dimensions included seed volume, area, perimeters, length, width, length to width ratio, circularity, horizontal deviation from ellipse (HDEV), vertical deviation from ellipse (VDEV), factor form density (FFD) etc. The thousand grain weight (TGW) was positively correlated with grain size direct measurements; however, VDEV, FFD and other derived grain attributes showed no or negative correlation with TGW. Digital imaging divided the genotypes correctly into well-defined clusters. The wheat genotypes studied were further grouped into two sub-clusters by the Bayesian structure analysis using unlinked SSR markers. A number of loci over various chromosomal regions were found associated to grain morphology by the genome wide analysis using mixed linear model (MLM) approach. A considerable number of marker-trait associations (MTAs) on chromosomes 1D and 2D may carry new alleles with potential to enhance grain weight due to the use of untapped wild accessions of Aegilops tauschii. Conclusively, we demonstrated the application of multiple approaches including high throughput phenotyping using DI complemented with genome wide association studies to identify candidate genomic regions underlying these traits, which allows a better understanding on molecular genetics of wheat grain weight.

ANÁLISIS DE MARCADORES MOLECULARES PARA EL ÍNDICE DE FERTILIDAD DE ESPIGA Y CARACTERES ASOCIADOS EN UNA POBLACIÓN DE LÍNEAS ENDOCRIADAS RECOMBINANTES DE TRIGO PAN.

Spike fertility index (SF) has been well established as an ecophysiological trait related to grain number per unit area and a promising selection target in wheat breeding programs. Scarce information on the molecular basis of SF is available thus far. In this study, a preliminary molecular marker analysis was carried out in a RIL population derived from the cross between two Argentinean cultivars with contrasting SF to identify candidate genomic regions associated with SF. Twenty-four microsatellites and two functional markers that had been found to co-segregate with SF in a bulked-segregant analysis of the F3 generation of the population were analyzed. Phenotypic data were collected from three field experiments carried out during 2013, 2014 and 2015 growing seasons at Balcarce, Argentina. Two genomic regions associated with SF in chromosomes 5BS and 7AS were detected, which merit further investigation. Key words: selection, genomic regions, grain number, yield, QTL, spike fertility index, fruiting efficiency

Next-generation sequencing-based bulked segregant analysis for QTL mapping in the heterozygous species Brassica rapa.

An improved protocol of QTL-seq, an NGS-based method for bulked segregant analysis we previously developed in rice, allowed successful mapping of QTLs of interest in the highly heterozygous genome of B. rapa, demonstrating the power of this elegant method for genetic analyses in heterozygous species of economic importance. Recent advances in next-generation sequencing (NGS) and the various NGS-based methods developed for rapidly identifying candidate genes of interest have accelerated genetic analysis mainly in the model plants rice and Arabidopsis. Brassica rapa includes several economically important crops such as Chinese cabbage, turnip and various leafy vegetables. The application of NGS-based approaches for the analysis of B. rapa has been limited mainly due to its highly heterozygous genome and poor quality of the reference genome sequence currently available for this species. In this study, we have improved QTL-seq, a method for NGS-based bulked segregant analysis we previously developed in rice, extending its applicability for accelerating the genetic analysis and molecular breeding of B. rapa. Addition of new filters to the original QTL-seq pipeline allowed removal of spurious single-nucleotide polymorphisms caused by alignment/sequencing errors and variability between parents, significantly improving accuracy of the analysis. As proof of principle, we successfully applied the new approach to identify candidate genomic regions controlling flowering and trichome formation using segregating F2 progeny obtained from crosses made between cultivars of B. rapa showing contrasting phenotypes for these traits. We strongly believe that the improved QTL-seq method reported here will extend the applicability of NGS-based genetic analysis not only to B. rapa but also to other plant species of economic importance with heterozygous genomes.

Discovery of genomic regions and candidate genes controlling shelling percentage using QTL-seq approach in cultivated peanut (Arachis hypogaea L.).

SummaryCultivated peanut (Arachis hypogaea L.) is an important grain legume providing high‐quality cooking oil, rich proteins and other nutrients. Shelling percentage (SP) is the 2nd most important agronomic trait after pod yield and this trait significantly affects the economic value of peanut in the market. Deployment of diagnostic markers through genomics‐assisted breeding (GAB) can accelerate the process of developing improved varieties with enhanced SP. In this context, we deployed the QTL‐seq approach to identify genomic regions and candidate genes controlling SP in a recombinant inbred line population (Yuanza 9102 × Xuzhou 68‐4). Four libraries (two parents and two extreme bulks) were constructed and sequenced, generating 456.89–790.32 million reads and achieving 91.85%–93.18% genome coverage and 14.04–21.37 mean read depth. Comprehensive analysis of two sets of data (Yuanza 9102/two bulks and Xuzhou 68‐4/two bulks) using the QTL‐seq pipeline resulted in discovery of two overlapped genomic regions (2.75 Mb on A09 and 1.1 Mb on B02). Nine candidate genes affected by 10 SNPs with non‐synonymous effects or in UTRs were identified in these regions for SP. Cost‐effective KASP (Kompetitive Allele‐Specific PCR) markers were developed for one SNP from A09 and three SNPs from B02 chromosome. Genotyping of the mapping population with these newly developed KASP markers confirmed the major control and stable expressions of these genomic regions across five environments. The identified candidate genomic regions and genes for SP further provide opportunity for gene cloning and deployment of diagnostic markers in molecular breeding for achieving high SP in improved varieties.

Pooled whole-genome sequencing of interspecific chestnut (Castanea) hybrids reveals loci associated with differences in caching behavior of fox squirrels (Sciurus niger L.).

Dispersal of seeds by scatter‐hoarding rodents is common among tropical and temperate tree species, including chestnuts in the genus Castanea. Backcrossed (BC) interspecific hybrid chestnuts exhibit wide variation in seed traits: as the parent species (Castanea dentata and C. mollissima) have distinct seed phenotypes and tend to be handled differently by seed dispersers, phenotypic variation in BC trees is likely due to inheritance of genes that have undergone divergent evolution in the parent species. To identify candidate genomic regions for interspecific differences in seed dispersal, we used tagged seeds to measure average dispersal distance for seeds of third‐generation BC chestnuts and sequenced pooled whole genomes of mother trees with contrasting seed dispersal: high caching rate/long distance; low caching rate/short distance; no caching. Candidate regions affecting seed dispersal were identified as loci with more C. mollissima alleles in the high caching rate/ long‐distance pool than expected by chance and observed in the other two pools. Functional annotations of candidate regions included predicted lipid metabolism, dormancy regulation, seed development, and carbohydrate metabolism genes. The results support the hypothesis that perception of seed dormancy is a predominant factor in squirrel caching decisions, and also indicate profitable directions for future work on the evolutionary genomics of trees and coevolved seed dispersers.

QTL-seq for rapid identification of candidate genes for flowering time in broccoli×cabbage.

A major QTL controlling early flowering in broccoli×cabbage was identified by marker analysis and next-generation sequencing, corresponding to GRF6 gene conditioning flowering time in Arabidopsis. Flowering is an important agronomic trait for hybrid production in broccoli and cabbage, but the genetic mechanism underlying this process is unknown. In this study, segregation analysis with BC1P1, BC1P2, F2, and F2:3 populations derived from a cross between two inbred lines "195" (late-flowering) and "93219" (early flowering) suggested that flowering time is a quantitative trait. Next, employing a next-generation sequencing-based whole-genome QTL-seq strategy, we identified a major genomic region harboring a robust flowering time QTL using an F2 mapping population, designated Ef2.1 on cabbage chromosome 2 for early flowering. Ef2.1 was further validated by indel (insertion or deletion) marker-based classical QTL mapping, explaining 51.5% (LOD=37.67) and 54.0% (LOD=40.5) of the phenotypic variation in F2 and F2:3 populations, respectively. Combined QTL-seq and classical QTL analysis narrowed down Ef1.1 to a 228-kb genomic region containing 29 genes. A cabbage gene, Bol024659, was identified in this region, which is a homolog of GRF6, a major gene regulating flowering in Arabidopsis, and was designated BolGRF6. qRT-PCR study of the expression level of BolGRF6 revealed significantly higher expression in the early flowering genotypes. Taken together, our results provide support for BolGRF6 as a possible candidate gene for early flowering in the broccoli line 93219. The identified candidate genomic regions and genes may be useful for molecular breeding to improve broccoli and cabbage flowering times.