Sequencing-based Bulked Segregant Analysis Research Articles

Conjunctive BSA-Seq and BSR-Seq to Map the Genes of Yellow Leaf Mutations in Hot Peppers (Capsicum annuum L.).

Yellow leaf mutations have been widely used to study the chloroplast structures, the pigment synthesis, the photosynthesis mechanisms and the chlorophyll biosynthesis pathways across various species. For this study, a spontaneous mutant with the yellow leaf color named 96-140YBM was employed to explore the primary genetic elements that lead to the variations in the leaf color of hot peppers. To identify the pathways and genes associated with yellow leaf phenotypes, we applied sequencing-based Bulked Segregant Analysis (BSA-Seq) combined with BSR-Seq. We identified 4167 differentially expressed genes (DEGs) in the mutant pool compared with the wild-type pool. The results indicated that DEGs were involved in zeatin biosynthesis, plant hormone signal transduction, signal transduction mechanisms, post-translational modification and protein turnover. A total of 437 candidates were identified by the BSA-Seq, while the BSR-Seq pinpointed four candidate regions in chromosomes 8 and 9, containing 222 candidate genes. Additionally, the combination of BSA-Seq and BSR-Seq showed that there were 113 overlapping candidate genes between the two methods, among which 8 common candidates have been previously reported to be related to the development of chloroplasts, the photomorphogenesis and chlorophyll formation of plant chloroplasts and chlorophyll biogenesis. qRT-PCR analysis of the 8 common candidates showed higher expression levels in the mutant pool compared with the wild-type pool. Among the overlapping candidates, the DEG analysis showed that the CaKAS2 and CaMPH2 genes were down-regulated in the mutant pool compared to the wild type, suggesting that these genes may be key contributors to the yellow leaf phenotype of 96-140YBM. This research will deepen our understanding of the genetic basis of leaf color formation and provide valuable information for the breeding of hot peppers with diverse leaf colors.

The GmSNAP11 Contributes to Resistance to Soybean Cyst Nematode Race 4 in Glycine max.

Soybean cyst nematode (SCN) has devastating effects on soybean production, making it crucial to identify genes conferring SCN resistance. Here we employed next-generation sequencing-based bulked segregant analysis (BSA) to discover genomic regions, candidate genes, and diagnostic markers for resistance to SCN race 4 (SCN4) in soybean. Phenotypic analysis revealed highly significant differences among the reactions of 145 recombinant inbred lines (RILs) to SCN4. In combination with euclidean distance (ED) and Δsingle-nucleotide polymorphism (SNP)-index analyses, we identified a genomic region on Gm11 (designated as rhg1-paralog) associated with SCN4 resistance. Overexpression and RNA interference analyzes of the two candidate genes identified in this region (GmPLAC8 and GmSNAP11) revealed that only GmSNAP11 significantly contributes to SCN4 resistance. We developed a diagnostic marker for GmSNAP11. Using this marker, together with previously developed markers for SCN-resistant loci, rhg1 and Rhg4, we evaluated the relationship between genotypes and SCN4 resistance in 145 RILs and 30 soybean accessions. The results showed that all the SCN4-resistant lines harbored all the three loci, however, some lines harboring the three loci were still susceptible to SCN4. This suggests that these three loci are necessary for the resistance to SCN4, but they alone cannot confer full resistance. The GmSNAP11 and the diagnostic markers developed could be used in genomic-assisted breeding to develop soybean varieties with increased resistance to SCN4.

Fine Mapping of Clubroot Resistance Loci CRA8.1 and Candidate Gene Analysis in Chinese Cabbage (Brassica rapa L.).

Clubroot is caused by Plasmodiophora brassicae, which threatens Brassicaceae crop production worldwide. In recent years, there has been an outbreak and rapid spread of clubroot in many major cruciferous crop-producing areas of China. In this study, we identified a cabbage material DingWen (DW) with different resistant capabilities from Huashuang5R (H5R) and Huayouza62R of Brassica napus, which are currently used as the main resistant cultivars for clubroot management in China. We used a next-generation sequencing-based bulked segregant analysis approach, combined with genetic mapping to identify clubroot-resistant (CR) genes from F1 population generated from a cross between the DW (CR) and HZSX (clubroot susceptible). The CR locus of DW (named CRA8.1) was mapped to a region between markers A08-4346 and A08-4853, which contains two different loci CRA8.1a and CRA8.1b after fine mapping. The CRA8.1b loci contain a fragment of 395 kb between markers A08-4624 and A08-4853 on A08 chromosome, and it is responsible for the resistance to PbZj and PbXm isolates. However, together with CRA8.1a, corresponding to a 765-kb region between markers A08-4346 and A08-4624, then it can confer resistance to PbXm+. Finally, through expression analysis between resistant and susceptible materials, two genes encoding TIR-NBS-LRR proteins (BraA08g039211E and BraA08g039212E) and one gene encoding an RLP protein (BraA08g039193E) were identified to be the most likely CR candidates for the peculiar resistance in DW.

Identification and Characterization of an Early Leaf Senescence Gene ELS1 in Soybean.

Early leaf senescence phenotype in soybean could be helpful to shorten the maturation period and prevent green stem disorder. From a high-density mutation library, we identified two early leaf senescence soybean mutant lines, els1-1 (early leaf senescence 1) and els1-2. The chlorophyll contents of both els1-1 and els1-2 were low in pre-senescent leaves. They degraded rapidly in senescent leaves, revealing that ELS1 is involved in chlorophyll biosynthesis during leaf development and chlorophyll degradation during leaf senescence. The causal mutations in els1 were identified by next-generation sequencing-based bulked segregant analysis. ELS1 encodes the ortholog of the Arabidopsis CaaX-like protease BCM1, which is localized in chloroplasts. Soybean ELS1 was highly expressed in green tissue, especially in mature leaves. The accumulation of photosystem I core proteins and light-harvesting proteins in els1 was low even in pre-senescent leaves, and their degradation was accelerated during leaf senescence. These results suggest that soybean ELS1 is involved in both chlorophyll synthesis and degradation, consistent with the findings in Arabidopsis BCM1. The gene els1, characterized by early leaf senescence and subsequent early maturation, does not affect the flowering time. Hence, the early leaf senescence trait regulated by els1 helps shorten the harvesting period because of early maturation characteristics. The els1-1 allele with weakly impaired function of ELS1 has only a small effect on agricultural traits and could contribute to practical breeding.

Next-generation sequencing-based bulked segregant analysis without sequencing the parental genomes.

Genomic regions that control traits of interest can be rapidly identified using BSA-Seq, a technology in which next-generation sequencing is applied to bulked segregant analysis (BSA). We recently developed the significant structural variant method for BSA-Seq data analysis that exhibits higher detection power than standard BSA-Seq analysis methods. Our original algorithm was developed to analyze BSA-Seq data in which genome sequences of one parent served as the reference sequences in genotype calling and, thus, required the availability of high-quality assembled parental genome sequences. Here, we modified the original script to effectively detect the genomic region–trait associations using only bulk genome sequences. We analyzed two public BSA-Seq datasets using our modified method and the standard allele frequency and G-statistic methods with and without the aid of the parental genome sequences. Our results demonstrate that the genomic region(s) associated with the trait of interest could be reliably identified via the significant structural variant method without using the parental genome sequences.

Optimization of BSA-seq experiment for QTL mapping.

Deep sequencing-based bulked segregant analysis (BSA-seq) has become a popular approach for quantitative trait loci (QTL) mapping in recent years. Effective statistical methods for BSA-seq have been developed, but how to design a suitable experiment for BSA-seq remains unclear. In this paper, we show in theory how the major experimental factors (including population size, pool proportion, pool balance, and generation) and the intrinsic factors of a QTL (including heritability and degree of dominance) affect the power of QTL detection and the precision of QTL mapping in BSA-seq. Increasing population size can improve the power and precision, depending on the QTL heritability. The best proportion of each pool in the population is around 0.25. So, 0.25 is generally applicable in BSA-seq. Small pool proportion can greatly reduce the power and precision. Imbalance of pool pair in size also causes decrease of the power and precision. Additive effect is more important than dominance effect for QTL mapping. Increasing the generation of filial population produced by selfing can significantly increase the power and precision, especially from F2 to F3. These findings enable researchers to optimize the experimental design for BSA-seq. A web-based program named BSA-seq Design Tool is available at http://124.71.74.135/BSA-seqDesignTool/ and https://github.com/huanglikun/BSA-seqDesignTool.

A Robust and Rapid Candidate Gene Mapping Pipeline Based on M2 Populations

The whole-genome sequencing-based bulked segregant analysis (WGS-BSA) has facilitated the mapping candidate causal variations for cloning target plant genes. Here, we report an improved WGS-BSA method termed as M2-seq to expedite the mapping candidate mutant loci by studying just M2 generation. It is an efficient mutant gene mapping tool, rapid, and comparable to the previously reported approaches, such as Mutmap and Mutmap+ that require studying M3 or advanced selfed generations. In M2-seq, background variations among the M2 populations can be removed efficiently without knowledge of the variations of the wild-type progenitor plant. Furthermore, the use of absolute delta single-nucleotide polymorphism (SNP) index values can effectively remove the background variation caused by repulsion phase linkages of adjacent mutant alleles; and thereby facilitating the identification of the causal mutation in target genes. Here, we demonstrated the application of M2-seq in successfully mapping the genomic regions harboring causal mutations for mutant phenotypes among 10 independent M2 populations of soybean. The mapping candidate mutant genes just in M2 generation with the aid of the M2-seq method should be particularly useful in expediting gene cloning especially among the plant species with long generation time.

Characterization on a Novel Rolled Leaves and Short Petioles Soybean Mutant Based on Seq-BSA and RNA-seq Analysis

A soybean (Glycine max (Linn.) Merr.) mutant displaying rolled leaves and short petioles was identified as being caused by multiple sites, named rlsp1. Compared to wild-type (WT), the phloem structure of petioles in rlsp1 was not obvious, while the thickness of leaves was significantly greater but the arrangement of leaves cells was relatively looser. Sequencing-based bulked segregant analysis (Seq-BSA) identified 10 candidate regions on chromosomes 3, 6, 8, 13, and 17 with total 6.47 Mb, containing 790 genes. 7946 and 5402 differentially expressed genes (DEGs) were also identified in leaves and petioles, respectively, by RNA-sequencing (RNA-seq). Glyma.03G128600 as orthologs of Arabidopsis BOP (BLADE ON PETIOLES) gene may be the key candidate gene causing the variation of rlsp1. The abnormal development of petiole phloem structure, inhibition of auxin signal transduction pathway, promotion of microtubule-related pathways, and repressed of carbohydrate processes were also closely associated with the formation of rlsp1. These results were further validated by the changes of phytohormones, sucrose, Ca2+, and chlorophyll. The candidate genes found in this study enrich the understanding of the molecular basis for the formation of rolled leaves and short petioles in soybean.

Setaria viridis chlorotic and seedling-lethal mutants define critical functions for chloroplast gene expression.

Deep insights into chloroplast biogenesis have been obtained by mutant analysis; however, in C4 plants a relevant mutant collection has only been developed and exploited for maize. Here, we report the initial characterization of an ethyl methyl sulfonate-induced mutant population for the C4 model Setaria viridis. Approximately 1000 M2 families were screened for the segregation of pale-green seedlings in the M3 generation, and a subset of these was identified to be deficient in post-transcriptional steps of chloroplast gene expression. Causative mutations were identified for three lines using deep sequencing-based bulked segregant analysis, and in one case confirmed by transgenic complementation. Using chloroplast RNA-sequencing and other molecular assays, we describe phenotypes of mutants deficient in PSRP7, a plastid-specific ribosomal protein, OTP86, an RNA editing factor, and cpPNP, the chloroplast isozyme of polynucleotide phosphorylase. The psrp mutant is globally defective in chloroplast translation, and has varying deficiencies in the accumulation of chloroplast-encoded proteins. The otp86 mutant, like its Arabidopsis counterpart, is specifically defective in editing of the rps14 mRNA; however, the conditional pale-green mutant phenotype contrasts with the normal growth of the Arabidopsis mutant. The pnp mutant exhibited multiple defects in 3' end maturation as well as other qualitative changes in the chloroplast RNA population. Overall, our collection opens the door to global analysis of photosynthesis and early seedling development in an emerging C4 model.

Mapping of QTLs conferring high grain length-breadth relative expansion during cooking in rice cultivar Paw San Hmwe.

Paw San Hmwe (PSH) is a high-quality rice cultivar from Myanmar. PSH has short and broad grains, but the grains become slender after cooking. This desirable feature can be described as a high value of grain length-breadth relative expansion index (GREI). To understand the genetic basis of high GREI in PSH, we crossed PSH with Guang 8B (G8B), a rice cultivar from China with low GREI, to develop an F2 population and a subsequent F2:3 population. Based on the phenotypes of these two populations measured in two years and using the method of sequencing-based bulked segregant analysis followed by verification with conventional linkage-based QTL mapping method, we mapped three QTLs for GREI. The three QTLs were located on chromosomes 3, 5 and 12, respectively, with the trait-increasing alleles all from PSH, and could explain a total of 62.5% of the phenotypic variance and 84.1% of the additive genetic variance. The results suggest that the three QTLs would be useful for the genetic improvement of GREI in rice, and the linked markers will facilitate the selection of the favorable alleles from PSH in breeding.

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.

RNA Sequencing-Based Bulked Segregant Analysis Facilitates Efficient D-genome Marker Development for a Specific Chromosomal Region of Synthetic Hexaploid Wheat.

Common wheat originated from interspecific hybridization between cultivated tetraploid wheat and its wild diploid relative Aegilops tauschii followed by amphidiploidization. This evolutionary process can be reproduced artificially, resulting in synthetic hexaploid wheat lines. Here we performed RNA sequencing (RNA-seq)-based bulked segregant analysis (BSA) using a bi-parental mapping population of two synthetic hexaploid wheat lines that shared identical A and B genomes but included with D-genomes of distinct origins. This analysis permitted identification of D-genome-specific polymorphisms around the Net2 gene, a causative locus to hybrid necrosis. The resulting single nucleotide polymorphisms (SNPs) were classified into homoeologous polymorphisms and D-genome allelic variations, based on the RNA-seq results of a parental tetraploid and two Ae. tauschii accessions. The difference in allele frequency at the D-genome-specific SNP sites between the contrasting bulks (ΔSNP-index) was higher on the target chromosome than on the other chromosomes. Several SNPs with the highest ΔSNP-indices were converted into molecular markers and assigned to the Net2 chromosomal region. These results indicated that RNA-seq-based BSA can be applied efficiently to a synthetic hexaploid wheat population to permit molecular marker development in a specific chromosomal region of the D genome.

Identification and Mapping of the Clubroot Resistance Gene CRd in Chinese Cabbage (Brassica rapa ssp. pekinensis).

The rapid spread of clubroot disease, which is caused by Plasmodiophora brassicae, threatens Brassicaceae crop production worldwide. Breeding plants that have broad-spectrum disease resistance is one of the best ways to prevent clubroot. In the present study, eight Chinese cabbage germplasms were screened using published clubroot-resistant (CR) loci-/gene-linked markers. A CR gene Crr3 potential carrier “85-74” was detected which linked to marker BRSTS61; however, “85-74” shows different responses to local pathogens “LAB-19,” “LNND-2,” and “LAB-10” from “CR-73” which harbors Crr3. We used a next-generation sequencing-based bulked segregant analysis approach combined with genetic mapping to detect CR genes in an F2 segregant population generated from a cross between the Chinese cabbage inbred lines “85-74” (CR) and “BJN3-1” (clubroot susceptible). The “85-74” line showed resistance to a local pathogen “LAB-19” which was identified as race 4; a genetic analysis revealed that the resistance was conferred by a single dominant gene. The CR gene which we named CRd was mapped to a 60 kb (1 cM) region between markers yau389 and yau376 on chromosome A03. CRd is located upstream of Crr3 which was confirmed based on the physical positions of Crr3 linked markers. The identification of CRd linked markers can be applied to marker-assisted selection in the breeding of new CR cultivars of Chinese cabbage and other Brassica crops.

Rapid mapping of candidate genes for cold tolerance in Oryza rufipogon Griff. by QTL-seq of seedlings

Cold stress is a major problem in rice production. To rapidly identify genes for cold tolerance in Dongxiang wild rice (DWR, Oryza rufipogon Griff.), sequencing-based bulked segregant analysis of QTL-seq method was used to resequence the extremely resistant (R) and susceptible (S) bulks of a backcross inbred lines (BILs) population (derived from Oryza sativa×O. rufipogon) and their parents. Single nucleotide polymorphisms (SNP)-index graphs and corresponding ρ(SNP-index) graphs (at 99 and 95% confidence levels) for R- and S-bulks detected a total of 2 609 candidate SNPs, including 58 candidate cold-tolerance genes. Quantitative real-time PCR analysis revealed that 5 out of the 58 candidate genes had significant differences in expression between O. sativa and O. rufipogon. Structural variation and functional annotations of the 5 candidate genes were also analyzed, and allowed us to identify 2 insertion-deletion (InDel) markers (12-7 and 12-16) that were linked with candidate genes on chromosome 12 in DWR. These results are helpful for cloning and using cold tolerance genes from common wild rice in cultivated rice.

Exploring DNA variant segregation types in pooled genome sequencing enables effective mapping of weeping trait in Malus.

To unlock the power of next generation sequencing-based bulked segregant analysis in allele discovery in out-crossing woody species, and to understand the genetic control of the weeping trait, an F1 population from the cross 'Cheal's Weeping' × 'Evereste' was used to create two genomic DNA pools 'weeping' (17 progeny) and 'standard' (16 progeny). Illumina pair-end (2 × 151 bp) sequencing of the pools to a 27.1× (weeping) and a 30.4× (standard) genome (742.3 Mb) coverage allowed detection of 84562 DNA variants specific to 'weeping', 92148 specific to 'standard', and 173169 common to both pools. A detailed analysis of the DNA variant genotypes in the pools predicted three informative segregation types of variants: <lm×mm> (type I) in weeping pool-specific variants, and <lm×ll> (type II) and <hk×hk> (type III) in variants common to both pools, where the first allele is assumed to be weeping linked and the allele shown in bold is a variant in relation to the reference genome. Conducting variant allele frequency and density-based mappings revealed four genomic regions with a significant association with weeping: a major locus, Weeping (W), on chromosome 13 and others on chromosomes 10 (W2), 16 (W3), and 5 (W4). The results from type I variants were noisier and less certain than those from type II and type III variants, demonstrating that although type I variants are often the first choice, type II and type III variants represent an important source of DNA variants that can be exploited for genetic mapping in out-crossing woody species. Confirmation of the mapping of W and W2, investigation into their genetic interactions, and identification of expressed genes in the W and W2 regions provided insight into the genetic control of weeping and its expressivity in Malus.

Coupling Seq-BSA and RNA-Seq Analyses Reveal the Molecular Pathway and Genes Associated with Heading Type in Chinese Cabbage.

In Chinese cabbage, heading type is a key agricultural trait of significant economic importance. Using a natural microspore-derived doubled haploid plant, we generated self-crossed progeny with overlapping or outward curling head morphotypes. Sequencing-based bulked segregant analysis (Seq-BSA) revealed a candidate region of 0.52 Mb (A06: 1,824,886~2,347,097 bp) containing genes enriched for plant hormone signal transduction. RNA Sequencing (RNA-Seq) analysis supported the hormone pathway enrichment leading to the identification of two key candidate genes, BrGH3.12 and BrABF1. The regulated homologous genes and the relationship between genes in this pathway were also revealed. Expression of BrGH3.12 varied significantly in the apical portion of the leaf, consistent with the morphological differences between overlapping and outward curling leaves. Transcript levels of BrABF1 in the top, middle and basal segments of the leaf were significantly different between the two types. The two morphotypes contained different concentrations of IAA in the apical portion of their leaves while levels of ABA differed significantly between plant types in the top, middle, and basal leaf segments. Results from Seq-BSA, RNA-Seq and metabolite analyses all support a role for IAA and ABA in heading type formation. These findings increase our understanding of the molecular basis for pattern formation of the leafy head in Chinese cabbage and will contribute to future work developing more desirable leafy head patterns.

Fast neutron-induced structural rearrangements at a soybean NAP1 locus result in gnarled trichomes.

Key messageThree adjacent and distinct sequence rearrangements were identified at a NAP1 locus in a soybean mutant. Genetic dissection and validation revealed the function of this gene in soybean trichome development.A soybean (Glycine max (L.) Merr.) gnarled trichome mutant, exhibiting stunted trichomes compared to wild-type, was identified in a fast neutron mutant population. Genetic mapping using whole genome sequencing-based bulked segregant analysis identified a 26.6 megabase interval on chromosome 20 that co-segregated with the phenotype. Comparative genomic hybridization analysis of the mutant indicated that the chromosome 20 interval included a small structural variant within the coding region of a soybean ortholog (Glyma.20G019300) of Arabidopsis Nck-Associated Protein 1 (NAP1), a regulator of actin nucleation during trichome morphogenesis. Sequence analysis of the candidate allele revealed multiple rearrangements within the coding region, including two deletions (approximately 1–2 kb each), a translocation, and an inversion. Further analyses revealed that the mutant allele perfectly co-segregated with the phenotype, and a wild-type soybean NAP1 transgene functionally complemented an Arabidopsis nap1 mutant. In addition, mapping and exon sequencing of NAP1 in a spontaneous soybean gnarled trichome mutant (T31) identified a frame shift mutation resulting in a truncation of the coding region. These data indicate that the soybean NAP1 gene is essential for proper trichome development and show the utility of the soybean fast neutron population for forward genetic approaches for identifying genes.Electronic supplementary materialThe online version of this article (doi:10.1007/s00122-016-2735-x) contains supplementary material, which is available to authorized users.

Next-generation sequencing for identification of candidate genes for Fusarium wilt and sterility mosaic disease in pigeonpea (Cajanus cajan).

SummaryTo map resistance genes for Fusarium wilt (FW) and sterility mosaic disease (SMD) in pigeonpea, sequencing‐based bulked segregant analysis (Seq‐BSA) was used. Resistant (R) and susceptible (S) bulks from the extreme recombinant inbred lines of ICPL 20096 × ICPL 332 were sequenced. Subsequently, SNP index was calculated between R‐ and S‐bulks with the help of draft genome sequence and reference‐guided assembly of ICPL 20096 (resistant parent). Seq‐BSA has provided seven candidate SNPs for FW and SMD resistance in pigeonpea. In parallel, four additional genotypes were re‐sequenced and their combined analysis with R‐ and S‐bulks has provided a total of 8362 nonsynonymous (ns) SNPs. Of 8362 nsSNPs, 60 were found within the 2‐Mb flanking regions of seven candidate SNPs identified through Seq‐BSA. Haplotype analysis narrowed down to eight nsSNPs in seven genes. These eight nsSNPs were further validated by re‐sequencing 11 genotypes that are resistant and susceptible to FW and SMD. This analysis revealed association of four candidate nsSNPs in four genes with FW resistance and four candidate nsSNPs in three genes with SMD resistance. Further, In silico protein analysis and expression profiling identified two most promising candidate genes namely C.cajan_01839 for SMD resistance and C.cajan_03203 for FW resistance. Identified candidate genomic regions/SNPs will be useful for genomics‐assisted breeding in pigeonpea.