Project description:The growth habit of lateral shoots (also termed "branching habit") is an important descriptive and agronomic character of peanut. Yet, both the inheritance of branching habit and the genetic mechanism that controls it in this crop remain unclear. In addition, the low degree of polymorphism among cultivated peanut varieties hinders fine-mapping of this and other traits in non-homozygous genetic structures. Here, we combined high-throughput sequencing with a well-defined genetic system to study these issues in peanut. Initially, segregating F2 populations derived from a reciprocal cross between very closely related Virginia-type peanut cultivars with spreading and bunch growth habits were examined. The spreading/bunch trait was shown to be controlled by a single gene with no cytoplasmic effect. That gene was named Bunch1 and was significantly correlated with pod yield per plant, time to maturation and the ratio of "dead-end" pods. Subsequently, bulked segregant analysis was performed on 52 completely bunch, and 47 completely spreading F3 families. In order to facilitate the process of SNP detection and candidate-gene analysis, the transcriptome was used instead of genomic DNA. Young leaves were sampled and bulked. Reads from Illumina sequencing were aligned against the peanut reference transcriptome and the diploid genomes. Inter-varietal SNPs were detected, scored and quality-filtered. Thirty-four candidate SNPs were found to have a bulk frequency ratio value >10 and 6 of those SNPs were found to be located in the genomic region of linkage group B5. Three best hits from that over-represented region were further analyzed in the segregating population. The trait locus was found to be located in a ~1.1 Mbp segment between markers M875 (B5:145,553,897; 1.9 cM) and M255 (B5:146,649,943; 2.25 cM). The method was validated using a population of recombinant inbreed lines of the same cross and a new DNA SNP-array. This study demonstrates the relatively straight-forward utilization of bulk segregant analysis for trait fine-mapping in the low polymeric and heterozygous germplasm of cultivated peanut and provides a baseline for candidate gene discovery and map-based cloning of Bunch1.
Project description:Peanut (Arachis hypogaea. L) is an important oil crop worldwide. The common testa colours of peanut varieties are pink or red. But the peanut varieties with dark purple testa have been focused in recent years due to the potential high levels of anthocyanin, an added nutritional value of antioxidant. However, the genetic mechanism regulating testa colour of peanut is unknown. In this study, we found that the purple testa was decided by the female parent and controlled by a single major gene named AhTc1. To identify the candidate gene controlling peanut purple testa, whole-genome resequencing-based approach (QTL-seq) was applied, and a total of 260.9 Gb of data were generated from the parental and bulked lines. SNP index analysis indicated that AhTc1 located in a 4.7 Mb region in chromosome A10, which was confirmed by bulked segregant RNA sequencing (BSR) analysis in three segregation populations derived from the crosses between pink and purple testa varieties. Allele-specific markers were developed and demonstrated that the marker pTesta1089 was closely linked with purple testa. Further, AhTc1 encoding a R2R3-MYB gene was positional cloned. The expression of AhTc1 was significantly up-regulated in the purple testa parent YH29. Overexpression of AhTc1 in transgenic tobacco plants led to purple colour of leaves, flowers, pods and seeds. In conclusion, AhTc1, encoding a R2R3-MYB transcription factor and conferring peanut purple testa, was identified, which will be useful for peanut molecular breeding selection for cultivars with purple testa colour for potential increased nutritional value to consumers.
Project description:BACKGROUND: The application of RNA-seq has accelerated gene expression profiling and identification of gene-associated SNPs in many species. However, the integrated studies of gene expression along with SNP mapping have been lacking. Coupling of RNA-seq with bulked segregant analysis (BSA) should allow correlation of expression patterns and associated SNPs with the phenotypes. RESULTS: In this study, we demonstrated the use of bulked segregant RNA-seq (BSR-Seq) for the analysis of differentially expressed genes and associated SNPs with disease resistance against enteric septicemia of catfish (ESC). A total of 1,255 differentially expressed genes were found between resistant and susceptible fish. In addition, 56,419 SNPs residing on 4,304 unique genes were identified as significant SNPs between susceptible and resistant fish. Detailed analysis of these significant SNPs allowed differentiation of significant SNPs caused by genetic segregation and those caused by allele-specific expression. Mapping of the significant SNPs, along with analysis of differentially expressed genes, allowed identification of candidate genes underlining disease resistance against ESC disease. CONCLUSIONS: This study demonstrated the use of BSR-Seq for the identification of genes involved in disease resistance against ESC through expression profiling and mapping of significantly associated SNPs. BSR-Seq is applicable to analysis of genes underlining various performance and production traits without significant investment in the development of large genotyping platforms such as SNP arrays.
Project description:KEY MESSAGE:Using bulked segregant analysis of exome sequence, we fine-mapped the ABA-hypersensitive mutant ERA8 in a wheat backcross population to the TaMKK3-A locus of chromosome 4A. Preharvest sprouting (PHS) is the germination of mature grain on the mother plant when it rains before harvest. The ENHANCED RESPONSE TO ABA8 (ERA8) mutant increases seed dormancy and, consequently, PHS tolerance in soft white wheat 'Zak.' ERA8 was mapped to chromosome 4A in a Zak/'ZakERA8' backcross population using bulked segregant analysis of exome sequenced DNA (BSA-exome-seq). ERA8 was fine-mapped relative to mutagen-induced SNPs to a 4.6 Mb region containing 70 genes. In the backcross population, the ERA8 ABA-hypersensitive phenotype was strongly linked to a missense mutation in TaMKK3-A-G1093A (LOD 16.5), a gene associated with natural PHS tolerance in barley and wheat. The map position of ERA8 was confirmed in an 'Otis'/ZakERA8 but not in a 'Louise'/ZakERA8 mapping population. This is likely because Otis carries the same natural PHS susceptible MKK3-A-A660S allele as Zak, whereas Louise carries the PHS-tolerant MKK3-A-C660R allele. Thus, the variation for grain dormancy and PHS tolerance in the Louise/ZakERA8 population likely resulted from segregation of other loci rather than segregation for PHS tolerance at the MKK3 locus. This inadvertent complementation test suggests that the MKK3-A-G1093A mutation causes the ERA8 phenotype. Moreover, MKK3 was a known ABA signaling gene in the 70-gene 4.6 Mb ERA8 interval. None of these 70 genes showed the differential regulation in wild-type Zak versus ERA8 expected of a promoter mutation. Thus, the working model is that the ERA8 phenotype results from the MKK3-A-G1093A mutation.
Project description:Bacterial wilt (BW) caused by Ralstonia solanacearum is a serious, global, disease of peanut (Arachis hypogaea L.), but it is especially destructive in China. Identification of DNA markers linked to the resistance to this disease will help peanut breeders efficiently develop resistant cultivars through molecular breeding. A F2 population, from a cross between disease-resistant and disease-susceptible cultivars, was used to detect quantitative trait loci (QTL) associated with the resistance to this disease in the cultivated peanut. Genome-wide SNPs were identified from restriction-site-associated DNA sequencing tags using next-generation DNA sequencing technology. SNPs linked to disease resistance were determined in two bulks of 30 resistant and 30 susceptible plants along with two parental plants using bulk segregant analysis. Polymorphic SSR and SNP markers were utilized for construction of a linkage map and for performing the QTL analysis, and a moderately dense linkage map was constructed in the F2 population. Two QTL (qBW-1 and qBW-2) detected for resistance to BW disease were located in the linkage groups LG1 and LG10 and account for 21 and 12 % of the bacterial wilt phenotypic variance. To confirm these QTL, the F8 RIL population with 223 plants was utilized for genotyping and phenotyping plants by year and location as compared to the F2 population. The QTL qBW-1 was consistent in the location of LG1 in the F8 population though the QTL qBW-2 could not be clarified due to fewer markers used and mapped in LG10. The QTL qBW-1, including four linked SNP markers and one SSR marker within 14.4-cM interval in the F8, was closely related to a disease resistance gene homolog and was considered as a candidate gene for resistance to BW. QTL identified in this study would be useful to conduct marker-assisted selection and may permit cloning of resistance genes. Our study shows that bulk segregant analysis of genome-wide SNPs is a useful approach to expedite the identification of genetic markers linked to disease resistance traits in the allotetraploidy species peanut.
Project description:BACKGROUND:Although rapeseed (Brassica napus L.) mutant forming multiple siliques was morphologically described and considered to increase the silique number per plant, an important agronomic trait in this crop, the molecular mechanism underlying this beneficial trait remains unclear. Here, we combined bulked-segregant analysis (BSA) and whole genome re-sequencing (WGR) to map the genomic regions responsible for the multi-silique trait using two pools of DNA from the near-isogenic lines (NILs) zws-ms (multi-silique) and zws-217 (single-silique). We used the Euclidean Distance (ED) to identify genomic regions associated with this trait based on both SNPs and InDels. We also conducted transcriptome sequencing to identify differentially expressed genes (DEGs) between zws-ms and zws-217. RESULTS:Genetic analysis using the ED algorithm identified three SNP- and two InDel-associated regions for the multi-silique trait. Two highly overlapped parts of the SNP- and InDel-associated regions were identified as important intersecting regions, which are located on chromosomes A09 and C08, respectively, including 2044 genes in 10.20-MB length totally. Transcriptome sequencing revealed 129 DEGs between zws-ms and zws-217 in buds, including 39 DEGs located in the two abovementioned associated regions. We identified candidate genes involved in multi-silique formation in rapeseed based on the results of functional annotation. CONCLUSIONS:This study identified the genomic regions and candidate genes related to the multi-silique trait in rapeseed.
Project description:BACKGROUND:Analysis of mutants isolated from forward-genetic screens has revealed key components of several plant signalling pathways. Mapping mutations by position, either using classical methods or whole genome high-throughput sequencing (HTS), largely relies on the analysis of genome-wide polymorphisms in F2 recombinant populations. Combining bulk segregant analysis with HTS has accelerated the identification of causative mutations and has been widely adopted in many research programmes. A major advantage of HTS is the ability to perform bulk segregant analysis after back-crossing to the parental line rather than out-crossing to a polymorphic ecotype, which reduces genetic complexity and avoids issues with phenotype penetrance in different ecotypes. Plotting the positions of homozygous polymorphisms in a mutant genome identifies areas of low recombination and is an effective way to detect molecular linkage to a phenotype of interest. RESULTS:We describe the use of single nucleotide polymorphism (SNP) density plots as a mapping strategy to identify and refine chromosomal positions of causative mutations from screened plant populations. We developed a web application called CandiSNP that generates density plots from user-provided SNP data obtained from HTS. Candidate causative mutations, defined as SNPs causing non-synonymous changes in annotated coding regions are highlighted on the plots and listed in a table. We use data generated from a recent mutant screen in the model plant Arabidopsis thaliana as proof-of-concept for the validity of our tool. CONCLUSIONS:CandiSNP is a user-friendly application that will aid in novel discoveries from forward-genetic mutant screens. It is particularly useful for analysing HTS data from bulked back-crossed mutants, which contain fewer polymorphisms than data generated from out-crosses. The web-application is freely available online at http://candisnp.tsl.ac.uk.
Project description:We have identified 645,088 candidate polymorphisms in zebrafish and observe a single nucleotide polymorphism (SNP) validation rate of 71% to 86%, improving with polymorphism confidence score. Variant sites are non-random, with an excess of specific novel T- and A-rich motifs. We positioned half of the polymorphisms on zebrafish genetic and physical maps as a resource for positional cloning. We further demonstrate bulked segregant analysis using the anchored SNPs as a method for high-throughput genetic mapping in zebrafish.
Project description:Host plant resistance has been widely used for controlling the major rice pest brown planthopper (BPH, Nilaparvata lugens). However, adaptation of the wild BPH population to resistance limits the effective use of resistant rice varieties. Quantitative trait locus (QTL) analysis was conducted to identify resistance-breaking genes against the anti-feeding mechanism mediated by the rice resistance gene Bph1. QTL analysis in iso-female BPH lines with single-nucleotide polymorphism (SNP) markers detected a single region on the 10th linkage group responsible for the virulence. The QTL explained from 57 to 84% of the total phenotypic variation. Bulked segregant analysis with next-generation sequencing in F2 progenies identified five SNPs genetically linked to the virulence. These analyses showed that virulence to Bph1 was controlled by a single recessive gene. In contrast to previous studies, the gene-for-gene relationship between the major resistance gene Bph1 and virulence gene of BPH was confirmed. Identified markers are available for map-based cloning of the major gene controlling BPH virulence to rice resistance.
Project description:Soybean is one of the most important food and oil crops in the world. Plant height (PH) and the number of nodes on the main stem (NNMS) are quantitative traits closely related to soybean yield. In this study, we used 208 chromosome segment substitution lines (CSSL) populations constructed using "SN14" and "ZYD00006" for quantitative trait locus (QTL) mapping of PH and NNMS. Combined with bulked segregant analysis (BSA) by extreme materials, 8 consistent QTLs were identified. According to the gene annotation of the QTL interval, a total of 335 genes were obtained. Five of which were associated with PH and NNMS, potentially representing candidate genes. RT-qPCR of these 5 candidate genes revealed two genes with differential relative expression levels in the stems of different materials. Haplotype analysis showed that different single nucleotide polymorphisms (SNPs) between the excellent haplotypes in Glyma.04G251900 and Glyma.16G156700 may be the cause of changes in these traits. These results provide the basis for research on candidate genes and marker-assisted selection (MAS) in soybean breeding.