Bulked Segregant Analysis to Detect QTL Related to Heat Tolerance in Rice(Oryza sativa L.) Using SSR Markers

The study was undertaken to assess the genetic effect of quantitative trait loci （QTLs） conferring heat tolerance at flowering stage in rice. A population consisting of 279 F2 individuals from the cross between 996, a heat tolerant cultivar and 4628, a heat-sensitive cultivar, was analyzed for their segregation pattern of the difference of seed set rate under optimal temperature condition and high temperature condition. The difference of seed set rate under optimal temperature condition and high temperature condition showed normal distribution, indicating the polygenic control over the trait. To identify main effect of QTL for heat tolerance, the parents were surveyed with 200 primer pairs of simple sequence repeats （SSR）. The parental survey revealed 30% polymorphism between parents. In order to detect the main QTL association with heat tolerance, a strategy of combining the DNA pooling from selected segregants and genotyping was adopted. The association of putative markers identified based on DNA pooling from selected segregants was established by single marker analysis （SMA）. The results of SMA revealed that SSR markers, RM3735 on chromosome 4 and RM3586 on chromosome 3 showed significant association with heat tolerance respectively, accounted for 17 and 3% of the total variation respectively. The heat tolerance during flowering stage in rice was controlled by multiple gene. The SSR markers, RM3735 on chromosome 4 and RM3586 on chromosome 3 showed significant association with heat tolerance respectively, accounted for 17 and 3% of the total variation respectively. The two genetic loci, especially for RM3735 on chromosome 4, can be used in marker-assistant-selected method in heat tolerance breeding in rice.

Evaluation and Bulked Segregant Analysis of Major Yield QTL qtl12.1 Introgressed into Indigenous Elite Line for Low Water Availability under Water Stress

Near isogenic lines carrying large-effect QTL （qtl2.1）, which has a consistent influence on grain yield under upland drought stress conditions in a wide range of environments, were evaluated under water stress in the fields. The line which gave higher yield under drought was crossed with a local elite line, PMK3, and forwarded to F2：3 generation. Significant variation was found among the F2：3 lines for agronomic traits under water stress in the fields. Low to high broad sense heritability （H） for investigated traits was also found. Water stress indicators such as leaf rolling and leaf drying were negatively correlated with plant height, biomass and grain yield under stress. Bulked segregant analysis （BSA） was performed with the markers in the vicinity of qUl2.1, and RM27933 was found to be segregated perfectly well in individual components of drought resistant and drought susceptible bulks which were bulked based on yield under water stress among F2：3 lines. Hence, this simple and breeder friendly marker, RM27933, may be useful as a potentially valuable candidate marker for the transfer of the QTL qtl12.1 in the regional breeding program. Bioinformatic analysis of the DNA sequence of the qtl12.1 region was also done to identify and analyze positional candidate genes associated with this QTL and to ascertain the putative molecular basis of qUl2.1.

Tagging of Brown Planthopper Resistance Genes in F_2s of IR50 × Ptb33 of Rice by Using Bulked Segregant Analysis

Brown planthopper （Nilaparvata lugens Stal） is one of the most damaging pests causing hopper burn in rice, and thereby reducing the productivity and also the quality of the product. The effective management strategy to control this pest is the identification and transfer of desirable genes to local rice cultivars. The most important approach for developing resistant cultivars is the identification of markers, which can help in marker-assisted selection of more durable resistant genotype. The susceptible parent IR50 and the resistant parent Ptb33, and their F2 populations were used in bulked segregant analysis for identification of resistant genes with random amplified polymorphic DNA marker （RAPD） primers. The primers OPC7 and OPAG14 showed both dominant and susceptible specific banding pattern so called co-dominant markers. Moreover, OPC7697 and OPAG14680 showed resistant specific bands and thus being in coupling phase, whereas OPC7846 and OPAG14650 showed susceptible specific genotypic bands in bulked segregant analysis. Therefore, the coupling phase markers, OPC7697 and OPAG14680, are considered to be more useful in marker-assisted selection of rice genotypes in crop improvement.

DeepBSA: A deep-learning algorithm improves bulked segregant analysis for dissecting complex traits

Bulked segregant analysis(BSA)is a rapid,cost-effective method for mapping mutations and quantitative trait loci(QTLs)in animals and plants based on high-throughput sequencing.However,the algorithms currently used for BSA have not been systematically evaluated and are complex and fallible to operate.We developed a BSA method driven by deep learning,DeepBSA,for QTL mapping and functional gene cloning.DeepBSA is compatible with a variable number of bulked pools and performed well with various simulated and real datasets in both animals and plants.DeepBSA outperformed all other algorithms when comparing absolute bias and signal-to-noise ratio.Moreover,we applied DeepBSA to an F2 segregating maize population of 7160 individuals and uncovered five candidate QTLs,including three well-known plant-height genes.Finally,we developed a user-friendly graphical user interface for DeepBSA,by integrating five widely used BSA algorithms and our two newly developed algorithms,that is easy to operate and can quickly map QTLs and functional genes.The DeepBSA software is freely available to noncommercial users at http://zeasystemsbio.hzau.edu.cn/tools.html and https://github.com/lizhao007/DeepBSA.

Fine-mapping and primary analysis of candidate genes associated with seed coat color in mung bean(Vigna radiata L.)

Seed coat color affects the appearance and commodity quality of mung beans(Vigna radiata L.).The substances that affect mung bean seed coat color are mainly flavonoids,which have important medicinal value.Mapping the seed coat color gene in mung beans would facilitate the development of new varieties and improve their value.In this study,an F2 mapping population consisting of 546 plants was constructed using Jilv9(black seed coat)and BIS9805(green seed coat).Using bulk segregated analysis(BSA)sequencing and kompetitive allele-specific PCR(KASP)markers,the candidate region related to seed coat color was finally narrowed to 0.66 Mb on chromosome(Chr.)4 and included eight candidate genes.Combined transcriptome and metabolome analyses showed that three of the eight candidate genes(LOC106758748,LOC106758747,and LOC106759075)were differentially expressed,which may have caused the differences in flavonoid metabolite content between Jilv9 and BIS9805.These findings can provide a research basis for cloning the genes related to seed coat color and accelerate molecular markerassisted selection breeding in mung beans.

Integrated genomic and transcriptomic analysis reveals genes associated with plant height of foxtail millet

Foxtail millet(Setaria italica)is an important C4 model crop;however,due to its high-density planting and high stature,lodging at the filling stage resulted in a serious reduction in yield and quality.Therefore,it is imperative to identify and deploy the genes controlling foxtail millet plant height.In this study,we used a semi-dwarf line 263A and an elite high-stalk breeding variety,Chuang 29 to construct an F2 population to identify dwarf genes.We performed transcriptome analysis(RNA-seq)using internode tissues sampled at three jointing stages of 263A and Chuang 29,as well as bulk segregant analysis(BSA)on their F2 population.A total of 8918 differentially expressed genes(DEGs)were obtained from RNA-seq analysis,and GO analysis showed that DEGs were enriched in functions such as‘‘gibberellin metabolic process”and‘‘oxidoreductase activity”,which have previously been shown to be associated with plant height.A total 593 mutated genes were screened by BSA-seq method.One hundred and seventy-six out of the 593 mutated genes showed differential expression levels between the two parental lines,and seven genes not only showed differential expression in two or three internode tissues but also showed high genomic variation in coding regions,which indicated they play a crucial role in plant height determination.Among them,we found a gibberellin biosynthesis related GA20 oxidase gene(Seita.5G404900),which had a single-base at the third exon,leading to the frameshift mutation at 263A.Cleaved amplified polymorphic sequence assay and association analysis proved the single-base in Seita.5G404900 co-segregated with dwarf phenotype in two independent F2 populations planted in entirely different environments.Taken together,the candidate genes identified in this study will help to elucidate the genetic basis of foxtail millet plant height,and the molecular marker will be useful for marker-assisted dwarf breeding.

Fine-mapping of a candidate gene for web blotch resistance in Arachis hypogaea L.

Peanut(Arachis hypogaea L.)is a globally important oil crop.Web blotch is one of the most important foliar diseases affecting peanut,which results in serious yield losses worldwide.Breeding web blotch-resistant peanut varieties is the most effective and economically viable method for minimizing yield losses due to web blotch.In the current study,a bulked segregant analysis with next-generation sequencing was used to analyze an F2:3 segregating population and identify candidate loci related to web blotch resistance.Based on the fine-mapping of the candidate genomic interval using kompetitive allele-specific PCR(KASP)markers,we identified a novel web blotch resistance-related locus spanning approximately 169 kb on chromosome 16.This region included four annotated genes,of which only Arahy.35VVQ3 had a non-synonymous single nucleotide polymorphism in the coding region between the two parents.Two markers(Chr.16.12872635 and Chr.16.12966357)linked to this gene were shown to be co-segregated with the resistance of peanut web blotch by 72 randomly selected recombinant inbred lines(RIL),which could be used in marker-assisted breeding of resistant peanut varieties.

Mapping of wheat stripe rust resistance gene Yr041133 by BSR-Seq analysis

Puccinia striiformis Westend. f. sp. tritici(Pst) pathotype CYR34 is widely virulent and prevalent in China.Here, we report identification of a strpie rust resistance(Yr) gene, designated Yr041133, in winter wheat line 041133. This line produced a hypersensitive reaction to CYR34 and conferred resistance to 13 other pathotypes. Resistance to CYR34 in line 041133 was controlled by a single dominant gene. Bulked segregant RNA sequencing(BSR-Seq) was performed on a pair of RNA bulks generated by pooling resistant and susceptible recombinant inbred lines. Yr041133 was mapped to a 1.7 c M genetic interval on the chromosome arm 7 BL that corresponded to a 0.8 Mb physical interval(608.9–609.7 Mb) in the Chinese Spring reference genome. Based on its unique physical location Yr041133 differred from the other Yr genes on this chromosome arm.

A 1-bp deletion in the MC04g1399 is highly associated with failure to produce fruit wart in bitter gourd

Fruit wart is an important appearance trait influencing consumer preferences of bitter gourd(Momordica charantia L.).The molecular genetic mechanisms underlying fruit wart formation in bitter gourd are largely unknown.In this study,genetic analysis based on four generations showed that fruit wart formation in bitter gourd was controlled by a single dominant locus named as Fwa.The Fwa locus was initially mapped into a 4.82 Mb region on pseudochromosome 4 by BSA-seq analysis and subsequently narrowed down to a 286.30 kb region by linkage analysis.A large F2population consisting of 2360 individuals was used to screen recombinants,and the Fwa locus was finally fine mapped into a 22.70 kb region harboring four protein-coding genes through recombination analysis.MC04g1399,encoding an epidermal patterning factor 2-like protein,was proposed as the best candidate gene for Fwa via sequence variation and expression analysis.In addition,a 1-bp insertion and deletion(InDel)variation within MC04g1399 was converted to a cleaved amplified polymorphic sequence(CAPS)marker that could precisely distinguish between the warty and non-warty types with an accuracy rate of 100%among a wide panel of 126 bitter gourd germplasm resources.Our results not only provide a scientific basis for deciphering the molecular mechanisms underlying fruit wart formation but also provide a powerful tool for efficient genetic improvement of fruit wart via marker-assisted selection.

Development of Sequence Characterized Amplified Region (SCAR) Primers for the Detection of Resistance to Sporisorium reiliana in Maize

Head smut of maize （Zea mays L.）, which was caused by Sporisorium reiliana, occurred in most of the maize growing areas of the world. The purpose of this study was to develop SCAR markers for map-based cloning of resistance genes and MAS. Two sets of BC3 progenies, one （BC3Q） derived from the cross Qi319 （resistance）×Huangzao 4 （susceptible）, the other （BC3M） from Mol7 （resistance）× Huangzao 4 （susceptible）, were generated. Huangzao 4 was the recurrent parent in both progenies. A combination of BSA （bulked segregant analysis） with AFLP （amplified fragment length polymorphism） method was applied to map the genes involving the resistance to S. reiliana, and corresponding resistant and susceptible bulks and their parental lines were used for screening polymorphic AFLP primer pairs. One fragment of PI3M61-152 was converted into SCAR （sequence charactered amplified fragment） marker S130. The marker was mapped at chromosome bin 2.09, the interval of a major QTL region previously reported to contribute to S. reiliana resistance. Furthermore, S130 was highly and facilitate map-based cloni associated with resistance to S. reiliana, and could be useful for marker-assisted selection ng of resistance genes.

A Co-Dominant Marker BoE332 Applied to Marker-Assisted Selection of Homozygous Male-Sterile Plants in Cabbage (Brassica oleracea var.capitata L.)

The dominant genic male sterility （DGMS） gene CDMs399-3 derived from a spontaneous mutation in the line 79-399-3 of spring cabbage （Brassica oleracea var. capitata L.）, has been successfully applied in hybrid seed production of several cabbage cultivars in China. During the development of dominant male sterility lines in cabbage, the conventional identification of homozygous male-sterile plants （CDMs399-3/CDMs399-3） is a laborious and time-consuming process. For marker-assisted selection （MAS） of the gene CDMs399-3 transferred into key spring cabbage line 397, expressed sequence tag-simple sequence repeats （EST-SSR） and SSR technology were used to identify markers that were linked to CDMs399-3 based on method of bulked segregant analysis （BSA）. By screening a set of 978 EST-SSRs and 395 SSRs, a marker BoE332 linked to the CDMs399-3 at a distance of 3.6 cM in the genetic background of cabbage line 397 were identified. 7 homozygons male-sterile plants in population P1170 with 20 plants were obtained finally via MAS of BoE332. Thus, BoE332 will greatly facilitate the transferring of the gene CDMs399-3 into the key spring cabbage line 397 and improve the application of DGMS in cabbage hybrid breeding.

Identification RAPD Markers Linked to Texture Quality of Chinese Cabbage

[ Objectives] This study was conducted to identify the random amplification of polymorphic DNA （RAPD） markers linked to chewy texture-controlling gene of Chinese cabbage. [ Methods] The RAPD markers associated with chewy texture of Chinese cabbage were identified via bulked segregant analysis （BSA） in an F2 population derived from the cross between Hua 273 （female parent） and 114 Fushan （male parent）. [ Results] OPA06-1400 was identified to he linked to the chewy texture-controlling gene of Chinese cabbage. The genetic distance between the target gene and the RAPD marker was 24.8 cM. [ Conclusions] The resuits provide experimental evidence for breeding of Chinese cabbage.

Mapping a leaf senescence gene els1 by BSR-Seq in common wheat

Leaf senescence is normally the last stage of plant development. Early senescence of functional leaves significantly reduces the photosynthetic time and efficiency, seriously affecting grain yield and quality in wheat. Discovering genes responsible for early leaf senescence(els) are necessary for developing novel germplasms and cultivars with delayed leaf-senescence through molecular manipulation and marker assisted selection. In this study, we identified an early leaf senescence line M114 in a derivative of a wheat breeding population. Genetic analysis indicated that early leaf senescence in M114 is controlled by a single recessive gene, provisionally designated els1. By applying bulked segregant analysis and RNA-Seq(BSR-Seq), seven polymorphic markers linked to els1 were developed and the gene was located on chromosome arm 2 BS in a 1.5 c M genetic interval between markers WGGB303 and WGGB305. A co-segregating marker, WGGB302, provide a starting point for fine mapping and map-based cloning of els1.

Identifying a melanogenesis-related candidate gene by a high-quality genome assembly and population diversity analysis in Hypsizygus marmoreus

Hypsizygus marmoreus is one of the most important edible fungi in Basidiomycete division and includes white and gray strains.However,very limited knowledge is known about the genomic structures and the genetic basis for the white/gray diversity of this mushroom.Here,we report the near-complete high-quality H.marmoreus genome at the chromosomal level.Comparative genomics analysis indicates that chromosome structures were relatively conserved,and variations in collinearity and chromosome number were mainly attributed by chromosome split/fusion events in Aragicales,whereas the fungi genome experienced many genomic chromosome fracture,fusion,and genomic replication events after the split of Aragicales from Basidiomycetes.Resequencing of 57 strains allows us to classify the population into four major groups and associate genetic variations with morphological features,indicating that white strains were not originated independently.We further generated genetic populations and identified a cytochrome P450 as the candidate causal gene for the melanogenesis in H.marmoreus based on bulked segregant analysis (BSA)and comparative transcriptome analysis.The high-quality H.marmoreus genome and diversity data compiled in this study provide new knowledge and resources for the molecular breeding of H.marmoreus as well as the evolution of Basidiomycete.

Inheritance and Gene Mapping of Resistance to Soybean Mosaic Virus Strain SC14 in Soybean

Soybean mosaic virus （SMV） is one of the most broadly distributed diseases worldwide. It causes severe yield loss and seed quality deficiency in soybean （Glycine max （L.） Merr.）. SMV Strain SC14 isolated from Shanxi Province, China, was a newly identified virulent strain and can infect Kefeng No. 1, a source with wide spectrum resistance. In the present study, soybean accessions, PI96983, Qihuang No. 1 and Qihuang No. 22 were identified to be resistant （R） and Nannong 1138-2, Pixianchadou susceptible （S） to SC14. Segregation analysis of PI96983 x Nannong 1138-2 indicated that a single dominant gene （designated as Rsc14） controlled the resistance to SC14 at both V2 and R1 developmental stages. The same results were obtained for the crosses of Qihuang No. 1 × Nannong 1138-2 and Qihuang No. 22 x Nannong 1138-2 as in PI96983 x×Nannong 1138-2 at V2 stage, but at R1 stage, the F1 performed as necrosis （a susceptible symptom other than mosaic）, F2 segregated in a ratio of 1R：2N：IS, and the progenies of necrotic （N） F2 individuals segregated also in R, N and S. It indicated that a single gene （designated as Rsc140, to be different from that of PI96983） controlled the resistance to SC14, its dominance was the same as in PI96983 × Nannong 1138-2 （without symptoms） at V2 stage and not the same at R1 stage. The tightly linked co-dominant simple sequence repeat （SSR） marker Satt334 indicated that all the heterozygous bands were completely corresponding to the necrotic F2 individuals, or all the necrotic F2 individuals were heterozygotes. It was inferred that necrosis might be due to the interaction among SMV strains, resistance genes, genetic background of the resistance genes, and plant development stage. Furthermore, the bulked segregant analysis （BSA） of SSR markers was conducted to map the resistance genes. In F2 of PI96983 × Nannong 1138-2, five SSR markers, Sat_297, Sat_234, Sat_154, Sct_033 and Sat_120, were found closely linked to Rsc14, with genetic distances of 14.5 cM, 11.3 cM, 4.3 cM, 3.2 cM and 6 cM, respectively. In F2 of Qihuang No. 1 × Nannong 1138-2,three SSR markers, Sat_234, Satt334 and Sct_033, tightly linked to Rsc140 with genetic distances of 7.2 cM, 1.4 cM and 2.8 cM, respectively. Based on the integrated joint map by Cregan et al. （1999）, both Rsc14 and Rsc140 were located between Sat_234 and Sct_033 on linkage with group F of soybean, with their distances from Sct_033 at the same side being 3.2 cM and 2.8 cM, respectively. Therefore, Rsc14 and Rsc140 might be on a same locus. The obtained information provides a basic knowledge for marker-assisted selection of the resistance gene in soybean breeding programs and fine mapping and map-based cloning of the resistance gene.

A combinatorial strategy to identify various types of QTLs for quantitative traits using extreme phenotype individuals in an F_(2)population

Theoretical and applied studies demonstrate the difficulty of detecting extremely over-dominant and smalleffect genes for quantitative traits via bulked segregant analysis(BSA)in an F_(2)population.To address this issue,we proposed an integrated strategy for mapping various types of quantitative trait loci(QTLs)for quantitative traits via a combination of BSA and whole-genome sequencing.In this strategy,the numbers of read counts of marker alleles in two extreme pools were used to predict the numbers of read counts of marker genotypes.These observed and predicted numbers were used to construct a new statistic,G_(w),for detecting quantitative trait genes(QTGs),and the method was named dQTG-seq1.This method was significantly better than existing BSA methods.If the goal was to identify extremely over-dominant and smalleffect genes,another reserved DNA/RNA sample from each extreme phenotype F_(2)plant was sequenced,and the observed numbers of marker alleles and genotypes were used to calculate G_(w)to detect QTGs;this method was named dQTG-seq2.In simulated and real rice dataset analyses,dQTG-seq2 could identify many more extremely over-dominant and small-effect genes than BSA and QTL mapping methods.dQTGseq2 may be extended to other heterogeneous mapping populations.The significance threshold of G_(w)in this study was determined by permutation experiments.In addition,a handbook for the R software dQTG.seq,which is available at https://cran.r-project.org/web/packages/dQTG.seq/index.html,has been provided in the supplemental materials for the users’convenience.This study provides a new strategy for identifying all types of QTLs for quantitative traits in an F_(2)population.

An Exome-seq Based Tool for Mapping and Selection of Candidate Genes in Maize Deletion Mutants

Despite the large number of genomic and transcriptomic resources in maize, there is still much to learn about the function of genes in developmental and biochemical processes. Some maize mutants that were generated by gamma-irradiation showed clear segregation for the kernel phenotypes in B73 ? Mo17 F2 ears. To better understand the functional genomics of kernel development,we developed a mapping and gene identi?cation pipeline, bulked segregant exome sequencing(BSEx-seq), to map mutants with kernel phenotypes including opaque endosperm and reduced kernel size. BSEx-seq generates and compares the sequence of the exon fraction from mutant and normal plant F2 DNA pools. The comparison can derive mapping peaks, identify deletions within the mapping peak, and suggest candidate genes within the deleted regions. We then used the public kernel-speci?c expression data to narrow down the list of candidate genes/mutations and identi?ed deletions ranging from several kb to more than 1 Mb. A full deletion allele of the Opaque-2 gene was identi?ed in mutant 531, which occurs within a $200-kb deletion. Opaque mutant 1486 has a 6248-bp deletion in the mapping interval containing two candidate genes encoding RNA-directed DNA methylation 4(RdDM4) and AMP-binding protein, respectively. This study demonstrates the ef?-ciency and cost-effectiveness of BSEx-seq for causal mutation mapping and candidate gene selection,providing a new option in mapping-by-sequencing for maize functional genomics studies.