Rapid identification of lettuce seed germination mutants by bulked segregant analysis and whole genome sequencing

Abstract

Lettuce (Lactuca sativa) seeds exhibit thermoinhibition, or failure to complete germination when imbibed at warm temperatures. Chemical mutagenesis was employed to develop lettuce lines that exhibit germination thermotolerance. Two independent thermotolerant lettuce seed mutant lines, TG01 and TG10, were generated through ethyl methanesulfonate mutagenesis. Genetic and physiological analyses indicated that these two mutations were allelic and recessive. To identify the causal gene(s), we applied bulked segregant analysis by whole genome sequencing. For each mutant, bulked DNA samples of segregating thermotolerant (mutant) seeds were sequenced and analyzed for homozygous single-nucleotide polymorphisms. Two independent candidate mutations were identified at different physical positions in the zeaxanthin epoxidase gene (ABSCISIC ACID DEFICIENT 1/ZEAXANTHIN EPOXIDASE, or ABA1/ZEP) in TG01 and TG10. The mutation in TG01 caused an amino acid replacement, whereas the mutation in TG10 resulted in alternative mRNA splicing. Endogenous abscisic acid contents were reduced in both mutants, and expression of the ABA1 gene from wild-type lettuce under its own promoter fully complemented the TG01 mutant. Conventional genetic mapping confirmed that the causal mutations were located near the ZEP/ABA1 gene, but the bulked segregant whole genome sequencing approach more efficiently identified the specific gene responsible for the phenotype.

The cloning and characterization of hypersensitive to salt stress mutant, affected in quinolinate synthase, highlights the involvement of NAD in stress‐induced accumulation of ABA and proline

“…Ethyl methanesulfonate‐mediated mutagenesis and genetic screens are the most powerful approaches to identify key genes involved in plant response to abiotic stress (Zhu, ; Papdi et al , ). Recently, the mapping‐by‐sequencing (MBS) method has emerged as a rapid, successful and more popular way to identify the mutation site in genomes of Arabidopsis, rice, barley and other species (Schneeberger, ; Qin et al , ; Takagi et al , ; Huo et al , ). We combined these two methods to identify the hss mutant in Arabidopsis, which is affected in the QS gene.…”

Next-Generation Sequencing from Bulked-Segregant Analysis Accelerates the Simultaneous Identification of Two Qualitative Genes in Soybean

“…For this reason, only a few mutant libraries have been developed in soybean to date, using fast neutron or ethyl methanesulfonate methods (Bolon et al, 2011; Tsuda et al, 2015; Li et al, 2017), far fewer than mutant resources of Arabidopsis and rice (Wang et al, 2013). Although NGS-based BSA approaches have been shown to be efficient in isolating a gene controlling a given mutant phenotype by backcrossing the mutant to the non-mutagenized parental genotype, it is difficult to unequivocally identify the causal mutation due to limited polymorphic markers between mutant and wild-type (Abe et al, 2012; Hartwig et al, 2012; James et al, 2013; Schneeberger, 2014; Huo et al, 2016). Another challenge for crops with large or complex genomes is that some short sequence reads may not be mapped to unique positions in the reference genome and identified nucleotide variations cannot be distinguished from differences among closely related paralogous sequences (Xu et al, 2013).…”

“…The other strategy is to backcross the mutant to its parental genotype and the size of a backcrossing population has less influence on the mapping power (James et al, 2013; Schneeberger, 2014). Some successful applications were reported in species with large genomes such as lettuce and wheat (Trick et al, 2012; Huo et al, 2016). In order to fine-map target loci to small regions, subsequently genotyping the progeny of the segregating population is cost-efficient and rapid.…”

Apple fruit acidity is genetically diversified by natural variations in three hierarchical epistatic genes: MdSAUR37, MdPP2CH and MdALMTII

“…Pedigree-based quantitative trait locus (QTL) mapping and genome-wide association studies are powerful tools for mapping both qualitative and quantitative traits (Hamblin et al, 2011;Longhi et al, 2012;Farneti et al, 2017;Ye et al, 2017). Recently, bulk segregant analysis (BSA)-seq, a next-generation sequencing (NGS)-based QTL mapping strategy analysis, in which two phenotypically distinct subpopulations (bulks) (BSA extremity bulks) of recombinant progeny (segregants) are isolated from a genetic cross and genotyped, has proved successful for rapidly mapping genes in several cereal and vegetable plants (Trick et al, 2012;Takagi et al, 2013;Huo et al, 2016;Dougherty et al, 2018). By combining MapQTL and BSA-seq, an AAA ATPase gene CsARN6.1 and the Ef1.1 gene for waterlogging tolerance and early flowering, respectively, in cucumber (Cucumis sativus), and the ABA1/ZEP gene for thermotolerance in lettuce (Lactuca sativa) were efficiently identified (Lu et al, 2014;Huo et al, 2016;Xu et al, 2018).…”

“…Recently, bulk segregant analysis (BSA)-seq, a next-generation sequencing (NGS)-based QTL mapping strategy analysis, in which two phenotypically distinct subpopulations (bulks) (BSA extremity bulks) of recombinant progeny (segregants) are isolated from a genetic cross and genotyped, has proved successful for rapidly mapping genes in several cereal and vegetable plants (Trick et al, 2012;Takagi et al, 2013;Huo et al, 2016;Dougherty et al, 2018). By combining MapQTL and BSA-seq, an AAA ATPase gene CsARN6.1 and the Ef1.1 gene for waterlogging tolerance and early flowering, respectively, in cucumber (Cucumis sativus), and the ABA1/ZEP gene for thermotolerance in lettuce (Lactuca sativa) were efficiently identified (Lu et al, 2014;Huo et al, 2016;Xu et al, 2018). However, MAS is still difficult to practically apply in breeding programmes of perennial plants such as apples (Malus domestica Borkh.)…”