Govindarajulu, R. | bioRxiv Science

bioRxiv2018

Bulked-Segregant Analysis Coupled to Whole Genome Sequencing (BSA-Seq) for Rapid Gene Cloning in Maize

Forward genetics remains a powerful method for revealing the genes underpinning organismal form and function, and for revealing how these genes are tied together in gene networks. In maize, forward genetics has been tremendously successful, but the size and complexity of the maize genome made identifying mutant genes an often arduous process with traditional methods. The next generation sequencing revolution has allowed for the gene cloning process to be significantly accelerated in many organisms, even when genomes are large and complex. Here, we describe a bulked-segregant analysis sequencing (BSA-Seq) protocol for cloning mutant genes in maize. Our simple strategy can be used to quickly identify a mapping interval and candidate single nucleotide polymorphisms (SNPs) from whole genome sequencing of pooled F2 individuals. We employed this strategy to identify narrow odd dwarf as an enhancer of teosinte branched1, and to identify a new allele of defective kernel1. Our method provides a quick, simple way to clone genes in maize.\n\nReferences numbers for supporting data:figshare and NCBI SRA links to follow

plant biology↗

bioRxiv2017

Repeated translocation of a gene cassette drives sex chromosome turnover in strawberries

Turnovers of sex-determining systems represent important diversifying forces across eukaryotes. Shifts in sex chromosomes, but conservation of the master sex-determining genes, characterize distantly-related animal lineages. Yet in plants, where separate sexes have evolved repeatedly and sex chromosomes are typically homomorphic, we do not know whether such translocations drive turnovers within closely related groups. This phenomenon can only be demonstrated by identifying sex-associated nucleotide sequences, still largely unknown in plants. The wild North American octoploid strawberries (Fragaria) exhibit separate sexes (dioecy) with homomorphic, female heterogametic (ZW) inheritance, yet sex maps to at least three different chromosomes. To characterize these turnovers, we sequenced the complete genomes of 60 plants of known sex from five Fragaria taxa. We identified 31-mers unique to females and assembled their reads into contigs. Remarkably, a short (13 kb) sequence is observed in nearly all females and never in male-fertile individuals, implicating it as the sex-determining region (SDR). This female-specific \"SDR cassette\" contains both a gene with a known role in fruit and pollen production and a novel retrogene absent on Z and autosomal chromosomes. Comparing SDR cassettes across taxa reveals a history of repeated translocation, which can be ordered temporally due to the capture of adjacent sequence with each successive move. The accumulation of these \"souvenirs\" suggests an adaptive basis for the expanding (up to at least 23 kb) hemizygous region. This is the first plant SDR known to be translocated, and it suggests a new mechanism (\"move-lock-grow\") for expansion and diversification of incipient sex chromosomes.\n\nSignificance StatementSex chromosomes frequently restructure themselves during organismal evolution, often becoming highly differentiated. This dynamic process is poorly understood for most taxa, especially during the early stages typical of many dioecious plants. In wild strawberries, a sex-determining region of DNA has repeatedly changed its genomic location, each time increasing the size of the hemizygous female-specific sequence. This observation shows for the first time that plant sex regions can \"jump\", and suggests that this phenomenon may be adaptive by gathering and locking new genes into linkage with sex. This conserved and presumed causal sequence with a variable genomic location presents a unique opportunity to understand how sex chromosomes first begin to differentiate.

evolutionary biology↗