Identifying the growth associating genes of Nannochloropsis oceanica by bulked mutant analysis (BMA) and RNA sequencing (BMR-seq).

Microalgal strains (lines), either economically or ecologically important or both, are usually clones derived from a single cell. The functions of their genes are verified usually through either loss of function or gain of function approaches, for example, overexpression, knockout, silencing, and editing. In comparison with plants and animals, the genetic studies of microalgae are scarce, and less genes were de novo cloned. Bulked segregant analysis (BSA) and bulked segregant RNA sequencing (BSR-seq) are widely used to identify the linking markers or control genes of sexually reproducing organisms. In order to avoid genotyping, a large amount of individuals in genome-wide association study (GWAS), we modified BSA and BSR-seq in this study to bulked mutant analysis (BMR) and bulked mutant RNA sequencing (BMR-seq). In order to generate stable variations, we used Zeocin to cause the double-strand breaks of DNA (DSBs), and generate rich InDel variations during DNA repairing. We identified a set of growth performance associating SNPs and InDels among the bulked DNA and bulked RNA. We found that the function-known genes either containing these variations or locating upstream or downstream of these variations are highly related to the complex growth performance trait of Nannochloropsis oceanica. Our findings verified that Zeocin is effective to create rich InDels in monoploid and asexual N. oceanica genome, and BMA and BMR-seq are applicable for identifying the associating SNPs and InDels of the growth performance trait. We prefer to use BMA to identify trait associating genes and variations; microalgae are fast growing with a short generation time, and mRNAs are the transcripts of genes and share the variations with DNA. We believe that the methods we developed in this study will facilitate the genetic studies of the traits of diverse microalgae. [ABSTRACT FROM AUTHOR]