Recently, Liu Shengyi, a researcher at the Institute of Oil Materials, Chinese Academy of Agricultural Sciences, and Yang Qingyong, a professor at the School of Information, Huazhong Agricultural University, collaborated to develop a new idea/method for the functional identification of multi-copy genes, and based on this identification, the important transporter BnaA06. GTR2 of glucosinolates in rapeseed was analyzed, and CRISPR/cas9 was also used to create germplasm resources for seed low-sulfur glycosides and vegetative tissue high-sulfur glycosides that can be directly utilized in rapeseed breeding. The paper was published in Plant Physiology.
In crop genetic breeding studies, genetic variation is the basis of forward genetic studies, such as QTL mapping and genome-wide association study (GWAS) in bi-parental segregating populations. Forward genetics is unable to study trait functional genes that lack natural variation. To this end, researchers have developed reverse genetics research methods (such as the construction of artificial mutant libraries, etc.) as a supplement.
However, in the polyploid species such as rapeseed, cotton, and wheat, most of the genes exist in the form of multiple copies, and the functions of multiple copies of genes are both differentiated and redundant, as well as the lack of variant copies in germplasm resources. Therefore, in polyploid species, how to accurately select the target copy for genetic manipulation is an important challenge in current genetic studies.
Glucosinolates (thioglycosides) are unique secondary metabolites of cruciferous plants. Most kinds of thioglycosides are synthesized in the vegetative tissues of plants and then transported into seeds. Their metabolites play an important role in plant pest defense. However, the high content of thioglycosides and their degradation products in cake will cause goiter and other diseases of fed livestock, reducing the economic value of rapeseed meal high-quality protein.
With the popularization of rapeseed "double low" (seed low sulfur glycosides and low erucic acid) breeding, the amount of thiosidic acid in vegetative tissues is also reduced while reducing seed thioglycoside content, which leads to decreased plant disease and insect resistance. Although the genes and mechanisms related to the synthesis and transport of thioglycosides in Arabidopsis have been relatively clearly studied, and mutations in thioglycoside transporter-encoding genes (GTRs) can significantly reduce seed thioglycoside content in some cruciferous plants, the developed rapeseed BnGTR2s gene editing mutants have a large negative impact on agronomic traits such as yield and are difficult to apply in breeding practice.
In response to the above problems, this study comprehensively identified the transport-related gene BnaGTRs of thiosides from 12 de novo assembled B. napus genomes, and then further examined the expression patterns of these genes in 20 stages/tissues of growth and development of representative varieties with high and low seed thiosidic content, and found that the expression levels of BnaA06.GTR2 were generally higher than those of other BnaGTRs. Interestingly, BnaA06.GTR2 did not detect a relevant effect with seed thiosides in both GWAS analysis of seed thioside content and published QTL/GWAS mapping as well as whole transcriptome association analysis (TWAS) studies, indicating a lack of variation in rapeseed germplasm. Further, CRISPR/cas9 gene editing demonstrated that BnaA06.GTR2 played an important role in the seed thioglycoside transport process, and its seed thioglycoside content in loss-of-function mutants decreased by 76.05% compared with transgenic negative controls, and no negative effects on other agronomic traits were observed.
This study finally provides an excellent germplasm resource for cultivating high resistance and high-quality varieties with high thiosides in rapeseed leaves and low thiosides in seeds. The method provides a new idea and method for the study of the lack of genetic variation in crop resources, especially the function of polyploid multicopy genes, and provides a method strategy for plant breeding.