Development of mitochondrial simple sequence repeat markers to simultaneously distinguish cytoplasmic male sterile sources in cotton

Deleterious effects on anther development and main economy traits caused by sterile genes or cytoplasms are one of the important genetic characteristics of cytoplasmic male sterility (CMS) systems in cotton, which severely hinder the large-scale application of three-line hybrids in production. Therefore, distinct characterization of each cytoplasmic type is mandatory to improve the breeding efficiency of cotton hybrids. In this study, four isonuclear-alloplasmic cotton male sterile lines with G. hirsutum (CMS-(AD)1), G. barbadense (CMS-(AD)2), G. harknessii (CMS-D2), and G. trilobum (CMS-D8) cytoplasms were first created by multiple backcrosses with common genotype Shikang126. Then, 64 pairs of mitochondrial simple sequence repeat (mtSSR) markers were designed to explore the mitochondrial DNA diversities among four isonuclear-alloplasmic cotton male sterile lines, and a total of nine pairs of polymorphic mtSSR molecular markers were successfully developed. Polymorphism analysis indicated that mtSSR59 marker correlated to the atp1 gene could effectively divide the CMS-D2, CMS-(AD)1, and CMS-(AD)2 in one category while the CMS-D8 in another category. Further cytological observation and determination of ATP contents also confirmed the accurate classification of CMS-D2 and CMS-D8 lines. Moreover, the mtSSR59 marker was successfully applied in the marker-assisted selection (MAS) for breeding new male sterile lines and precise differentiation or purity identification of different CMS-based three-line and conventional cotton hybrids. This study provides new technical measures for classifying various cytoplasmic sterile lines, and our results will significantly improve the efficiency of there-line hybrid breeding in cotton.

Automated summary

The deleterious effects of sterile genes or cytoplasms on anther development and main economy traits are hindrances to the large-scale application of three-line hybrids in cotton. This study created four isonuclear-alloplasmic cotton male sterile lines and identified mitochondrial DNA diversities using mitochondrial simple sequence repeat (mtSSR) markers. The mtSSR59 marker effectively classified the CMS-D2, CMS-(AD)1, and CMS-(AD)2 into one category and the CMS-D8 into another category. The marker was also successfully used in marker-assisted selection (MAS) for breeding new male sterile lines and precise differentiation of different CMS-based three-line and conventional cotton hybrids.