Marker-Assisted Selection of Male-Sterile and Maintainer Line in Chili Improvement by Backcross Breeding

Cytoplasmic genic male sterility (CGMS) is a male sterility system that uses the maternal line for hybrid production, ensuring no obscurity of F₁ seed purity and reducing the cost of hybrid seed production. Identification of the male sterility gene is important for plant improvement and classification when using the molecular marker-assisted selection (MAS) method. This study aimed to produce a new maternal line (A-line) and its maintainer line (B-line) by transferring a male-sterile line (A-line) and its maintainer line (B-line) gene from another variety to restorer lines (C-line) to achieve future hybrid seed production. In the process, the CGMS line (A-line) and B-line transferred to C1 and C3 lines, which finally resulted in new A-line (BC₂F₂A1 × C1, BC₂F₂A1 × C3), and B-line (BC₁F₂B1 × C1, BC₁F₂B1 × C3), and then used the MAS method for detecting genes and pollen viability test in the newly improved lines. The results indicated that the 3336-last2-SCAR (1639 bp) and 4162-SCAR (1046 bp) DNA markers classified the <i>Rf</i> locus, and the CMS-SCAR130/140 marker confirmed the S or N cytoplasm. The BC₂F₂A1 × C1 and BC₂F₂A1 × C3 lines represented both male-fertile (S<i>Rf_</i>) and male-sterile (S<i>rfrf</i>) progenies in a Mendelian ratio of 3:1. Moreover, stained pollen grains with 1% acetocarmine confirmed abnormal pollen in male-sterile plants. The molecular markers that detect maintainer lines (N<i>rfrf</i>) are BC₁F₂B1 × C1-14, BC₁F₂B1 × C3-10, and BC₁F₂B1 × C3-11. The 3336-last2-SCAR (1639 bp) and CMS-SCAR130/140 markers successfully identified the male-sterile line (S<i>rfrf</i>) and maintainer line (N<i>rfrf</i>), and 4162-SCAR (1046 bp) detected the presence of the <i>RfRf</i> or <i>Rfrf</i> genotype in chilies at the seedling stage. The use of these markers was highly accurate and confirmed the results at the early generation stage of a conventional breeding program. It can be concluded that the CGMS and maintainer gene in chilies were successfully transferred during early generation using the backcross method.