Development and application of a KASP marker for marker-assisted selection against the male-sterile gene MALE STERILITY 4 (MS4) in Japanese cedar (Cryptomeria japonica D. Don)

A causal gene for MALE STERILITY 4 (MS4), the CjTKPR1 gene, was recently identified in Cryptomeria japonica. To apply marker-assisted selection (MAS) to breeding of male-sterile C. japonica with MS4, we developed a Kompetitive Allele-Specific polymerase chain reaction (KASP) marker, as a highly cost-effective and accurate genotyping method based on the CjTKPR1 sequence. The accuracy of genotyping using the KASP marker was verified with a backcross family for MS4, and MS4 genotypes were accurately identified. Next, we attempted to select individuals carrying ms4 from 1,500 breeding materials collected throughout Japan. No new individuals with ms4 were detected beyond those already identified. Individuals with ms4 were estimated to be geographically restricted and distributed at extremely low frequency (0.13%). We also attempted to produce new breeding materials with double heterozygotes for MS1 and MS4 (Ms1/ms1, MS4/ms4). Artificial crossings were performed between ‘Shindai 11’ (ms1/ms1, Ms4/Ms4) or ‘Shindai 12’ (ms1/ms1, Ms4/Ms4) and ‘S8-94_sb1’ (Ms1/Ms1, Ms4/ms4). Of the 56 and 35 progenies produced from each family, 31 and 13 double-heterozygous individuals with MS1 and MS4 (Ms1/ms1, Ms4/ms4) were selected, respectively. The segregation ratio of genotypes for MS4 did not deviate from the expected 1:1 ratio in either family. The KASP marker developed in this study facilitates accurate genotyping that is less labor-intensive and less costly than conventional methods. This method is anticipated to contribute to greater efficiency in the breeding and seed production of male-sterile C. japonica with MS4.