Pyramiding of male-sterile genes in Cryptomeria japonica D. Don with the aid of closely linked markers

Gene pyramiding is a breeding method used to combine multiple useful genes. Although several genes have been pyramided in certain crops, gene pyramiding has not previously been applied to forest trees. In this study, we used the markers closely linked to the two male-sterile genes MS1 and MS2 for the effective development of individuals doubly heterozygous for these two genes. This is the first example of gene pyramiding through marker-assisted selection (MAS) in forest trees. The markers gSNP06239, which is closely linked to the MS1 gene, and estSNP00695, which is closely linked to MS2, were used in MAS. On the basis of the linkage phase between the markers and male-sterile loci, we selected five F₁ individuals (S3-64 from Shindai-3 × Kamikiri-31, S3-70 from Shindai-3 × Kamikiri-38, S3-77 from Shindai-3 × Kamikiri-47, S1-22 from Shindai-1 × Nakakubiki-4, and S1-56 from Shindai-1 × Setsugai-20) as parents for artificial crossing. The 268 seedlings obtained from six artificial cross combinations were used in this study. Chi-squared tests showed no significant deviation from the expected Mendelian ratios of genotypes, indicating that MAS using markers closely linked to the male-sterile genes worked very well. Fifteen individuals that showed unexpected genotypes were probably recombinants, because the map distances between the male-sterile locus and the DNA markers were 4.1 cM (gSNP06239 to MS1) and 6.9 cM (estSNP00695 to MS2). Development of markers more closely linked to the male-sterile loci will facilitate precise gene pyramiding in the future.