Development of simple DNA markers for selecting trees with the male-sterile gene of cryptomeriajaponica "Sosyun"

We report the development of two types of simple DNA markers that can be used to easily identify genotypes of both the male-sterile and wild-type individuals at a locus, which is tightly linked to the causative gene of male sterility in the “Sosyun” clone of Cryptomeria japonica. Both the allele-specific primer (ASP) PCR marker and cleaved amplified polymorphic sequence (CAPS) marker were designed using previously reported single nucleotide polymorphism (SNP) information. The cDNA and genomic DNA of the EST reCj 19250, which is linked to the ms-1 locus, were sequenced for both the “Sosyun” and wild-type plus-tree “Usui 2” clones. Primers were designed for detecting both the sterile and wild-type SNPs using the genomic DNA sequences. For the ASP-PCR marker, homozygous recessive male-sterile individuals yielded a single 157-bp fragment, whereas the homozygous wild-type individuals yielded a single 345-bp fragment, and heterozygotes yielded both fragments. Similarly, for the CAPS marker, male-sterile individuals yielded a single 101-bp fragment after digestion, whereas wild-type ones yielded an intact single 125 -bp fragment, and heterozygotes yielded fragments of both sizes. These markers could be utilized for identifying male-sterile individuals during seedling production and for identifying heterozygous trees for future tree breeding.