S-Locus Genotyping in Japanese Plum by High Throughput Sequencing Using a Synthetic S-Loci Reference Sequence

Area of Science:

• Plant genetics
• Reproductive biology
• Horticulture

Background:

• Self-incompatibility (SI) in *Prunus* species is controlled by linked F-box (SFB) and S-RNase genes, crucial for breeding and pollination.
• Traditional genotyping methods like gel-based PCR are limited with high polymorphism in the S-locus region.
• Advancements in sequencing necessitate new approaches for accurate S-locus genotyping.

Purpose of the Study:

• To develop and validate a novel genotyping-by-sequencing procedure for accurately determining S-genotypes in Japanese plum (*Prunus*).
• To analyze the S-genotype and incompatibility groups in a large collection of Japanese plum cultivars.
• To identify new S-alleles and incompatibility groups within the *Prunus* species.

Main Methods:

• Developed a synthetic reference sequence using concatenated Japanese plum S-loci.
• Applied a genotyping-by-sequencing procedure using the synthetic reference for S-locus analysis.
• Genotyped 88 Japanese plum cultivars, including 74 not previously reported.

Main Results:

• Successfully genotyped 88 Japanese plum cultivars, with 74 reported for the first time.
• Identified at least two S-alleles in 74 cultivars and uncovered two new S-alleles from reference genomes.
• Assigned cultivars to 22 incompatibility groups, including nine newly identified groups (XXVII-XXXV).

Conclusions:

• The developed method accurately genotypes S-alleles in Japanese plum, overcoming limitations of traditional techniques.
• This research expands the knowledge of S-allele diversity and incompatibility groups in Japanese plum.
• The findings provide essential genetic information for optimizing breeding strategies and managing pollination in *Prunus* species.