Objective To identify the number and distribution of SSR loci on different chromosomes in the whole genome of walnut (Juglans regia L., 2n = 2x = 32), and to develop and validate the monomorphic SSR primers.

Method In this study, walnut whole genome sequencing data were used as experimental materials, and the whole genome microsatellites were screened and analyzed by bioinformatics software MISA. Primer 3.0 was employed to design monomorphic SSR primers. SSR primers were evaluated by electronic PCR and some of the monomorphic ones were synthesized randomly to detect their usefulness and verify the effectiveness of the method.

Result (1) A total of 357 629 SSR loci were identified in the walnut genome, with a distribution density of 662.28 SSRs/MB. The dominant repeat units were mainly A/T bases, showing significant base preference. These SSR sequences were mainly short sequences with a length of 10～30 bp, up to more than 95.00%. The number of SSR loci on different chromosomes varied greatly. Among them, the number of SSR loci on chromosome 1 was the largest, and the numer of SSR loci on chromosome 16 was the least. The number and type of SSRs showed positively correlated with chromosome sequence length. Most of the 644 rare SSR units were hexa-nucleotides. (2) Based on cluster analysis, all the 16 chromosomes could be divided into 4 groups, of which the number of members in group 4 was the most (11), and there was only chromosome 10 in group 1. In general, chromosome 10 forms a main branch, indicating that it may have experienced a relatively conservative evolutionary history. (3) 303 009 pairs of SSR primers were designed by using the conservative sequence flanking the SSR locus. And then 32 pairs of monomorphic primers clarified by electronic PCR were randomly screened and synthesized for wet PCR experiments, of which 30 pairs (93.75%) were amplified in 6 walnut varieties. The PCR amplification results of 28 pairs (87.50%) were consistent with that of electronic PCR.

Conclusion In this study, SSR loci in different chromosome sequences of ‘Zhongmucha-1’ walnut reference genome are identified. Their amounts and repeat types are found to be highly variable among different chromosome sequences and show a highly significant positive correlation with chromosome length. Mono-nucleotide repeat SSRs are the most common type. A novel protocol combining electronic PCR and traditional screening methods are established and validated, which provide an effective strategy for the personalized and rapid development of walnut SSR primers. The developed 28 pairs of monomorphic primers can provide scientific basis for “Illegitimacy Testing” of hybrid offspring in molecular marker assisted breeding.