Abstract:

Objective: In this study, species-specific KASP primers were developed to identify the interspecific hybrids from Salix suchowensis and S. triandra, which would lay a foundation for the authenticity identification of breeding new germplasm of fast growth and insect resistance in willow. Methods: Whole genome SNPs were analyzed for selecting species-specific SNPs, and designing KASP primers based on the resequencing data of S. suchowensis and S. triandra. SeqHunter2 was used to detect the versatility of the designed primers in S. suchowensis, and natural population materials from S. suchowensis and S. triandra were used to test validation of the primers for species-specific and versatility identification by PCR amplification test. The selected species-specific KASP primers were used for the authentication identify of their hybrids. Results: The all resequencing data of S. suchowensis and S. triandra were aligned to the S. triandra genome, and a total of 6 598 144 SNPs were detected over the whole genome. After filtering by a house perl script, 674 144 homozygous SNP sites were identified in S. suchowensis, which were homozygous mutation sites with S. triandra. One hundred SNPs were randomly selected from each chromosome for KASP primer design based on the genome sequence of S. triandra and 750 primer pairs were successful designed among the 1 900 randomly selected SNP sites. SeqHunter2 detection revealed that 11 primers were versatile in S. suchowensis and S. triandra from the 38 primer pairs randomly selected from different chromosomes. Ten synthesized versatile primer pairs were used for for intra species conservation and inter species difference detection, and four of them could get an obvious clustering in S. suchowensis, S. triandra and its hybrids. Primer pairs of Stri08_82809, Stri14_11602, Stri14_12274 and Stri17_10731 amplified homozygous site of G/G, T/T, A/A and T/T respectively in the population of S. suchowensis, and homozygous site of A/A, C/C, G/G and C/C respectively in the population of S. triandra. For the hybrids identification, the real interspecific hybrids amplified heterozygous sites of G/A, T/C, A/G and T/C respectively. The above four primers were used to identify the inter-specific hybrids of S. suchowensis × S. triandra, the result showed that 29 out of 31 hybrids were real interspecific hybrids because they could amplify heterozygous sites which were inherited from the two parents used for hybridization. The remained two hybrids were none inter-specific because they could only amplify homozygous sites in the four SNPs sites that were identical to the female parent, indicating that these two offspring were descendants contaminated with pollen from S. suchowensis. Conclusion: In this study, four groups of species-specific KASP markers have been obtained for identifying conservative between S. suchowensis and S. triandra. The developed KASP primers can identify the authority of inter-specific hybrids from the cross or backcross breeding of S. suchowensis and S. triandra, which would provide technological support for the insect resistance germplasm innovation by inter-specific hybridization between S. suchowensis and S. triandra.

Key words: Salix suchowensis, S. triandra, interspecific hybridization, species-specific KASP primers, molecular identification of hybrids