Abstract: Objective Distant hybridization is an important way for germplasm innovation which can achieve the aggregation of favorable genes. Cross-incompatibility often occurs in some of Populus sects, and interspecific hybrids are difficult to distinguish from its morphology. In this study, species-specific InDel markers were developed to identify the interspecific hybrids from different sects of Populus, so as to lay a foundation for the new germplasm innovation in poplar. Method The primers were developed from species-specific InDel sites in P. deltoides and P. simonii and were used to blast sequentially the genomes of P. euphratica, P. davidiana, P. simonii and P. lasiocarpa using SeqHunter2. Natural individuals from different sects were used to verify the species-specific primers in intra species conservation and inter species polymorphism by PCR amplification test, and the identified species-specific primer sequences for different sects of Populus were screened, and the authenticity of interspecies hybrid progenies among any combinations from the five species in different Populus sects was identified.Result Five thousands of InDel primers were designed from the genome of P. deltoides and used to blast sequentially the genomes of P. euphratica, P. davidiana, P. simonii and P. lasiocarpa. A total of 341 primer pairs were detected as universal markers among the five species. A total of 48 primer pairs from different chromosome were selected and synthesized, and 43 were identified as universal in five species from different sects, of which 19 primer pairs were conservative within individual species and produced length polymorphism of amplified bands in different species. The mixed DNA from natural population was used to test the 19 pairs of universal primers for interspecific polymorphism and intraspecific conservation by PCR amplification, and 4 pairs of polymorphic primers were preliminarily obtained. Finally, natural population DNA was used to amplify and detect these 4 pairs of primers, and it was found that primer pairs of Chr12_4341229 and Chr13_10582756 were species-specific, and could be used to amplify clear and unique bands in P. deltoides, P. simonii, P. davidiana, P. lasiocarpa and P. euphratica. Primer pairs of Chr12_4341229 could be used to amplify the bands with length of 148, 152, 113, 152 and 159 bp, and primer pairs of Chr13_10582756 could be used to amplify the bands with length of 140, 144, 116, 140 and 159 bp separately in the species of P. deltoides, P. simonii, P. davidiana, P. lasiocarpa and P. euphratica. Primers Chr12_4341229 and Chr13_10582756 were used to identify the inter-specific hybrids of P. simonii × P. euphratica, P. deltoides × P. euphratica, and P. deltoides × P. simonii, showing that all the three inter-specific hybrids could amplify two bands which were inherited from the parents used for hybridization. The above two primers were able to be used for verifying the authenticity of progenies from any combination of the five species in different Populus sects.Conclusion In this study, two pairs of species-specific InDel markers have been identified conservative within species and with length polymorphism in the five species from different Populus sects, and can be used to identify inter-specific hybrids from any combination among the five species in Populus. The development of the two species-specific InDel markers will provide technical support for the further germplasm innovation by distant hybridization in Populus.

Key words: Populus, distant hybridization, species-specific primers, molecular identification of hybrids