关键词: 美洲黑杨, 小叶杨, 种间杂交, 物种特异性引物, 分子鉴别

Abstract:

Objective: The identification of interspecific poplar hybrids at seedling stage is challenging because of the morphological similarity. Here, we developed species-specific InDel markers to identify the interspecific hybrids between Populus deltoides and P. simonii, in order to lay a foundation for the identification of hybrids and molecular marker-assisted breeding in poplar. Method: Ten individuals of each P. deltoides and P. simonii were resequenced to screen species-specific InDels, which were further applied for primer designing. The resulted primers were tested for their versatility and specificity using 8% non-denature polyacrylamide gel electrophoresis. The selected species-specific polymorphic primers were used to identify interspecific hybrid progeny between P. deltoides and P. simonii. Result: Resequencing reads from each ten individuals of P. deltoides and P. simonii were mapped onto the genome of P. deltoides for comparisons, and a total of 359 733 InDel sites with a length difference larger than 5 bp were obtained, of which 18 418 sites are conserved within species but different between species. Based on the genome sequence of P. deltoides, 17 956 primer pairs were successfully designed using Primer3. Ten primer pairs from different chromosomes were selected and tested for their versatile in P. simonii by SeqHunter2 software. A total of six primer pairs were versatile between P. deltoides and P. simonii. The electrophoresis with 8% non-denature polyacrylamide gel showed that primers Psi2-1 and Psi13-14 were polymorphic between P. deltoides and P. simonii. Both primer pairs were then screened in natural population of P. deltoides and P. simonii each with fifty clones separately. The primers Psi2-1 amplified specific bands of 128 bp and 139 bp respectively in P. deltoides and P. simonii. And both of these bands were amplified in interspecific hybrids. Primers Psi13-14 amplified specific bands of 132 bp and 144 bp respectively in P. deltoides and P. simonii. And both of these two bands were successfully amplified in the interspecific hybrids. The two pairs of primers can be used to identify the hybrids between P. deltoides and P. simonii with an accuracy of 100%. Conclusion: This study confirmed that species-specific InDel markers can be used for the identification of interspecific hybrids of P. deltoides and P. simonii, which is of great significance for the identification of interspecific hybrid and breeding of new varieties of poplar.

Key words: Populus deltoides, Populus simonii, interspecific-cross, species-specific marker, molecular identification