Application of InDel markers on progeny identification in Actinidia arguta

Abstract:【Objective】Identification of the hybrid progenies is a prerequisite for molecular genetic research in kiwifruit.Moreover, identification of the hybrid progenies is an important step in hybrid breeding, because there are mechanical mixing and biological mixing during the course of hybrid production, resulting in inaccuracy of male parent.InDel (Insertion/Deletion) , as an important source of genetic marker, has been used in hybrid progeny identification.In this study, the authenticity of hybrid progenies of Actinidia arguta were identified by InDel molecular markers.【Methods】Four Actinidia arguta cultivars ('Yongfeng No.1' 'RB-3' '11-17' and 'Kuilü male') were used to construct two hybrid populations'RB-3'×'Kuilü male'and'Yongfeng No.1'×'11-17'.'Yongfeng No.1' and 'Kuilü male' are highly resistant to the cold, both were originated from northeast of China, while'RB-3'and'11-17'are poorly resistant to the cold, both came from central part of China.The crosses were conducted in the summer of 2015.Fresh leaves of the hybrid progenies and parents were collected and frozen in liquid nitrogen in August2016.The DNA of four A.arguta cultivars ('Yongfeng No.1''RB-3''11-17'and'Kuilü male') and the progenies of'RB-3'×'Kuilü male' (a total of 175 plants) and'Yongfeng No.1'×'11-17' (a total of531 plants) were extracted using high throughput magnetic bead DNA extraction approach.Then, the In-Del primers were designed according to the data of resequencing.Fifty different InDel loci were chosen randomly based on the resequencing for designing primers.Fifty pairs of primers were used to amplify bands among four parents.In order to identify the authenticity of progeny populations, the special InDel markers were chosen among four parents through polyacrylamide gel electrophoresis methods.Primer screening principle was that the polymorphism primers must have clear and stable bands which were specific in male parent.The authenticity of those two hybrid populations ('RB-3'×'Kuilü male'and'Yongfeng No.1'×'11-17') were identified by agarose gel electrophoresis or polyacrylamide gel electrophoresis methods according to the InDel fragment length, Finally, we counted the number of hybrid progenies with the male specific band and calculated the rate of true hybrid progenies.【Results】For the'RB-3'×'Kuilü male'hybrids, 4 pairs of primers were selected as marker of hybrid identification.At the first, primer 42 and primer 5 were used to identify all hybrid progenies, a total of 126 offsprings had the male specific band.Primer 52 was used to identify the rest 49 offsprings, a total of 26 offsprings had the male specific band.Then, primer 134 was used to identify the rest 23 offsprings.Among them, a total of 9 offsprings had the male specific band.Finally, a total of 14 offsprings did not have the male specific band, they were considered as false hybrid individuals.4 pairs of primers in the hybrid combination were53.14%, 46.94%, 53.06% and 39.13%, respectively.175 hybrid individuals were identified in the experiment, and the number of true hybrid offsprings was 161.The results showed that the true hybrids rate was about 92%.For the'Yongfeng No.1'×'11-17'hybrids, another 4 pairs of primers were selected as marker of hybrid identification.Firstly, primer 44 was used for identifying all hybrid progenies, a total of 439 offsprings had the specific bands, they were considered as true hybrid, and the true hybrid rate was82.67%.Then primer 3 was used toidentify the rest 92 offsprings.Among them 71 offsprings had the male parent specific bands, the true hybrid rate was 77.17%.Primer 100 was used to identify the rest 21 offsprings.Among them, 16 offspring had the male parent specific bands, the true hybrid rate was 76.19%.Finally, primer 55 was used to identify the rest 5 offsprings and 1 offsprings had the male specific band.4 offsprin without the male specific bands were considered as false hybrids.527 true hybrid offsprings were identified using 4 pairs of primers from the progenies of'Yongfeng No.1'×'11-17'.The true hybrid rate was 99.24%.【Conclusion】The InDel markers developed by resequencing were highly effective for identifying hybrid offspring in kiwifruit.3-5 InDel markers should be used for comprehensive analysis to ensure the authenticity of offspring in the identification process.