核桃类植物SRAP-PCR反应体系的建立及遗传多样性分析OA

This study aimed to establish the best reaction system of walnut SRAP-PCR,screen the polymor-phic primer combination,explore the sequence polymorphism among germplasm,clarify the sequence char-acteristics of high-quality germplasm,and provide genetic basis for walnut breeding.The improved CTAB method was used to extract DNA from 34 walnut plant resources.The L16(45)orthogonal design was used to optimize five SRAP-PCR systems,establish the best reaction system,screen primer combinations with high polymorphism,and analyze the genetic diversity of 34 walnut DNA.The results showed that the opti-mal reaction system for walnut SRAP-PCR was(25 μL):Taq DNA polymerase 1.0 U,Mg2+2.0 mmol/L,dNTPs 0.15 mmol/L,template DNA 40 ng,primer 0.8 μmol/L,10×PCR Buffer 2.5 μL.The effects of various factors on walnut SRAP-PCR reaction system from small to large were as follows:dNTPs<tem-plate DNA<Taq DNA polymerase<Mg2+<primer.20 pairs of primer combinations with high polymor-phism were screened,and 220 loci were amplified,including 168 polymorphic loci,accounting for 76.36%.The average number of alleles(Na),effective alleles(Ne),Nei's genetic diversity index(h)and Shan-non's diversity index(I)were 1.750 7,1.493 7,0.258 4 and 0.384 2,respectively.UPGMA cluster analy-sis based on genetic distance showed that the genetic distance between different walnut germplasm ranged from 0.051 6 to 0.384 2,which could be roughly divided into four groups.In this study,a stable and relia-ble SRAP-PCR system of walnut was established,and the results showed that there were high genetic differences in 34 walnut samples at the molecular level.