Construction Of DNA Fingerprints Of Different Walnut Cultivars Based On SSR Markers And Its Application In Paternity Identification

Walnut is one of the main economic tree species in Shaanxi.In the long-term cultivation process,due to the diversity of terrain,climate and other different ecological environment,some cultivars have little difference in morphology,and it is difficult to accurately identify from the appearance,which brings great difficulties to the collection and promotion of excellent germplasm.Fingerprint is a useful tool to identify cultivars and strains.SSR molecular markers have been widely used in plant variety identification and genetic relationship analysis due to their high polymorphism,good experimental repeatability,large number,codominance,and immunity from environmental factors.Therefore,in order to protect walnut resources correctly and make full use of walnut resources,it is necessary to use SSR molecular marker technology to identify walnut cultivars and build their fingerprints.The application of SSR technology in walnut parent-child relationship identification was explored to shorten the breeding period of walnut.21 common walnut cultivars in Shaanxi were collected in this study,high-quality SSR primers were screened by polyacrylamide gel electrophoresis,genomic polymorphism was analyzed by fluorescence capillary electrophoresis,SSR fingerprints of different walnut cultivars were constructed by combining core primers,and genetic relationship was analyzed,in order to provide more comprehensive information for parents’ selection in future cross breeding provide theoretical basis for germplasm innovation and intellectual property protection;SSR technology was used to test the relationship between walnut parents and offspring,and the offspring polluted by exogenous pollen and apomixis of female parent were eliminated at the seedling stage,in order to improve the efficiency of walnut breeding.The main results are as follows:(1)Identification of walnut cultivars and construction of fingerprintsSix pairs of core primers were screened out of 20 pairs of SSR primers,and the SSR fingerprints of different walnut cultivars were constructed.The results showed that 6 pairs of fluorescent primers detected a total of 54 allelic loci,with an average of 9 amplifying loci per pair of primers.The genetic similarity coefficients among the 21 walnut cultivars ranged from 0.59 to 0.93,and the effective allelic variation ranged from 3.7373 to 5.6903,with an average of 4.4599.The Shannon’s information index ranged from 1.6287 to 1.8715,with an average of 1.7901.There is relatively rich genetic diversity among walnut cultivars.Further analysis found that the combination of primer WJR265 and primer WGA331,and then any combination of any of the four primers WJR031,WJR281,WGA321 or WGA032 could completely distinguish 21 walnut cultivars.Twenty-one walnut materials were identified using 3 pairs of high-efficiency primers,and 4 different combinations were possible.The molecular map of the tested walnut cultivars was constructed with 6 pairs of primers,which provided data for the rapid identification of walnut cultivars and the establishment of walnut fingerprint database.(2)F1 hybrid authenticityThe fruit setting rate of the ‘Xiangling’ × ‘Xiluo 2’ hybrid combination was 81.9%,and the seedling emergence rate was 58.2%.Three pairs of SSR primers with specificity between parents were screened for hybrid identification.Among them,two pairs of primers WGA118 and WJR265 had high identification efficiency.There were 32 progeny strains verified by these two pairs of primers,and the true hybrid rate was 71.1%.