DNA Fingerprinting And Genetic Diversity In Apple Cultivars

Apple is one of the most important temperate deciduous fruit trees. It possesses abundant germplasm resources and diverse cultivars. These cultivars are easily to be confused, due to the homonym or synonym by its asexual reproduction and wide variety. The development and application of DNA molecular markers provide a tool for cultivar identification, cross parent choosing and breeding selection.Optimization of PCR reaction, primer screening and marker detection for micro-satellite analysis in 40 apple cultivar have been done in this study as to establish a suitable protocol for DNA fingerprinting, to analyze the genetic diversity and relationship in apples, and then to provide a scientific basis for parent choosing and parent combination for apple breeding. The main results are as follows:1. A suitable protocol to extract high-quality genome DNA from apple young leaves for SSR analysis in apples was developed based on the traditional CTAB methods. The main improvements were: 2% PVP, and more NaCl were added to the extraction buffer.2. A protocol for PCR was established. PCR reactions were performed in single 20 L reactions comprising 20ng DNA, 4.0mM MgCl2, 0.4mM dNTPs, 0.5μM primers, 1.0U Taq Polymerase and PCR .3. Twelve SSR markers were screened out of 87 primers as to amplify micro-satellites for genetic analysis in 40 apple. 114 alleles, averaging 9.5 alleles were obtained. The heterozygosity of loci was among 0.2378 and 0.6827, genetic diversity indexes was 0.5344.4. Finger printing of'Qinguan'and'Pinklady'were analyzed using micro-satellites from amplification of 12 pairs SSR primers. 40 varieties can be distinguished completely by 3 pairs SSR primers (Hi01c11, Hi03d06 and GD162).5. Cluster analysis revealed that similarity coefficients (SC) were among 0.705 and 0.982 in the 40 apple cultivars, showing a high genetic diversity in apple cultivars, but SC was low in the related cultivars. All 40 cultuivars can be clustered into 5 groups, which was similar to that of their pedigree data.