Isolation And Identification Of Malus Resources Based On SSR Molecular Markers

This study is a preliminary study and discussion for the final achievement of the accurate identification of Malus and the systematic classification of all kinds of Malus.In this study,SSR molecular marker technology is used to isolate and identify the germplasm of Malus from Shandong province.In this study,18 pairs of SSR primers was selected from the primers which developed from the Rosaceae(Malus).96 copies of Malus materials was collected from the professional nursery stock cooperative,Changyi district,Weifang city,Shandong province;the High-tech Innovation forestry Park of Rizhao city,Shandong province;the courtyard of Forestry Department of Shandong Province;Wang Zhuang sand area forest farm of Lijin County,Dongying,Shandong Province;the green area of the Yellow River delta comprehensive training base military area in Ji’nan military region;and the base of forest tree germplasm resources of Shandong Agricultural University.According to these primers,96 copies of Malus materials were isolated and identified,and genetic diversity analysis was carried out.The experimental materials were systematically analyzed and constructed from the DNA point of view,and the fingerprint was constructed to preserve the germplasm of Malus,avoid repeated collection and new varieties of the same germplasm.The research is of great significance to foster evolution and standardize the market names of Malus.The main results shows as the following:1.The five main factors affecting the SSR reaction system of Malus were optimized at different levels.Finally,the reaction system of the molecular marker of the Malus was established to be 20μL: Upstream primers and downstream primers are equal 0.15μL,1μL DNA original liquid,10ng/μL DNA template,0.5U TaqDNA polymerase 0.2μL,200 μmol/L dNTP1.6 L,200 m mol/L MgCl2 2μL.Using the double distilled water to adjust the final volume of the system to 20 μ L.Touchdown PCR amplification program is: predenaturation at 95℃ for 5 min;denaturation at 95℃ for 30s;refolding at Tm ℃(primers vary)for 30s;extending at 72℃ for 30s;after 35 cycles;extension at 72 ℃ for 10 min.Cryopreservated at 4 ℃.2.Choosing 18 pairs of primers with good stability,clear strip and good polymorphism from 40 pairs of SSR primers of Malus,which were used for genetic diversity analysis of the samples of Malus.After data analysis by capillary electrophoresis,18 pairs of polymorphic primers were detected,228 polymorphic loci were detected,and the polymorphic locus detection rate was 80.85%.Samples collected from the same place were taken as the same population.All samples were considered as six populations.The following results were obtained: The average number of alleles(Na)was 1.8085,the average effective allele number(Ne)was 1.1544,the average Nei genetic diversity index(H)was 0.1099,and the average Shannon diversity index(I)was 0.1920,the total gene diversity(Ht)was 0.1179,and the average gene differentiation coefficient(Gst)was0.2696.Based on comprehensive analysis of the data,Malus has high genetic diversity.3.Through the combination of genetic coefficient and related traits of new varieties of Malus,96 samples of Malus were clustered and analyzed.At the genetic coefficient 0.79-0.82,it was divided into the ‘Malus hupenensis group’,the ‘new hybrid breeding group’,the ‘Malus spectabilis group’,the ‘Malus baccata group’,the ‘Malus asiatica group’,the ‘Malus micromalus group’ and the ‘Malus Hubei group’.According to their relationship,the ‘new hybrid breeding group’,the ‘Malus baccata group’ and the ‘Malus asiatica group’ were divided into 6,2 and 4 groups,in order to distinguish the close relationship between the new cultivar of the Malus material collected.4.Through the amplification results of the experiment,96 samples were analyzed,and 88 samples of Malus germplasm were obtained.On this basis,18 pairs of highly polymorphic amplified primers and 88 germplasms were selected to construct the fingerprint of Malus.On this basis,86% of all the germplasms were identified by combination of five pairs of primers only,that is,five pairs of primers could be used to identify 76 Germplasms of Malus.These primers can be used as priority selection primers in the next research.Another 8 germplasms failed to detect differences,and whether the same germplasm or undetected differences still need further research and verification.