| In this study, the walnut germplasm resources in 6 areas(Minhe County, Xunhua County, Jianzha County, Hualong County, Ledu district, Guide county) were investigated in Qinghai Province. A total of 211 walnut plants as the object of study, main economic characters of walnut index were measured, including: fat content, protein content, fatty acid content and content of amino acid, also with DNA genetic markers, analyzes the differences between different areas of walnut. The main results are as follows:1.Main economic characters of Walnut were measured and analyzed among the 6 different original. The result show that the substances diversity was high among germplasm in Qinghai. The fat content varied from 9.69% to 31.21%, the average was 61.92%, and the variation coefficient of fat content was 9.96%. Monounsaturated fatty acid(Oleic) form 7.14 to 51.91; Polyunsaturated fatty acid(Linoleic, Linolenic) from 41.93 to 84.24; Unsaturated fatty acidfrom 88.24 to 95.56%. The protein content in walnut kernel varied from 10.02 to 25.23, the average was 16.94%, and the variation coefficient of protein content was 14.76%; The total amino acids content in walnut kernel varied from 11.1453 to 28.5423, the average was 17.4518%, and the variation coefficient of total amino acids content was 5.6582%; The content has much difference in different kinds of amino acids, content from 0.4802 to 3.7614, the average was 1.1635%, and the variation coefficient from 14.5814 to 26.3897, the average variation coefficient was 18.0956.2. The OD260 /OD280 value was between 1.72~1.97 of the extracted genomic DNA, the concentration was between 120ng/μL ~ 580 ng/μL.According to the orthogonal design, in separate system, the optimal level of the 5 factors that affect the PCR reaction was: 0.45 mmol·L-1 dNTP, 0.5 umol·L-1 Primer, 1.75 mmol·L-1 Mg2+, 0.75 U Taq Polymerase and 80 ng DNA. In 10μL mix system, the optimal PCR reaction level was 5μL 2×Taq PCR MasterMix 、1.5 umol·L-1 Primer 、100 ng DNA and 1.5μL H2 O. There were 12 primers were screened out used for PCR amplification. The range of amplified bands were between 100-2000 bp, the number of bands from 11-19, There are 167 bands were amplified, the average was13.92, of which 127 were polymorphic loci. The average PPL(percentage of polymorphic loci) was 76.05%. The ISSR-PCR results showed that: At population level, NP(The number of polymorphic loic) was 100.67, the average vaule of PPL was 60.28%, A(Observed number of alleles) was 1.6028, Ae from1.3456 to 1.4028,the average was 1.3665, H from 0.2071 to 0.2377, the average was 0.2156, I was 0.3223. At species level, NP was 127, the average vaule of PPL was 76.05%, A was 1.7605, Ae was 1.3866, H was 0.2339, I was 0.3585. Gst(Genetic coefficient of differentiation) among populations based Nei’s gene diversity was 0.0826, It indicated that 8.26% genetic differentiation existenced among populations, and 91.74% genetic differentiation existence within populations, the genetic differentiation within populations was higher than that among populations. The Nm(gene flow) was 5.5527, It indicated that there was a large gene exchange among the populations of walnut. The difference among populations and within populations were significant(P < 0.001). The genetic distances among populations varied from 0.0154(XH and MH) to 0.0414(MH and JZ) according to Nei’s(1978) method, the average was 0.0257. Analysis of UPGMA cluster for 6 populations by using genetic consistency of walnut showed they were divided into 3 groups at 0.98, first group included XH and MH, second group included JZ and HL, GD and LD were in third group. 12 primers could separate the 211 walnut plants, The similarity coefficient was between 0.9521 and 0.6048, the average was 0.8023. UPGMA cluster analysis showed that 211 samples were clustered into 3 groups at 0.76. The tested results of UPGMA clustering by Cophenetic Correlation analysis showed the correlation coefficient r was 0.8159, it indicated clustering results was fine. |
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