| Vitis amurensis Rupr.is an important resource of wild fruit in China.The rich resources of Vitis amurensis were identified mainly on morphological markers.This method is simple,but it was affected by the environmental and subjective judgment.DNA barcoding is a kind of molecular biology technique based on the principle of DNA molecular evolution and it is similar to barcodes of commodity in the supermarket,using a standard DNA fragment for rapid,accurate and automated identification of species.The problem on the identification varieties of Vitis amurensis will be solved by DNA barcoding which may provide an accurate basis for classification and identification,relationship analysis,phylogenetic analysis,genetic map construction,gene localization and early selection of breeding materials.At the same time,screening and establishment of Vitis amurensis DNA barcoding will be carried out.In the study,the materials came from the National Field Gene Bank for Amur Grapevine.The nuclear DNA ITS and ITS2 gene fragments as well as chloroplast DNA psbA-trnH,rbcL,matK gene fragments were selected.The DNAs of 33 samples from 11 germplasm in Vitis amurensis were extracted,amplified by PCR and sequenced.The amplification efficiency and success rate of sequencing were used to evaluate universality of five DNA barcodes in Vitis amurensis,and analyze the sequence features.Finally,two key DNA barcodes as ITS2 and psbA-trnH were selected and used in the study on the analysis and identification of DNA barcoding in 290 samples.Results were as follow:1.Universality evaluation of five barcodes in Vitis amurensis were: The amplication efficiency and success rate of sequencing are two important indicator for evaluating DNA barcode sequences.The amplification efficiency of ITS2,psbA-trnH,rbcL and matK were 96.9 %-100 %,and success rate of sequencing were 90.9 %-100 %,which showed good generality in Vitis amurensis.They can be uesed as candidate DNA barcode fragments to carry on further research.In addition,the amplification efficiency of ITS was 45.5 %,and success rate of sequencing was 30.3 %.2.Analysis of features of four DNA barcodes were: alignment length of fragments was 422-896 bp,and the numbers of variable loci were 44-297.And the average content of GC was 27.5 %-64.5 %.ITS2 and psbA-trnH have 297 and 238 variation sites,which means that the evolutionary rate is fast and could be used in the identification of Vitis amurensis.3.The screening and identification of DNA barcoding in Vitis amurensis were: Effectiveness of ITS2 and psbA-trnH sequences were evaluated by calculating specification of the inter-germplasm and intra-germplasm variation,barcoding gap analysis,and the identification effciency untill we screened the right candidate DNA barcoding for Vitis amurensis identification.Following results were:The mean value of All intra-germplasm distance was 1.036 %.The mean value of Theta was 0.970 %.The mean value of Coalescent depth was 9.870 %.The mean value of All inter-germplasm distance was 11.375 %.The mean value of Theta prime was 10.203 %.The mean value Minimum inter-germplasm distance was 0.265 %.The Barcoding gap diagram showed barcoding gap among ITS2.The identification efficiency was 55 % by BLAST,and the identification efficiency of NJ sequence analysis was 75 %.The mean value of All intra-germplasm distance was 1.442 %.The mean value of Theta was 1.296 %.The mean value of Coalescent depth was 2.426 %.The mean value of All inter-germplasm distance was 4.800 %.The mean value of Theta prime was 4.320 %.The mean value Minimum inter-germplasm distance was 2.650 %.The Barcoding gap diagram showed sequences some overlap,but it depicted barcoding gap among psbA-trn H.The identification efficiency was 60.5 % by BLAST,and the identification efficiency of NJ sequence analysis was 85 %,identification of Vitis amurensis in different flower types and regions。The psbA-trnH sequence was used as the DNA barcode candidate sequence of Vitis amurensis,and the ITS2 sequence was used as a complementary sequence. |
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