Identification Of Actinidia Arguta Germplasm Resources Using DNA Barcoding And SRAP Markers

Actinidia arguta,also called ‘Ruan Zao Zi’,‘Teng Li’ or ‘Teng Gua’ in China,belonging to Actinidiaceae,Actinidia Lindl.is perennial defoliation vine and diecious plant.As a burgeoning fruit of high quality,Actinidia arguta is smooth,glabrous and directly consumable with a unique flavor,full of nutriments and Vc.DNA barcoding technique is a molecular biotechnology base on molecular evolution utilizing a short and standard sequence of DNA to identify species accurately and rapidly.Sequence related amplified polymorphism(SRAP)marker has been applied as the new random primer labeling since 2001,with a series of advantages of easy operation,high repeatability and lower cost.Currently,SRAP marker has been successfully applied to genetic diversity analysis and genetic maps construction.With advantages of being intuitional,clear and practicable,manual cultivar identification diagram(MCID)developed by Fang et al.can identify plant varieties rapidly in combination with molecular marker.The research performs a molecular identification study on 44 Actinidia arguta germplasm resources utilizing DNA barcoding and SRAP marker,the main results of the test are as follows:1.Barcoding sequence analysis: The research selects five candidate barcoding sequences,and the sequencing result of ITS fragment is poor.ITS sequence has been eliminated due to the low success rate(27.78%)of sequencing.The sequence-acquisition rate of the rest four candidate barcoding sequences is higher than ITS,which can be used in the identification test.2.The evaluation of identification method of DNA barcoding for Actinidia arguta: the genetic distance among different germplasm is relatively small.Barcoding gap analysis indicates that there are no significant ‘gap’ areas between inter-varieties and intra-varieties.NJ phylogenetic tree analysis indicates only a small part of germplasm can be identified but many germplasms cluster to the same clade.The overall evaluation indicates that the identification method is of low efficiency for different test Actinidia arguta resources.3.Resource identification analysis of DNA barcoding combined with SNP loci: It is found that there are 4 steady variable sites on ITS2 sequence among 44 germplasm resources,and one steady variable site on matK sequence.Construct MCID map utilizing DNA barcoding combined with SNP analysis,which can achieve identification for partial resources;however the overall identification efficiency is relatively low.4.Identification evaluation of DNA barcoding at the level of species: The barcoding gap analysis indicates there are significant ‘gap’ areas of the genetic distance distribution of inter-and intra-species.The phylogenetic tree analysis shows the Actinidia arguta can be cluster into individual branch,which indicate DNA barcoding method is able to identify the seedling of Actinidia arguta accurately at the level of species.5.Screening of SRAP labeled primer: we screen for SRAP labeled primer by using the genome DNA of ‘Kuilv’ and ‘Fenglv’(new varieties of Actinidia arguta),and screen out five pairs of primer combinations with high polymorphism,clear bands and steady difference.6.Construct MCID identification diagram of 44 resources using SRAP marker: screening out five pairs of primer combinations for PCR amplification of 44 germplasm resources.The MCID of 44 Actinidia arguta germplasm resources is constructed as per the results of electrophoretic band.7.Comparison and application of the two methods of molecular marker: DNA barcoding can identify species at the level of inter-species,and shows worse effects at the level of intra-species;SRAP marker can identify different germplasm resources at the level of variety.We should choose appropriate methods in practical identification task.