Construction And Application Of DNA Fingerprinting Traceability System Of The Main Japonica Rice Varieties In Heilongjiang Province

Heilongjiang Province is the main producing area of japonica rice in China.To improve the protection,quality traceability and management of rice germplasm resources in the province,a method for constructing the DNA fingerprinting map and traceability system of Japonica rice in Heilongjiang Province whihc is based on SSR molecular markers and capillary electrophoresis is proposed.The province provides technical support for the breeding of japonica rice varieties,the identification of the purity of varieties,and the protection of counterfeit and shoddy products.Through literature surveys,the Chinese Ministry of Agriculture's "DNA fingerprinting method for identifying rice varieties"(NY/T 1433-2007)(hereinafter referred to as the "national standard method")and the public database www.gramene.org as standards,12 pairs of recommended SSR primers were used to analyze the genetic diversity and DNA fingerprinting of 50 major japonica rice varieties in Heilongjiang Province(Chahayang,Jiansanjiang,Wuchang,Fangzheng and Xiangshui).According to the online barcode generator and QR code generative software,a proprietary bar code map and QR code map were constructed,meanwhile a traceability system for rice varieties was formed.The main research achievements are as follows:1.The design of rice SSR primer sequences:Through the methods of literature research,it summarized the characteristics of 3 388 rice germplasm resources in 17 provinces,cities,and regions from 2007 to 2016 using SSR markers.Analysis of the characteristics and research status of superior SSR Primers in China.The results showed that among the 354 pairs of primers in the survey,there were 24 pairs of primers that appeared more than 8 times.Compared with the 24 primers recommended by the national standard method,only RM273 appeared at a low frequency,only 6 times;The frequencies of all the 23 national standard primers were appeared more than 8 times,which showed that the goodness of fit between 24 common primers and national standard could be reach 96% in the last ten years.Therefore,the first 12 prs SSR primers which was recommended by the national standard method were tested.2.The analysis of genetic diversity of japonica rice in Heilongjiang Province:(1)SSR marker polymorphism analysis: 40 alleles are detected by 12 pairs of primers and capillary electrophoresis,and each pair of primers amplified 3.3 alleles with good polymorphism.The range of FP value of the polymorphic frequency of the marker is ranged from 0 to 0.667 with an average of 0.259,the PIC value of the polymorphism information of the marker is ranged from 0.093 to 0.493 with an average value of 0.266,the Ne number of effective alleles is ranged from 1.000(RM72)to 1.779(RM19)with a mean of 1.341,the Nielsen genetic diversity is ranged from 0 to 0.442 with an average of 0.204,and the Shannon information index is ranged from 0 to 0.608 with an average of 0.319.It turns out that the genetic background of the main japonica rice cultivars in Heilongjiang Province was narrow and the genetic diversity was low.The genetic background could be expanded by introducing new genetic resources.(2)The genetic similarity coefficient and genetic distance of the five rice habitats: The range of genetic similarity between five rice producing areas varied from 0.850 to 0.982 with an average of 0.928.The genetic similarity of the rice varieties was the highest(0.982)between Jiansanjiang area and Chahayang area,while the genetic similarity of the rice varieties was the lowest(0.850)between Wuchaang area and Chahayang area.The results showed that the genetic variation was relatively small,and the rice varieties in the five regions had higher genetic similarities and narrow genetic basis.(3)Cluster analysis of rice varieties: 50 clusters of indica rice cultivars were calculated by using the Origin Pro software upon Nearest Neighbor and Euclidean.Using the similarity coefficient of 3.4 as the boundary,50 varieties can be divided into eight groups.According to the genetic similarity coefficient,the rice in the five japonica rice growing areas in Heilongjiang Province can be further distinguished,in which the first category contains a maximum of 12 species,while the third category has a minimum of 3 species,even all four species are distributed in Wuchang.5 rice varieties in Xiangshui district and 4 in Fangzheng district are all respectively distributed in category IV and VIII.The results showed that the genetic information content of Heilongjiang japonica rice is complex,but Wuchang rice with geographical indication still can be accurately identified.3.The construction of DNA fingerprinting:Based on the results of the 12 preferred pairs of rice primers amplified and determined by capillary electrophoresis,the presence or absence of polymorphic fragments was recorded at a rate of 0/1,and 50 major japonica rice varieties were constructed in Heilongjiang Province.4.The construction of molecular identification of rice varieties:To construct unique identification cards for rice varieties,DNA fingerprints of 50 rice varieties were coded,and combined with the basic information of the varieties.They take the national technical standard ?GB/T 18347-2001 128 barcode? and the ?GB/T 18284-2000 rapid response matrix code ? as reference,using online barcode generator and QR code generative software to form the bar code and QR code,thus to build the rice varieties information.5.The construction and application of rice traceability system:In order to achieve internet-based technology of the sharing of DNA fingerprinting data of the main japonica rice in Heilongjiang province,it was used as the main body of the traceability system,to build the traceability system which based on the traceable information repository.