Development Of Method And Tool For Rice Variety Homogeneity Analysis Based On Whole Genome Resequencing

Precise evaluation of genetic background of rice(Oryza sativa L.)varieties/lines is essential for variety certification and identification.Traditional variety identification techniques were based on molecular markers,which have limitation of genomic coverage and poor representation of genetic characteristics.In this study,we conducted genome re-sequencing of multiple individuals from representative rice varieties and estimated intra-variety genetic variation as well as validating by genomic sequencing data from public datasets.The method of rice homogeneity assessment based on genome re-sequencing was proposed.A platform with 4,500 public rice varieties dataset(http://ricediversity.org/data/)for rice variety homogeneity evaluation was constructed based on the methods mentioned above.Detailed results are as follows:1.Five indica and japonica rice varieties(10 varieties in total)were selected from China National Rice Research Institute(CNRRI),and cultivated in the field for observation of growth uniformity;Five single plants from each variety were randomly selected for genome re-sequencing which generated a total of approximately 360 Gb data,with an average sequencing depth of 16×.Meanwhile,re-sequencing data of 15 rice varieties from public databases were downloaded(the number of institutes that uploaded the same variety ≥ 3 and average sequencing depth ≥ 3×)for validation of our proposed method.2.Inter-and intra-variety genetic variation analysis was carried out on the selected 10 CNRRI varieties.Phylogenetic tree and IBS distance analysis indicate that inter-variety variation was significantly greater than intra-variety variation and intravariety variation was limited in a certain threshold.Nucleotide diversity(π)was considered as a good parameter for homogeneity assessment.Among our resequenced varieties,the average π of 5 japonica varieties was suggested to be lower than 0.003(thus π was considered as assessment threshold)and 0.008 for indica varieties.Average π of the 15 public varieties was generally consistent with the obtained threshold.In addition,the heterozygosity of each sample based on whole genome sliding windows exhibited distribution consistency of intra-variety and significant variation of inter-variety.The concept of "whole-genome heterozygosity polymorphism fingerprint" was proposed,providing new insights into rice variety homogeneity evaluation and variety certification.Application of homogeneity analysis to public varieties revealed issues in certain varieties from public database.3.Based on the methods mentioned above,rice variety homogeneity assessment platform Variety Facer(http://ibi.zju.edu.cn/g Breeder/Variety Facer)was constructed with integration of genome sequencing data from 4,500 rice varieties.Two main functions were realized in our platform.Function 1: when variety of submitted sample was known,homogeneity comparison would be carried out between user sample and selected variety;Function 2: while variety of submitted sample was unknown,the platform can search against all of the collected varieties to compare the homogeneity.Currently we have collected approximately 600 genomic datasets from Chinese rice varieties and homogeneity analysis among them could be accomplishable.What’s more,1,200 Chinese rice varieties would be added into the platform in the near future.