Genetic diversity analysis and fingerprinting of citrus hybrid varieties based on SV markers

【Objective】Citrus hybrids have gained significant popularity among consumers and hold broad market prospects due to their desirable characteristics, including juiciness, easy-to-separate segments, rich flavor, low-seed or seedless flesh, and easy peeling. However, with the increasing number of commercial varieties and the expansion of planting areas, issues such as variety mixing and counterfeit labeling have become increasingly prominent. These problems are particularly difficult to identify at the seedling stage, which severely restricts the healthy development of the citrus industry. Therefore, it is urgent to establish rapid and accurate variety identification techniques to protect breeders' rights. To address this need, the present study utilized structural variation (SV) molecular markers to analyze the genetic diversity of 16 citrus hybrids and constructed their corresponding molecular identification profiles. This research aims to provide technical support for the identification and protection of citrus hybrids germplasm resources, while also offering data references for further exploration of their genetic background.【Methods】This study selected 16 popular citrus hybrids as materials. Genetic diversity analysis and molecular fingerprint construction were based on 7 qualitative traits and 34 pairs of SV marker primers. Data for qualitative traits were processed using Microsoft Excel 2016, and the genetic diversity index (H) was calculated using SPSS 25 following the Shannon-Weaver method. PCR products amplified by the 34 SV marker primer pairs were detected via agarose gel electrophoresis, and banding patterns were scored as“0”and“1”. The Data Formatter software was used to convert the binary (0/1) data into fragment size (bp) data. Popgene 32 software was used to calculate the number of alleles (Na), the effective number of alleles (Ne), the Shannon's information (I), the observed heterozygosity (Ho), the expected heterozygosity (He) and the Nei's gene diversity (H). The Power Marker V3.25 was used to calculate the major allele frequency (MAF) and polymorphism information content (PIC). Cluster analysis was performed using NTSYSpc2.10e software to calculate genetic similarity coefficients, and a dendrogram was constructed using the UPGMA method.【Results】The results showed that the 7 qualitative traits across the 16 citrus hybrids exhibited 16 variation types, with an average diversity index of 0.517 5. While there was some variation among the 16 citrus hybrids accessions in traits such as leaf blade shape, leaf base shape, wing leaf shape, and leaf margin, these traits were insufficient to fully distinguishing the tangor varieties at the seedling stage. Furthermore, seedling phenotypic traits are easily influenced by environment and cultivation conditions, indicating that morphological identification can only serve as an auxiliary method for variety identification. Eight SV marker primers exhibiting good polymorphism were selected from the successfully 34 amplified primers; these eight primers could collectively distinguish 14 citrus hybrids. Among them, the varieties Nishinoka, Asumi, and Yangguang No. 1 possessed unique genotypes distinguishable using just one primer pair. A total of 19 alleles were detected by the eight SV marker primers. The number of alleles (Na) was 2.375, the effective number of alleles (Ne) was 2.062, the major allele frequency (MAF) was 0.582, the polymorphism information content (PIC) was 0.393, the Nei's gene diversity (H) was 0.492, the average Shannon's information (I) was 0.742, the observed heterozygosity (Ho) was 0.625, and the expected heterozygosity (He) was 0.508. These results indicate that the eight SV marker primers possess good polymorphism and that the tested 16 citrus hybrids have a high degree of heterozygosity and relatively rich genetic diversity. Cluster analysis revealed that at a genetic similarity coefficient of 0.64, the 16 citrus hybrids could be divided into five groups. Varieties bred using Kiyomi (a monoembryonic trait) as a female parent or its descendants as parents, were mainly clustered into the first group. Varieties without Kiyomi ancestry were primarily clustered into the second group. Yangguang No.1, Haruka, and Jinqiu Shatangju each formed separate groups, indicating significant differences in their genetic backgrounds compared to the other 13 citrus hybrids accessions. Using the amplification results from the eight selected SV marker primers, unique molecular fingerprints were constructed for 14 citrus hybrids. Subsequently, six samples with established molecular fingerprint information provided by an external institution were tested, verifying that the established citrus hybrids molecular fingerprints can be used for authenticity identification of citrus hybrids at the seedling stage.【Conclusion】This study statistically analyzed the seedling phenotypic traits of 16 citrus hybrids and found them to be relatively consistent, making it difficult to directly identify varieties based on morphological characteristics at the seedling stage. Based on citrus whole-genome sequencing data, eight SV markers were developed and screened. 14 citrus hybrids could be distinguished combinations of these eight SV marker primers. Furthermore, the amplification results of the eight SV markers were converted into character strings and combined to successfully construct the unique DNA molecular fingerprints for 14 citrus hybrids. Corresponding barcode and QR code molecular fingerprints were generated using a QR code generator. Additionally, the results of genetic diversity and cluster analysis of the 16 citrus hybrids indicated that the tested varieties have complex genetic backgrounds and rich genetic diversity. In summary, the above research findings provide effective technical support for the identification and protection of citrus hybrids at the seedling stage.