Identification of Local Citrus Breeds Using the Start-Codon Targeted Polymorphism Technique Combined with Clonal Sequencing

Abstract

This study explored the genetic diversity of 35 citrus accessions using start-codon targeted (SCoT) markers. Total 15 primers were used to amplify products ranging in length from 100 to 2000 bp. A total of 133 fragments were amplified and found that 126 (95%) were polymorphic. The genetic similarity coefficients among the 35 accessions ranged from 0.67 to 1.00, indicating that the SCoT markers could reveal high genetic diversity in the citrus germplasm. A cluster analysis showed that local citrus breeds of Sihui city fell into one cluster, possibly reflecting the geographical distribution of the tested samples. The sizesof the fragments of eight local citrus cultivars amplified by one of the primers ranged from 951 to 1001 bp, and the similitude was 95.17%high. Both single-base mutations, insertions and deletions were identified among the fragments and comparison with asequence database suggested that the amplified region was part of a ribosomal protein-coding sequence. Using Scot marks combined with clonal sequencing, each of the test samples had one or more mutation sites that could be used as markers to differentiate from the other seven test samples. Thus, the resolution of the genetic diversity among the local citrus breeds revealed by the SCoT technique was enhanced by the subsequent sequencing analysis of specific fragments. The experimental results also provide evidence that the relationship between CitrusnobilisL our. ‘gonggan’ and C. hanianaHort. ex Tseng ‘Sihuihanggan’ is that between parent and offspring hybrids and not between bud-mutation strains. The SCoT marker is a targeted gene molecular marker, based on the characteristic bands of primers, it can be used as a marker to isolate genes of local citrus varieties and also for investigating mutational hotspots of the segene.© 2021Friends Science Publishers