关键词: 柑橘, 芽变, Target SSR-seq

Abstract: 【Background】Bud sports mutation is a DNA mutation occured in somatic meristem, it often display visible morphological and other charactistics changes different from its mother plants in branches, leaves, flowers and fruits. However, discrimating bud sports mutation from the epigenetic variation caused by environmental conditions and cultivation measures etc. external factors was still mostly depended on the morlecular fingerprint detection. 【Objective】This study objective was to identifiy citrus bud sports mutant through Target-SSR sequencing thechology.【Method】Firsly the genome of clementine mandarin (Citrus reticulata Blanco) and satsuma (Citrus unshiu Macf.), as well as GSS and EST sequences of satsuma were used to scan SSRs loci with GMATA, the highly polymorphic SSRs loci were screened out to design primers, multiplex PCR with optimized primers were amplified on 60 citrus bud sports mutants to construct high-thoughout sequencing library, the ampification products were then sequenced on illumina Minseq platform, the clean sequencing short reads were mapped to reference target sequences to find differentiated SSRs loci presented in citrus bud sports mutants.【Result】 A total of 77 pairs of SSR primers were designed from highly polymorphic SSRs loci. The primers pairs combination were optimized and 18 multiplex PCR amplification products were sequenced. Target SSR-seq analysis showed that the genotyping data of SSRs could divided 60 citrus accessions into two groups correspongding to sweet orange and mandarin, and mandarin group could be further subdivided into different citrus cultivar such as Orah, ponkan etc. 11 SSR loci containing mostly ATT motif were found in 7 Tarroco blood orange mutants, 8 SSR loci containing mostly TAA motif were found in 2 ‘Wu Yue Hong’ mutants and 5 navel orange, 16 SSR loci containg mostly GA motif were found in 9 Bing Tang Cheng mutants, 9 SSR loci containg mostly AAT motif were found in 2 Sha Tang Ju mutants, and 15 SSR loci with mostly AAT motif were found in 4 satsuma mutants. This study showed that Target-SSR sequecning technology provided an excellent resolving approach to discriminate citrus bud mutations.【Conclusion】In this study, an effictive method for citrus bud mutant identification using Target SSR-seq technology were established, 60 citrus germplasm accessions could be discriminated. The precision and reliability of SSR genotyping information could be utilizaed in citrus germplasm resources managemant and variety intellectual property protection.

Key words: citrus, bud sports, Target SSR-seq