【Objective】Huyou (Citrus changshanensis K. S. Chen et C. X. Fu) is a local characteristic citrus in China with a history of over a hundred years, and its main production area is Changshan County, Quzhou City, Zhejiang Province. After a long process of propagation by seeds and selection by seedlings, new cultivars/lines such as ‘Cuihong’, Hongrou huyou, ‘Xiahong’, Huyou elite line a, Huyou elite line b, and ‘01-7’ were obtained from the ordinary huyou. Among them, ‘01-7’ has become the main huyou cultivar to be extended because of its strong cold resistance for the tree, as well as long storage longevity and slight-but-special bitter flavor for the fruit. However, there is a high degree of similarity between different accessions of huyou, and it is difficult to distinguish between them with naked eyes in terms of sapling morphology. With the progresses in techniques on genome sequencing and associated single nucleotide polymorphism (SNP) analysis, SNP mining and marker development are becoming an efficient and accurate way to discriminate close cultivars. However, to date, there has been no study related to SNP based molecular markers for discriminating huyou accessions. The aim of this study was to mine homozygous SNP(s) present between ‘01-7’ and ordinary huyou, and to develop efficient and accurate molecular markers for identifying ‘01-7’.【Methods】Whole genome resequencing was performed on ‘01-7a’ and ordinary huyou ZZ (ancestral tree), and after obtaining high quality sequencing data, bioinformatics analysis tools were utilized to identify homozygous SNPs between these two accessions. The authenticity of SNPs predicted in silico was verified by PCR, cloning combined with Sanger sequencing. Then allele-specific polymerase chain reaction (AS-PCR) and derived cleaved amplified polymorphic sequences (dCAPS) molecular markers was developed based on the obtained SNP information, and finally, the applicability of molecular markers was evaluated by the performance of AS-PCR and dCAPS molecular markers in 12 huyou accessions covering main production bases and main cultivars/lines.【Results】After removal of adaptor sequences, contamination and low-quality reads from raw reads, a total of 120.21 M of high-quality reads were obtained, and 36.06 Gb of high-quality bases were obtained. The average scores of Q20 and Q30 were 96.55% and 89.97%, respectively, indicating a high quality of the data. After mapping reads to the reference pummelo genome, the average mapping rate was 98.54%, and the 1´ genome coverage was above 95%, which was close to the whole genome coverage. A homozygous SNP, i.e., Chr1_7111834_G/A, was identified through bioinformatics analysis. The genotype of ‘01-7a’ at this SNP site was A/A, while that of the ordinary huyou ZZ was G/G, which was consistent with the results obtained by traditional gene cloning and Sanger sequencing. Based on this SNP, AS-PCR and dCAPS molecular markers were designed, and the performance of these two markers in 12 huyou accessions was evaluated. As expected, for AS-PCR marker, with AS-PCR-F1 primer, a specific band of 205 bp in size was amplified in all four ‘01-7’ accessions, while no band was amplified in the other huyou accessions; with AS-PCR-F2 primer, no band was amplified in neither of four ‘01-7’ accessions, while the amplification of the specific band of 205 bp was successful in other huyou accessions except for ‘Cuihong’. The result indicated that both pairs of allele-specific primers could identify '01-7'. However, an exception occurred where ‘Cuihong’ did not show amplification bands with both pairs of allele-specific primers, and the Sanger sequencing results of SNP flanking sequence showed that the above phenomenon is due to the presence of three nucleotide mutations in the forward primer region of ‘Cuihong’, which resulted in tremendous loss of complementarity for the primers to pair with the DNA sequence. For dCAPS marker, PCR product of 176 bp can be amplified from all four ‘01-7’ accessions and can be cleaved by restriction endonuclease FspBI into 24 bp and 152 bp bands, while that from the other huyou accessions, except for ‘Cuihong’, can be amplified but cannot be cleaved by the enzyme. For ‘Cuihong’ dCAPS, the PCR product was cleaved into 62 bp and 117 bp bands due to a mutation that created a natural cleavage recognition site as revealed by the Sanger sequencing. The results of Sanger sequencing of 12 huyou accessions further demonstrated that the genotype prediction by the two markers were correct. 【Conclusion】In this study, using the whole-genome resequencing data obtained from ‘01-7a’ huyou and ordinary huyou ZZ, we identified a homozygous SNP, i.e., Chr1_7111834_G/A, with the genotype A/A for ‘01-7’ and G/G for the ordinary huyou. Subsequently, based on this SNP, AS-PCR and dCAPS markers were developed. These markers not only distinguish ‘01-7’ from ordinary huyou but also differentiate it from Huyou elite line a, Huyou elite line b, ‘Cuihong’, and ‘Xiahong’, demonstrating stable performance. The application of these molecular markers enables the authentication of ‘01-7’ saplings, ensures sapling purity and thereby supporting the extension of ‘01-7’.
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