Development and application of AS-PCR and dCAPS markers for discriminating new cultivar 01-7 Huyou (Citrus changshanensis)

Abstract: 【Objective】Huyou (Citrus changshanensis K. S. Chen et C. X. Fu) is a local characteristic citrus species in China with a history of over a hundred years, and its main production area is in 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 plant a, Huyou elite plant b and 01-7 were obtained from the ordinary huyou population. Among them, 01-7 has become the main huyou cultivar to be extended because of its strong cold resistance, as well as long storage longevity and slight-but-special bitter flavor in the fruit. However, there is a high degree of simi-larity between different accessions of huyou, and it is difficult to distinguish among 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 on 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 were developed based on the obtained SNP information, and finally, the applicability of molecular markers was evaluated by the performance of ASPCR and dCAPS molecular markers in 12 huyou accessions covering main production bases and main cultivars/lines/plants.【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 highquality bases were obtained. The average scores of Q20 and Q30 were 96.55% and 89.97%, respectively, indicating a high quality of data. After mapping reads to the pummelo reference 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 ASPCR-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 successfully seen 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 was 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 template. 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 was 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 can not only distinguish 01-7 from ordinary huyou but also differentiate it from Huyou elite plant a, Huyou elite plant 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 supports the extension of 01-7.