胡柚新品种01-7的AS-PCR和d CAPS标记开发与应用

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

【目的】挖掘胡柚新品种01-7和普通胡柚间的差异单核苷酸多态性(SNP),据此开发可区分01-7与其他胡柚的分子标记。【方法】对01-7a和普通胡柚ZZ(祖宗树)进行全基因组重测序,利用生物信息学分析方法挖掘两份材料间的纯合SNP。采用PCR、克隆结合Sanger测序对SNP的正确性进行验证,进而开发等位基因特异PCR(AS-PCR)和衍生酶切扩增多态性(dCAPS)分子标记体系,最后应用12份胡柚材料就AS-PCR和dCAPS分子标记的普适性进行评估。【结果】对01-7a和普通胡柚ZZ重测序原始数据进行过滤,共获得高质量碱基数36.06 Gb。利用生物信息学手段挖掘出一个纯合SNP:Chr1_7111834_G/A。01-7a和普通胡柚ZZ在该SNP位点的基因型分别是A/A和G/G,这一结果得到了经典的基因克隆-Sanger测序确认。基于此SNP开发了AS-PCR和dCAPS分子标记,经对12份胡柚材料测试,表现符合预期:AS-PCR-F1在所有4份01-7中扩增出特异条带,而在其他胡柚材料中无扩增;AS-PCR-F2在01-7中无扩增,在其他胡柚材料(脆红除外)中扩增出特异条带;所有4份01-7材料在dCAPS分析中出现酶切条带,而在其他胡柚材料(脆红除外)中不被酶切。脆红因突变产生天然的酶切识别位点而被酶切。经典的基因克隆-Sanger测序结果确认两种标记正确区分了基因型,并发现脆红有着不同于其他胡柚的分子标记条带是由于它在该SNP位点侧翼还有额外的序列变异。【结论】基于全基因组重测序数据挖掘出01-7a胡柚和普通胡柚ZZ间的一个纯合SNP,据此开发了ASPCR和dCAPS分子标记,该两标记可区分01-7、脆红、夏红和其他胡柚。

【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 similarity 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.