基于简化基因组测序的越橘杂交后代鉴定

Identification of F1 Hybrids in Blueberry(Vaccinium corymbosum L.)Based on Specific-Locus Amplified Fragment Sequencing(SLAF-seq)

【目的】针对遗传群体测序数据开发一种杂交后代鉴定方法,以获得继承双亲基因的真杂种后代,为果树杂交育种、遗传分析及遗传图谱构建奠定基础。【方法】本研究以越橘正反交群体共计318个F1子代和2个亲本为试材,利用SLAF技术进行简化基因组测序并比对越橘参考基因组获得群体SNP数据,通过稀有等位变异分析和基于PCA、K-means聚类的遗传关系分析鉴定供试群体中的非杂交后代,结果利用双亲纯合显性SNP标记进行验证。【结果】SLAF简化测序共获得65.89 Gb数据,GC含量39.72%,平均Q30为95.04%,亲本和子代平均测序深度为12.86×和5.41×。参照四倍体越橘基因组信息,正、反交组合分别获得73543个和114851个SNP,利用次等位基因频率(MAF)>0.05的SNP数据集分别对正、反交群体进行PCA和K-means聚类分析,鉴定出4个离群个体;利用MAF<0.05的SNP数据集对正、反交群体进行个体稀有等位变异和个体特有的稀有等位变异数统计,共鉴定出10个离群个体(包含了MAF>0.05的SNP数据集鉴定的4个离群个体)。通过双亲纯合显性SNP标记进行验证,正、反交群体双亲纯合SNP位点分别占群体总SNP的34.56%和38.95%,除H194-123个体外,其余非杂交后代在验证结果中同为离群个体,即准确通过验证。【结论】对于有参考基因组物种的杂交群体,利用基于测序的SNP次等位基因频率(MAF)数据集,采用遗传关系和个体特有的稀有等位变异分析方法,从不同角度反映群体子代间的遗传关系以鉴别离群个体,是一种鉴定群体假杂交后代的有效方法。

【Objective】The aim of this investigation was to develop a method of identifying true F1 hybrids based on next-generation sequencing data,so as to provide an alternative strategy for fruit breeding,genetic analysis and genetic map construction.【Method】A total of 318 F1 population derived from reciprocal cross between two blueberry(Vaccinium corymbosum L.)accessions were sequenced by specific-locus amplified fragment sequencing(SLAF-seq).Based on the blueberry reference genome,the rare allelic variation,principal component analysis(PCA)and k-means clustering were used to identify the accidental hybrid in blueberry population.Furthermore,homozygous SNP were used to confirm the data.【Result】A total of 65.89 Gb sequence data was generated by Illumina sequencing,in which the GC content was 38.63%and Q30 value was 95.44%.The average sequencing depth of the two parents and F1 progenies were 12.86×and 5.41×,respectively.By aligning the sequencing reads onto the reference genome of Vaccinium corymbosum,a total of 73543 and 114851 SNPs were called in two reciprocal cross populations,respectively.According to these SNPs with minor allele frequency(MAF)more than 0.05,PCA,and k-means analysis,it was yielded four outlier individuals regarded as accidental hybrid progenies.Moreover,ten outliers were identified after total rare-alleles analysis and the private rare-alleles analysis by using SNP with MAF﹤0.05,including the above four individuals.By verifying the accidental hybrid progenies,homozygous SNP between the two parents was accounted for 34.56%and 38.95%of the total SNP,respectively,which were subjected to genotype the ten outliers and conflicted genotypes,and which were observed in individuals except H194-123 with the two parents,and the accuracy of our developed methods was validated.【Conclusion】For species with a reference genome,it was an effective strategy by using allele frequency(MAF)data set to identify the accidental hybrid progeny based on genetic relationship analysis and unique rare allele variation analysis.