基于RAD-seq技术的鹅掌楸基因组SNP标记开发

Development of genomic SNP markers based on RAD-seq and genome data in Liriodendron

【目的】开发大量可靠的SNP标记,为鹅掌楸高密度遗传连锁图谱的构建和基于基因组的林木选择育种提供分子基础。【方法】从北美鹅掌楸NK基因型为母本、鹅掌楸LS基因型为父本的F1代杂交群体中,选取198株个体为作图群体。用限制性内切酶EcoR I对包括2个亲本和198个子代在内的200个单株的基因组DNA进行酶切,构建RAD(restriction-site associated DNA)文库并进行RAD-seq测序。采用读长为91 bp的双末端测序。2个亲本的平均测序深度为2×,198个子代的平均测序深度为0.8×,平均产量为1.94 Gb,共获得约387.21 Gb数据。用Stacks软件将每个样品的RAD-reads作生物信息学分析,对候选位点进行卡方检验和缺失率检验,再将符合孟德尔遗传的标记及与之相对应的RAD-tag序列和鹅掌楸参考基因组序列进行比对。最后,从本研究开发的SNP标记中选取27个候选SNP位点,设计引物,对随机挑选的16个F1代进行PCR扩增并将结果进行测序,同时验证SNP的有效性。【结果】从候选群体中共鉴定到22 019个SNP位点,符合孟德尔遗传规律的标记为4 233个,最终获得3 501个候选SNP标记。SNP验证中,共有293个SNP标记完成测序并能判读结果,所有位点都为SNP位点,共有194(66.2%)个SNP变异类型得到了验证。【结论】基于RAD-seq技术和鹅掌楸参考基因组序列为基础的策略,能够作为一种快速有效的手段,实现大规模的分子标记开发,可用于鹅掌楸等林木的高密度遗传图谱的构建。

【Objective】 The large-scale development of reliable SNP markers is the key to obtaining molecular markers for the construction of high-density genetic linkage maps and for innovative selection and breeding strategies based on genomic information in Liriodendron.【Method】One hundred and ninety-eight F1 trees were selected as the mapping population, which was obtained by intraspecific cross between the NK(♀)(Liriodendron tulipifera Linn.) and LS(♂)[L. chinense(Hemsl.) Sargent] genotypes. Genomic DNA of 200 individuals, including 2 parents and 198 offspring, was extracted and digested using the restriction endonuclease EcoR I. RAD libraries were constructed using genomic DNA from both parents and F1 trees. The read length of paired-end sequencing is 91 bp. In total, 387.21 Gb of data were obtained, with an average sequencing depth of 2× in the parents RAD libraries and 0.8× in their progeny RAD libraries, and the average production was 1.94 Gb. RAD-reads from each sample were analyzed using Stacks software. The distribution of candidate loci was tested by chi-squared test and the deletion rate was evaluated. Then each RAD-tag sequence containing genetic markers,whose segregation ratios were consistent with the expected Mendelian inheritance rations, was aligned to the reference genome of L. chinense. In the end, we selected 27 pairs of SNP markers then designed primers for amplification within random 16 F1 by PCR in order to validate the accuracy of the candidate SNPs.【Result】A total of 22 019 SNPs were observed in the candidate population, and 4 233 markers conformed to Mendelian inheritance. Finally, 3 501 candidate SNP markers were identified. In the validation experiment, 293 SNPs were successfully sequenced and all of these were identified as SNP loci. A total of 194(66.2%) SNPs were validated in variation types.【Conclusion】RAD-seq is a fast and effective technique for the large-scale development of SNPs and construction of genetic linkage maps in Liriodendron and other taxa.