火炬松叶绿体基因组遗传多样性及育种群体亲缘关系调控

Based on the previously accomplished sequence of loblolly pine (pinus taeda) chloroplast genome, which has been submitted to NCBI database and been released, in this project, mixed sample from the first-generation nucleus breeding population will be sequenced by using high-throughput sequencing technology, with the above mentioned submission as the reference sequence. After comparison, genetic diversity and variation in the chloroplast genome of loblolly pine will be profiled, and diversity loci will be screened. Featured primer PCR based single nucleotide polymorphism (SNP) markers will be developed with the high frequent mutation loci. The new developed SNP markers will be used to detect the second-generation nucleus population, in order to test the stability of the polymorphic loci among different generations under complete pedigree mating design. The tested SNP markers will be used to detect and profile the genetic relationship in the main populations of two generations and then, inheritance model of genetic relationship among different generations under incomplete pedigree mating design will be established. this will be the basis of regulation for the genetic relationship in the breeding population. The project will systematically explain the polymorphism and variation in the chloroplast genome sequence of loblolly pine, establish a new technology system to regulate the genetic relationship in the breeding populations of loblolly pine, innovate the model of multi-generation recurrent selection. This is significantly important to increase the level of genetic improvement in loblolly pine.

以课题组精确测序并提交到NCBI数据库的一个火炬松叶绿体基因组序列为参考序列，采用高通量测序技术测定火炬松第一代核心育种群体混合样品的叶绿体基因组序列，经过比对分析，绘制火炬松叶绿体基因组个体水平突变图谱，筛选多态性位点，开发基于特异引物PCR的SNP标记，用于检测火炬松第二代核心群体的亲缘关系，验证多态性位点在完全谱系交配设计条件下世代传递的遗传稳定性，经验证的标记用于检测火炬松两个世代主群体的亲缘关系，建立育种群体亲缘关系图谱，确立亲缘关系在不完全谱系交配设计条件下在不同世代间的传递模式，作为群体亲缘关系调控的依据。本项目将全面阐明火炬松叶绿体基因组序列多态性以及在个体水平的变异模式，建立新型的松树育种群体亲缘关系调控技术体系，创新松树多世代轮回选择育种模式，对提高火炬松遗传改良水平具有重要意义。

在火炬松高世代实际育种中，为了全面了解杂交组合的效应和避免群体内亲缘关系过快增加，采用完全谱系交配设计和人工控制授粉，这样不仅杂交组合多，而且耗时耗力、投入大。为了解决高世代育种面临的问题，本研究利用叶绿体序列鉴定两代亲本间的关系，为控制育种群体亲缘关系提供理论和技术支撑。.对核心育种群体54个火炬松单株的叶绿体进行测序，得到的叶绿体基因组序列与NCBI数据库中的火炬松叶绿体序列（编号：NC_021440.1）比对，发现有81个SNP位点，SNP位点分布广泛，不均地分布在3990bp到119714bp之间（火炬松叶绿体大小为121530bp）。对等位基因频率大于5%的32个SNP所在序列进行注释，14个定位在基因间隔区，18个定位在9个基因内。多数基因都只有一个SNP位点，而个别基因有多个SNP，如ycf2基因有3个SNP，占总SNP的3.70%，占基因内SNP的16.67%；ycf1基因检测到9个SNP，占总SNP位点的11.11%，占基因内SNP位点的50%，表明ycf1基因很活跃。.根据变异频率大于5%的SNP位点设计23对特异引物，对火炬松测交系群体45个子代及其母本和父本共75个材料进行扩增，将所有扩增得到的产物进行Sanger测序。在火炬松75份种内材料中，存在19个SNP位点，表明这些火炬松的叶绿体基因组变异丰富。用测交系的母本和父本与子代的19个SNP进行比对，所有母本的SNP都与子代不一致，说明火炬松叶绿体非母系遗传。在父本N4的子代中，有7个子代的19个变异位点与父本一致。这表明火炬松叶绿体不是母系遗传，而是父系遗传。.当前我国火炬松遗传改良已经完成了第二代，正向第三代过渡，利用叶绿体基因组技术检测并建立育种群体亲缘关系图谱，可为火炬松高世代育种实施自由授粉交配设计、鉴定优树父本以及控制育种群体亲缘关系提供理论和技术支撑。