大花序桉心边材变异规律和候选基因挖掘研究

Sapwood and Heartwood Variation Pattern and Candidate Gene Discovery in Eucalyptus cloeziana

为探究大花序桉(Eucalyptus cloeziana)心材比例差异显著的不同家系间心边材变异规律,挖掘心材变异相关的候选基因,为珍贵用材树种高效培育及育种利用提供基因资源。以18 a生的2个心材比例差异显著的大花序桉家系为材料(家系1和2),各制作解析木3株,沿着树干以1 m为区间分段截取圆盘,测量东西和南北2个方向的带皮直径、去皮直径、总年轮数、边材年轮数、边材直径,并开展心材和边材径向和轴向分析。同时利用各解析木胸径处初生木质部样品进行DNA混池测序,发掘等位基因频率差异显著的SNP位点并挖掘相关功能基因。结果表明,大花序桉边材宽度和心材半径的方位变异中家系2大于家系1,平均差值分别为0.7和5.5 cm,在随树高的变异中,家系1和2的心材半径和心材年轮数的下降速率分别为0.40和0.64及0.43和0.36。两家系间基本密度差异显著,家系1为0.80~0.82 g/cm^(3),家系2为0.75~0.78 g/cm^(3)。基本密度与树高、横截面半径和心材半径呈显著负相关,与顺纹抗拉强度、弦面硬度和部分力学性质呈显著正相关。利用DNA混池测序共筛选到两家系间基因频率显著差异的SNP位点1842个,SNP注释分析表明位于基因间、上游区域、下游、外显子和内含子区域的SNP位点分别为55.8%、18.3%、16.3%、5.1%和4.4%。基因功能注释及富集分析发现SNP位点区域的基因主要与植物细胞分裂、植物细胞膜和植物蛋白激酶相关。通过大花序桉心材比例差异大的两家系间解析木径向和轴向分析,探讨了它们的心边材变异规律,结合DNA混池测序,挖掘了心材变异相关的SNP位点并筛选出一些木材形成相关候选功能基因。

In order to provide the genetic resource for accelerating the breeding in Eucalytpus,the variation of heartwood and the related candidate genes were explored in E.cloeziana.Wood discs were cut at intervals of 1 meter along the stem of felled tree in two 18-year-old families with contrasting high and low heartwood width,and each family has three individuals.The diameter outside and inside bark,overall ring number,sapwood ring number,sapwood width at two directions of northeast and southwest were measured.Radial and axial analysis were analyzed and the DNA from developing xylem tissues were harvested at breast height and used for bulk DNA sequencing,the SNP loci and the functional genes highly related with the variation of heartwood were discovered.The variation of sapwood width and heartwood radius among orientations were the highest in family 2,the average difference in family 1 and 2 were 0.7 and 5.5 cm,respectively.The decrease rate of heartwood radius and ring number with tree height were 0.40 and 0.64 in family 1,and 0.43 and 0.36 in family 2.The average basic density of E.cloeziana was significantly different between two families,with family 1 ranged from 0.80 to 0.82 g/cm^(3),and family 2 from 0.75 to 0.78 g/cm^(3).The basic density was negatively correlated with tree height,cross-section radius and heartwood radius,and positively correlated with tensile strength parallel to the grain and the hardness of tangential section,along some mechanical properties.Using Bulk DNA sequencing,a total of 1842 SNPs with high difference allele frequency were screened.Approximately 55.8%of these SNPs were distributed between genes,18.3%in the upstream region,16.3%in the downstream region,5.1%in exons and 4.4%in introns using SnpEff annotation.The identified SNPs were used to locate the genes belonging to 50 terms,mainly related to plant cell division,plant cell membrane and plant protein kinase by GO enrichment analysis.By conducting the radial and axial analysis of these two families with contrasting high and low heartwood width,the trend of sapwood and heartwood variation were discussed.The SNPs and candidate genes were screened by bulk DNA sequencing,and the functions of genes involved to the process of sapwood formation were explored.