马尾松松材线虫病抗性无性系的筛选和遗传多样性分析

【目的】 松材线虫病是松属致命性的世界检疫性森林病害。筛选和创新抗性遗传资源是马尾松抗松材线虫病育种的重要基础。松材线虫病重灾疫区自然淘汰存留下来的马尾松资源,可能是开展马尾松抗松材线虫病育种潜在的重要遗传基础,值得进一步深入挖掘、系统评价和开发利用。本研究通过对110个对松材线虫病具有抗性表型的无性系进行遗传多样性、生长性状测定和保存率调查,综合评价其在抗性育种中的潜在价值。【方法】 通过接种松材线虫对马尾松的1 201个初选无性系进行筛选,并对110个具有抗线虫表型的重选无性系进行遗传多样性RAPD和生长性状评价。从14对SCAR分子标记引物中筛选通用性强、多态性位点高的两组引物,用于MuPS(Multiplex-PCR of SCAR markers) 反应扩增和遗传多态性位点检测。基于79个MuPS唯一型无性系群体,采用Popgene 32软件估算的遗传多样性,结合生长量和存活率指标,评估该批遗传资源在马尾松抗性育种中的潜在价值。【结果】 初选的1 201个无性系(来自251个马尾松初选家系),经松材线虫接种筛选后,获得110个无性系(来自初选家系中的81个),由两组引物扩增得到92种MuPS分型。其中,有79个无性系为单一的MuPS分型所完全准确识别(占71.81%)。基于79个具单一MuPS分型的无性系群体遗传多样性分析表明,群体所有位点的平均有效等位基因数(N_e)为1.508 1个,Nei’s基因多样度(H)为0.302 3,Shannon多样性指数(I)为0.459 1。无性系体间遗传参数差异明显,N_e变幅为1.012 8~1.998 1,H变幅为0.012 6~0.499 5,I变幅为0.038 5~0.692 7。具有抗松材线虫病表型的110个马尾松无性系存活83个。保存无性系间其生长性状存在显著差异,树高、胸径和单株材积生长量变幅分别为4.6~10.7 m,6.7~21.7 cm和0.012 3~0.189 4 m³,生长量最大的休3-3号无性系立木材积(0.189 4m³)为最小的无性系广27-2的(0.012 3 m³)15倍以上。【结论】 马尾松1 201个初选无性系,经野外人工接种松材线虫的抗性筛选后,110个无性系表现出对松材线虫病有显著的抗性表型,占初选无性系的9.09%。这些经过抗性筛选的无性系在材积生长性状上达到了极显著差异水平。这意味着在抗性改良基础上,进行生长量改良的策略是可行的。与已有的天然和人工育种群体的遗传多样性参数相比,本研究虽然经初选和抗性复选淘汰了90%以上的无性系,仍保留了群体中较高的遗传多样性。综上,笔者认为这批初选资源经抗性筛选存留的遗传资源,在马尾松抗松材线虫病育种研究中具有明显的潜在价值。

Growth and genetic diversity analysis of clones screened by phenotypical resistant to pine wilt disease in Pinus massoniana

【Objective】 Pine wilt disease caused by pine wood nematode is one of the most deadly forest quarantine diseases in the world. Screening and innovation of resistance genetic resources is an essential basis for breeding for pine wood nematode disease resistance of Pinus massoniana. The remained genetic resources in the severely disease-affected areas in natural distribution of masson pine, may be an important and potential genetic basis for resistant breeding, which is worth further exploration, systematic evaluation, exploring and utilization. The aim of this study was to comprehensively evaluate the genetic resources of masson pine with phenotypical resistance to pine nematode and its potential value in resistance breeding by analyzing the genetic diversity, growth traits and surveil rate of the 110 selected clones.【Method】 All 1 201 primary-selected clones of masson pine were screened by inoculated pine wood nematode, and 110 re-selected clones with pine wood nematode phenotypic resistance were involved in the evaluation of genetic diversity by random amplified polymorphic DNA markers (RAPD)], and growth performance. From 14 pairs of SCAR markers, two pairs of primers with strong versatility and high polymorphic loci were selected for MuPS (Multiplex PCR of SCAR Markers) amplification and genetic polymorphic loci detection. The potential value of these masson’s pine genetic resources in resistance breeding was evaluated based on the genetic diversity parameters (estimated by Popgene 32 software) of the 79 clones with unique MuPS type, combined with the traits of single-tree volume and survival rate. 【Result】 There were 110 clones (from 81 families out of primary ) survived among the 1 201 primary selected clones (selected from the 251 primary families), after the inoculation with pine wood nematode. And 92 MuPS types were amplified from the 110 cloned by two pairs of primers, and 79 of the 110 clones were accurately identified by a unique MuPS type (71.81%). Based on the each unique MuPS type’s information of the 79 clones, the average number of effective alleles (N_e) at all loci was 1.508 1, the Nei’s gene diversity (H) was 0.302 3, and the Shannon’s diversity index (I) was 0.459 1. The N_e, H, I for the re-selected population was variated from 1.012 8 to 1.998 1, 0.012 6 to 0.499 5, and 0.038 5 to 0.692 7, respectively. There were 83 clones well survived out of the 110 re-selected clones after pine wood nematode inoculation. There were 83 clones well survived out of the 110 re-selected clones,after the pine wood nematode inoculation. The tree height, DBH and tree volume growth was ranged from 4.6 to 10.7 m, 6.7 to 21.7 cm, and 0.012 3 to 0.189 4 m ³, respectively. And the tree volume of the best performed clone (clone Xiu 3-3, 0.189 4 m³) was more than 15-fold over the lowest clone (clone Guang 27-2, 0.012 3 m³). 【Conclusion】 There were 110 clone among the 1 201 primary clones of masson’s pine, showed significant resistance to pine wood nematode disease after manual inoculation in field, which was around 9% of the primary selected clones. A significant variation/differentiation on tree volume growth was also found among the 110 re-selected clones with phenotypic pine resistance to wilt disease, which may indicated that it was possible to improve tree volume growth based on the disease resistance improvement background. It was worth to notice that, a higher genetic diversity was still existed in the reserved populations in this study, even though ninety percentage of the primary selected clones were eliminated through the disease resistance screening in this study. In conclusion, we believe that these precious genetic resources through resistance screening, have obvious potential value in P. massoniana growth and nematode disease resistance breeding.