摘要
翅荚木是我国特有的珍稀、速生、多功能树种,具有广阔的开发利用前景。翅荚木作为我国西南各省石漠化治理及困难地造林的先锋树种,野生资源已处于濒危状态。但该树种被引种到福建、江西、浙江等地后,其速生性显著优于当地其他乡土树种,与杨树、桉树相当。过去对翅荚木的研究主要集中在田间栽培技术上,其他基础研究工作均未见报道。本研究以调查收集的翅荚木主要分布区的9个种源为材料,于2005年-2007年开展了种源遗传多样性及其抗低温胁迫能力研究,利用ISSR分子标记技术结合苗期种源试验分析了其遗传多样性,分析了不同种源的抗寒性,以便为今后的良种选育和丰产栽培提供科学理论依据。本项研究的主要结果如下：
1、翅荚木不同种源的苗期生长规律。湖南通道、江华、广东英德、翁源、广西桂林、平果、忻城、靖西、贵州兴义等9个种源苗期试验表明,翅荚木不同种源的种子大小及小叶叶片大小存在极显著的差异,可作为区分种源的重要特征。不同种源苗期生长量不存在极显著差异,但田间试验表明不同种源的抗寒能力存在极显著差异。湖南通道种源苗木在连续七天的日极端最低气温均低于零度,最低气温达-6.1℃的情况下也能安全越冬,表现了较高的抗低温胁迫能力。因此认为抗寒性等特异性状的选择应该成为翅荚木种源选择的重要内容,单株选择是速生良种选育的关键。研究还观察了翅荚木不同种源的苗期生长节律,并利用Logistic方程拟合建立了生长模型,发现翅荚木小苗移植后6月份生长加速,7月-8月为苗木的速生期,径生长高峰期稍早于高生长高峰期,显示了翅荚木的喜阳耐旱的特性。
2、翅荚木不同种源的抗低温胁迫能力。结果表明,翅荚木幼苗经低温诱导后体内生理物质发生变化,保护物质(可溶性蛋白、可溶性糖)和渗透调节物质(游离脯氨酸)含量增加,体内抗氧化酶(SOD、POD)活性增强；在7℃时可溶性蛋白含量、可溶性糖含量、脯氨酸含量和SOD、POD酶活性均处于最高值,而丙二醛(MDA)含量处于最低值,此时翅荚木的最大抗寒力已经形成。翅荚木枝条经低温冷冻处理后,生理指标发生变化并在一定的低温下存在显著差异；-8℃处理后可溶性蛋白含量有较大的增加,MDA含量回升,SOD活性种源之间开始出现明显差异；-12℃处理后,相对电导率急剧增加,除通道种源外所有种源的相对电导率达到了50%以上,细胞质膜产生了严重的伤害。叶片解剖结构分析表明,不同种源翅荚木叶片细胞结构紧密度(CTR)和疏松度(SR)与种源的抗寒性有一定的关系,CTR值越大、SR值越小,种源的抗寒性越强。采用Logistic方程拟合出湖南通道种源枝条的半致死温度为-13.06℃,低于其它种源0.89℃-2.74℃。利用差示扫描量热仪(DSC)测量的湖南通道种源叶片组织细胞的结晶温度为-15.04℃,低于其他种源0.42℃-1.34℃。综合分析表明翅荚木不同种源的抗寒性强弱的排序为：湖南通道>贵州兴义>广西忻城>广东英德>湖南江华>广西靖西。
3、翅荚木不同种源的遗传多样性。成功地建立了翅荚木的ISSR-PCR反应体系,筛选出的10个引物平均扩增条带5.4条,引物平均多态位点比率为64.29%。6个翅荚木种源平均多态位点比率达到56.48%,Shannon指数(I)为0.3048,Nei指数(H)为0.2052,表明种源间存在一定程度的遗传分化,其遗传多样性水平高低为：忻城>江华>英德>靖西>通道>兴义。翅荚木物种的基因流Nm=0.4840,种源间的基因多样度Dst=0.1058,种源间存在一定的基因流动,遗传分化处于中等水平；种源间的遗传变异占总遗传变异的34.03%,65.97%的遗传变异分布在种源内的个体之间。翅荚木种源的遗传距离与地理距离没有显著的相关性,多态位点比率与叶片形态显著相关,Shannon信息指数(I)、Nei's基因多样性指数(H)与生长性状显著相关,但均与抗寒生理指标无关,因此可以认为翅荚木的抗寒性状主要由特异基因所控制。根据种源间遗传距离聚类结果可将翅荚木6个种源大体分为4大类：第Ⅰ类为英德、忻城、通道种源：第Ⅱ类为靖西种源；第Ⅲ类为兴义种源：第Ⅳ类为江华种源。翅荚木各大类群种源之间存在地形隔离,与地理分布格局基本一致。
Zenia insignis, one of the most valuable, rare, rapid growing species with multi-functions, has a bright prospect of utilization. As pioneer tree species in difficult forestation regions, the wild resources of Z. insignis were getting extinct. Z. insignis has been introduced to Fujian, Jiangxi and Zhejiang provinces, and its capability of rapid growing excelled indigenous trees and equaled to poplar and eucalyptus. Previous researches on Z. insignis focus on cultivation techniques, while no other basic researches were reported. This paper investigated genetic diversities of Z. insignis provenances and their resistance to low temperature stress using nine Z. insignis provenances collected from their main distribution areas as materials from 2005 to 2007. Moreover, genetic diversity of the Z. insignis provenances was estimated by combination of ISSR marker and provenance experiments in seedling stage. This study would provide a scientific base for cultivation and breeding of Z. insignis. The main results are as follows:
1. The growth pattern in seedling stage of different Z. insignis provenances. Experiments were involved in nine provenances including Tongdao and Jianghua in Hunan, Yingde and Wongyuan in Guangdong, Guilin, Pingguo, Xincheng and Jingxi in Guangxi, and Xingyi in Guizhou. Results showed that there were extremely significant differences in seed sizes and leaf sizes between different provenances which were considered to be important characters distinguishing different provenances. Extremely significant differences were not present in production volume in different seedling stages, but in capability of cold resistance which was indicated by field experiments. Provenance of Tongdao, Hunan, could safely winter under the conditions of daily lowest temperature below 0℃, even by -6.1℃within durative seven days, showing good capability of resistance to stress. Therefore, special characters such as cold resistance should be considered as indexes when performing Z. insignis provenance selection. Individual selection played a key role in selection and breeding of superior and rapid growing varieties of Z. insignis. Growth pattern in seedling stage of different Z insignis provenances was investigated. The growth model was established by Logistic equation. From this equation it could be found that small seedlings began to accelerate growth from June after transplanting, and then rapidly grew from July to August. The peak stage of stem diameter growth was earlier than that of height growth, suggesting that Z. insignis is drought resistant.
2. The cold resistance of different Z. insignis provenances. Results showed that physiological substances in Z. insignis seedlings changed after low temperature treatment. For instance, contents of protective substances such as soluble proteins and soluble carbohydrates, and osmotic adjustment substances such as free praline increased, and activities of antioxidative Enzymes were also increased. At 7℃the contents of soluble proteins, soluble carbohydrates and praline, and the activities of SOD and POD reached by the maximum values, whereas the content of MDA by the minimum value when the strongest cold resistance was developed. After treatment with low temperature, physiological indexes in Z. insignis shoots varied and showed significant differences at certain value of low temperature. There was a big increase in the contents of soluble proteins after being treated with -8℃, and the contents of MDA increased a little. The SOD activities between different Z. insignis provenances began to show significant differences. Relative electric conductivity sharply increased after treatment with -12℃, and that of all provenances apart from Tongdao provenance reached over 50%. The membrane of cytoplasms was damaged. Leaf anatomical analysis indicated that cell tense ratio (CTR) and spongy ratio (SR) of Z. insignis leaf were correlated with its cold resistance; the cold resistance ability became stronger with the increase of CTR and the decrease of SR. According to the Logistic equation, it could be calculated the semi-lethal temperature of Tongdao provenance was -13.06℃which is 0.89℃～2.74℃lower than that of the other provenances. The crystallization temperature of leaf of Tongdao provenance was -15.04℃indicated by DSC which is 0.42℃～1.34℃lower than that of the other provenances. Integrated analyses showed that the cold resistance of Tongdao provenance planting stock was the strongest, followed by Xinyi, Xincheng, Yingde, jianghua and Jingxi provenances.
3. The genetic diversity of different Z. insignis provenances. ISSR-PCR reaction system for genetic diversity analysis of Z. insignis was successfully established for the first time. Averagely, each of the 10 primers screened amplified 5.4 bands, and the mean Percentage of Polymorphic Bands (PPB) was 64.29%. Among the six provenances, the mean PPB was 56.48%, Shannon's Information index 0.3048, Nei's index 0.2052, suggesting certain degree of genetic divergence exists among provenances. The genetic diversity of Xincheng provenance was the highest followed by Jianghua, Yingde, Jingxi, Tongdao and Xingyi provenances. Geneflow and Gene diversity were 0.4840 and 0.1058, respectively, indicating that certain degree of gene flow was present in Z. insignis provenances and the degree of genetic differentiation was medium. Genetic variations among provenances accounted for 34.03% of total genetic variations, and the genetic variations of 65.97% distributed in individuals of Z. insignis.
Genetic distance and geographic distance matrices were not significantly correlated, while the PPB and leaf morphological characters were significantly correlated, and Shannon's Information index, Nei's index and growth characteristics were significantly correlated. While all these indexes were not correlated with physiological index of cold resistance, thus it could come to the conclusion that the cold resistance of Z. insignis was controlled by some specific genes. According to cluster analysis, Z. insighnis provenances were divided into four groups. GroupⅠ:Yingde, Xincheng and Tongdao provenances. GroupⅡ:Jingxi provenance. GroupⅢ:Xingyi provenance. Group IV:Jianghua provenance. There existed geographical isolation among the four groups, which is consistent with geographical distribution patterns.
引文
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