摘要
土壤盐碱化现已成为危害农业发展和生态环境的全球性问题。棉花作为我国主要的经济作物,在国民经济发展中占有举足轻重的地位。由于我国人均耕地面积较少、粮棉争地矛盾突出,棉花的种植和生产受到严重影响。因此培育棉花耐盐品种,将是有效开发利用盐碱地、推进农业可持续发展的一条有效途径。棉花的耐盐性鉴定工作对耐盐品种的选育起着至关重要的作用,目前生产上常用的形态学的耐盐性鉴定方法费时费力,且易受外界环境变化影响和季节限制。本研究选用25份耐盐和23份盐敏感棉花种质为实验材料,采用SSR技术,展开了棉花耐盐性鉴定技术的有关研究,初步建立了棉花耐盐性的SSR鉴定方法。
1.利用95对SSR引物对48份棉花耐盐相关种质的遗传多样性进行分析,研究结果表明大多数种质之间的亲缘关系较近。棉花种内遗传基础狭窄,种间遗传差异较大。利用类平均法(UPGMA)聚类将48份棉花种质分成3个类群,聚类分析表明类群的划分和材料的地域来源关系不大。
2.从5053对SSR引物中筛选了26对清晰度高、稳定性好的SSR核心引物,构建了48份棉花耐盐相关种质的DNA指纹图谱,为棉花遗传多样性研究、纯度检测、种质鉴定和评价等奠定了基础。
3.针对棉花耐盐性复杂,耐盐基因显性标记难以筛选,单标记鉴定棉花耐盐性有困难等问题,提出用多标记组合法鉴定棉花耐盐性。通过分析比较单标记和多标记组合的鉴定效率,确定Y190、Y159和Y258三标记组合鉴定棉花耐盐性最为经济有效。
4.从DNA快速提取到PCR扩增和产物检测以及多标记组合鉴定等环节进行分析探讨,初步制定了一套适于棉花耐盐性分子鉴定的方法,即多标记组合鉴定法。并用11份材料对该方法进行了验证,结果表明和盐池鉴定结果的相符率达90.91%。初步研究结果表明多标记组合鉴定法可用于棉花耐盐分子标记辅助鉴定。
本研究所建立的棉花耐盐性的SSR鉴定方法—多标记组合鉴定法,为实现棉种耐盐性分子鉴定奠定了基础,为棉花耐盐育种提供理论依据。但是本方法目前仅能将耐盐和盐敏感材料进行初步区分,关于棉花耐盐性详细的分级标准还有待于进一步研究。
Soil salinization has become a serious global problem affecting the agricultural development and the ecological environment. Cotton, the major cash crop in China, is playing a crucial role in national economic development. China, with less cultivated lands and more people, faces the contradiction between food and cotton, which seriously affects the cultivation and production of cotton. Therefore, it is an effective way to farm saline land and to enhance the sustainable agricultural development by develop the salinity-tolerant varieties of cotton. Identification of salinity-tolerance also plays a vital role on cotton breeding. The salinity-tolerant identification methods used before, mainly based on morphological characters, were usually restricted for time-wasting, labor-costing, environment influence, and seasonal cultivation. 25 salinity- tolerant and 23 salinity-sensitive cotton accessions were used in this study to identify salinity-tolerance of cotton by SSR markers. A SSR molecular marker method was initially established to identify salinity-tolerance of cotton.
1. The genetic diversity of cotton germlasm was analyzed among 48 cotton salinity-tolerance relevant accessions using 95 SSR molecular markers, which showed that most of germplasm had close genetic relationship, the cotton germplasm were of close relationship with narrow background of the intra-specific genetic diversity and broad hereditary gap between inter-specific cotton accessions. 48 cotton accessions could be classified into 3 groups by cluster analysis by UPGMA, which revealed that there was similar background among the dividing group in territorial background.
2. 26 pairs of SSR core primers were screened from 5053 pairs of primers. The fingerprints of 48 salinity-tolerance relevant cotton accessions were constructed by 26 pairs of core SSR primers, which enhenced the researches of the genetic diversity, purity test, identification and evaluation of cotton germplasm.
3. A new SSR molecular marker identification method was suggested for the complex of the salinity-tolerance of cotton, for the difficulty to screen dominant markers related to the salinity-tolerant genes, and for the difficulty to identify salinity-tolerance by single marker. To analyze the identification efficiency between single marker and multi-markers, the most economical and effective way to identify the salinity tolerance was the combination of 3 markers Y190, Y159 and Y258.
4. A new preliminary methods, called SSR multi-markers salinity-identification method, was initially established to identify salinity-tolerance of cotton by the standardization of the whole process of DNA extraction, PCR amplification, amplification products detecting, and marker-combination. Another 11 materials were used to testify this method, which showed the coincidence of 90.91% in consistence with the identification result of 0.4%NaCl identification method. This study showed that the multi-markers identification method was proved to be used to assist identify the salinity-tolerance of cotton germplasm.
The multi-markers identification method lays the foundation for molecular identification of salinity-tolerance on cotton, and provided theoretical basis for screening and breeding of salinity-tolerant cotton varieties. This method might be restricted only to be used to distinguish salinity-tolerant accessions from salinity sensitive accessions, which suggests that it need further in-depth studying to research how to grading salinity-tolerance of cotton germplasm.
引文
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