Based on pre-census survey, 36 representative walnut seedlings were selected to analyze the top leaf’s five morphological traits and nut’s eight morphological traits. The results showed: (1) In Tibet, the traits of nuts and leaves of walnuts changed in a large range, the ratio of the maximum and minimum values of the traits could be as high as 3.06, showing the leaf traits and nut traits with certain diversity. (2) The variation coefficients of leaf area, suture line height, husk thickness and nut weight were about 20%, the genetic diversity and these traits had greater selectivity in breeding, the variation coefficients of the indexes of leaf shape, nut length, nut height and the indexes of nut shape did not reach 10%, these traits kept stable. (3) There was a significantly positive correlation between leaf area and nut weight and suture line height, the correlation coefficients were 0.505 and 0.440, which noted that the larger the leaf area, the greater the height and the larger the suture, there was a significant positive correlation between leaf width and nut weight, the correlation coefficient was 0.561.
To obtain high-quality DNA suitable for methylation-sensitive amplification polymorphism (MSAP), analysis, the mature cotyledon and tissue cultured cotyledon of walnut cultivar‘Liaoning-1’were used as the testing materials in the research of DNA isolation by 3 methods (SDS, high salt CTAB and modified high salt CTAB) and the mature leaf tissue was taken as the control. The concentration and intactness of the extracted DNA were determined using a spectrometer and by electrophoresis in 0.8% (w/v) agarose gels against standard solutions of lambda DNA. High quality DNA was extracted from leaf tissues using the high salt CTAB method and the modified high salt CTAB method. No detectable DNA was extracted from the mature cotyledon and cultured cotyledon using SDS method. The DNA with high quality but little quantity was extracted from the mature cotyledon but not the extract cultured cotyledon using the high salt CTAB method. The DNA with high quantity and quality suitable for MSAP analysis from both mature cotyledon and cultured cotyledon was extracted using the modified high salt CTAB method. The DNA extraction method of walnut cotyledon suitable for MSAP analysis was established, which could lay foundation for downstream molecular biology experiment and provide a reference to the polysaccharide removal research in DNA extraction procedure.
以‘中宁强’核桃品种成熟叶片为材料,采用优化的 MSAP 反应体系,在全基因组水平对核DNA 进行甲基化修饰位点分析,结果表明:选用20对引物组合,共扩增出1 060条清晰、重复性好的谱带,平均每对引物扩增出53条谱带,其中,甲基化位点241个,甲基化修饰比例为22.73%. 对部分核桃 DNA 甲基化修饰位点进行回收测序,得到10条存在 DNA 甲基化修饰的 DNA 序列,BLASTn分析表明,核桃基因组中多种类型的DNA序列存在甲基化修饰现象.
'Liaoning 2' is a dwarf walnut variety bred by Chinese scientists through artificial hybridization, but little is known about its dwarfing mechanism. In this study, the dwarfing mechanism of 'Liaoning 2' is discussed through the comparison of biological characteristics, genomics and the expression of growth-related gene JrGAI,with a growth normally, closely related(half-sib families) variety of 'Liaoning 1' as control. By observing the phenology and tree phenotypes, it was found that there was no significant difference in phenological phase between 'Liaoning 1' and 'Liaoning 2', but 'Liaoning 2' was obviously dwarf, the height, crown and trunk diameter were smaller, the average length of internode reduced significantly, but there was no significant difference in the number of development branch internode. The dwarf phenotype of 'Liaoning 2' is mainly caused by inter-shortening. The qRT-PCR technique was performed to analyze the expression of GA negative regulation gene JrGAI and the results showed that during the shoot development, the expression of JrGAI gene exhibited decreasing expression in the shoot rapid growing period in both varieties and the decline in 'Liaoning 1' was remarkably reduced. The SSR analysis with 25 pairs of primers showed that SSR loci of two varieties were different, especially the homozygosity of 'Liaoning 2' SSR loci was higher. It is concluded that different expressions of JrGAI gene and homozygosity of SSR loci could be important internal reasons of dwarf phenotype of 'Liaoning 2'.