越橘变异株生物学特性及ISSR分子标记研究

发布时间：2018-01-15 01:24

本文关键词：越橘变异株生物学特性及ISSR分子标记研究　出处：《西南大学》2017年硕士论文　论文类型：学位论文

【摘要】：越橘(Vaccinium vitis-idaea Linn.),俗称蓝莓,为杜鹃花科(Ericaceae)越橘属(Vaccinium Linn.)灌木或小乔木。全世界越橘属植物约有450个种,我国已知有91个种,28个变种,主要分布在我国东北和西南地区。北美地区既是越橘原产地也是主要产区。越橘果实为蓝色小浆果,富含多种维生素、黄酮、花青素,具有显著提高视力和抗癌保健功效,近年来世界各地需求量不断增加。南高丛越橘品种V3及Legacy引种西南地区多年,V3长势旺盛,但果实很小;Legacy果实中等,长势较为旺盛,但在重庆每年春夏梅雨季节,叶片易出现大量褐色病斑,抗病性较差。由于多倍体植株具有器官巨大性、抗性增强及营养成分增加等特点,本实验室连续若干年采用秋水仙碱对北高丛、南高丛越橘不同品种进行加倍,陆续得到了不同表型的变异植株。在诱变初期虽已经对变异株的形态学及细胞学进行了分析鉴定,但在田间栽培过程中,变异株还陆续有新的表型特征出现。加倍后的多倍体回复突变问题一直是研究中的空白领域,植株在回复突变中的生物学特性及分子水平的变化等指标的记录是进行基础研究的重要数据。因此,本研究运用ISSR分子标记技术在DNA水平对变异株进行分析,并结合形态学和细胞学检测方法对变异植株与正常植株的解剖结构、生理指标、染色体数目进行分析研究和差异显著性比较,进一步确认变异株在回复突变过程中的各方面指标的变化,旨在为越橘的多倍体育种提供基础研究数据。主要研究结果如下:1.V3变异株移栽田间生长三年后,形态学鉴定显示:平均高度为对照的110%,茎直径为对照的132%,叶长、叶宽为对照的144%、137%。与对照相比,变异株长势更好但果实较小,三棵变异株之间无显著差异。Legacy经秋水仙素诱变后,根据表型特征分为L1、L2两种类型。L1变异株表现多倍体的特性,经鉴定:茎粗,叶长、叶宽与对照相比分别增加了40%、56%、52%,为显著差异;L2变异株为分枝量增大类型,移栽田间后株高相比对照下降32.5%,分枝量增加194.4%,为极显著差异,叶长宽分别增加34%、38%,与对照相比差异显著。2.经叶表皮气孔观察,气孔密度方面:V3变异株下降20.5%;L1变异株下降11.6%,与对照相比差异显著;保卫细胞叶绿体数目:V3变异株增加14.6%,L1变异株增加35.6%,L2变异株增加24.6%,与对照相比均为显著差异。气孔保卫细胞平均长度、宽度:L1变异株分别增加16.7%、18.4%,与对照差异显著;V3、L2与对照相比差异不显著。两个品种的变异株叶片横切结构显示:叶片主脉直径、上表皮厚度、栅栏组织及海绵组织厚度均显著增加,Legacy变异株的叶片结构紧密度(CTR)与对照在5%水平上存在差异,栅/海比明显高于对照,表明Legacy变异株抗旱能力较对照有增强。3.去壁低渗火焰干燥法对变异株茎尖染色体鉴定结果表明:越橘V3及L1变异株均为二倍体细胞和四倍体细胞共存的嵌合体,多倍体细胞比率分别为:45.52%、38.62%,较其诱导初期鉴定结果(70%、49.06%)相比呈下降趋势,表明在越橘异倍型嵌合体中,二倍体细胞分裂增殖速度较四倍体细胞快,加倍后有回复突变倾向。4.V3变异株的叶绿素含量、可溶性糖含量、脯氨酸含量均较对照显著增加,丙二醛含量显著下降;Legacy变异株的叶绿素含量、可溶性糖含量、脯氨酸含量均较对照增加,丙二醛含量下降,呈极显著差异。表明变异株具有生长优势,抗逆性更强。5.采用改良CTAB法获取高质量越橘DNA。通过直观分析和方差分析结合,建立了越橘ISSR-PCR(20μl)最佳反应体系,各组分的浓度为:10×buffer 2μl;Taq酶0.75U;Mg2+1.875mmol.L-1;DNA 80ng;dNTP 0.1mmol.L-1;引物0.2μmol.L-1。在此基础上探讨了引物UBC835的最佳退火温度、循环次数和延伸时间,结果分别为54.2℃、35次、60s。电泳结果显示优化体系稳定性较高。6.对V3、Legacy变异株及在田间栽培中出现表型变异的南高丛品种-南好变异株(B1、B2、B3)及10个由本实验室长期栽培保存的北高丛和南高丛越橘品种共21个样品进行ISSR多态性扩增。从70个ISSR引物中筛选出10个多态性高且较稳定的引物,共扩增出95个位点,多态性位点90个,多态比率为94.70%。UPGMA聚类显示21个样品的遗传距离介于0.57~0.83,均值0.7,在系数0.57处材料被分为南北高丛两大分支。3个品种的变异株均表现出很高的多态性。与对照株相比,变异株在ISSR-PCR扩增中的条带差异为缺失、新增、既缺失又新增三种类型。A1、A2、A3变异株与对照V3之间遗传相似系数为:0.800、0.711、0.722;L1、L2变异株与对照Legacy之间遗传相似系数为:0.800、0.767;B1、B2、B3与对照南好之间遗传相似系数为:0.633、0.589、0.578。结合变异株生物学差异和ISSR多态性分析,表明V3、Legacy及南好的变异植株在DNA分子水平上发生改变。
[Abstract]:Blueberry (Vaccinium vitis-idaea Linn.), commonly known as blueberry, Vaccinium Ericaceae (Ericaceae) (Vaccinium Linn.). The whole world shrubs or small trees of the genus Vaccinium about 450 species known in China, there are 91 species, 28 varieties, mainly distributed in the northeast and southwest China. North America is blueberry the origin is the main producing area. Blueberry fruit is blue berries, rich in vitamins, flavonoids, anthocyanins, can significantly improve the visual acuity and anticancer efficacy of health care, demand all over the world is increasing in recent years. Many years of highbush blueberry varieties of V3 and Legacy in the southwest region, V3 growing strong, but the fruit is small; the fruit Legacy medium. The growth is strong, but in Chongqing, the annual spring and summer rainy season, leaves to the emergence of a large number of brown spots, the resistance is poor. Because of polyploid plants with great organ, enhance the resistance characteristics and nutrition increase, The laboratory for several years by colchicine in northern highbush, highbush blueberry cultivars were doubled, gradually got a different phenotype in variant plants. Although the initial mutation variants have on cytological and morphological analysis were carried out in the field, but in the process of cultivation, mutants have also been appeared new phenotypic characteristics after the double mutants. Polyploidy has been studied in the blank field, plants in response to biological characteristics and molecular level of mutation in the other indicators of change records are important data for basic research. Therefore, this research uses the ISSR molecular marker technology at the DNA level of variation were analyzed, combined with morphological and cytological detection method of anatomical structure, mutated and normal plant physiological index, chromosome number comparison research and analysis of the significant differences, to further confirm the change Changes in the different strains mutation of the indicators in the process, in order to provide basic data for the polyploid breeding of blueberry. The main results are as follows: three years of growth of 1.V3 mutant transplanting field, morphological identification showed that the average height is 110% of the control, the stem diameter was 132% of the control, leaf length, leaf width in 144%, 137%. compared with the control, mutant grew better but the fruit is small, three tree variants did not differ significantly between.Legacy by colchicine, based on phenotype characteristics, divided into L1, L2 two types of.L1 mutants showed polyploid were identified: stem diameter, leaf length, leaf width compared with the control group increased by 40%, 56%, 52%, was significantly different; L2 mutants for increasing the amount of branching type, transplanting field height after 32.5% lower than the control, branching amount increased 194.4%, extremely significant differences, leaf length and width were increased by 34% and 38%, compared with the control group Significant.2. difference after leaf stomata observed, stomatal density: V3 mutation decreased 20.5%; mutation of L1 decreased by 11.6%, compared with CK; guard cell chloroplast number: V3 mutation increased 14.6%, L1 mutation increased 35.6%, L2 mutation increased 24.6%, compared with the control were significantly different. The stomatal defend the average length, width: L1 mutant cells were increased by 16.7%, 18.4%, and the difference was significant; V3, L2 had no significant difference compared with control. Two varieties of mutant leaf leaf main vein crosscutting structure: diameter, thickness of upper epidermis, palisade tissue and spongy tissue thickness increased significantly, leaf structure the Legacy mutant (CTR) with the control of close at 5% level between palisade tissue and spongy tissue's ratio was significantly higher than the control, showed that the Legacy mutants have enhanced drought resistance to low permeability of wall.3. variant stem tip chromosome in flame drying method compared with the control The results showed that: V3 and L1 mutant blueberry were chimera diploid and tetraploid cells coexist, polyploid cell ratio were 45.52% and 38.62%, compared with its initial induction identification results (70%, 49.06%) compared to a downward trend, that in different times the chimeric blueberry, diploid cell proliferation rate tetraploid cells quickly, doubling response to chlorophyll content mutations of.4.V3 strains, the content of soluble sugar, proline content increased, MDA content decreased significantly; Legacy mutant of chlorophyll content, soluble sugar content, proline content increased, MDA content decreased, indicating that the variation appeared significant difference. Strains with growth advantage, stronger resistance of.5. by using the modified CTAB method to obtain the high quality of blueberry DNA. through the combination of intuitive analysis and variance analysis, the establishment of Vaccinium ISSR-PCR (20 L) best The reaction system, the concentration of each component is: 10 * buffer 2 L; Taq 0.75U Mg2+1.875mmol.L-1; DNA enzyme; 80ng; dNTP 0.1mmol.L-1; 0.2 mol.L-1. primers on the basis of the optimal annealing temperature and primer UBC835, cycle number and extension of time, the results were 54.2 degrees, 35 times, 60s. electrophoresis results showed that the optimized the stability of the system of higher.6. V3, Legacy mutant strains and in field cultivation in the phenotypic variation of southern highbush varieties - South strains (B1, B2, B3) and 10 from the laboratory preservation of long-term cultivation of northern highbush and highbush blueberry varieties a total of 21 samples were amplified ISSR polymorphism screening. 10 highly polymorphic and stable primers from 70 ISSR primers, 95 loci were amplified, 90 polymorphic loci, polymorphic ratio of 94.70%.UPGMA clustering indicated that genetic distance of 21 samples ranged from 0.57~0.83, average 0.7, was in the coefficient of 0.57 materials For the north and South highbush varieties of two branches of.3 mutant showed high polymorphism. Compared with the control strain, mutant in ISSR-PCR amplification bands between lack of new, both lack of added three types of.A1, A2, A3 mutation and V3 control between the coefficient of genetic similarity is 0.800,0.711,0.722; L1, L2 mutation and genetic similarity coefficient between Legacy control: 0.800,0.767; B1, B2, B3 and control the south good genetic similarity coefficient was: 0.633,0.589,0.578. binding analysis, mutation and biological differences between ISSR polymorphism showed that V3, Legacy and the south good mutants changed at DNA molecular level.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S663.9

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