本文關(guān)鍵詞：越橘變異株生物學(xué)特性及ISSR分子標(biāo)記研究　出處：《西南大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 越橘 V3變異株 Legacy變異株 秋水仙素 ISSR

【摘要】：越橘(Vaccinium vitis-idaea Linn.),俗稱藍(lán)莓,為杜鵑花科(Ericaceae)越橘屬(Vaccinium Linn.)灌木或小喬木。全世界越橘屬植物約有450個(gè)種,我國已知有91個(gè)種,28個(gè)變種,主要分布在我國東北和西南地區(qū)。北美地區(qū)既是越橘原產(chǎn)地也是主要產(chǎn)區(qū)。越橘果實(shí)為藍(lán)色小漿果,富含多種維生素、黃酮、花青素,具有顯著提高視力和抗癌保健功效,近年來世界各地需求量不斷增加。南高叢越橘品種V3及Legacy引種西南地區(qū)多年,V3長勢旺盛,但果實(shí)很小;Legacy果實(shí)中等,長勢較為旺盛,但在重慶每年春夏梅雨季節(jié),葉片易出現(xiàn)大量褐色病斑,抗病性較差。由于多倍體植株具有器官巨大性、抗性增強(qiáng)及營養(yǎng)成分增加等特點(diǎn),本實(shí)驗(yàn)室連續(xù)若干年采用秋水仙堿對北高叢、南高叢越橘不同品種進(jìn)行加倍,陸續(xù)得到了不同表型的變異植株。在誘變初期雖已經(jīng)對變異株的形態(tài)學(xué)及細(xì)胞學(xué)進(jìn)行了分析鑒定,但在田間栽培過程中,變異株還陸續(xù)有新的表型特征出現(xiàn)。加倍后的多倍體回復(fù)突變問題一直是研究中的空白領(lǐng)域,植株在回復(fù)突變中的生物學(xué)特性及分子水平的變化等指標(biāo)的記錄是進(jìn)行基礎(chǔ)研究的重要數(shù)據(jù)。因此,本研究運(yùn)用ISSR分子標(biāo)記技術(shù)在DNA水平對變異株進(jìn)行分析,并結(jié)合形態(tài)學(xué)和細(xì)胞學(xué)檢測方法對變異植株與正常植株的解剖結(jié)構(gòu)、生理指標(biāo)、染色體數(shù)目進(jìn)行分析研究和差異顯著性比較,進(jìn)一步確認(rèn)變異株在回復(fù)突變過程中的各方面指標(biāo)的變化,旨在為越橘的多倍體育種提供基礎(chǔ)研究數(shù)據(jù)。主要研究結(jié)果如下:1.V3變異株移栽田間生長三年后,形態(tài)學(xué)鑒定顯示:平均高度為對照的110%,莖直徑為對照的132%,葉長、葉寬為對照的144%、137%。與對照相比,變異株長勢更好但果實(shí)較小,三棵變異株之間無顯著差異。Legacy經(jīng)秋水仙素誘變后,根據(jù)表型特征分為L1、L2兩種類型。L1變異株表現(xiàn)多倍體的特性,經(jīng)鑒定:莖粗,葉長、葉寬與對照相比分別增加了40%、56%、52%,為顯著差異;L2變異株為分枝量增大類型,移栽田間后株高相比對照下降32.5%,分枝量增加194.4%,為極顯著差異,葉長寬分別增加34%、38%,與對照相比差異顯著。2.經(jīng)葉表皮氣孔觀察,氣孔密度方面:V3變異株下降20.5%;L1變異株下降11.6%,與對照相比差異顯著;保衛(wèi)細(xì)胞葉綠體數(shù)目:V3變異株增加14.6%,L1變異株增加35.6%,L2變異株增加24.6%,與對照相比均為顯著差異。氣孔保衛(wèi)細(xì)胞平均長度、寬度:L1變異株分別增加16.7%、18.4%,與對照差異顯著;V3、L2與對照相比差異不顯著。兩個(gè)品種的變異株葉片橫切結(jié)構(gòu)顯示:葉片主脈直徑、上表皮厚度、柵欄組織及海綿組織厚度均顯著增加,Legacy變異株的葉片結(jié)構(gòu)緊密度(CTR)與對照在5%水平上存在差異,柵/海比明顯高于對照,表明Legacy變異株抗旱能力較對照有增強(qiáng)。3.去壁低滲火焰干燥法對變異株莖尖染色體鑒定結(jié)果表明:越橘V3及L1變異株均為二倍體細(xì)胞和四倍體細(xì)胞共存的嵌合體,多倍體細(xì)胞比率分別為:45.52%、38.62%,較其誘導(dǎo)初期鑒定結(jié)果(70%、49.06%)相比呈下降趨勢,表明在越橘異倍型嵌合體中,二倍體細(xì)胞分裂增殖速度較四倍體細(xì)胞快,加倍后有回復(fù)突變傾向。4.V3變異株的葉綠素含量、可溶性糖含量、脯氨酸含量均較對照顯著增加,丙二醛含量顯著下降;Legacy變異株的葉綠素含量、可溶性糖含量、脯氨酸含量均較對照增加,丙二醛含量下降,呈極顯著差異。表明變異株具有生長優(yōu)勢,抗逆性更強(qiáng)。5.采用改良CTAB法獲取高質(zhì)量越橘DNA。通過直觀分析和方差分析結(jié)合,建立了越橘ISSR-PCR(20μl)最佳反應(yīng)體系,各組分的濃度為: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。在此基礎(chǔ)上探討了引物UBC835的最佳退火溫度、循環(huán)次數(shù)和延伸時(shí)間,結(jié)果分別為54.2℃、35次、60s。電泳結(jié)果顯示優(yōu)化體系穩(wěn)定性較高。6.對V3、Legacy變異株及在田間栽培中出現(xiàn)表型變異的南高叢品種-南好變異株(B1、B2、B3)及10個(gè)由本實(shí)驗(yàn)室長期栽培保存的北高叢和南高叢越橘品種共21個(gè)樣品進(jìn)行ISSR多態(tài)性擴(kuò)增。從70個(gè)ISSR引物中篩選出10個(gè)多態(tài)性高且較穩(wěn)定的引物,共擴(kuò)增出95個(gè)位點(diǎn),多態(tài)性位點(diǎn)90個(gè),多態(tài)比率為94.70%。UPGMA聚類顯示21個(gè)樣品的遺傳距離介于0.57~0.83,均值0.7,在系數(shù)0.57處材料被分為南北高叢兩大分支。3個(gè)品種的變異株均表現(xiàn)出很高的多態(tài)性。與對照株相比,變異株在ISSR-PCR擴(kuò)增中的條帶差異為缺失、新增、既缺失又新增三種類型。A1、A2、A3變異株與對照V3之間遺傳相似系數(shù)為:0.800、0.711、0.722;L1、L2變異株與對照Legacy之間遺傳相似系數(shù)為:0.800、0.767;B1、B2、B3與對照南好之間遺傳相似系數(shù)為:0.633、0.589、0.578。結(jié)合變異株生物學(xué)差異和ISSR多態(tài)性分析,表明V3、Legacy及南好的變異植株在DNA分子水平上發(fā)生改變。
[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.

【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S663.9

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