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糯玉米种质资源遗传多样性研究

Studies on Genetic Diversity of Waxy Corn Germplasms

【作者】 蔚荣海

【导师】 王玉兰;

【作者基本信息】 吉林农业大学 , 作物遗传育种学, 2006, 博士

【摘要】 遗传多样性是种内不同群体之间或一个群体内不同个体遗传变异的总和。在生物遗传资源多样性的研究中经历了由农艺性状形态标记、品质性状比较分析、生化(同工酶、种子贮藏蛋白等)标记,以及近年开展的对其内部遗传物质差异的各种分子生物学上的分子标记。遗传多样性的研究对生物种质资源的收集、评价、保存和利用具有十分重要的意义。通过对表型不同的80份糯玉米自交系(其中黄糯40份、白糯25份、黑糯13份、红糯2份)调查17个农艺性状,进行遗传距离及聚类分析;主要生化品质成分含量的测定,种子贮减蛋白(盐溶蛋白)聚丙烯酰胺凝胶电泳图谱的聚类分析;SSR分子标记指纹图谱及聚类分析。随机选取7份自交系进行完全双列杂交,对其配合力、杂种优势测定验证,得到如下结论:(1)无论是在农艺性状、内部生化品质性状、DNA遗传物质上,80份糯玉米种质资源都具有丰富的遗传多样性。在对17个农艺性状的调查和主要品质性状成分含量的测定中,变幅较大,方差分析差异显著。(2)将17重要性状进行主成分分析,按贡献率的85%计算,得到7个主成分因子,两个自交系之间的7个主成分因子之间的差异即为遗传距离;80个糯玉米自交系以遗传距离4.15分为九大类。,第Ⅰ类包括2个糯玉米自交系:N76,N09;第Ⅱ类仅有1个糯玉米自交系:N66;第Ⅲ类共有5个糯玉米自交系:N68,N67,N49,N74,N42;第Ⅳ包括2个糯玉米自交系:N05,N37;第Ⅴ类仅有1个糯玉米自交系N57;第Ⅵ类包括19个糯玉米自交系,分布在两个亚群中,第Ⅵ1亚群中包括:N80,N62,N72,N71,N10,第Ⅵ2亚群中包括N41,N77,N44,N63,N70,N46,N60,N43,N15,N30,N26,N78,N19,N06;第Ⅶ类共有7个糯玉米自交系,包括:N56,N55,N54,N53,N48,N31,N18;第Ⅷ类包括2个自交系:N04,N21;第Ⅸ类共有41个糯玉米自交系分布在4大亚群中,第Ⅸ1亚群中包括:N73,N58,N52;第Ⅸ2亚群包括:N39,N17,N16,N47,N12,N61,N24,N38,N33,N14,N07;第Ⅸ3亚群包括:N29,N13;第Ⅸ4亚群包括:N75,N65,N45,N69,N35,N79,N27,N20,N11,N08,N64,N25,N51,N03,N59,N02,N32,N28,N50,N36,N23,N40,N24,N22,N01。农艺性状的聚类结果表明:表型不同的自交系分布在不同的类群里。(3)糯玉米种质资源的主要营养品质成分含量变幅较大。80份糯玉米自交系的淀粉、蛋白质、脂肪含量的变幅分别在58.05%~76.20%、7.3%—13.97%、3.00%—6.68%;平均值分别为67.79%、11.15%、4.93%,淀粉为100%支链淀粉。淀粉与蛋白质呈显著负相关,相关系数为-0.3784;蛋白质与脂肪之间呈负相关,相关系数为-0.1908;脂肪与淀粉之间呈正相关,相关系数为0.1786。(4) 80份糯玉米自交系盐溶蛋白电泳图谱按迁移率划分,可分为13条特征谱带,进行聚类分析共聚成6类(以遗传距离0.27为标准)。其中第Ⅲ类在遗传距离0.14处又分为2个亚群;第Ⅳ类在遗传距离0.22处分为3个亚群,第Ⅰ类仅有一个糯玉米自交系N16;第Ⅱ类也仅有一个糯玉米自交系:N51;第Ⅲ类有7个糯玉米自交系分布在2个亚群中,在第Ⅲ1亚群包括:N71,N70,N68,N67;第Ⅲ2亚群包括:N65,N66,N39;第Ⅳ类有68个糯玉米自交系分布在3个亚群中,第Ⅳ1亚群包括:N10,N06,N05;第Ⅳ2亚群包括:N74,N69,N11,N46,N43,N45,N19,N28,N18,N17,N08,N15,N13,N07,N14,N04;第Ⅳ3亚群包括:N63,N62,N56,N55,N57,N50,N38,N27,N25,N24,N12,N49,N47,N80,N79,N76,N44,N42,N41,N78,N77,N61,N60,N22,N21,N54,N53,N64,N58,N52,N59,N37,N36,N35,N34,N33,N32,N31,N30,N23,N20,N72,N40,N29,N26,N48,N09,N75,N03;第Ⅴ大类仅有1个糯玉米自交系:N73;第Ⅵ大类包括2个糯玉米自交系:N02,N01。遗传距离变幅在0.00—0.46之间。(5) 22对SSR核心引物分布在玉米基因组的10条染色体上,每对引物可以检测到2--4个数目不等的多态性片断(等位基因),共60个多态性片断,平均为2.73个。80个糯玉米自交系间的遗传距离范围为0.045-0.946。(6) SSR的聚类结果将80份糯玉米自交系聚成9类(以遗传距离0.55为标准)。其中第Ⅱ类在遗传距离0.46处又分为3个亚群;第Ⅴ类在遗传距离0.50处分为2个亚群,第Ⅶ类在遗传距离0.46处又分为2个亚群,第Ⅷ类在遗传距离0.46处又分为2个亚群,第Ⅸ类在遗传距离0.44处分为4个亚群。各类及亚类包含的糯玉米自交系如下:第Ⅰ类包括:N12;第Ⅱ类含三个亚群包括:N69,N80,N79,N73,N71,N70,N74,N68,N67,N65,N63,N61,N66,N52;第Ⅲ类包括:N50,N48;第Ⅳ类包括:N72;第Ⅴ类含两个亚群包括:N64,N59,N58,N46,N44,N62,N57,N54,N53,N60,N56,N55,N43;第Ⅵ类包括:N16,N42,N16,N41;第Ⅶ类含两个亚群包括:N40,N36,N25,N39,N37,N78,N77,N09;第Ⅷ类含两个亚群包括:N76,N45,N47,N11,N49,N07,N06;第Ⅸ类含四个亚群包括:N75,N51,N30,N13,N14,N29,N28,N23,N33,N38,N19,N27,N34,N35,N18,N15,N10,N26,N05,N24,N22,N32,N31,N21,N03,N04,N02,N08,N20,N01。黄色糯玉米自交系除N12以外其余全部分布在Ⅵ,Ⅶ,Ⅷ,Ⅸ里面,在第Ⅸ大类30个自交系里面黄色糯玉米有28个,占93%,其余11个黄色糯玉米自交系分布在Ⅵ,Ⅶ,Ⅷ里面;白色糯玉米自交系几乎全部分布在Ⅱ的第三亚群,Ⅲ,Ⅴ,Ⅵ,共21个,占84%;粒色为黑色的糯玉米自交系中N72自成一类群,N75,N76,N78分散在Ⅶ,Ⅷ,Ⅸ类群中,其余全部集中在第Ⅱ类群中,占57%;红色的两个糯玉米自交系分布在第Ⅱ类群的第Ⅱ2亚群中,遗传距离没有差异。(7)不同粒色的糯玉米自交系间存在着遗传上的差异,粒色相同的类群之间遗传距离(黄糯自交系、白糯自交系、黑糯自交系之间)明显小于不同粒色类群之间遗传距离(黄糯与白糯自交系、黄糯与黑糯自交系、白糯与黑糯自交系之间)。不同粒色自交系之间和相同粒色自交系之间的最小遗传距离差异显著。40份黄糯玉米间的遗传距离变幅为0.087—0.865之间,遗传距离平均值为0.632,最大遗传距离为N36和N12之间,最小遗传距离为N35和N18、N35和N34之间。白糯玉米的遗传距离在0.091—0.946之间,遗传距离平均值为0.680,最大遗传距离为N49和N65之间,最小遗传距离为N53和N54之间。13份黑糯玉米间的遗传距离在0.045-0.895之间,遗传距离平均值为0.655,最大遗传距离为N69和N72之间,最小遗传距离为N77和N78之间。40份黄糯玉米自交系与25份白糯玉米自交系间的遗传距离变幅为0.464—0.927之间,平均值为0.747,最大遗传距离为N46和N08之间,最小遗传距离为N41和N17。40份黄糯玉米自交系与13份黑糯玉米自交系间的遗传距离变幅为0.517-0.897之间,平均值为0.749,最大遗传距离为N72和30之间,最小遗传距离为N78和N09之间。25份白糯玉米自交系和13份黑糯玉米自交系间的遗传距离变幅为0.483-0.892,平均值为0.721,最大为N50和N69之间,最小为N51和N70之间。(8)根据22对SSR核心引物指纹号码可构建80份糯玉米自交系标准的数字化指纹图谱。两个自交系之间出现相同谱图的概率是8.7×10-19,微乎其微,因此可广泛用于鉴定自交系的真伪性。(9)随机抽取的7个自交系经田间杂交组合验证:一般配合力、特殊配合力及杂种优势差异明显,得到优良自交系N69、N51、N45、N73,并由其为亲本组配的杂交组合都具有较强杂种优势。评价出的优良自交系和优良杂交组合符合遗传差异的聚类分析,尤其是SSR聚类分析。(10)贯穿整个试验上,我们看出,在整个物种的遗传标记发展过程中,从表型标记、生化标记、DNA分子标记是经历了由简单到复杂、由可靠性低到准确性高、从由揭示生命现象到揭示生命本质的过程。通过三种聚类方法对糯玉米种质资源进行遗传物质的差异分析,尚属首次,三种聚类方法中,以SSR分子标记进行类群间鉴定最为可靠。

【Abstract】 Genetic diversity is the summation of different colonies in one species or different individual genetic variation in one colony. The research on genetic diversity experienced the following stages: agronomic character morphological markers, comparative analysis of quality character,, biochemical marker (isozyme and seed store protein), and latest research on DNA molecular marker in the field of molecular biology, that indicates difference of internal germplasm. The study on genetic diversity study plays a significant role in the collection, valuation, preservation and utilization of biotic germplasm resources. The following conclusions were made via investigate 17 agronomic characters of 80 waxy corn inbred lines(40 yellow, 20 white, 13 black and 2 red ones), cluster analysis and genetic distance, measure content of chief biochemical component, PAGE cluster analysis of seed store protein(salt-soluble protein), SSR molecular marker fingerprint, total diallel cross of randomly chose 7 inbred lines, measure the combining ability and heterosis.1. Abundant genetic diversity of 80 waxy corn germplasm were showed on agronomic character, quality character, DNA inheritance material. The 17 agronomic characters and quality characters express great range of variance, different significant of variance analysis.2. By analyzing the chief components of 17 important quality characters and calculating according to the 85% contribution rate, we got 7 chief component factors. The geometrical distance difference among these 7 between the 2 inbred lines i. e. genetic distance. 80 inbred lines are divided 9 groups, according to the 4.15 genetic distance. The 1 st included 2 inbred lines, which are N76 and N09. There is only N66 in the 2nd. There are 5 in the 3rd, which are N68, N67, N49, N74 and N42. There are N05 and N37 in the 4th. There is only N57 in the 5th. In the 6th, there are 19 inbred lines, which are distributing into 2 sub—groups. In the 1st sub—group, it included N80, N62, N72, N71 and N10. In the 2nd sub—group, it included N41, N77, N44, N63, N70, N46, N60, N43, N15, N30, N26, N78, N19, N06. In the 7th group, there are 7 inbred lines, which are N56, N55, N54, N53, N48, N31, and N18. In the 8th group contains 2 inbred lines which are N04, N21, there are 41 inbred lines in the group 9th, which are divided into 4 sub—groups. In the 1st, it include N73, N58, and N52. In the 2nd, they are N39, N17, N16, N47, N12, N61, N24, N38, N33, N14, and N07. In the 3rd, there are 2 inbred lines, which are N29, and N13. In the 4th, it include N75, N65, N69, N35, N79, N27, N20, N11, N08, N64, N25, N51, N03, N59, N02, N32, N28, N50, N36, N23, N40, N24, N22, N01. The clustering analysis results of agronomic character shows that different phenotypes of inbred lines are distributing in different groups.3. There are great range in the content of chief nutritious quality component of waxy corn germplasm resources. The content of starch, protein and fat variance range is 58.05—76.20%, 7.31—13.97%, and 3.00—6.68% respectively, the average value is 67.69%, 11.15% and 4.93%. The starch is 100% amylopectin, which is remarkable negative correlation with protein, and the correlation coefficient is -0.1908. The fat and starch is positive correlation, and the correlation coefficient is 0.1786.4. The PAGE map of 80 waxy corn inbred lines was divided 13 special bands according migrate rate, and cluster 6 groups(genetic distance 0.27 as standard), the 3rd group was divided 2 sub-groups further at the site of genetic distance 0.14, the 4th group was divided 3 sub-groups at site 0.22, there is only 1 line in the 1st groups i. e. N16, and so is the 2nd group that is N51, in the 3rd group, there are 7 lines divided 2 sub-groups, which is N71, N70, N68, N67; and N39, N65, N66. In the 4th group there are 68, which divided 3 sub-groups, In the first sub-groups, they are N10, N06, N05. In the second, they are N74, N69, N11, N46, N43, N45, N19, N28, N17, N08, N15, N13, N07, N14, and N04. In the third, it includes the following: N63, N62, N56, N55, N57, N50, N38, N27, N25, N24, N12, N49, N47, N80, N79, N76, N44, N42, N41, N78, N77, N61, N60, N22, N21, N54, N53, N64, N58, N52, N59, N37, N36, N35, N34, N33, N32, N31, N30, N23, N20, N72, N40, N29, N26, N48, N09, N75, and N03. In the 5th group, only N73 is included. In the 6th, N02, and NO 1 are included. The genetic distance variance range from 0.00 to 0.46.5. 22 pair primers of SSR distribute in 10 chromosomes of maize genome, each of which can check and measure 2-4 alleles. Altogether there are 60 morphological fragment, average of which is 2.73. The variance range of genetic distance of 80 waxy corn inbred lines is from 0.045 to 0.946.6. As the standard of genetic distance 0.55, the 80 waxy corn inbred lines cluster 9 groups via cluster analysis of SSR. Among them, the 2nd group is divided into 3 sub—group at the site of 0.46. The 5th is into 2 at 0.490. So is the 7th and 8th at 0.46, 0.46 respectively. The 9th is into 4 at 0.493. The following is the distribution situations: the 1st group include N12. The 2nd are N69, N80, N79, N73, N71, N70, N74, N68, N67, N65, N63, N61, N66, and N52. The 3rd are N50 and N48. The 4th is N72. The 5th are N64, N59, N58, N46, N44, N62, N57, N54, N53, N60, N56, N55, and N43. The 6th are N16, N42, N16, and N41. The 7th are N40, N36, N25, N39, N37, N78, N77, and N09. The 8th are N76, N45, N47, N11, N49, N07, and N06. The 9th are N75, N51, N30, N13, N14, N29, N28, N23, N33, N38, N19, N27, N34, N35, N18, N15, N10, N26, N05, N24, N22, N32, N31, N21, N03, N04, N02, N08, N20, and N01.For the yellow lines, except N12, the others distribute in the 6th, 7th, 8th and 9th group. In the 9th group, 28 yellow lines in it, which account for 93%. The others yellow lines distribute 6, 7, 8th group. The white ones almost distribute in the 3rd sub-group of 2nd group, the 3rd, 5th and 7th group, the number of lines is 21, account for 84%. The black ones, except N72 being one group, N75, N76, and N78 distribute in the 7th, 8th and 9th group respectively, the others are all in the 2nd group, which covers 57%. The red ones distribute in the 2nd sub-group of 2nd group, with no different of genetic distance.7. There is remarkable genetic difference among different colors of waxy corn inbred lines. The genetic distance of same color inbred lines is less than different ones. There is remarkable different of the least genetic distance between the inbred lines of different colors and same color lines. The 40 yellow inbred lines, the genetic distance variance range from 0.087 to 0.865, average of which is 0.632. The maximum is between N36 and N12, while the minimum is between N35 and N18, N35 and N34. For the white ones, the genetic distance variance range from 0.091 to 0.946, average of which is 0.680. The maximum is between N49 and N65, while the minimum is between N53 and N54. For 13 black ones, the genetic distance is from 0.045 to 0.895, average of which is 0.655. The maximum is between N69 and N72, while the minimum is between N77 and N78. The genetic distance between 40 yellow ones and 25 white is from 0.464 to 0.927, average of which is 0.747. The maximum is from N46 to N08, while the minimum is from N41 to N17. The genetic distance between 40 yellow ones and 13 black is from 0.517 to 0.897, average of which is 0.749. The maximum is from N72 to N30, while the minimum is from N78 to N09. The genetic difference between 25 white ones and 13 black is from 0.483 to 0.892, average of which is 0.721. The maximum is from N50 to N69, while the minimum is from N51 to N70.8. According to 22 pairs primers of SSR, the digitized fingerprint was established of 80 waxy corn inbred lines. The probability of the same map of 2 inbred lines is 8.7×10-19, so it can be adopted to identify truth from falsehood.9. The general combing ability, special combing ability and heterosis of 7 lines which randomly choice and cross combination in the field are remarkable difference. The cross combination of inbred lines N69, N51, N45 and N73 express strength heterosis. The appraised choiceness inbred lines and cross combination ones accord with the clustering analysis of genetic discrepancy, especially SSR clustering analysis.10. Through the whole experiment, during the process of the whole species genetic marker development, the morphological marker, biochemical marker, and DNA molecular marker all experienced the process from simple to complex, low reliability to high accuracy, and revealing vital phenomena to essence. It is the first to adopt 3 kinds cluster analyse of waxy corn gerrnplasm in this paper, among these three methods, the SSR molecular marker is the most reliable.

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