【作者】 姚启伦；

【导师】 杨克诚； 潘光堂；

【作者基本信息】 四川农业大学 ， 作物遗传育种， 2008， 博士

【摘要】 根据西南地区玉米地方品种的主要地域分布,本研究以来自四川、重庆、云南和贵州四省（市）的玉米地方种质群体为材料,对54个玉米地方品种群体做遗传多样性的SSR分析和B染色体的细胞学鉴定;并从中选取50个品种群体进行田间实验,分析玉米地方品种农艺、经济性状的差异表现;结合室内分析,研究低磷胁迫下玉米地方种质的主要形态和生理特性,探讨耐低磷玉米地方种质的筛选指标。主要结果如下:1.农艺、经济性状的差异表现分析结果表明,玉米地方品种在各农艺、经济性状上存在极显著差异。分析各性状的变异幅度,农艺性状株高、穗位高、总叶片数、散粉期、抽丝期和全生育期的变幅分别为213.25～322.98、87.30～198.59、15.50～24.38、69.50～94.50、70.50～93.50和114.00～142.00;经济性状穗长、秃尖长、穗粗、轴粗、穗行数、行粒数、穗粒重、百粒重和容重的变幅分别是8.40～18.32、0.30～2.28、2.49～4.88、1.51～2.74、8.70～17.05、16.17～31.39、26.57～161.68、9.00～36.03和245.00～739.00。农艺性状与经济性状的变异系数比较表明,经济性状的变异程度较农艺性状的变异程度高。农艺性状变异程度大小排序为穗位高、总叶数、抽丝期、散粉期、株高、全生育期;经济性状变异程度的大小排序为秃尖长、穗粒重、百粒重、行粒数、穗长、穗行数、穗粗、容重、轴粗。根据“主成分”分析结果,结合玉米育种目标,分析各玉米地方品种农艺、经济性状的分量值,在供试材料中评选出综合性状优良的地方品种有DP-11、DP-44、DP-42、DP-31、DP-65、DP-19、DP-60、DP-15、DP-57和DP-13。农艺、经济性状的聚类分析表明,同一产地来源玉米地方品种的农艺、经济性状存在较大差异,而不同产地来源的玉米地方品种可能具有相似的农艺、经济性状。2.基于SSR标记的遗传多样性分析结果,均匀覆盖玉米染色体组的42对SSR引物,在作DNA混合取样的54个玉米地方品种中检测到256个等位基因,每个SSR标记的等位基因数为2～9个,平均6.1个,多态信息量0.30～0.85,平均0.76,说明玉米地方品种群体遗传多样性丰富。根据遗传相似系数矩阵做出的树状图,将54个玉米地方品种大致划分成4类,来源于同一地区的多数玉米地方品种划分在同一类中,其遗传相似系数在0.60以上,表明玉米地方品种的地理分布与其遗传背景存在内在联系。比较SSR聚类与农艺经济性状聚类结果,2种聚类结果差异很大,这表明SSR分子标记所揭示的DNA的结构差异与“主成分”分析所揭示的表型差异是不一致的。从54个玉米地方品种中选出11个,每个品种随机抽取15个单株,共165个DNA单株样品,分析玉米地方品种的遗传结构及其品种内的遗传多样性。对于检测玉米地方品种的遗传多样性,DNA单株样品分析优于DNA混合样品分析,42对相同的SSR引物在11个玉米地方品种中检测到330个等位基因,平均等位基因数A=7.86,有效等位基因数=3.90,平均期望杂合度H_e=0.69,实际观察杂合度H₀=0.37。据遗传结构分析结果,固定指数（F）为0.25～0.79,表明玉米地方品种是典型的混合繁育系统;由于杂合体不足,玉米地方品种群体内的遗传结构偏离了Hardy-Weinberg平衡;杂合性基因多样度比率(F_st)平均为0.07,表明品种间和品种内的遗传变异分别占总遗传变异的7%和93%。“主成分”分析（PCA）结果与品种间遗传距离分析结果相一致,同一品种群体内的个体以及来源于同一地区不同品种群体间的个体距离较近,相邻分布;来源于不同地区品种群体间的个体距离较远,产生明显分离。品种群体内遗传多样性分析结果表明,四川玉米地方品种的遗传变异水平、等位基因频率以及基因杂合度均为最高,贵州玉米地方品种的最低,表明在我国西南地区的四省（市）中,四川的玉米地方品种具有最丰富的遗传变异。3.B染色体细胞学鉴定结果表明,玉米地方品种的B染色体具有常见B染色体的基本特征,B染色体在地方品种细胞中的异常分布是细胞和植株个体存在B染色体数目变化的主要原因。在检测的54个玉米地方品种中有9个存在B染色体,B染色体数目在品种间发生0B～7B的数目变化,在同一品种的不同细胞间发生0B～3B的变化;具有1B、2B和3B染色体的地方品种分别占玉米地方品种总数的12.96%、5.56%和3.70%;在对每个玉米地方品种所检测的30个细胞中,具有0B、1B、2B和3B染色体的细胞比例分别为86.81%、5.42%、4.44%和3.33%。分析含B染色体玉米地方品种的地理分布,四川的东南部地区是玉米地方品种B染色体的集中分布区。尽管本研究未发现B染色体与基于SSR的DNA多态性的相关关系。然而,综合分析B染色体的地理分布和玉米地方品种群体的遗传多样性,支持西南地区玉米地方品种的地理演变途径为最早引进到四川的假说。4.分析低磷胁迫在苗期对玉米地方品种的主要生物学效应,结果表明,低磷胁迫下各地方品种的根体积、总叶面积、根干重和地上部干重均显著降低,而根冠比和根毛密度明显增加;同样,土壤缺磷诱导植株体内磷利用率、酸性磷酸酯酶活性、过氧化氢酶活性、过氧化物歧化酶活性以及丙二醛和脯氨酸含量的显著提高,但显著降低磷含量和可溶性蛋白质含量。不同玉米地方品种的耐低磷性存在明显的基因型差异,与低磷敏感玉米地方品种DP-36和DP-27比,低磷胁迫对耐低磷玉米地方品种DP-60、DP-02和DP-40影响较小。分析低磷胁迫下各生物学性状的变化,干物重和植株吸磷量的变化较大,表明干物重和吸磷量是苗期筛选玉米地方品种耐低磷种质的可靠指标。此外,酸性磷酸酯酶活性可作为筛选耐低磷种质的生化指标。低磷胁迫下干物重与缺磷症状的显著相关表明以植株缺磷症状为依据对玉米地方品种耐低磷等级的划分是可行的。基于上述结果,结合低磷胁迫下玉米地方品种后期的经济性状表现,在供试的50个玉米地方品种中,筛选出3个耐低磷地方品种DP-60、DP-02和DP-41,5个中耐低磷地方品种DP-12、DP-48、DP-54、DP-59和DP-65。5.综合分析供试玉米地方品种的农艺、经济性状,遗传多样性及其耐低磷营养特性。西南地区玉米地方品种抗病力强,多数地方品种高抗大、小斑病和锈病;群体遗传变异水平高,42个SSR标记在54个品种群体中共检测到256个等位位点,在11个群体内的165个个体中检测到330个等位位点,有效基因杂合度达0.67;品种类型丰富多样,生育期从早熟（114 d）到晚熟（142d）,粒型有硬粒、马齿和半马齿,粒色有黄、白、红、蓝;耐低磷种质丰富。从供试的50个玉米地方品种中筛选到与育种目标相关、主要农艺经济性状优良的品种10个,品种群体内遗传变异丰富（等位位点数在6.00以上）的品种7个,可供作理论研究的含B染色体的玉米地方品种9,耐低磷品种3个,中耐低磷品种5个。多数玉米地方品种植株和穗位较高,株型松散,平均株高和穗高分别高达2.64和1.38 m;植株抗倒性差,表现程度不同的倒伏和倒折。鉴于地方品种遗传上的复杂性和适应性与丰产性的矛盾,建议采用优良地方种质群体改良、地方种质与外引种质组建群体和地方种质与热带种质进行相互改良这三种方法,对玉米地方种质进行间接利用。更多还原

【Abstract】 Based on their geographical distribution, the maize landraces from Sichuan, Chongqing, Guizhou and Yunan in southwest China were used in the study. Genetic diversity of 54 maize landraces was tested by using microsatellite (SSR) loci and their B chromosomes were observed cytologically. In addition, a field trial was conducted to analysis agronomic and economic traits, and investigate main morphological and physiological changes of different maize landraces to low-P stress at the stage of seedling. The main results as following:1. Variances of maize landraces in all agronomic and economic traits were significant at 0.01 level. With regard to their ranges among landraces, plant height, ear height, total leaves, flowering period, silking and growth period were 213.25～322.98, 87.30～198.59, 15.50～24.38, 69.50～94.50, 70.50～93.50 and 114.00～142.00, respectively. The ranges of ear length, sterile length, ear diameter, axis ear, rows per ear, kernels per row, kernel weight per ear, 100-kernel weight and unit weight were 8.40～18.32, 0.30～2.28, 2.49～4.88, 1.51～2.74, 8.70～17.05, 16.17～31.39, 26.57～161.68, 9.00～36.03 and 245.00～739.00, respectively. In comparison of the variation coefficients between agronomic and economic traits, the latter was higher than the former. The ranges of ear height, total leaves, silking period, flowering period, plant height and growth period were, in turns, higher. Accordingly, those of sterile length, kernel weight per ear, 100-kernel weight, kernels per row, ear length, rows per ear, ear diameter, axis ear and unit weigh, in turns, higher. On the whole, maize landraces exhibited various strains, a large phynotypic varition, high plant individuals and resistance to disease. Based on the principal component of all agronomic and economic traits by Principal Component Analysis (PCA) as well as the goals of maize breeding, 10 landraces with excellent traits DP-11, DP-44, DP-42, DP-31, DP-65, DP-19, DP-60, DP-15, DP-57 and DP-13 were chosen from the materials studied. The results of the clustering analysis indicated that obvious differences existed in agronomic and economic traits of landraces with the same geographical origin, and there maight be similar traits in landraces with the different geographical origin.2. Genetic diversity of 54 maize landraces was tested by using bulk DNA samples and 42 microsatellite (SSR) loci distributed uniformly on 10 chromosomes of maize. A total of 256 alleles were detected among 54 landraces. At each locus, the number of alleles varied from 2 to 9, with an average of 6.1. Mean polymorphism information content was 0.78 ranging from 0.34 to 0.78. 54 landraces could be clustered into four groups by the clustering analysis based on the genetic similarity coefficients. The landraces collected from the same region could mostly be grouped together and their genetic similar coefficients were over 0.6. Comparison of the clustering result of agronomic and economic traits and that of SSR, no consistent relationship was found. This implied that DNA differences revealed by SSR were different from those by PCA. To reveal the genetic structure and genetic diversity within landraces, 165 individuals in total from 11 out of 54 landraces were analysed on the basis of the same 42 SSR loci. The analysis of individual DNA samples was proved superior to that of bulk DNA samples to identify genetic diversity of landraces. A total of 330 alleles were found in 11 landraces. Estimates of the mean number of alleles ’A’, the effective allelic number ’A_e’, the observed heterozygosity ’H_o’ and expected heterozygosity ’H_e’ were 7.86, 3.90, 0.69 and 0.37, respectively. An obvious genetic deviation from Hardy-Weinberg expectation was observed both among and within landraces and a considerable genetic variation was revealed within rather than among landraces. The results of Principal Component Analysis (PCA) were consistent with those of the clustering analysis and the genetic distance. Individuals within a region and a landrace were grouped more closely while the individuals from the different landraces and regions were located more distantly. According to genetic diversity within landraces, the landraces from Sichuan were the highest genetic variation, allelic frequency and gene heterozygosity. It indicated genetic diversity of landraces was more plentiful in Sichuan than in other 3 regions.3. B chromosomes (Bs) in 54 maize landraces from the four regions was tested by means of cytological observations. General B characteristics was found in the B chromosome of maize landraces. The aberrant distribution and separation of B chromosome might result in their differences in plant individuals and cells. Out of 54 maize landraces, 9 landraces with Bs were observed. the number of Bs was found variable. The number of Bs in the landraces ranged from 0 to 7 and varied from 0 to 3 in a single cell. The 12.96, 5.56 and 3.70% of the total landraces were found with 1B, 2B and 3Bs. It was indicated that southeastern Sichuan was the main distribution area of the landraces with Bs in southwest China. The relationship between B chromosomes and DNA polymorphism based on SSRs was not found in the study. However, the geographical distribution of B chromosomes, together with the genetic diversity of the maize landraces, supported that maize landraces in southwest China were firstly introduced to Sichuan from India via Tibet to a certain degree.4. Using two P treatment, a randomized complete block design with two replications was designed to investigate biological changes of different maize landraces. The results showed that P-deficiency significantly decreased root volume, total leaf area, and plant dry weight, but greatly increased density of root hairs and root top ratio. In addition, P-deficiency induced the significant enhancement of phosphorus utilization efficiency and the amount of proline, malondialdehye (MDA), acid phosphatase (APase), peroxidase ( POD) and superoxide dismutase (SOD), but the significant reduction of P uptake and soluable protein content. Since P-deficiency had smaller effects on the P-tolerant maize landraces DP-60, DP-02 and DP-40 as compared with P-sensitive landraces DP-36 and DP-27, it was demonstrated that differences of tolerance to P-deficiency existed among different maize landraces. Dry matter weight and P uptake were suggested as reliable screening standards to identify low-P intolerant germplasm in landraces and Apase activity as physiological one. The results based on correlation analysis also indicated that it was feasible to rate low-P intolerant levels by P-deficiency symptoms. According to the effects of low-P stress on economic traits and the results discussed above, low-P intolerant landraces DP-60, DP-02, and DP-41 were found. At the same time, medium-P intolerant DP-12, DP-48, DP-54, DP-59 and DP-65 were also selected.5. On the whole, maize landraces exhibited a large phynotypic varition and resistance to leaf blight, leaf spot as well as brown spot of corn. Using 42 SSR loci, high genetic variation was found among landraces with a total of 256 alleles being detected. At the same SSR loci, 330 alleles in total were found in 11 landraces with a high expected heterozygosity ’H_e’ (0.67). There were various strains in landraces. Their growth period varied from the early to late maturing and the kernels had yellow, red, white and blue flint, as well as yellow, red, white and blue dent. Rich low-P intolerant germplasm was also found. 10, 7, 9, 3 and 5 landraces with excellent traits, high genetic variation, B chromosomes, low-P intolerant and medium-P intolerant, respectively, were chosen from the materials studied. However, most landraces exhibited poor in plant height and resistance to lodging. Their plant and ear height were 2.64 and 1.38 m, respectively. Since direct utilization on maize landrace germplasm is difficult in maize breeding, it is suggested that the germplasm is utilized indirectly by using genetic innovation of landrace populations, mass selection between landrace and exotic germplasm, and recurrent selection between the landrace and tropic germplasm.更多还原