【作者】 库丽霞；

【导师】 陈彦惠；

【作者基本信息】 河南农业大学 ， 作物遗传育种， 2010， 博士

【摘要】 玉米不仅是重要的粮食作物,而且也是重要的饲料、经济和能源作物。高产是现代玉米育种追求的最重要目标。以改良玉米株型、增强抗耐高密度逆境、提高群体光合效率为目标的株型育种,是实现玉米高产再高产的重要途径。因此,研究玉米株型相关性状的分子遗传机理,不仅对促进玉米耐密型高产育种研究和分子标记辅助选择技术的发展,而且对解析玉米株型性状分子生物学机制都具有重要的理论和实践意义。本研究以玉米株型主要性状QTL为主要研究对象,分以下4个步骤开展研究。首先,以紧凑型和松散型玉米自交系为亲本,构建了一套F2作图群体及其衍生的F2:3家系的定位群体,在3个环境下对玉米田间表型进行鉴定,利用Mapmaker version 3.0构建了具有222个SSR标记的连锁图谱,对玉米株型相关性状进行了QTL初步定位及效应分析。其次,利用多群体联合QTL分析的方法,将具有不同亲本的2套F2群体基因型数据进一步整合成为一张包含352个分子标记的遗传图谱,再结合2套定位群体多个环境田间鉴定的表现型数据,对玉米株型相关性状的QTL进一步定位和印证,明确了控制株型性状主效QTL集中的染色体区域。此外,又利用生物信息学和比较基因组学的方法,预测了控制主要性状QTL所在染色体区段的候选基因。再次,根据QTL初步定位的结果,将研究目标集中控制玉米叶夹角主效QTL qLA1-1基因位点上,利用连续多次回交和开发分子标记等方法,成功构建了qLA1-1的近等基因系群体,利用BC3F2:3家系进行定位验证qLA1-1的准确性并利用BC3F2分离群体进行精细定位,逐步缩小QTL目标区域,最终将qLA1-1的QTL目标区域限定在0.75Mb范围之内。最后,以双亲为材料利用同源克隆的方法,对定位主效QTL目标区域预测的候选基因进行克隆。成功克隆了与调控水稻叶夹角基因OsTAC1高度同源的玉米ZmTAC1基因,根据双亲DNA序列的差异开发了一个CAPs标记,将其作为基因的功能标记,重新对定位群体进行了叶夹角QTL基因定位分析;另外,根据双亲序列差异性分析了31个不同叶夹角表型自交系的DNA序列,初步验证了ZmTAC1与叶夹角之间的关系和功能。具体研究结果如下:1、利用豫82×沈137构建的F2群体构建了一张包含有222个标记的遗传连锁图,覆盖玉米10条染色体,图谱总长度为1864.7 cM,两标记间平均距离为8.40 cM。利用3个环境鉴定的表型值的均值对株型相关性状进行QTL定位及效应分析,共检测到了25个QTL,除第6、8染色体外分布在其余8条染色体上;其中,检测到叶夹角QTL3个,叶向值QTL5个,叶长QTL3个,叶宽QTL4个,株高QTL3个,穗位高QTL7个。发现了2个调控叶夹角和叶向值的主要染色体区域:①位于第1染色体的1.02区域,介于标记umc1166和umc2226之间,解释表型遗传变异的20.4%;②位于第5染色体的5.04-5.05区域,介于标记bnlg1287和mmc0282之间,贡献率为9.7%;这两个位点具有减小叶夹角、增大叶向值作用,基因效应均来自紧凑型亲本豫82。2、联合2个F2作图群体整合了一张具有333个SSR标记、3个新开发的CAPS标记和16个候选基因位点的遗传图谱,整合的遗传图谱总长度为1766.4cM,相邻两标记间的平均距离为5.0cM。利用该遗传图谱通过MCQTL软件分析,共检测到与叶夹角、叶向值、叶长、叶宽有关的QTL 33个。其中与叶夹角相关的QTL 7个,位于第1,2,3,5,7和8染色体上,单个QTL的贡献率变异范围是7.3%-19.0%;检测到11个影响叶向值的QTL,位于第1,2,3,4,5,7,8和9染色体上,单个QTL的贡献率变异范围5.03%-23.2%;检测到7个与叶长相关的QTL,分布在第3,5,7和10染色体上,单个QTL的贡献率变异范围7.14%-20.6%;检测到8个QTL与叶宽有关,分布在第1,3,4,7,8和9染色体上,单个QTL贡献率的变异范围4.86%-20.4%。结果表明控制叶型性状QTL主要集中的染色体区域有2个(ZmLAR1和ZmLAR2):ZmLAR1位于第3染色体的umc1608-umc1674,除叶夹角的贡献率为9.26%外,其它3个性状的贡献率范围14.07%-20.6%;ZmLAR2位于第7染色体的umc1380-umc1936,4个性状的贡献率范围6.8%-14.3%。此外,也发现了3个调控叶夹角、1个调控叶长和1个调控叶宽的主要位点。这些主要位点/区域都包含的相应候选基因为调控叶夹角的基因DWARF4、TAC1、LIC、YABBY15、ligueless1和ligueless2;一个调控叶长的基因lng1和一个调控叶宽的基因NAL7。3、在初步定位的基础上,对调控叶夹角的位于第1染色体1.02区域内的QTL qLA1-1通过多次回交采用“双卡”的方法,即表型选择结合基因型选择,“一卡”遗传背景回复率(RRGB)大于95%以上;“二卡”目标性状(叶夹角)小于12.5°以下的单株等构建了近等基因系。利用BC3F2:3家系验证了该QTL的准确性及构建的近等基因系是单个QTL位点控制叶夹角的大小。根据定位结果,在目标区域内开发了43对SSR引物,筛选到在亲本间具有多态性强且带型清晰的SSR引物9对。通过对近等基因系的BC3F2分离大群体精细定位,将qLA1-1限定在标记LA25与bnlg1484之间,其标记距离在0.75Mb范围之内。4、利用同源克隆方法,首次克隆了与叶夹角发育调控有关ZmTAC1基因。该基因序列中792bp的开放性阅读框可编码263个氨基酸,编码的氨基酸序列与水稻的同源性为68%,cDNA 5’端非编码区碱基序列在豫82与沈137两个自交系中一个单碱基的差异可能引起mRNA表达水平的差异。应用实时荧光定量PCR对该基因时空表达分析表明,叶枕和叶鞘中表达量最高,叶片和茎尖其次,根中很低;4叶期开始表达,9-10叶期达到高峰,11叶期以后逐渐下降;豫82的时空表达量均明显低于沈137。在31个自交系中,豫82和13个穗上部叶夹角小的自交系在5’端非编码区碱基序列均为CTCCT,而沈137和18个穗上部叶夹角的自交系在5’端非编码区碱基序列均为CCCCT。利用开发的基因内CAPS标记和利用豫82与沈137构建的F2:3家系进行基因内定位,ZmTAC1被定位在qLA1-2的区间内。更多还原

【Abstract】 Maize is a not only important cereal crop but alse important forage, economic and energy crop. High yield is a most important aim that modern maize breeders pursue today. Maize plant-type breeding with the aim by improving plant-type, strengthening resistance high-density adversity, and improving group photosynthetic efficiency is a main approache to achieve high yield of corn. Therefore, researches related to the characters of corn plant molecular genetic mechanism has the important theoretical and practical significance to not only promote maize resistance high-density breeding and molecular marker assisted selection technology development but aslo analyze maize plant traits molecular biology mechanism.In this study, maize architecture traits QTL as the main research object carry out research as following four steps. Firstly, a set of F2:3 families derived from a compact and expanded maize inbred line cross were developed.a set of 229 F2:3 families derived from compact and spread-out inbred line cross was evaluated for three environments. Genetic linkage maps with 222 SSR markers were constructed using Mapmaker version 3.0. Were detected for the six measured morphological traits using composite interval mapping (CIM). The performinance data of plant architecture traits in the F2:3 families were used for QTL mapping and analysis to genetic effect. Secondly, the genetic linkage maps with 352 SSR markers integrated using the two sets of genotypes data from F2 population of different parents and the performinance data of plant architecture traits from the two populations evaluated in three environments were used for further QTL mapping and confirmation by analysis technique of multiple population joint QTL and the chromosomal regions that main effect QTL with the traits concentrated were identified. In addition, the cadidate genes of QTL located on chromosome regions were speculated using comparative genomics and bioinformatics. Thirdly, our research objective concentrated on the major effect QTL qLA1-1 with leaf angle by preliminary results of QTL mapping. The near-isogenic line (NIL) of qLA1-1 were successfully constructed using repeatedly backcross and molecular marker assisted selection. qLA1-1 were confirmed by BC3F2:3 families. Fine mapping of qLA1-1 by the BC3F2 segregation population and the latest molecular marker developed shortened the distance of QTL region that were limited in 0.75 Mb scope. Finally, the candidate gene speculated in the scope of major effect QTL was cloned through homology-based cloning. ZmTAC1 was successfully cloned that the gene was high homologous to OsTAC1 with rice leaf angle. A CAPs marker was developed on the basis of the differences of parents DNA sequences. As the function marker of the gene, the performinance data of leaf angle were again used for QTL mapping. In addition, the DNA sequences that 31 inbred lines had difference of leaf angle were cloned and analysed. The relationship of ZmTAC1 and leaf angle was identified. The main results obtained in this research were concluded as follow:1. The linkage map of 222 SSR markers constructed using F2 population of Yu82×Shen137 consisted of all ten maize chromosomes allocated to ten linkage groups, spanning a total length of 1864.7 cM with an average marker interval of 8.40 cM. QTL for plant architecture traits were mapped using a set of 229 F2:3 families derived from the cross between compact and expanded inbred lines, evaluated in three environments. Twenty-five QTL were detected in total on the rest 8 chromosomes besides chromosome 6 and 8. 3 QTL of leaf angle were located, 5 QTL of leaf orientation value were detected, 3 QTL of leaf length were detected, 4 QTL of blade width were detected, 3 QTL of plant height were located, and 7 QTL of ear height were located. Two key genome regions controlling leaf angle and leaf orientation were identified from our study. The QTL between umc1166 and umc2226 in the 1.02 region of chromosome 1 explained 20.4% of the phenotypic variance and another QTL between umc1166 and umc2226 in the 5.04-5.05 region of chromosome 5 explained 9.7% of the phenotypic variance. two QTL associated with decreased leaf angle and increased leaf orientation value were contributed by Yu82.2. The linkage map of 333 SSR markers, 3 CAPs markers developed, and 16 candidate genes were integrated using two F2 population3 of Yu82×Shen137 and Yu82×Yu87-1, spanning a total length of 1766.4 cM with an average marker interval of 5.0 cM. 33 QTL were detected for leaf angle, leaf orientation value, leag length, and blade width using MCQTL 4.0. Seven QTL for leaf angle were located on chromosome 1, 2, 3, 5, 7, and 8 with individual effects ranging from 7.3% to 19.0%. 11 QTL for leaf orientation value were detected on chromosome 1, 2, 3, 4, 5, 7, 8, and 9 with individual effects ranging from 5.03% to 23.2%. 7 QTL associated with leaf length were located on chromosome 3, 5, 7, and 1 with individual effects rangingfrom 7.14% to 20.6%. 8 QTL of leaf width were detected on chromosome 1, 3, 4, 7, 8, and 9 with individual effects ranging from 4.86% to 20.4%. Two key genome regions(ZmLAR1and ZmLAR2)controlling leaf architecture traits were identified from our study. ZmLAR1 were between umc1608 and umc1674 on chromosome 3 with individual effect of four traits ranging from 9.26% to 20.4% and ZmLAR2 were between umc1380 and umc1936 on chromosome 7 with individual effect of the traits ranging from 6.8% to 14.3%. Moreover, 3 genome regions of leaf angle, a genome region of leaf length, and 1 genome region of leaf width were identified. All these regions encompass candidate genes DWARF4, TAC1, LIC, YABBY15, ligueless1and ligueless2 for leaf angle, lng1 for leaf length, and NAL7 for leaf width.3. According to the results of QTL mapping, QTL qLA1-1-NIL in bin 1.02 of chromosome 1 for leaf angle was constructed by more once backcrosses and“Double Card”.“One Card”was RRGB greater than 95% and“Two Card”was leaf angle lesser than 12.5o. qLA1-1 was idengtified again using BC3F2:3famalies and that the NIL had controled leaf angle for single locus segregation. 43 SSR markers were developed in the target region. 9 SSR markers had polymorphism strong and clear banding. qLA1-1 was defined by BC3F2 population between LA25 and bnlg1484, spanning a total length of 0.75 Mb.4. ZmTAC gene for leaf angle was cloned by homology-based cloning in maize inbred lines Yu82 and Shen137, a predicated 792-bp open reading frame was detected in the cloned sequence, which encoded 263 amino acids. The protein sequence was 91% homology to rice TAC1. The difference in the inbred lines which a single DNA letter change in the 5’- untranslated region(UTR) caused the difference in expression level of mRNA. Real-time PCR analysis revealed that ZmTAC1 was expressed superlatively in leaf sheath and pulvinus, secondly in leaf and stem apex, and expressioned from 4-leaf stage, with the peak at 9-11 leaf stage, the expression decreased gradually from 11-leaf stage. Yu82 was lower than Shen137 in expression. The sequence of Yu82(CTCCT) in 5’- UTR was always associated with 13 compact plant architecture inbred lines and the sequence of Shen137(CCCCT) in 5’- UTR was always associated with 18 expended plant architecture inbred lines. ZmTAC1 was defined in the region of qLA1-2 by the CAPs developed and the F2:3 families of Yu82×Shen137.更多还原