【作者】 郭营；

【导师】 李斯深；

【作者基本信息】 山东农业大学 ， 作物遗传育种， 2011， 博士

【摘要】 小麦是我国主要的粮食作物之一,在粮食生产中具有举足轻重的作用,种植面积大,对肥料的需求量高。小麦对矿质营养元素的吸收利用大多为受多基因控制的数量性状,易受环境影响。QTL作图技术为数量性状遗传研究开辟了新的途径。它不仅有利于分子标记辅助选择和相关基因的克隆,也为未来的分子设计育种奠定了理论基础。因此,开展小麦矿质营养利用效率相关性状的QTL分析具有重要意义。本研究利用课题组创制的“川35050×山农483”RIL群体(2009年为F17)为材料,对小麦苗期相关性状(生物量,形态性状,N、P、K的吸收利用效率)和产量/籽粒性状(株高、穗数、千粒重等)进行了测定和QTL定位分析,并对N代谢过程中的关键酶谷氨酰胺合成酶基因TaGS1a与苗期、产量/籽粒性状进行QTL分析,以期了解营养吸收利用的遗传控制机制。获得了以下主要结果。(1)小麦苗期不同N、P、K处理下营养相关性状的QTL分析。本试验中,在水培条件下,设置3个N水平、2个P水平、2个K水平,完全组合,包含12个处理(简称为HC-1)。共检测到373个QTL位点(590个性状-处理QTL),位于小麦的18条染色体上,除3D,4D,7D外。在不同处理条件下,单一QTL可解释表型变异的2.9-73.6%,LOD值最大值为16.2(SKUE)。有22个QTL位点是相对出现频率比较高的位点(relative high frequency QTLs,RHF-QTLs),这些位点均可在4个以上的处理中检测到。其中,16个RHF-QTLs的平均贡献率都达到20%以上,包括QSnc-1B.2,QSpc-1B.1,QSkc-1B.3,QSkue-1B.1,QRdw-4A.1,QRdw-4A.2,QSdw-4A.1,QTdw-4A.1,QRnc-4A.1,QSnc-4A.1,QRpc-4A.3,QRkc-4A,QTnue-4A.1,QTpue-4A,QTkue-4A.1和QMrl-5D.2,说明这些位点是比较重要的RHF-QTLs位点。涉及5个以上性状的位点形成了22个QTL簇,位于染色体1A,1B,1D,2B,3B,4A,4B,5D,6A,6B,7A和7B上。其中,有5个QTL簇涉及到218个QTLs(36.9%=218/590×100%)。并发现了N、P、K协同吸收利用的QTLs。(2)小麦不同N处理下苗期营养相关性状和产量、籽粒相关性状的QTL分析。本试验中,设置苗期和成熟期2个试验:在水培条件下,设置6个N水平(简称为HC-2);在盆栽条件下,设置3个N水平。水培试验:共检测到203个QTL位点(243个性状-处理的QTL),位于小麦的20条染色体上,除3D外。在不同处理条件下,单一QTL可解释表型变异的3.2-56.9%,LOD值最大值为12.3。其中,有6个QTL位点是相对出现频率比较高的位点(relative high frequency QTLs,RHF-QTLs),这些位点均可在3个以上的处理中检测到,包括QRdw-1A.1,QSdw-1D,QTdw-7A,QTpc-1A,QTkc-1A.1,QSnue-1A.2,平均贡献率在9.6-25.7%。盆栽试验:共检测到92个QTL位点(107个性状-处理的QTL),位于小麦的18条染色体上,除3D,5D,7D外。在不同处理条件下,单一QTL可解释表型变异的3.5-50.0%,LOD值最大值为10.6。其中,有11个QTL位点是相对出现频率比较高的位点(RHF-QTLs),这些位点均可在2个以上的处理中检测到,包括QSn-1B,QSl-2D,QTgw-3A,QGh-3A.2,QGh-6A.1,QGh-6A.3,QFfd-1D,QGv-1D,QGv-3A,QGa-1D,QGa-3A,平均贡献率在7.1-24.0%。苗期、籽粒性状的共定位:QTL簇包括20个QTL位点(包括C2–3,6–9,12,15–17,20–22,25,27,31,35,36,38和43)。其中,有3个QTL簇的位点是比较重要的,包括C6,C17,C36,主要涉及TGW和籽粒大小性状。我们推测该位点存在基因之间的相互作用,增加N、P、K含量或者利用效率可以提高籽粒相关性状,如千粒重、粒宽、粒高等。(3)在水培试验HC-1和HC-2中,我们发现了一种N、P、K3种元素协同吸收利用的现象。我们定义,当某一个位点包含根、地上部、总植株的N、P、K其中的2种或3种元素的含量的QTL(QRnc, QSnc,QTnc,QRpc,QSpc,QTpc,QRkc,QSkc和QTkc)时,该位点为协同吸收(cooperative uptake)的位点。同样地,当某一个位点包含根、地上部、总植株的N、P、K其中的2种或3种元素的利用效率的QTL(QRnue,QSnue,QTnue,QRpue,QSpue,QTpue,QRkue,QSkue和QTkue)时,该位点为协同利用(cooperative utilization)的位点。两次水培试验中,HC-1有33个CUU-QTLs位点(L1-33),HC-1有20个CUU-QTLs位点(L34-53)。这53个CUU-QTLs位点中,仅仅涉及协同吸收(cooperative uptake)的位点有13个(包括L3,L5,L8,L11,L16,L21,L23,L26,L38,L42,L44,L45和L51),仅仅涉及协同利用(cooperative utilization)的位点有21个(包括L10,L12,L13,L18,L28,L31,L32,L33,L35,L36,L37,L39,L40,L41,L43,L47,L48,L49,L50,L52和L53),涉及协同吸收利用的位点有19个(包括L1,L2,L4,L6,L7,L9,L14,L15,L17,L19,L20,L22,L24,L25,L27,L29,L30,L34和L46)。(4)小麦N代谢相关基因TaGS1a的单倍型分析及其与小麦苗期、籽粒性状的QTL分析。本试验中,对60份小麦品种(系)的TaGS1a的gDNA序列进行测序、比对,进而将多态性位点转化为功能标记并进行功能鉴定。结果显示,通过PCR扩增,获得约3.0kb的PCR产物,经转化大肠杆菌、质粒鉴定后进行测序。为分析测序结果的可靠性,比对TaGS1a mRNA多处与其他2个GS1基因(GS1b和GS1c)同源性高的非等位基因序列特征性SNP(single nucleotide polymorphism)和Indels(small insertion or deletion)位点信息,结果表明上述序列为TaGS1a的gDNA全长序列。对TaGS1a基因的结构分析表明,编码区自起始密码子(ATG)至终止密码子(TGA)长3415bp,由11个外显子组成,外显子中间有10个内含子。其中,有一段162bp的序列是TaGS1a基因特有的。通过对60份小麦材料的TaGS1a基因的gDNA序列进行比对分析,共发现11个位点变异,其中包括9个SNPs和2个InDels。对上述变异位点进行分析,发现TaGS1a基因共有2种单倍型,命名为Hap1和Hap2。根据1777bp处的SNP多态性转换为一种分子标记:酶切扩增多态性序列(Cleaved Amplified Polymorphic Sequences,CAPS),命名为TaGS1a-CAPS。利用中国春缺体-四体系对TaGS1a-CAPS标记进行定位,结果显示,该标记位于6D染色体上。通过连锁分析,该标记与SSR标记barc1112b连锁,遗传距离为2.5cM,由于找到的与TaGS1a-CAPS连锁的标记比较少,所以仅仅构建了一条包含3个标记的6D染色体图谱。更多还原

【Abstract】 Wheat is one major crop and plays an important role in grain production, with the large growing area. Nitrogen (N) is often considered to be one of the three most important mineral nutrient elements limiting wheat growth. The nutrient-related traits of N metabolism and yield-related traits are complicated quantitative traits. To improve nitrogen use efficicency (NUE), an elaborate understanding of the genetic basis of traits that manifest at different stages of plant development under varying nutrient conditions is required. In wheat (Triticum aestivum L.), genotypic differences in the NUE of N have been well documented, suggesting that it is possible to improve NUE through a genetic approach. Quantitative trait locus (QTL) analysis provides an effective approach to dissect complicated traits into component loci and study their relative effects on a specific trait.In order to improve wheat breeding level and study the genetic mechanism of nitrogen use efficicency, quantitative trait locus (QTL) analysis was conducted in common wheat using a set of 131 recombinant inbred lines (RIL) derived from‘Chuan 35050’בShannong 483’. The objective of this study was to detect QTLs at the seedling stage grown in a variety of N (P, K) concentrations in hydroponic culture trial, and at maturity under moderate, high and low N concentrations in a pot trial. Furthermore, we sought to isolate and characterize the full-length gDNA sequence of TaGS1a and identify polymorphisms among 60 wheat varieties. We also explored a functional marker based on diversities of the gDNA sequence and investigated the role of the TaGS1a in the control of NUE at the seedling stage under hydroponic culture trial and the grain traits under field trial.The mian results are as follows.(1) Nutrient use efficiency, comprising of nutrient uptake and utilization efficiency, is regarded as one of the most important factors for wheat yield. In the present study, 6 morphological, 9 nutrient content, and 9 nutrient utilization efficiency traits were investigated at the seedling stage using a set of recombinant inbred lines (RILs), under hydroponic culture of 12 treatments including single nutrient levels and two- and three-nutrient combinations treatments of N, P and K. A total of 373 quantitative trait loci (QTLs) on 18 chromosomes for the 24 traits (590 QTLs for trait-treatments) were detected. Most QTLs were located in new marker regions. Twenty-two important QTL clusters were mapped on 12 chromosomes, 1A, 1B, 1D, 2B, 3B, 4A, 4B, 5D, 6A, 6B, 7A and 7B. Of these, 5 clusters involved 214 QTLs (36.8%) for trait-treatments, indicating that these 5 loci were important for NuUE of N, P and K. We found evidence for cooperative uptake and utilization (CUU) of N, P and K in the early growth period at both phenotype and QTL level. The correlation coefficients (r) between nutrient content and nutrient utilization efficiency traits for N, P and K were almost all significantly positive correlations. A total of 33 cooperative CUU loci (L1-L33) were found, which included 277 out of the 417 QTLs (66.4%) for trait-treatments, indicating that the CUU-QTLs were common for N, P and K. The CUU-QTLs in L4, L6, L7, L15 and L17 were relatively stable. The CUU-QTLs may explain the CUU phenotype at the QTL level.(2) Twenty-one seedling traits under hydroponic culture trial of six N treatments, and 16 yield traits and grain size traits in a pot trial of three N concentrations were investigated. A total of 203 QTLs for seedling traits and 92 QTLs for yield and grain size traits were detected. Forty-seven QTL clusters were mapped, which involved 291 out of 350 QTLs (83.1%) for treatment-traits. We found cooperative uptake and utilization (CUU) of N, P and K under hydroponic culture trial at both the phenotype and QTL level. A total of 20 CUU loci were found, which included 112 out of 243 QTLs (46.1%) for treatment-traits under hydroponic culture trial. Surprisingly, 20 QTL clusters (42.6%) were referred simultaneously to seedling traits and yield or grain size traits, which should be the constitutive expression loci at the whole stage of plant growth and development. Consequently, most QTLs in this study were located in new marker regions. The founding of constitutive expression loci provided genetic evidence for that seedling traits influenced yield and grain size improvement at maturity.(3) We defined a cooperative uptake locus when QTLs were detected for more than 2 elements of the N, P and K contents in roots, shoots or total plants (QRnc, QSnc, QTnc, QRpc, QSpc, QTpc, QRkc, QSkc and QTkc). Analogously, a cooperative utilization locus was defined when QTLs were detected for more than 2 elements of the N, P and K utilization efficiencies in roots, shoots or total plants (QRnue, QSnue, QTnue, QRpue, QSpue, QTpue, QRkue, QSkue and QTkue). In this study, a total of 53 CUU loci (L1-L53) were found. Of these, 13 loci (L3, L5, L8, L11, L16, L21, L23, L26, L38, L42, L44, L45 and L51) were related to cooperative uptake only, 21 loci (L10, L12, L13, L18, L28, L31, L32, L33, L35, L36, L37, L39, L40, L41, L43, L47, L48, L49, L50, L52 and L53) were related to cooperative utilization only, and 19 loci were related to cooperative uptake and utilization simultaneously (L1, L2, L4, L6, L7, L9, L14, L15, L17, L19, L20, L22, L24, L25, L27, L29, L30, L34 and L46).(4) Glutamine synthetase (GS) is a key enzyme in the formation of the amino acid glutamine during N assimilation. The characterization of GS genes and the development of functional markers are very important for marker-assisted selection (MAS) in wheat breeding. In the present study, the full-length genomic DNA (gDNA) sequence of TaGS1a was obtained from 60 wheat varieties. TaGS1a comprises 3,415 bp and has eleven exons and ten introns. Nine single nucleotide polymorphisms (SNPs) and two insertions and deletions of DNA segments (InDels) were detected in introns, resulting two haplotypes: Hap 1 and Hap 2. A cleaved amplified polymorphic sequence (CAPS) marker was developed to distinguish the two haplotypes. The CAPS marker was located on chromosome 6D using Chinese Spring nullisomic–tetrasomic lines, and mapped at 2.5 cM from the marker barc1121b in a RIL population. The completely corresponding results between quantitative trait locus (QTL) analysis and association analysis suggested that the TaGS1a gene had functions for the ratio of root and shoot for dry weight and N, P and K contents during seedling stage, and for grain size traits during maturity stage. Four QTLs for seedling traits and seven QTLs for grain traits were detected.更多还原