【作者】 陈亮；

【导师】 胡银岗；

【作者基本信息】 西北农林科技大学 ， 作物遗传育种， 2014， 博士

【摘要】 株高是影响小麦产量的主要因子之一，矮秆基因的发现和利用大幅提升了世界小麦产量，并引发了第一次“绿色革命”，为解决世界粮食问题做出了巨大贡献。然而，目前生产上应用的小麦矮秆基因比较单一，主要为赤霉素不敏感型矮秆基因Rht1和Rht2，且其潜力已发挥殆尽，不利于产量和品质的持续提升；同时，研究也发现Rht1、Rht2会缩短胚芽鞘长度，影响苗期活力，尤其在干旱情况下会影响出苗和幼苗群体的建成；而赤霉素敏感型矮秆基因（Rht4、Rht5、Rht12、Rht13等）在降低株高的同时，对胚芽鞘长度等性状没有负面影响，重新认识这些矮秆基因，促进其合理利用，对进一步提高小麦生产潜力具有重要的理论和实践意义。赤霉素敏感型矮秆基因Rht12可显著降低株高（约45％），目前对其遗传效应、致矮机理等还不清楚，应用价值尚不明确。因此，本研究对Rht12进行较为全面的分析，为其合理利用奠定基础。研究以小麦赤霉素敏感型矮秆基因Rht12的供体亲本Karcagi分别与中国春性小麦品种宁春45（含Vrn-B1）和冬性品种晋麦47（含Ppd-D）进行杂交，通过分子标记辅助选择，以目的基因纯合的株系构建F2:3及F3:4遗传群体，调查各基因型的发育进程、株高、穗粒数、千粒重等相关农艺性状，以明确Rht12对各性状的效应及Rht12与Vrn-B1和Ppd-D的互作。同时，利用外源赤霉素（GA3）对Karcagi×宁春45的群体进行处理，分析了Rht12对外源GA3的反应，以揭示Rht12和赤霉素合成或信号转导间的可能关系，分析其致矮机理。另外，为了发掘更多的矮秆材料或矮秆基因，研究还构建了一个小麦EMS诱变群体（TILLING群体），并对其突变表型和突变频率进行了分析，初步鉴选了一批综合性状较好的矮秆突变体，为矮秆基因的深入研究和新矮秆材料的创制奠定了基础。本研究的主要结果如下：1.矮秆基因Rht12的遗传效应及其与Vrn-B1的互作通过两年、三个播期的试验，对Karcagi×宁春45的F2:3及F3:4各基因型纯合株系农艺性状的分析结果表明，Rht12可显著降低株高，矮秆系的株高比高秆系平均降低了47.6cm (38.3％)，且这种致矮效应在苗期就表现出来；Rht12对茎节个数没有影响，其通过缩短每一茎节的长度来降低最终株高；其中，穗下茎的缩短幅度最大，较高秆系平均缩短了22cm (45％)；Rht12在缩短茎节长度的同时，不影响茎节直径，但增加了茎节壁厚，有利于提高其抗倒伏能力。在产量组成性状方面，Rht12可显著增加有效分蘖数（2个）；增加可育小花个数（10％），提高小花育性（13％），并最终增加了穗粒数，矮秆系的穗粒数比高秆系平均多4.1个（9.7％）；然而，Rht12显著减小了种子体积，降低了千粒重（20％）；最终，矮秆系的单株产量与高秆系无显著差异，但由于生物量较小，矮秆系的收获系数显著高于高秆系。同时，研究还发现Rht12可显著推迟小麦的幼穗发育进程，尤其是推迟了二棱期的出现，延长了营养生长的时间，其中，矮秆系（RRVV、RRvv）比高秆系（rrvv）晚到达二棱期约16天，且显性Vrn-B1不能打破Rht12的这一效应，最终，矮秆系比高秆系晚开花5～6天，导致其灌浆期较短，不利于充分灌浆。室内试验分析结果表明，Rht12对小麦胚芽鞘长度没有不利影响；苗期根系分析结果发现，Rht12尽管减少了初生根个数（2条）和初生根总长（6cm，9％），但显著增加了最大初生根长度（3cm，15％），有利于矮秆系在苗期吸取深层土壤中的水分。对Rht12与春化基因Vrn-B1的互作分析表明，春化基因Vrn-B1在高秆系中，可促进植株提前进入二棱期，表现出春性特性；而在矮秆系中，Vrn-B1促进幼穗发育的特性被完全掩盖，表明Rht12对春化基因Vrn-B1有上位性效应。2.矮秆基因Rht12与光周期基因Ppd-D1的互作效应为了改良Rht12的晚熟特性，试验引入了显性光周期基因Ppd-D1。对Karcagi×晋麦47的F2:3各纯合基因型株系的分析结果表明，Ppd-D1对营养生长时间（播种至二棱期）没有影响，矮秆系（无论含显性Ppd-D1还是隐性ppd-D1）仍需要较长的时间才能进入生殖生长阶段，其到达二棱期所经历的时间比高秆系多8天；然而，含显性Ppd-D1的矮秆系在生殖生长后期发育加快，仅需56.0天便到达开花期，而含隐性ppd-D1的矮秆系需60.5天才能到达开花期；含显性Ppd-D1的矮秆系要比含隐性ppd-D1的矮秆系早开花5天，仅比高秆系晚开花1～2天，基本上克服了Rht12延迟开花期的不利效应。在高秆系中，Ppd-D1也同样表现出促进早开花的特性，但其对植株到达二棱期所需的时长也没有影响。结果表明，尽管Ppd-D1不能打破Rht12延迟二棱期出现的不利效应，但其显著缩短了二棱期至开花期所用的时间，并促进植株早开花，因此，利用Ppd-D1来改良Rht12矮秆系的开花期是可行的。另外，在Karcagi×晋麦47群体中，Rht12同样显著的降低了株高（约40cm；38％），其中，含显性Ppd-D1的矮秆系（RRPP）最矮，可能是因为Ppd-D1与矮秆基因Rht8紧密连锁的原因；Rht12增加了有效分蘖个数，并显著提高了主茎穗粒数，矮秆系的主茎穗粒数比高秆系平均多2.5粒（5.8％），其中，RRPP、rrPP的穗粒数分别小于RRpp和rrpp,可能是由于显性Ppd-D1缩短了生殖生长后期所经历的时间，使小花发育的时间有所减少，影响了可育小花的分化；Rht12降低了千粒重，矮秆系的千粒重比高秆系平均小6.7g（14.9％），其中，显性Ppd-D1不同程度的增加了千粒重，可能是因为其促进植株早开花，有利于充分灌浆；最终，矮秆系的单株产量较高秆系稍低，但其收获系数显著大于高秆系；这些结果与Karcagi×宁春45群体的一致。因此，在利用Rht12提高小麦抗倒伏性、增加穗粒数和有效分蘖育种时，还可利用显性光周期基因Ppd-D1改良其千粒重较小的不利效应。3.矮秆基因Rht12对外源赤霉素（GA3）的响应通过两年、三个播期的赤霉素喷施试验，分析了Rht12对外源GA3的响应，结果表明，外源GA3可在一定程度上抵消Rht12的效应，使矮秆系恢复高秆表型。GA3打破了Rht12对Vrn-B1的掩盖作用，促进矮秆系较早进入二棱期，使含显性Vrn-B1的矮秆材料（RRVV）表现出春性特性；GA3处理的矮秆系较未处理的早开花约7天，且仅比未处理的高秆系晚开花1天；而GA3处理的高秆系只比未处理的早开花1.5天，反应程度显著小于矮秆系。另外，GA3处理后，矮秆系表现出类似于高秆系的生长能力，苗期株高和干物质量的增长速度都显著高于未处理的矮秆系；同时，GA3处理恢复了矮秆系失去的获得较长茎节的能力，增加了各茎节长度，其中，穗下茎的长度比未处理的平均增长19.3cm（81.5％）；GA3处理在增加茎节长度的同时，降低了茎节直径和壁厚，尤其是基部第一、第二茎节的直径和壁厚，显著降低了抗倒伏能力；GA3处理的矮秆系的株高比未处理的平均增加了40.7cm（53％），仅比高秆系矮5～10cm；而高秆系受GA3的影响要远小于矮秆系，株高仅增加了6.1cm（4.8％）。在产量组成性状方面，GA3处理弥补了Rht12降低千粒重的不利效应，增大了种子体积；不过，GA3处理减少了矮秆系的穗粒数和有效分蘖个数，并降低了单株产量和收获系数；而高秆系的产量组成性状并未受GA3显著影响。同时，油菜素内酯敏感性测定表明，Rht12不是BR缺陷型突变。因此，随着外源GA3对Rht12矮秆系各“矮化性状”的恢复，使其表现出类似于高秆系的表型，表明矮秆系的矮化特性很可能是其缺乏充足的内源赤霉素的结果，Rht12很可能是赤霉素合成缺陷型突变。4.小麦新矮秆突变体的筛选为发掘更多的矮秆突变体，构建了一个小麦品种晋麦47的EMS诱变群体（TILLING群体），对其表型变异和部分候选基因的突变体进行了调查与筛选。对M2突变群体的表型变异进行调查分析，发现了矮化、黄化、晚熟等丰富的变异类型，估算M2总的表型变异频率为4.2％；其中，矮秆半矮秆突变最多，占所有变异的32％。通过随机引物PCR扩增分析，估算M2在DNA水平上的突变频率约为1/35kb，即每35kb基因组序列中，就可能存在一个点突变；这些结果表明，该诱变群体不仅存在丰富的表型变异，其基因组水平上的变异更加丰富，为突变材料的选育及候选基因突变体的挖掘提供了有效的资源。进一步利用TILLING（Targeting Induced Local Lessions In Genomes，定向诱导基因组局部突变）技术对光周期基因Ppd-D1及矮秆基因Rht-D1等基因的突变位点进行了检测，发现了Ppd-D1的15个点突变，Rht-D1的3个点突变，这些突变主要为C到T、G到A的转换突变，个别突变引起了氨基酸的改变，可能会对蛋白功能产生破坏性影响；对Ppd-D1和Rht-D1的突变体进行分析，发现这些突变可能并不是引起表型变化的直接原因。同时，为了鉴选农艺性状较好的矮秆突变体，用野生型材料（晋麦47）与部分矮秆突变体进行回交，并从回交后代中选择择株高适宜且能稳定遗传、开花较早、农艺性状较好的矮秆材料，为进一步深入研究奠定了良好的基础；同时，部分矮化相关基因（如赤霉素合成或信号转导相关基因）的突变体TILLING筛选也正在进行。总之，以上研究结果表明，赤霉素敏感型矮秆基因Rht12可显著降低株高、增加有效分蘖、穗粒数和收获系数，且对苗期活力没有负面影响；但有较强的延迟幼穗发育的的不利效应，育种应用时可通过配合使用Ppd-D1等促进生长发育的基因，以克服其延迟开花的不利效应。Rht12对外源GA3反应敏感，且GA3可以恢复矮秆系的高秆表型，推测Rht12很可能作用于内源赤霉素合成相关途径，而非信号转导途径。另外，在构建的TILLING突变群体中发现了丰富的矮秆材料；相关基因的突变也在进一步筛选中，这些研究将促进小麦新矮秆基因的发现与利用。更多还原

【Abstract】 Plant height is an important factor in wheat production, the introduction of dwarf andsemi-dwarfing genes into wheat was significantly increased wheat yield of the world, andbring about the ‘Green Revolution’ which play a key role in solving the food problem.However, there is only a small number of dwarf genes available for wheat breeding to date,the genetic diversity are very narrow and its not good for the sustainable improvement ofyield and quality. Currently, the most widely used wheat dwarf genes are Rht1and Rht2(GAinsensitive), but they also reduced coleoptile length and seedling vigour which may reducecrop water-use efficiency, and performance in some unfavorable environments. So,opportunities exist for replacing Rht1and Rht2in wheat with alternative dwarfing genes, suchas the GA-responsive dwarfing genes (Rht4, Rht5, Rht12, Rht13) which can reduce plantheight without compromising early plant growth. So, a better understanding of theseGA-responsive dwarfing genes is very important for improving the yield potential of wheat.Rht12, a dominant GA-responsive dwarfing gene, can significantly decreased plant height(45％), but its genetic effect on wheat production, and the possible role of Rht12in GAbiosynthesis or signaling are still not clear, and its full potential remains uncertain. So, theobjectives of this work were to evaluate the effect of Rht12comprehensively and to promotethe proper utilization of dwarfing gene Rht12in wheat breeding.Two cross was made using Ningchun45(with Vrn-B1)、Jinmai47(with Ppd-D1) asfemales and Karcagi (with Rht12) as pollen donor respectively, then the homozygousgenotypes was chosen to develop the F2:3and F3:4populations by molecular marker-assistedselection. The phenological development, plant height, grain number per spike,1000-kernalweight and other agronomic traits were evaluated for analyzing the effect of Rht12on wheatand the interaction effects between Rht12and Vrn-B1. Additionally, the Karcagi×Ningchun45population was treated with exogenous GA3for examining the response of Rht12toexogenous GA3on plant development, agronomic traits and yield components and toinvestigate the possible role of Rht12in GA biosynthesis or signaling. Moreover, a wheat EMS induced mutant population (TILLING mutant population) was developed to find moredwarf materials or more dwarf genes. The mutant frequency of the TILLING population wascalculated and the visible mutant phenotype was also analyzed and some dwarf mutants withgood agronomic traits was chosen for further study. The main results of this study were asfollows:1. Genetic effects of Rht12and the interactive effects of Rht12and Vrn-B1Analysis was conducted in the F2:3and F3:4lines derived from the cross betweenNingchun45and Karcagi in three experiments, and found that plant was significantly reducedby Rht12. Plant height of the dwarf lines was reduced by47.6cm (38.3％) compared with thetall lines, and this dwarf effects was expressed from seedling stage. Rht12reduce plant heightthrough reducing the length of internodes without affect the number of internodes, and thepeduncle length was reduced most significantly by22cm (45%) in dwarf lines compared withthat of the tall lines. However, the diameter of the internodes was not affected by Rht12whilethe wall sickness of the internodes was significantly increased and this character was benefitfor improving lodging resistance. Additionally, Rht12can significantly increase the number ofeffective tillers, increase floret fertility (13%) and produced more fertile florets (10%), andfinally increase grain number per spike (4.1;9.7％）compared with the tall genotypes.However, Rht12significantly decreased seed size and1000-kernel weight (20％). Finally,there is no significant difference between the dwarf and tall lines on yield per plant, but due tothe reduced plant biomass, the dwarf lines achieved higher harvest index than the tall lines. Itwas also found that the spike development, especially the double ridge formation phase, wassignificantly delayed by Rht12, and the vegetative growth stage was elongated in the dwarflines. However, the dwarf lines (RRVV、RRvv) was delayed by16days to reach the doubleridge stage and the dominant Vrn-B1could not compensate this negative effect. The anthesisdate of the dwarf lines was about5～6days later than that of the tall lines which may havenegative effects on grain filling.Additionally, the laboratory test confirmed that Rht12have no negative effects oncoleoptile length. The total number of seminal roots was significantly decreased in the dwarflines and the total root length in the dwarf lines was reduced by6cm (9%) compared to thatof the tall lines. However, the maximum root length of the dwarf group was3cm (15%)longer than that of the tall group, which may be beneficial for water acquisition in deeper soilin dry environments.Moreover, it was found that the tall spring lines (with Vrn-B1) developed faster than thetall winter lines (vrn-B1) before terminal spikelet stage, suggesting that the dominant Vrn-B1 needed less time to undergo vernalization in tall lines. But, in the dwarf lines, the effect of thedominant vernalization gene Vrn-B1was masked, indicating that the dwarfing Rht12allelemight have an epistatic effect on Vrn-B1.2. The interactive effects of Rht12and Ppd-D1The photoperiod insensitivity gene Ppd-D1was introduced to improve the late flowingcharacter of Rht12. Analysis was conducted in the F2:3lines derived from the cross betweenJinmai47and Karcagi, and found that Ppd-D1has no effects on the duration of vegetativegrowth phase (from sowing to double ridge stage), the dwarf lines either with Ppd-D1orppd-D1are need more time (8days) to reach the reproductive growth stage compared with thetall lines. However, the dwarf lines with Ppd-D1development faster during the latereproductive phase, and need56.0days to reach to anthesis date while the dwarf lines withppd-D1need60.5days to reach to anthesis date. Finally, the dwarf lines with Ppd-D1flowingearlier5days than the dwarf lines with ppd-D1and only1～2days later than the tall lines.Ppd-D1was basically overcome the negative of Rht12on flowing time. In the tall lines,Ppd-D1showed the similar effect on spike development as in the dwarf lines. However,though Ppd-D1can not break the negative effect of Rht12on the initiation of double ridgestage, Ppd-D1has significantly reduced the duration time from double ridge stage to anthesis,and promotes earlier flowing. So, it is favorable to use Ppd-D1to improve the flowing time ofthe dwarf lines.Additionally, in the F2:3lines of Jinmai47×Karcagi population, Rht12has the same effecton plant height; plant height was reduced about40cm (38％) by Rht12. Because of Ppd-D1was linked closely to another dwarf gene Rht8, the dwarf lines with ppd-D1showed theshortest plant height in all the four genotypes. Rht12increased the number of effective tillersand grain number per spike (5.8％). Genotypes with Ppd-D1have less grain numbers thangenotypes with ppd-D1which may be caused by the shorter late reproductive phase ofPpd-D1lines that have negative effect on floret development. Moreover, possible due to theearlier flowing of Ppd-D1lines which benefit for grain filling, the dwarf lines with Ppd-D1achiebed bigger1000-grain weight than the dwarf lines with ppd-D1. Finally, the yield of thedwarf lines was slightly shorter than tall lines, but its harvest index was significantly than thetall lines. These results were similar with that in the ningchun45×Karcagi population. So,Ppd-D1can be used to improve grain size in the Rht12dwarf lines.3. The response of Rht12to exogenous GA3The responses of Rht12to exogenous GA3were investigated using F2:3and F3:4linesderived from the cross between Ningchun45and Karcagi in three experiments, and found that exogenous GA3can break the masking effect of Rht12on Vrn-B1and also restore othercharacters of Rht12to normal. GA3shortened the time to double ridge in the dwarf lines andmake the dwarf lines with dominant Vrn-B1displayed a spring-like phenotype. The dwarflines with GA3application flowered7days earlier than the dwarf lines without GA3application and only1day later than the tall lines. However, the GA3-treated tall linesflowered only1.5days earlier than the ones without GA3treatment, its response to GA3wasmuch weaker compared with the dwarf lines. Moreover, after exogenous GA3application, theRht12dwarf plants showed a faster stem elongation and dry weight accumulation rate, similarto that of tall plants, compared with the untreated Rht12dwarf plants. GA3applicationincreased the lengths of each internodes especially the peduncle length (19.3cm;81.5％).However, the diameter and wall thickness of those internodes were significantly reduced inthe GA3-treated dwarf lines, especially the first and the second, which significantly decreasedthe lodging resistance. Finally, compared with the untreated Rht12dwarf plants, plant heightwas increased by40.7cm (53%) in the GA3-treated ones and was only5～10cm shorter thanthe tall lines. For the tall lines, plant height was only increased6.1cm (4.8％) under GA3application, suggesting that the tall plants were not as sensitive as the Rht12dwarf plants toexogenous GA3. Additionally, exogenous GA3increased plant biomass but it reduced thenumber of fertile florets, which resulted in a lower grain number per spike, lower plant yieldand lower harvest index. Exogenous GA3increasing seed size in the Rht12dwarf lines and the1000-grain weight of the GA3-treated Rht12dwarf plants was significantly increasedcompared with untreated dwarf plants, indicating that GA3could partially compensate for thesubstantial negative effect of Rht12on yield components. Thus, it is clear that exogenous GA3can restore the ‘dwarfing characters’ of Rht12to normal. It suggested that Rht12mutants maybe deficient in GA biosynthesis rather than in GA signal transduction.4. Screening of new wheat dwarf mutantsTo develop more dwarf mutants, a wheat EMS induced mutant population (TILLINGmutant population) was developed using wheat cultivar Jinmai47, and the variation ofphenotypes and the mutation of candidate genes were analyzed. Firstly, altered morphologicaltraits were record, dwarf, albinism, late heading and other mutant phenotypes were found inM2individuals, and about4.2%of the surveyed lines displayed noticeable phenotypes(distinctive from Jinmai47). Moreover, variants in plant height comprised32%of all visiblephenotypes noted and mainly yielded different kinds of dwarfism. Additionally, the mutationfrequency at DNA level was1/35kb, which means that one mutation per34kb. It is suggestedthat the TILLING population not only has rich phenotypic mutations, but also has high mutation density in the genome which would be a valuable resource for screening desiredwheat mutants needed in forward and reverse genetic researches. Then, the target fragment ofphotoperiod gene Ppd-D1and the dwarf gene Rht-D1was screened for SNPs by TILLING(Targeting Induced Local Lessions In Genomes) method. The detection method was alsomodified by using agarose gel, non-denaturing polyacrylamide gel and HRM technology.Finally,15and3mutations were obtained in Ppd-D1and Rht-D1, and the average mutationfrequency is1/48kb. Sequencing confirmed that all of the mutations were C to T or G to Atransitions, and some mutations led to amino acid changes or have negative effects on proteinfunction. Mutants of Ppd-D1and Rht-D1were characterized and not found noticeable alteredphenotypes in these mutants. Furthermore, to select dwarf mutants with good agronomic traits,backcross was conducted using the mutants and the wild type. Now, some dwarf mutant withstable heredity, earlier flowing time and good agronomic traits were chosen for furtheranalysis and the screening of SNPs on dwarf related genes (genes involved in gibberellinsbiosynthesis or signaling) was ongoing.Conclusively, Rht12significantly reduced plant height, increased the number of effectivetillers, grain number and also the harvest index, and has no negative effects on seedling vigour;Rht12significantly delayed the spike development phase and it should be introduce otherdevelopment-promoting genes, like Ppd-D1, to promote spike development, and compensatefor the delay in spike development in Rht12lines. Additionally, Rht12is sensitive toexogenous GA, and exogenous GA3can restore the characters of Rht12to normal, suggestingthat Rht12mutants may be deficient in GA biosynthesis rather than in GA signal transduction.Moreover, many dwarf mutants and also some mutants of the candidate genes were found inthe TILLING population, and this TILLING population will be a useful resource for bothmutation breeding and functional genomics.更多还原