K-Domain Splicing Factor OsMADS1 Regulates Open Hull Male Sterility in Rice

We identified the rice floral organ development mutant, termed as open hull and male sterile 1(ohms1), from the progeny of the indica restorer line Zhonghui 8015 treated with 60 Co γ-ray irradiation. The ohms1 mutant exhibited an open hull and lemma- and palea-like structure conversion between the anthers and stigma, which resulted in the ohms1 mutant spikelet showing ‘tridentate lemma'. The ohms1 mutant was entirely sterile but had 60%–70% fertile pollen. Genetic analysis and gene mapping showed that ohms1 was controlled by a single recessive gene, and the mutant gene was fine-mapped to a 42-kb interval on the short arm of chromosome 3 between markers KY2 and KY29. Sequence analysis of the four open reading frames in this region revealed that the mutant carried a single nucleotide transformation(A to G) at the last base of the fifth intron, which was likely corresponded to ohms1 phynotype, in an MIKC type MADS-box gene OsMADS1(LOC_Os03g11614). Enzyme digestion and c DNA sequencing further indicated that the variable splicing was responsible for the deletion of the sixth exon in ohms1, but no structural changes in the MADS domain or amino acid frame shifts appeared. Additionally, real-time fluorescent quantitative PCR analysis showed that the OsMADS1 expression level decreased significantly in the ohms1 mutant. The expression levels of rice flowering factors and floral glume development-related genes also changed significantly. These results demonstrate that OsMADS1 may play an important role in rice floral organ development, particularly in floral glume development and floret primordium differentiation.     

SRG1, Encoding a Kinesin-4 Protein,Is an Important Factor for Determining Grain Shape in Rice

Grain shape is one of the important agronomic traits, which is closely related to the yield of rice. A new rice mutant, named small and round grain (srg1), was isolated from an indica cultivar Zhenong 34 by ethyl methane sulfonate (EMS) mutagenesis. The microscopic analysis showed that the cell length of spikelet in srg1 was decreased compared with that in wild type (WT), which caused the grain length short. Meanwhile, the grains of srg1 were wider than those of WT because of the increased cell layers in spikelet in the lateral direction. Therefore, the inhibition of cell expansion and increased cell proliferation collectively led to the small and round grain. By map-based cloning, the gene SRG1 was located on the short arm of chromosome 9, which encodes a kinesin-4 protein, represented by the gene LOC_Os09g02650. A single nucleotide polymorphism, occurred in the 16th exon of SRG1, led to premature translation stop in mutant. The cell cycle-related genes were up-regulated in srg1, which conferred that SRG1 controlled grain width through the cell proliferation. Since the role of SRG1 in regulating grain shape was not clarified well before, it is valuable to explore the mechanism of grain development. This study could, hence, provide a morphogenesis and molecular basis for elucidating the function of SRG1, as well as a new germplasm resource for the further study of grain development.     

Identification of a Gravitropism-Deficient Mutant in Rice

A gravitropism-deficient mutant M96 was isolated from a mutant bank, generated by ethyl methane sulfonate(EMS) mutagenesis of indica rice accession ZJ100. The mutant was characterized as prostrate growth at the beginning of germination, and the prostrate growth phenotype ran through the whole life duration. Tiller angle and tiller number of M96 increased significantly in comparison with the wild type. Tissue section observation analysis indicated that asymmetric stem growth around the second node occurred in M96. Genetic analysis and gene mapping showed that M96 was controlled by a single recessive nuclear gene, tentatively termed as gravitropism-deficient M96(gd M96), which was mapped to a region of 506 kb flanked by markers RM5960 and In Del8 on the long arm of chromosome 11. Sequencing analysis of the open reading frames in this region revealed a nucleotide substitution from G to T in the third exon of LOC_Os11g29840. Additionally, real-time fluorescence quantitative PCR analysis showed that the expression level of LOC_Os11g29840 in the stems was much higher than in the roots and leaves in M96. Furthermore, the expression level was more than four times in M96 stem than in the wild type stem. Our results suggested that the mutant gene was likely a new allele to the reported gene LAZY1. Isolation of this new allele would facilitate the further characterization of LAZY1.     

Characterization and Genetic Analysis of a Novel Mutant mst of Rice Defective in Flower Development

A spontaneous mutant with multiple stigmas (mst) was found in an indica rice line 466. The mst mutant exhibits normal at the vegetative development stage and produces normal inflorescence structures. The difference between the mutant and the wild type was observed when the stamen primordium began to develop. In the mst florets, palea and lemma opened, lodicules were homeotically transformed into palea/lemma-like structures, and stamens were homeotically transformed into carpel-like structures. It looked like multiple stigmas being full of the whole floret. The phenotypic changes of mst were very similar to that of B-like mutant spw1. Compared with other mutants with pistillate morphologies, the severe mst florets showed that the inner three floral organs were completely changed into palea/lemma-like structures. Moreover, the mutant was female sterile. Occasionally, with the changing environment, one or two stamens were fertile. Genetic analysis indicated that the mutant traits were controlled by a single recessive gene.     

水稻粒宽突变体gw4的鉴定与基因定位

[目的]水稻粒形是影响水稻产量和决定稻米外观品质的主要性状之一.筛选和鉴定新的粒形突变材料,可为研究水稻籽粒发育的调控机制奠定基础.[方法]粳稻品种中花11经1％的EMS处理,在诱变群体中获得一份窄粒突变体gw4(grain width on chromosome 4);分析粒形和其他主要农艺性状,在扫描电镜下观察颖壳...     

水稻矮化小穗基因DSP2的鉴定与克隆

[目的]水稻株高和穗型在产量形成中发挥着重要作用.鉴定与克隆水稻株高和穗型发育相关基因,可以丰富水稻株高穗型发育调控的分子机理,为分子设计育种奠定理论基础和提供基因资源.[方法]在粳稻日本晴T-DNA插入群体中筛选到矮化小穗突变体dsp2-D(dwarf and small panicle 2-Dominant),对其...     

水稻温敏转绿突变体osv15的鉴定和遗传分析

【目的】本研究旨在鉴定和克隆水稻温敏转绿新基因,揭示其参与叶绿体发生发育和光合作用的分子机制,为高光效育种提供理论支撑。【方法】利用辐射诱变的方法,从粳稻品种日本晴中筛选获得叶片黄化突变体osv15,并对其表型、农艺性状和遗传方式进行详细分析。构建了突变体与Kasalath的F₂群体,利用多态性分子标记对目的基因进行定位和测序分析。【结果】osv15幼苗期在22℃低温下叶片黄化,叶绿素含量仅为野生型的10%,光化学效率下降,叶绿体结构异常;随着温度的升高,osv15的叶色由黄转绿,30℃时叶绿素含量恢复到野生型的68%,光化学效率和叶绿体发育与野生型相近。在自然环境下,osv15突变体从苗期至成熟期均表现为叶片黄化,且株高、分蘖数和产量等农艺性状与野生型相比差异显著。遗传分析表明osv15突变体的表型由一对隐性核基因控制。将OsV15基因定位到第6染色体多态性标记S4和S5之间84 kb的区间内,定位区间测序发现突变体中编码分子伴侣蛋白的基因Cpn60β1(LOC_Os06g02380)发生单碱基缺失。【结论】osv15是一个新的水稻温敏转绿突变体,Cpn60β1可能为突变基因。     

水稻脆秆突变体bc1-wu3的鉴定与基因克隆

【目的】茎秆机械强度与植株抗倒伏性直接相关。发掘脆秆突变体,克隆其相关基因有助于了解茎秆机械强度遗传机制,为抗倒伏育种提供理论依据。【方法】在经~(60)Co-γ诱变的粳稻品种武育粳3号后代群体中获得一个脆秆突变体,命名为bc1-wu3(brittle culm 1 from Wuyujing3),以突变体bc1-wu3为母本,Kasalath为父本构建相应的F_2分离群体,采用图位克隆的方法定位相应脆秆基因。【结果】与野生型相比,突变体的叶片、叶鞘、茎秆等组织在全生育期始终表现为脆性,易折断;穗长和粒长显著降低,粒宽显著增加。茎秆细胞细胞壁糖成分测定表明,细胞壁中纤维素含量极显著下降,而木糖、葡萄糖及阿拉伯糖含量则显著增加。茎秆切片观察发现突变体茎秆的厚壁细胞层数减少,厚壁细胞细胞壁极显著变薄。遗传分析表明,bc1-wu3脆性性状受1对隐性核基因控制。采用图位克隆的技术将该基因定位于第3染色体标记MK12与MK18之间,物理距离为57 kb,在此定位区段内包含1个已克隆的脆性基因BC1(LOC_Os03g30250)。测序结果表明,突变体bc1-wu3中BC1基因第2外显子内(CDS 659处)有1个碱基的替换(G-T),导致编码氨基酸由半胱氨酸变异为苯丙氨酸。实时荧光定量PCR结果表明,BC1基因在脆秆突变体bc1-wu3茎秆中的表达量显著降低。【结论】据此推断本研究中定位的脆秆基因bc1-wu3为BC1新等位基因。相关结果加深了对BC1基因功能的认识,有助于阐明水稻茎秆强度遗传机制。     

水稻雌性败育基因FA的图位克隆

目的 对水稻胚囊突变体的表型观察、遗传定位和基因克隆,将为研究植物生殖发育奠定理论基础。方法 从粳稻品种宁粳4号突变体库中筛选出一个雌性败育突变体female abortion(fa),对野生型和突变体不同发育时期的胚囊进行细胞学观察并统计胚囊类型。以突变体杂合型为母本,N22为父本构建定位群体,对该表型进行遗传分析,采用图位克隆方法精确定位目的基因。结果 表型分析显示,突变体雌蕊在外观上没有表现出差异,但成熟时植株完全不育。与野生型相比, 该突变体胚囊发育异常,不能形成七细胞八核胚囊结构,而且形成多种类型的异常胚囊。遗传分析表明,该突变性状受一对隐性核基因控制。我们将该基因初定位在第1染色体的分子标记L1和L3之间,通过扩大定位群体,最终将基因定位在分子标记L10和L11之间,物理距离为117 kb。测序分析发现该区间内LOC_{_}Os01g68870外显子上有一个单碱基替换,由胸腺嘧啶(T)替换成胞嘧啶(C),氨基酸由亮氨酸变为脯氨酸,从而导致该表型的出现。qRT-PCR结果显示,相对于OsMADS13、OsAPC6、OsTDL1A基因在突变体fa胚囊中表达量而言,OsDEES1基因在突变体fa胚囊中表达量变化最为显著,FA可能是OsDEES1的上游基因。亚细胞定位结果显示,该蛋白定位于质膜。结论 FA是多孢囊基因MULTIPLE SPOROCYTE 1(MSP1)的新等位基因。本研究进一步明确了该基因对于水稻胚囊发育的重要性,可为探明它所在的调控网络体系提供新的线索。     

水稻条斑和颖花异常突变体st fon的鉴定与遗传分析

 在水稻遗传转化过程中发现一个不含外源基因的条斑和颖花异常的双突变体,暂命名为st fon。该突变体除茎、叶、穗出现白条斑外,花器数目增多,颖花开裂。其极端表型为同一颖花中长出几朵小花或小花中枝梗伸长为花序。利用透射电镜对苗期叶片白色组织细胞超微结构进行观察,发现质体结构异常,不能发育成正常叶绿体所具有的片层和类囊体,叶绿体的发育阻断在质体发育早期。利用扫描电镜和石蜡切片对花器官形态发生过程进行观察,发现花分生组织生长具有无序和无限性。对扭曲的叶片石蜡切片观察,发现维管束鞘细胞过度生长,或一些泡状细胞增大。将突变体与4个叶色正常的材料进行杂交和回交,遗传分析表明突变性状呈细胞质遗传。     

A Point Mutation in an F-Box Domain-Containing Protein Is Responsible for Brown Hull Phenotype in Rice

The accumulation of pigments affects the color of rice hulls while only limited information is known about its underlying mechanisms. In the present study, a rice brown hull 6(bh6) mutant was isolated from an ethane methyl sulfonate(EMS)-induced IR64 mutant bank. Brown pigments started to accumulate in bh6 rice hulls after heading and reached a higher level in mature seeds. Some major agronomic traits including panicle length and 1000-grain weight in bh6 were significantly lower than those in its corresponding wild type IR64, while other agronomic traits such as plant height, growth duration and seed-setting rate were largely similar between the two genotypes. The analysis of pigment content showed that the contents of total flavonoids and anthocyanin in bh6 hulls were significantly higher than those in IR64 hulls. Our results showed that the brown hull phenotype in bh6 was controlled by a single recessive gene which locates on the long arm of chromosome 9. Sequencing analysis detected a single base substitution(G/A) at position 1013 of the candidate gene(LOC_Os09g12150) encoding an F-box domain-containing protein(FBX310). Functional complementation experiment using the wild type allele can rescue the phenotype in bh6. Thus, we named this mutated gene as Os FBX310~(bh6), an allele of OsFBX310 functioning as an inhibitor of brown hull. The isolation of Os FBX310~(bh6) and its wild type allele can provide useful experimental materials and will facilitate the studies on revealing the mechanisms of flavonoid metabolism in monocot plants.     

水稻白条纹叶及白穗突变体wlp6的鉴定与基因定位

目的 叶色突变体是研究水稻光合作用,叶绿素生物合成和遗传发育调控机理的重要材料。发掘水稻叶色突变体,是水稻功能基因组学研究的重要遗传基础。方法 在昌恢121中发现了一份白条纹叶及抽穗期白穗突变体,经过连续多代自交能稳定遗传,暂命名为wlp6(white striped leaf and white panicle 6)。在南昌分早、中和晚3季播种wlp6与野生型种子,考查了中稻与晚稻的部分农艺性状;测定3叶期、分蘖期、抽穗期叶片及颖壳的叶绿素含量;通过电镜观察抽穗期叶肉细胞发育情况。在光照培养箱中进行温光敏感实验;将wlp6与昌恢121及02428正反交,观察F₁植株表型,对F₂分离群体进行卡方测验,分析突变体遗传规律;以wlp6/02428衍生的F₂群体为材料,利用BSA法进行基因定位。结果 wlp6自第1片叶到成熟,叶片均呈白条纹,抽穗期颖壳及枝梗失绿,高温天气穗转绿。突变体株高、有效穗数和每穗粒数在早稻季和中稻季均显著低于野生型,晚稻季wlp6的结实率和千粒重也显著低降低。叶绿素含量测定表明,wlp6叶片叶绿素含量在不同生育期及不同季均显著低于野生型,早稻和晚稻季种植的wlp6颖壳叶绿素含量也比野生型低。电镜观察抽穗期的叶肉细胞发现,wlp6叶绿体数目减少,体积变小,没有分化出明显的片层结构。温光敏感实验表明,突变体对光照强弱钝感,叶色受温度和日照长短影响,随着温度升高和日照时间变长突变体叶绿素含量有上升趋势。遗传分析表明,该性状受隐性核基因控制,利用wlp6/02428得到的616个F₂单株将WLP6定位于第6染色体短臂InDel标记R-7与R-8间,物理距离137 kb,此区间预测了21个候选基因。经候选基因分析及测序发现,其中LOC_Os06g14620编码一个核糖核酸还原酶小亚基,编码区第142和158位碱基由T替换为C,第288位插入了碱基A,碱基的插入导致翻译提前终止,因此推测LOC_Os06g14620是WLP6的候选基因。结论 LOC_Os06g14620是已经克隆的白条纹叶基因St1的候选基因,推测WLP6与St1等位,但突变位点不同,且表型也有差异。     

水稻花器官发育基因DPS2的鉴定和精细定位

【目的】鉴定和克隆花器官发育相关基因,为进一步研究水稻花发育的分子机制奠定基础。【方法】在大田常规种植条件下比较了突变体dps2 (Defective pistil and stamens 2)和野生型春江06的主要农艺性状及花器官形态特征差异;扫描电镜及石蜡切片观察花药结构并用染色法观察花粉和胚囊的育性;利用图位克隆方法进行基因精细定位;qRT-PCR分析了花发育相关基因在野生型和突变体中的表达水平。【结果】dps2突变体抽穗期变长,不能正常扬花,雄蕊和雌蕊皱缩且花药和柱头数目增多;进一步研究发现,dps2突变体花药腔室塌陷,内无可见小孢子,即使部分花药形成腔室,花粉粒也无淀粉积累呈干瘪状。此外,突变体胚囊育性也受到影响;遗传分析表明该突变性状受一对隐性核基因控制,该基因位于第4染色体短臂上91.2 kb的区间内,区间内未见花器官发育相关基因的报道。qRT-PCR检测发现,水稻ABCDE模型中的B类、C类和E类基因的表达在突变体中显著升高。【结论】dps2突变体的雄蕊及雌蕊均发育异常,最终导致完全不育,推测DPS2可能在水稻第3轮雄蕊发育和第4轮雌蕊发育调控中发挥重要作用。     

水稻类病斑突变体wy3的鉴定和基因定位

在粳稻品种武育粳3号栽培群体中获得一个类病斑突变体wy3。该突变体类病斑出现于苗期,分蘖期扩散至整张叶片,属于扩散型类病斑突变体。相比野生型,突变体wy3的株高明显降低,有效分蘖数减少,穗长、每穗粒数、结实率均显著降低。遮光处理表明,突变体wy3类病斑的产生受自然光诱导。台盼蓝染色结果表明,类病斑部位有大量的死亡细胞。突变体wy3的光合色素含量和净光合速率较野生型显著降低,SOD、POD、CAT活性和MDA含量均显著高于野生型。遗传分析表明突变体表型受单隐性核基因控制,采用BSA将该基因初步定位在第2染色体短臂端粒附近。采用F2群体中1099株类病斑单株将基因定位在标记W2-17和W2-18之间28kb的物理距离内。测序结果表明,突变体wy3中的LOC_Os02g02000编码区(CDS)第375位碱基C缺失,导致翻译提前终止,突变体中该候选基因为OsHPL3的一个新等位基因。     

花期高温胁迫对水稻颖花生理特性的影响

以野生型水稻中花11及其耐热型突变体(hst)为材料,于抽穗扬花期高温处理7d(40℃,每天处理6h),探讨开花期水稻颖花生理特性与耐热性之间的关系。研究表明,花期高温导致剑叶叶绿素含量、颖花可溶性蛋白和可溶性糖含量均显著下降,突变体hst的降幅明显小于野生型中花11;同时,高温下颖花中丙二醛(MDA)、过氧化氢(H_2O_2)及游离脯氨酸含量均显著增加,而突变体hst的增幅明显低于野生型中花11。在抗氧化酶活性方面,超氧化物歧化酶(SOD)、过氧化物酶(POD)及过氧化氢酶(CAT)活性对水稻开花期高温胁迫的响应有所不同,其变化与耐热性之间的相关性有待进一步验证。高温胁迫下水稻剑叶中保持较稳定的光合作用,颖花中保持较多的渗透调节物质及较低的MDA和H_2O_2含量,是突变体耐高温的生理基础。     

Pre-anthesis non-structural carbohydrate reserve in the stem enhances the sink strength of inferior spikelets during grain filling of rice

Sink strength plays an important role in grain filling of cereals but how it is related to the pre-anthesis non-structural carbohydrate (NSC) reserves is not clear. This study investigated if and how an increase in NSC reserves could enhance sink strength, and consequently improve grain filling of later-flowering inferior spikelets (in contrast to the earlier flowering superior spikelets) for rice varieties with large panicles. Two “super” rice varieties (the recently bred high-yielding rice) and two New Plant Type (NPT, named in IRRI for the extra-large panicle) rice lines were compared with two elite inbred varieties under field-grown conditions. Three nitrogen (N) treatments, applied at the stages of panicle initiation, spikelet differentiation or both, were adopted with no N application during the mid-season as control. Both super rice and NPT rice showed a greater yield capacity as a result of a larger panicle than the elite inbred rice. However, a lower percentage of filled grains limited the realization of higher yield potential in super rice and especially in NPT rice, due to their lower grain filling rate and the smaller grain weight of their inferior spikelets. The low grain filling rate and small grain weight of inferior spikelets are mainly attributed to a poor sink strength as a result of small sink size (small number of endosperm cells) and low sink activity, e.g. low activities of sucrose synthase (SuSase) and adenosine diphosphoglucose pyrophosphorylase (AGPase). The amounts of NSC in the stem and NSC per spikelet at the heading time are significantly and positively correlated with sink strength (number of endosperm cells and activities of SuSase and AGPase), grain filling rate, and grain weight of inferior spikelets. Nitrogen application at the spikelet differentiation stage significantly increased, whereas N application at the panicle initiation or at both panicle initiation and spikelet differentiation stages, significantly reduced, NSC per spikelet at the heading time, sink strength, grain filling rate, and grain weight of inferior spikelets in super rice. The results suggest that pre-anthesis NSC reserves in the stem are closely associated with the sink strength during grain filling of rice, and N application at the spikelet differentiation stage would be a good practice to increase pre-anthesis NSC reserves, and consequently to enhance sink strength for rice varieties with large panicles, such as super rice varieties.     

基于高产示范方的机插水稻群体特征研究

 考查了机插常规粳稻超高产群体产量构成、干物质积累以及氮素积累等群体特征。2008年对江苏武进区邹区（品种为武香粳9号）、漕桥和前黄镇（品种均为武运粳7号）等3个百亩连片超高产示范方,2009年对江苏省如东县(品种为宁粳3号)的百亩连片超高产示范方进行了调查。结果表明,机插稻产量与颖花量的相关达极显著,要达到10.5 t/hm2以上的产量,每1m2颖花量需≥ 45 000;籽粒产量10.5 t/hm2以上高产机插水稻的花后干物质积累占籽粒产量的70%～80%;高产机插稻氮素积累的优势在有效分蘖临界叶龄期前和抽穗后两个阶段。足够的颖花量是机插粳稻高产群体的基本特征;保证后期氮素供应、发挥后期干物质积累优势、促进大穗的形成是提高机插水稻产量的主要技术途径。     

甘蔗花芽分化及花序发育过程的石蜡切片显微观察与分析

常规杂交是甘蔗遗传改良的主要手段,开花诱导是亲本杂交的关键。花芽分化和花序发育对甘蔗开花至关重要,但目前笔者们对这一过程还缺少必要的了解。本研究以当前甘蔗主栽品种粤糖93-159为材料,对其茎尖分生区花芽分化及花序发育过程进行石蜡切片显微观察。结果显示,甘蔗开花过程可明显划分为6个阶段:营养生长阶段——甘蔗分生区维持营养生长;花芽分化阶段——生长锥分生区膨大,产生花序分生组织;枝梗分化阶段——分生区周围的褶皱分化产生一次、二次枝梗分生组织;小穗/小花原基分化阶段——沿枝梗产生的小穗分生组织分化出2朵小花分生组织;花器官分化阶段——小花分生组织分化形成典型花器官;成熟阶段——抽穗开花,可以授粉。此结果揭示了甘蔗花发育分化过程中的组织形态变化,可为甘蔗开花机理的深入研究提供参考。