High-resolution genetic mapping and candidate gene identification of the <Emphasis Type="Italic">SLP1</Emphasis> locus that controls glume development in rice

Stunted lemma palea 1 (slp1) is a rice mutant that displays dwarfism, shortened inflorescence lengths, severely degenerated lemmas/paleas, and sterility. The SLP1 locus was mapped between markers RM447 and D275 in the distal region of the long arm of chromosome 8, using the F2 progeny derived from the cross between the Slp1/slp1 mutant (Oryza sativa subsp. japonica) and the variety Taichung Native 1 (TN1, O. sativa subsp. indica). The SLP1 locus was further delimited to a 46.4-kb genomic region containing three putative genes: OsSPL16, OsMADS45, and OsMADS37. Comparisons of the sequence variations and expression levels of the three candidate genes between wild-type plants and homozygous slp1 mutants suggested that a missense mutation in the sixth amino acid of the OsSPL16 protein was likely responsible for the slp1 mutant phenotypes.     

一个水稻内颖退化突变体的形态特征及基因的精确定位

水稻产量和品质受花器官发育的直接影响,因此对水稻颖花发育机理的研究将有助于水稻产量提高和品质的改良。文章利用60Coγ射线辐照亲本8PW33(籼稻背景)获得一个性状能稳定遗传的内颖退化突变体(编号:MU102),并对其农艺性状和花器官进行了观察和分析。结果显示,相对于野生型,该突变体的株高、每穗总粒数及剑叶宽均显著增加,而结实率则显著降低,差异均达显著水平。解剖镜下观察表明,该突变体内颖退化,外颖弯曲呈现镰刀状,其余器官与野生型表型基本一致。扫描电镜观察显示,突变体与野生型叶片维管束的结构组成以及外颖表皮细胞组成、排列均正常,没有明显差异;与野生型相比,突变体内颖表皮细胞排列较为紧密,推测可能是内颖收缩退化导致的。遗传分析显示该突变性状是由隐性单基因控制,并命名为pd2。利用实验室现有的SSR分子标记将PD2基因定位于水稻第9号染色体上,通过进一步扩大群体和开发新的Indel标记,将PD2基因定位在2个Indel标记之间,两者间的物理距离大约是82 kb。在该物理区间内有一个已经克隆的内颖发育基因REP1,经过测序和比对分析,推测REP1与PD2为等位基因。     

BEAK LIKE SPIKELET1 is Required for Lateral Development of Lemma and Palea in Rice

Lemma and palea are unique floral structures found only in Poaceae, and are responsible for protecting the inner floral organs and kernels from environmental stresses. However, the mechanism underlying specification of their morphology remains unclear. In this study, we characterized a rice mutant, beak like spikelet1 (bls1), which specifically affects development of the lemma and palea. In bls1 mutant, floral-organ identity and floral-organ patterning are normal, and the defects occur at the stage of the lemma and palea expansion, whereas the other aspects of floral architecture and form are not affected. We isolated BLS1 by positional cloning and found that it encodes a protein with a conserved domain of unknown function. BLS1 is expressed strongly in young inflorescence, specifically the young lemmas and paleas of spikelets. Subcellular localization analysis showed that BLS1 is localized in the nucleus. Expression of the AP1-like and SEP-like floral homeotic genes were not changed in the bls1 mutant. Our study suggested that BLS1 is required for lateral development of the lemma and palea and does not function at stages of floral-organ initiation and patterning.     

Genetic and Phenotypic Analysis of <Emphasis Type="Italic">lax1-6</Emphasis>, a Mutant Allele of <Emphasis Type="Italic">LAX PANICLE1</Emphasis> in Rice

Proper function of the LAX1 gene is required for the development of axillary meristem in rice. Here, we report genetic and phenotypic characters of a novel recessive mutant allele of rice LAX1 gene, lax1-6, which showed abnormal panicle phenotypes with few numbers of elongated primary rachis branches. Beside typical lax mutant phenotype, abnormalities of lax1-6 mutant allele were observed with defect lemma and palea primordial in floral organs. The lax1-6 mutant locus was linked between SSR markers RM7594 and RM5389 on chromosome 1 with 1.02% and 1.0% recombination frequencies, respectively. Molecular analysis revealed that the lax1-6 mutant allele was caused by a transversion mutation of nucleotide T to G substitution that resulted in an amino acid substitution from serine (S) to alanine (A) at the 117^th position from amino terminus of a basic helix-loop-helix protein coded by LAX1 gene. Furthermore, we found that the Oryza sativa indica type cv. IRRI347 contained 24 nucleotide deletion in the upstream sequence in the LAX1 gene, but this deletion did not influence panicle morphology, which demonstrated that the deletion is a polymorphism in rice. All together, the lax1-6 mutant is a newly identified allele of LAX1 gene displaying the abnormal axillary meristems and inflorescences in rice.     

Ectopic expression of <Emphasis Type="Italic">OsMADS1</Emphasis> caused dwarfism and spikelet alteration in rice

In rice, an E-class gene, OsMADS1, acts to specify the identities of the lemma and palea. In this study, the OsMADS1 gene with a CaMV35S promoter was transformed into a japonica cultivar, Zhonghua 11. All transgenic plants successfully showed similar phenotypes, including dwarfism, distorted panicles, decreased numbers of branches and spikelets, and elongated sterile lemma. Histological analysis showed that the elongated sterile lemma developed with silicified epidermal and sclerenchymal cells, which were lacking in the wild-type sterile lemma, suggesting that the elongated sterile lemma had assumed the identity of the lemma or palea. Some marker genes were subjected to a detailed analysis of the distribution of their expression among the lemma, palea and sterile lemma. DROOPING LEAF (DL) and OsMADS6 genes were only expressed in the normal lemma or palea, respectively. In the elongated sterile lemma, a high level of DL gene expression was detected, while no expression of OsMADS6 was found, implying that the sterile lemma transformed into the lemma but not the palea. These results provide clues to elucidate the mechanism of evolution from lemma to sterile lemma in rice. qPCR analysis also suggested that the ectopic expression of OsMADS1 induced abnormal brassinosteroid and gibberellin acid activation, and then resulted in developmental defects in the stem and panicle.     

Identification of a novel <Emphasis Type="Italic">SPLIT</Emphasis>-<Emphasis Type="Italic">HULL</Emphasis> (<Emphasis Type="Italic">SPH</Emphasis>) gene associated with hull splitting in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Key message

The split-hull phenotype caused by reduced lemma width and low lignin content is under control of SPH encoding a type-2 13-lipoxygenase and contributes to high dehulling efficiency.

Abstract

Rice hulls consist of two bract-like structures, the lemma and palea. The hull is an important organ that helps to protect seeds from environmental stress, determines seed shape, and ensures grain filling. Achieving optimal hull size and morphology is beneficial for seed development. We characterized the split-hull (sph) mutant in rice, which exhibits hull splitting in the interlocking part between lemma and palea and/or the folded part of the lemma during the grain filling stage. Morphological and chemical analysis revealed that reduction in the width of the lemma and lignin content of the hull in the sph mutant might be the cause of hull splitting. Genetic analysis indicated that the mutant phenotype was controlled by a single recessive gene, sph (Os04g0447100), which encodes a type-2 13-lipoxygenase. SPH knockout and knockdown transgenic plants displayed the same split-hull phenotype as in the mutant. The sph mutant showed significantly higher linoleic and linolenic acid (substrates of lipoxygenase) contents in spikelets compared to the wild type. It is probably due to the genetic defect of SPH and subsequent decrease in lipoxygenase activity. In dehulling experiment, the sph mutant showed high dehulling efficiency even by a weak tearing force in a dehulling machine. Collectively, the results provide a basis for understanding of the functional role of lipoxygenase in structure and maintenance of hulls, and would facilitate breeding of easy-dehulling rice.

     

BEAK-SHAPED GRAIN 1/TRIANGULAR HULL 1, a DUF640 gene, is associated with grain shape, size and weight in rice

Grain shape and size both determine grain weight and therefore crop yield. However, the molecular mechanisms controlling grain shape and size are still largely unknown. Here, we isolated a rice mutant, beak-shaped grain1 (bsg1), which produced beak-shaped grains of decreased width, thickness and weight with a loosely interlocked lemma and palea that were unable to close tightly. Starch granules were also irregularly packaged in the bsg1 grains. Consistent with the lemma and palea shapes, the outer parenchyma cell layers of these bsg1 tissues developed fewer cells with decreased size. Map-based cloning revealed that BSG1 encoded a DUF640 domain protein, TRIANGULAR HULL 1, of unknown function. Quantitative PCR and GUS fusion reporter assays showed that BSG1 was expressed mainly in the young panicle and elongating stem. The BSG1 mutation affected the expression of genes potentially involved in the cell cycle and GW2, an important regulator of grain size in rice. Our results suggest that BSG1 determines grain shape and size probably by modifying cell division and expansion in the grain hull.     

Identification and fine mapping of a mutant gene for palealess spikelet in rice

In grass, the evolutionary relationship between lemma and palea, and their relationship to the flower organs in dicots have been variously interpreted and wildely debated. In the present study, we carried out morphological and genetic analysis of a palealess mutant (pal) from rice (Oryza sativa L.), and fine mapping the gene responsible for the mutated trait. Together, our findings indicate that the palea is replaced by two leaf-like structures in the pal flowers, and this trait is controlled by one recessive gene, termed palealess1 (pal1). With a large F2 segregating population, the pal1 gene was finally mapped into a physical region of 35 kb. Our results also suggest that the lemma and palea of rice are not homologous organs, palea is likely evolutionarily equivalent to the eudicot sepal, and the pal1 should be an A function gene for rice floral organ identity.     

Molecular mapping of split rice spikelet mutantsrs-1 and analysis of its homeotic function in rice

srs-1, a new floral organ identity gene in rice, was mapped using RAPD and RFLP markers. Firstly, the cross was made between "ZhaiYeQing 8" (ZYQ8, indica) and split rice spikelet (SRS, japonica) mutant. The ratio of wild-type individuals and mutant plants in F2 population is 3:1, which indicates that the mutant characteristics are controlled by single recessive gene, srs-1. Consequently, BSA method was adopted and 520 random 10-mer primers were used to screen polymorphic bands between two bulks. Six primers could amplify polymorphic bands, of which S465 generates the most stable RAPD patterns. Then, S465 that cosegregates in F2 population has been converted into an RFLP marker successfully. Furthermore, srs-1 gene was mapped on chromosome 3 using DH mapping population. The effect of srs-1 gene results in the mutant of split rice spikelet. The mutant has longer and softer palea/lemma than those of wild-type, and two small palea/lemma-like organs between palea and lemma. In addition, there is a flower wit     

Molecular mapping of split rice spikelet mutant srs-1 and analysis of its homeotic function in rice

srs-1, a new floral organ identity gene in rice, was mapped using RAPD and RFLP markers. Firstly, the cross was made between "ZhaiYeQing 8" (ZYQ8, indica) and split rice spikelet (SRS, japonica) mutant. The ratio of wild-type individuals and mutant plants in F2 population is 3:1, which indicates that the mutant characteristics are controlled by single recessive gene, srs-1. Consequently, BSA method was adopted and 520 random 10-mer primers were used to screen polymorphic bands between two bulks. Six primers could amplify polymorphic bands, of which S465 generates the most stable RAPD patterns. Then, S465 that cosegregates in F2 population has been converted into an RFLP marker successfully. Furthermore, srs-1 gene was mapped on chromosome 3 using DH mapping population. The effect of srs-1 gene results in the mutant of split rice spikelet. The mutant has longer and softer palea/lemma than those of wild-type, and two small palea/lemma-like organs between palea and lemma. In addition, there is a flower with three stamens and carpel in the axil of lemma. Thus, there are nine stamens and two carpels in the spikelet of mutant. srs-1 gene may belong to homeotic gene of class A according to the mutant characteristics and ABC model.     

水稻内颖畸形或缺失基因OsAPP1的图位克隆及表达分析

在育种基地材料中发现一株内颖畸形或缺失(abnormal or absent palea)突变体,将其命名为app1。该突变体在营养生长时期发育正常,但抽穗后突变体表现出内颖畸形(比外稃短导致颖壳不闭合,或者出现两个内稃)或缺失,其花粉育性为55.52%,结实率为6.48%,千粒重为10.811 g,种子发芽率为55.21%。以突变体app1与日本晴杂交构建了F1和F2群体,F1颖壳表型正常,F2群体出现内颖畸形和正常表型分离,内颖正常和突变表型分离比例为3∶1,表明app1内颖突变表型由单隐性核基因控制。以F2为分离群体,将app1精细定位于第3染色体上,位于分子标记ID4231和ID4246之间,遗传距离1.3 cM,对应物理距离为13.2 kb。该区段内完全包含1个开放阅读框,包含两个部分开放阅读框,经过测序分析发现候选基因LOC_Os03g11614启动子区发生点突变和245 bp缺失,qRT-PCR分析证实LOC_Os03g11614为OsAPP1基因。已有报道LOC_Os03g11614编码OsMADS1,是调控水稻花器官发育的重要明星基因,其不同位置的突变可以导致叶状颖壳和不育、以及控制籽粒大小。与3000份水稻种子资源SNP/Indel变异类型对比分析发现,突变体app1启动子的突变完全不同于现已OsMADS1研究报道突变类型,且与数据库中的自然突变类型多数不同。因此,本研究发现的app1突变体,是以往报道中从未出现的OsMADS1启动子发生突变的新型突变,且该类突变导致了其降低表达量,并产生了不同于前人研究的新表型,这为深入研究OsMADS1基因在水稻花器官发育中的功能提供了新的种质资源和思路。     

Molecular and genetic analyses of the silky1 gene reveal conservation in floral organ specification between eudicots and monocots

The degree to which the eudicot-based ABC model of flower organ identity applies to the other major subclass of angrosperms, the monocots, has yet to be fully explored. We cloned silky1 (si1), a male sterile mutant of Zea mays that has homeotic conversions of stamens into carpels and lodicules into palea/lemma-like structures. Our studies indicate that si1 is a monocot B function MADS box gene. Moreover, the si1 zag1 double mutant produces a striking spikelet phenotype where normal glumes enclose reiterated palea/lemma-like organs. These studies indicate that B function gene activity is conserved among monocots as well as eudicots. In addition, they provide compelling developmental evidence for recognizing lodicules as modified petals and, possibly, palea and lemma as modified sepals.