Sterile Lemma Research Articles

Agro-morphological and molecular characterization of traditional scented Radhatilak rice of lower Gangetic plains of West Bengal

Radhatilak, an indigenous tall-indica type scented rice, is presently cultivated in small pockets in 3-4 districts within lower gangetic plains of West Bengal, India. With a view to register as a farmers' variety, its agro-morphological characterization was done at 'C' Block Farm, B.C.K.V., Kalyani, West Bengal during kharif (wet) season of 2012, 2013 and 2014 following DUS test guidelines of Protection of Plant Varieties and Farmers' Rights Authority (PPV&FRA). The variety has late heading (115-120 days) and late maturity (140-150 days), which produces long-statured plants (130-140 cm height) with no anthocyanin colouration on leaf blade and sheath, nodes and internodes. The colour of lemma and palea is golden-yellow with red or reddish-purple spot at tip at maturity, while sterile lemma is red in colour. The awnless grains are short in length (6.3 mm) with very low test weight (10.95 g). The white- coloured kernels are short-bold in shape with low amylose (18.6%), medium gelatinization temperature (alkali value 3.3) and medium-strong aroma. Twenty three SSR markers were used for DNA amplification profiling of Radhatilak rice against non-aromatic international check variety IR 36. The study could identify two markers (RM 339 and RM 341) which can clearly distinguish Radhatilak and IR 36 (182.39 vs. 143.09 bp and 135.39 vs. 174.97 bp, respectively).

Characterization and fine mapping of nonstop glumes 2 (nsg2) mutant in rice (Oryza sativa L.).

In cereal crops, the grain number per panicle and the grain yield are greatly affected by the number of florets in a spikelet. In wild-type rice, a spikelet only produces one fertile floret and beneath the floret are a pair of sterile lemmas and a pair of rudimentary glumes. This study characterized a rice spikelet mutant nonstop glumes 2 (nsg2). In the nsg2 mutant, both the sterile lemmas and rudimentary glumes were elongated, and part of sterile lemma looked like a lemma in appearance, shape and size. Detailed histological analysis and qPCR analysis revealed that the sterile lemmas in the nsg2 mutant had homeotically transformed into lemma-like organs. This phenotype indicates that NSG2 is involved in the regulation of spikelet development and supports the long-held view that sterile lemmas were derived from the lemmas of the two lateral florets. This implies that the rice spikelet has the potential to be restored to the "three florets spikelet", which may have existed in its ancestors. Genetic analysis reveals that the nsg2 trait is controlled by a single recessive gene. The NSG2 gene was finally mapped between markers R-20 and R-39 on chromosome 7 with a physical region of 180 kb. The two MYB family factors LOC_Os07g44030 and LOC_Os07g44090 might be involved in the development of the spikelet and floral organ, and they were considered as candidate genes of NSG2. These results provide a foundation for map-based cloning and function analysis of NSG2, as well as evidence to support "three-florets spikelet" breeding in rice.

Synergistic roles of LAX1 and FZP in the development of rice sterile lemma

Rice florets are subtended by two sterile lemmas, whose origin and biological functions have not been studied extensively. Here we demonstrate that two putative transcription factors, LAX PANICLE1 (LAX1) and FRIZZY PANICLE (FZP), synergistically control the development of sterile lemmas. Both LAX1 and FZP are previously known for their roles in panicle and floret development. Disruption of either LAX1 or FZP greatly reduces the number of floret development. We generated new lax1 mutants (lax1-c) using CRISPR/Cas9 gene editing technology. In addition to the expected lax panicle phenotypes, we noticed that a significant number of spikelets of lax1-c developed elongated sterile lemmas. Moreover, our characterization of lax1-RNAi plants also revealed sterile lemma phenotypes similar to lax1-c mutants. We isolated a weak allele of fzp (fzp-14) in a genetic screen for lax1–1 enhancers. The fzp-14 lax1–1 double mutants completely eliminated flower development. Interestingly, the isolated fzp-14 produced spikelets with elongated sterile lemmas. Furthermore, fzp-14 was haploid-insufficient in the lax1–1 background whereas fzp-14 heterozygous plants were indistinguishable from wild type plants. The lax1–1 fzp-14+/− also developed elongated sterile lemma as observed in lax1-c, lax1-RNAi, and fzp-14, suggesting that LAX1 and FZP synergistically control sterile lemma development.

The rice pds1 locus genetically interacts with partner to cause panicle exsertion defects and ectopic tillers in spikelets

BackgroundRice (Oryza sativa L.) is a staple food crop worldwide. Its yield and quality are affected by its tillering pattern and spikelet development. Although many genes involved in the vegetative and reproductive development of rice have been characterized in previous studies, the genetic mechanisms that control axillary tillering, spikelet development, and panicle exsertion remain incompletely understood.ResultsHere, we characterized a novel rice recombinant inbred line (RIL), panicle exsertion defect and aberrant spikelet (pds). It was derived from a cross between two indica varieties, S142 and 430. Intriguingly, no abnormal phenotypes were observed in the parents of pds. This RIL exhibited sheathed panicles at heading stage. Still, a small number of tillers in pds plants were fully exserted from the flag leaves. Elongated sterile lemmas and rudimentary glumes (occurred occasionally) were observed in the spikelets of the exserted panicles and were transformed into palea/lemma-like structures. Furthermore, more interestingly, tillers occasionally grew from the axils of the elongated rudimentary glumes. Via genetic linkage analysis, we found that the abnormal phenotype of pds manifesting as genetic incompatibility or hybrid weakness was caused by genetic interaction between a recessive locus, pds1, which was derived from S142 and mapped to chromosome 8, and a locus pds2, which not yet mapped from 430. We fine-mapped pds1 to an approximately 55-kb interval delimited by the markers pds-4 and 8 M3.51. Six RGAP-annotated ORFs were included in this genomic region. qPCR analysis revealed that Loc_Os080595 might be the target of pds1 locus, and G1 gene might be involved in the genetic mechanism underlying the pds phenotype.ConclusionsIn this study, histological and genetic analyses revealed that the pyramided pds loci resulted in genetic incompatibility or hybrid weakness in rice might be caused by a genetic interaction between pds loci derived from different rice varieties. Further isolation of pds1 and its interactor pds2, would provide new insight into the molecular regulation of grass inflorescence development and exsertion, and the evolution history of the extant rice.

Characterization and Gene Mapping of non-open hull 1 (noh1) Mutant in Rice (Oryza sativa L.)

Hull opening is a key physiological process during reproductive development, strongly affecting the subsequent fertilization and seed development in rice. In this study, we characterized a rice mutant, non-open hull 1 (noh1), which was derived from ethylmethane-sulfonate (EMS)-treated Xinong 1B (Oryza sativa L.). All the spikelets of noh1 developed elongated and thin lodicules, which caused the failure of hull opening and the cleistogamy. In some spikelets of the noh1, sterile lemmas transformed into hull-like organs. qPCR analysis indicated that the expression of A- and E-function genes was significantly upregulated, while the expression of some B-function genes was downregulated in the lodicules of noh1. In addition, the expression of A-function genes was significantly upregulated, while the expression of some sterile-lemma maker genes was downregulated in the sterile lemma of noh1. These data suggested that the lodicule and sterile lemma in noh1 mutant gained glume-like and lemma-like identity, respectively. Genetic analysis showed that the noh1 trait was controlled by a single recessive gene. The NOH1 gene was mapped between the molecular markers ZJ-9 and ZJ-25 on chromosome 1 with a physical region of 60 kb, which contained nine annotated genes. These results provide a foundation for the cloning and functional research of NOH1 gene.

Agro-morphological and Physico-chemical Characterization of Indigenous Scented Lal Badshabhog Rice of West Bengal, India

The agro-morphological and physico-chemical characterization of traditional aromatic Lal Badshabhog rice landrace was done at BCKV, Kalyani, Nadia, West Bengal, India during kharif season of 2012, 2013 and 2014. The variety had long-statured plants (scale 7, 140–150 cm height) and late maturity (scale 7, 130–138 days). The lemma and palea of matured grain was brownish-red in colour, while sterile lemma was red and the awnless grains were short in length (5.98 mm) with very low test weight (10.6 g). The white-coloured kernels belonged to short-bold group (length 3.80 mm and width 1.87 mm), which had low amylose content (18.1%), medium alkali value (score 3.3) and medium aroma (score 2.1).

Genetic variability and association studies for yield and floral traits in Temperature Sensitive Male Sterile lines (TGMS) of rice (Oryza sativa L)

One hundred and nineteen rice TGMS lines were evaluated for ten morphological traits in fertility favouring environment and eight floral traits in sterility favouring environment respectively. The analysis of variance exhibited significant difference among the TGMS lines for all the traits studied except sterile lemma length indicates large amount of genetic variability was present in the experimental material. The morphological traits viz., pollen fertility, number of grains per panicle, single plant yield and floral traits namely anther length, anther breadth, stigma length, stigma breadth, glume opening angle, stigma exertion and pollen sterility were recorded high phenotypic and genotypic coefficient of variation. High heritability with high genetic advance as per cent of mean was observed in morphological traits namely, days to 50 per cent flowering, pollen fertility, spikelet fertility, number of grains per panicle, single plant yield and in floral traits viz., anther length, anther breadth, stigma length, stigma breadth, stigma exertion, glume opening angle and pollen sterility reveals presence of additive gene action and further genetic improvement through direct selection would be effective for these traits. The single plant yield observed significant and positive correlation with plant height, number of productive tillers per plant and spikelet fertility shows importance of these characters for yield improvement. In floral traits, stigma breadth and glume opening angle had non significant and positive correlation with pollen sterility. Path analysis showed that plant height and number of productive tillers had positive direct effect on single plant yield. In floral traits, stigma breadth and glume opening angle had small positive effects on pollen sterility.

FZP determines grain size and sterile lemma fate in rice.

In grass, the spikelet is a unique inflorescence structure that directly determines grain yield. Despite a great deal of research, the molecular mechanisms behind spikelet development are not fully understood. In the study, FZP encodes an ERF domain protein, and functions in grain size and sterile lemma identity. Mutation of FZP causes smaller grains and degenerated sterile lemmas. The small fzp-12 grains were caused by a reduction in cell number and size in the hulls. Interestingly, the sterile lemma underwent a homeotic transformation into a rudimentary glume in the fzp-12 and fzp-13 mutants, whereas the sterile lemma underwent a homeotic transformation into a lemma in FZP over-expressing plants, suggesting that FZP specifically determines the sterile lemma identity. We confirmed the function of FZP by complementation, CRISPR-Cas9 gene editing, and cytological and molecular tests. Additionally, FZP interacts specifically with the GCC-box and DRE motifs, and may be involved in regulation of the downstream genes. Our results revealed that FZP plays a vital role in the regulation of grain size, and first provides clear evidence in support of the hypothesis that the lemma, rudimentary glume, and sterile lemma are homologous organs.

Digitaria clarkei (Paniceae, Panicoideae, Poaceae) a new species with paniculate synflorescence and D. costaricensis a new record from Mexico

Digitaria clarkiae, a new species from the states of Guerrero, Morelos, and Puebla in México, is described and illustrated. The new species is compared to species of sect. Pennatae (D. arenicola, D. cognata and D. pubiflora) with so-called paniculate synflorescences, and to species of sect. Trichachne (e.g., D. patens), with which it shares an elongated rachilla between the glumes and the florets called the “stipe” and unequal spaces between the veins of the sterile lemma. Because the new species shares characteristics with both sections, its probable sectional affiliation based on morphological characters remains unclear, leaving it as incertae sedis in the genus. Digitaria costaricensis of section Debiles (Aequiglumae) is a new record from Veracruz, México.

Regulatory network and genetic interactions established by OsMADS34 in rice inflorescence and spikelet morphogenesis

Grasses display highly diversified inflorescence architectures that differ in the arrangement of spikelets and flowers and determine cereal yields. However, the molecular basis underlying grass inflorescence morphogenesis remains largely unknown. Here we investigate the role of a functionally diversified SEPALLATA MADS-box transcription factor, OsMADS34, in regulating rice (Oryza sativa L.) inflorescence and spikelet development. Microarray analysis showed that, at the very early stages of inflorescence formation, dysfunction of OsMADS34 caused altered expression of 379 genes that are associated with protein modification and degradation, transcriptional regulation, signaling and metabolism activity. Genetic analysis revealed that OsMADS34 controls different aspects of inflorescence structure, branching and meristem activity synergistically with LAX PANICLE1 (LAX1) and FLORAL ORGAN NUMBER4 (FON4), as evidenced by the enhanced phenotypes of osmads34 lax1 and osmads34 fon4 compared with the single mutants. Additionally, double mutant between osmads34 and the sterile lemma defective mutant elongated empty glume (ele) displayed an enhanced phenotype, that is, longer and wider sterile lemmas that were converted into lemma/palea-like organs, suggesting that ELE and OsMADS34 synergistically control the sterile lemma development. OsMADS34 may act together with OsMADS15 in controlling sterile lemma development. Collectively, these findings provide insights into the regulatory function of OsMADS34 in rice inflorescence and spikelet development.

LATERAL FLORET 1 induced the three-florets spikelet in rice

The spikelet is a unique inflorescence structure in grass. The molecular mechanisms behind the development and evolution of the spikelet are far from clear. In this study, a dominant rice mutant, lateral florets 1 (lf1), was characterized. In the lf1 spikelet, lateral floral meristems were promoted unexpectedly and could generally blossom into relatively normal florets. LF1 encoded a class III homeodomain-leucine zipper (HD-ZIP III) protein, and the site of mutation in lf1 was located in a putative miRNA165/166 target sequence. Ectopic expression of both LF1 and the meristem maintenance gene OSH1 was detected in the axil of the sterile lemma primordia of the lf1 spikelet. Furthermore, the promoter of OSH1 could be bound directly by LF1 protein. Collectively, these results indicate that the mutation of LF1 induces ectopic expression of OSH1, which results in the initiation of lateral meristems to generate lateral florets in the axil of the sterile lemma. This study thus offers strong evidence in support of the "three-florets spikelet" hypothesis in rice.

Mapping and allelic sequencing of a long sterile lemma trait in rice

ABSTRACT Some outer floral organs are unique in gramineous plants, like the sterile lemma and rudimentary glume in rice. However, their development mechanisms are still poorly understood. In this study, we used 4 mutants with long sterile lemma (LSL), named JF11, JF12, JF13 and JNY-7, to be crossed with Aijiaonante (AJNT) and Nipponbare (NIP), respectively. Genetic analysis indicated that LSL trait exhibited recessive heredity and was controlled by a common allele named sl-1(t). Using the method of bulk segregant analysis and linkage analysis between SSR markers and LSL trait based on F2 populations, the sl-1(t) gene was located at the interval between RM20903 and RM20948 on chromosome 7. The interval harbors a known G1 gene, which regulates the sterile lemma trait. The findings of allelic sequencing showed an 11-base deletion in gene G1 happened in the mutants of JF11, JF12 and JF13, which led to a frame-shifting mutation, whereas the mutant of JNY-7 had a base substitution that caused the change of the amino acid residue. Eight substitutions in the ORF and 10 in the upstream region from −1 to −824 were found between Indica and Japonica rice by DNA sequence analysis, but those polymorphisms have no effect on the gene function. In conclusion, we fine mapped the LSL gene, sl-1(t), and found 2 kinds of mutant alleles conferring the gene function and the DNA polymorphisms of G1 between Indica and Japonica rice.

イネ小穂の護穎、副護穎に関する変異体の遺伝解析(平成27年度年次講演会一般公演)

イネ小穂の護穎、副護穎に関する変異体の遺伝解析(平成27年度年次講演会一般公演)

Fine Mapping of a Novel defective glume 1 (dg1) Mutant, Which Affects Vegetative and Spikelet Development in Rice.

In cereal crops, vegetative and spikelet development play important roles in grain yield and quality, but the genetic mechanisms that control vegetative and spikelet development remain poorly understood in rice. Here, we identified a new rice mutant, defective glume 1 (dg1) mutant from cultivar Zhonghua11 after ethyl methanesulfonate treatment. The dg1 mutant displayed the dwarfism with small, rolled leaves, which resulted from smaller cells and more bulliform cells. The dg1 mutant also had an enlarged leaf angle and defects in brassinosteroid signaling. In the dg1 mutant, both the rudimentary glume and sterile lemma (glumes) were transformed into lemma-like organ and acquired the lemma identity. Additionally, the dg1 mutant produced slender grains. Further analysis revealed that DG1 affects grain size by regulating cell proliferation and expansion. We fine mapped the dg1 locus to a 31-kb region that includes eight open reading frames. We examined the DNA sequence and expression of these loci, but we were not able to identify the DG1 gene. Therefore, more work will be needed for cloning and functional analysis of DG1, which would contribute to our understanding of the molecular mechanisms behind whole-plant development in rice.

Infection Process of Burkholderia glumae in Rice Spikelets

AbstractBurkholderia glumae, which causes bacterial panicle blight of rice (BPBR), is a well‐known pathogen. The pathogen‐induced symptoms include seedling rot, grain rot and leaf‐sheath browning in rice plants. B. glumae can incubate in rice plants as endophytes before booting stage of rice. In this study, we constructed a gfp‐labelled system of B. glumae LMG 2196 and used SEM to clarify the colonization course of B. glumae at the heading stage. New locations of B. glumae were found. The pathogens initially distributed on the surface of the glumes and colonized in the glume hairs and cells of the edge of sterile lemma, palea and lemma. The base of glume hairs was the initial position for colonization. Bacterial population raised around glume hairs, penetrated into the inner surface of the palea and lemma, and spread on the gynoecium and stamens through contact. The spreading of B. glumae among the panicles mainly occurred through the contact or friction among glumes or leaf sheaths, but the inner spread of the stamens mainly occurred through the connective tissue of anther. We also detected the differences of bacterial content in stamens, gynoecia and glumes. The growing stage of B. glumae in spikelets could be divided into two sections. The biomass of all parts continued to increase to nearly 108 CFU/g at 10 DAI. This caused wilt symptoms and stopped the pollination. This work showed that glume hairs played an important role in the initial colonization of B. glumae, and provides a foundation for further studies of the infection manner of B. glumae and other pathogenic bacteria.

Regulatory Role of OsMADS34 in the Determination of Glumes Fate, Grain Yield, and Quality in Rice.

Grasses produce seeds on spikelets, a unique type of inflorescence. Despite the importance of grass crops for food, the genetic mechanisms that control spikelet development remain poorly understood. In this study, we used m34-z, a new mutant allele of the rice (Oryza sativa) E-class gene OsMADS34, to examine OsMADS34 function in determining the identities of glumes (rudimentary glume and sterile lemma) and grain size. In the m34-z mutant, both the rudimentary glume and sterile lemma were homeotically converted to the lemma-like organ and acquired the lemma identity, suggesting that OsMADS34 plays important roles in the development of glumes. In the m34-z mutant, most of the grains from the secondary panicle branches (spb) were decreased in size, compared with grains from wild-type, but no differences were observed in the grains from the primary panicle branches. The amylose content and gel consistency, and a seed-setting rate from the spb were reduced in the m34-z mutant. Interesting, transcriptional activity analysis revealed that OsMADS34 protein was a transcription repressor and it may influence grain yield by suppressing the expressions of BG1, GW8, GW2, and GL7 in the m34-z mutant. These findings revealed that OsMADS34 largely affects grain yield by affecting the size of grains from the secondary branches.

Loss of function of OsMADS34 leads to large sterile lemma and low grain yield in rice (Oryza sativa L.)

Rice (Oryza sativa L.) is one of the most important food crops, especially in Asia. The spikelet is a characteristic structure of grass inflorescences that determines crop output. However, the molecular mechanism that controls spikelet development and grain yield in rice remains unclear. In this study, we isolated a new osmads34 allelic mutant (i.e., osmads34-t). The osmads34-t mutant showed more primary branch numbers, short panicles, and long sterile lemmas. The sterile lemmas were transformed into the lemmas and had the lemma identity in the osmads34-t mutant, suggesting that the sterile lemma and lemma are homologous organs. Additionally, osmads34-t displayed smaller grains on its secondary branches of panicles and a lower seed-setting rate. These results suggest that OsMADS34 plays an important role in determination of grain size and yield in rice. OsMADS34 was expressed in tested organs and tissues, and its green fluorescent protein (GFP) signal was located in the nucleus. The result of this study will be used to understand the identity of unique organs in grass spikelets and may improve grain yield in breeding practice.

Ectopic expression of OsMADS1 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.

Identifying rice grains using image analysis and sparse-representation-based classification

Rice (Oryza sativa L.) is a major staple food worldwide, and is traded extensively. The objective of this study is to distinguish the rice grains of 30 varieties nondestructively using image processing and sparse-representation-based classification (SRC). SRC uses over-complete bases to capture the representative traits of rice grains. In the experiments, rice grain images were acquired by microscopy. The morphological, color, and textural traits of the grain body, sterile lemmas, and brush were quantified. An SRC classifier was subsequently developed to identify the varieties of the grains using the traits as the inputs. The proposed approach could discriminate rice grain varieties with an accuracy of 89.1%.

G1/ELE Functions in the Development of Rice Lemmas in Addition to Determining Identities of Empty Glumes.

Rice empty glumes, also named sterile lemmas or rudimentary lemmas according to different interpretations, are distinct from lemmas in morphology and cellular pattern. Consistently, the molecular mechanism to control the development of lemmas is different from that of empty glumes. Rice LEAFY HULL STERILE1(OsLHS1) and DROOPING LEAF(DL) regulate the cellular pattern and the number of vascular bundles of lemmas respectively, while LONG STERILE LEMMA1 (G1)/ELONGATED EMPTY GLUME (ELE) and PANICLE PHYTOMER2 (PAP2)/OsMADS34 determine identities of empty glumes. Though some progress has been made, identities of empty glumes remain unclear, and genetic interactions between lemma genes and glume genes have been rarely elucidated. In this research, a new G1/ELE mutant g1–6 was identified and the phenotype was analyzed. Similar to previously reported mutant lines of G1/ELE, empty glumes of g1–6 plants transform into lemma-like organs. Furthermore, Phenotypes of single and double mutant plants suggest that, in addition to their previously described gene-specific functions, G1/ELE and OsLHS1 play redundant roles in controlling vascular bundle number, cell volume, and cell layer number of empty glumes and lemmas. Meanwhile, expression patterns of G1/ELE in osmads1-z flowers and OsLHS1 in g1–6 flowers indicate they do not regulate each other at the level of transcription. Finally, down-regulation of the empty glume gene OsMADS34/PAP2 and ectopic expression of the lemma gene DL, in the g1–6 plants provide further evidence that empty glumes are sterile lemmas. Generally, our findings provided valuable information for better understanding functions of G1 and OsLHS1 in flower development and identities of empty glumes.