Bulked Segregant RNA-Seq Analysis of Pollinated Pistils Reveals Genes Influencing Spikelet Fertility in Rice

Prezygotic isolation is important for successful fertilization in rice, significantly affecting yield. This study focused on F_(5:6) generation plants derived from inter-subspecific crosses(Nipponbare × KDML105) with low(LS) and high seed-setting rates(HS), in which normal pollen fertility was observed. However, LS plants showed a reduced number of pollen grains adhering to the stigma and fewer pollen tubes reaching the ovules at 4-5 h post-pollination, compared with HS plants. Bulked segregant RNA-Seq analysis of pollinated pistils from the HS and LS groups revealed 249 and 473 differentially expressed genes(DEGs), respectively. Kyoto Encyclopedia of Genes and Genomes analysis of the HS and LS-specific DEGs indicated enrichment in metabolic pathways, pentose and glucuronate interconversions, and flavonoid biosynthesis. Several of these DEGs exhibited co-expression with pollen development genes and formed extensive clusters of co-expression networks. Compared with LS pistils, enzyme genes controlling pectin degradation, such as OsPME35 and OsPLL9, showed similar expression patterns, with higher levels in HS pistils pre-pollination. Os02g0467600, similar to cinnamate 4-hydroxylase gene(CYP73), involved in flavonoid biosynthesis, displayed higher expression in HS pistils post-pollination. Our findings suggest that OsPME35, OsPLL9, and Os02g0467600 contribute to prezygotic isolation by potentially modifying the stigma cell wall(OsPME35 and OsPLL9) and controlling later processes such as pollen-stigma adhesion(Os02g0467600) genes. Furthermore, several DEGs specific to HS and LS were co-localized with QTLs and functional genes associated with spikelet fertility. These findings provide valuable insights for further research on rice spikelet fertility, ultimately contributing to the development of high-yielding rice varieties.

OsPPG encodes a pseudouridine-5'-phosphate glycosidase and regulates rice spikelet development

Florets are the basic structural units of spikelets,and their morphogenesis determines the yield and quality of rice grains.However,whether and how pseudouridine-5’-phosphate glycosidase participates in rice spikelet development remains an open question.In this study,we identified a novel gene,OsPPG,which encodes a peroxisome-localized pseudouridine-5’-phosphate glycosidase and regulates the development of rice spikelets.osppg mutants exhibited abnormal sterile lemma,lemma,palea,lodicule,stamens,and pistils;male sterility;shorter panicles;and reduced plant height.OsPPG was found to regulate several OsMADS genes,thereby affecting the morphogenesis of rice spikelets.Furthermore,metabolomics revealed that the OsPPG gene was involved in the decomposition of pseudouridine via the pyrimidine metabolism pathway and may affect the jasmonic acid signaling pathway.These results suggest that OsPPG is a key regulator of rice spikelet development.

SUPER WOMAN 2(SPW2)maintains organ identity in spikelets by inhibiting the expression of floral homeotic genes OsMADS3,OsMADS58,OsMADS13,and DROOPING LEAF

Flower organ identity in rice is mainly determined by the A-,B-,C-and E-class genes,with the majority encoding MADS-box transcription factors.However,few studies have investigated how the expression of these floral organ identity genes is regulated during flower development.In this study,we identified a gene named SUPER WOMAN 2(SPW2),which is necessary for spikelet/floret development in rice by participating in the regulation of the expression of pistil identity genes such as OsMADS3,OsMADS13,OsMADS58 and DL.In the spw2 mutant,ectopic stigma/ovary-like tissues were observed in the non-pistil organs,including sterile lemma,lemma,palea,lodicule,and stamen,suggesting that the identities of these organs were severely affected by mutations in SPW2.SPW2 was shown to encode a plant-specific EMF1-like protein that is involved in H3K27me3 modification as an important component of the PRC2 complex.Expression analysis showed that the SPW2 mutation led to the ectopic expression of OsMADS3,OsMADS13,OsMADS58,and DL in non-pistil organs of the spikelet.The ChIP-qPCR results showed significant reductions in the levels of H3K27me3 modification on the chromatin of these genes.Thus,we demonstrated that SPW2 can mediate the process of H3K27me3 modification of pistil-related genes to regulate their expression in non-pistil organs of spikelets in rice.The results of this study expand our understanding of the molecular mechanism by which SPW2 regulates floral organ identity genes through epigenetic regulation.

Changes in Enzyme Activities Involved in Starch Synthesis and Hormone Concentrations in Superior and Inferior Spikelets and Their Association with Grain Filling of Super Rice

The changes in activities of key enzymes involved in sucrose-to-starch conversion and concentrations of hormones in superior and inferior spikelets of super rice were investigated and their association with grain filling was analyzed.Four super rice cultivars,Liangyoupeijiu,IIyou 084,Huaidao 9 and Wujing 15,and two high-yielding and elite check cultivars,Shanyou 63 and Yangfujing 8,were used.The activities of sucrose synthase （SuSase）,adenosine diphosphoglucose pyrophosphorylase （AGPase）,starch synthase （StSase） and starch branching enzyme （SBE）,and the concentrations of zeatin ＋ zeatin riboside （Z ＋ ZR）,indole-3-acetic acid （IAA） and abscisic acid （ABA） in superior and inferior spikelets were determined during the grain filling period and their relationships with grain filling rate were analyzed.Maximum grain filling rate,the time reaching the maximum grain-filling rate,mean grain filling rate and brown rice weight for superior spikelets showed a slight difference between the super and check rice cultivars,but were significantly lower in the super rice than in the check rice for inferior spikelets.Changes of enzyme activities and hormone concentrations in grains exhibited single peak curves during the grain filling period.The peak values and the mean activities of SuSase,AGPase,StSase and SBE were lower in inferior spikelets than in superior ones,as well as the peak values and the mean concentrations of Z ＋ ZR and IAA.However,the peak value and the mean concentration of ABA were significantly higher in inferior spikelets than in superior ones and greater in the super rice than in the check rice.The grain filling rate was positively and significantly correlated with the activities of SuSase,AGPase and StSase and the concentrations of Z ＋ ZR and IAA.The results suggested that the low activities of SuSase,AGPase and StSase and the low concentrations of Z ＋ ZR and IAA might be important physiological reasons for the slow grain filling rate and light grain weight of inferior spikelets in super rice.

Response of spikelet water status to high temperature and its relationship with heat tolerance in rice

In rice, high-temperature stress(HT) during flowering results in decreased grain yield via a reduction in spikelet fertility;however, the effect of plant water status on spikelet fertility under HT remains unknown. To investigate the relationship between spikelet water status and spikelet fertility under HT, two experiments were performed under temperature-controlled conditions using four genotypes with varying tolerance to HT. Rice plants were exposed to HT for seven consecutive days during the flowering stage under three soil water treatments(soil water potential 0, -20, and -40 kPa), as well as under hydroponic conditions in a separate experiment. HT significantly decreased spikelet fertility, pollen fertility, and anther dehiscence under each of the three water treatments. HT significantly increased the spikelet transpiration rate, and this change was accompanied by a significant decrease in the internal temperature of the spikelets. HT decreased pollen grain diameter in heat-sensitive genotypes. HT had varying effects on the water potential of panicles and anthers but increased anther soluble-sugar concentration. Different aquaporin genes showed different expression profiles under HT, and the expression levels of PIPs for plasma membrane intrinsic proteins and TIPs for tonoplast intrinsic proteins increased in anthers but decreased in glumes.Correlation analyses showed that anther dehiscence and pollen(spikelet) fertility were tightly associated with anther water status, and the expression levels of almost all anther aquaporin genes were significantly correlated with anther dehiscence under HT. In summary, an increased spikelet transpiration rate and decreased internal spikelet temperature were associated with alleviation of the effects of HT in rice genotypes with varying degrees of heat tolerance, and the response of spikelet water status to HT, involving increased total expression of aquaporins and soluble sugar content, thereby improved pollen fertility,anther dehiscence, and spikelet fertility, especially in heat-resistant genotypes. The heat-resistant genotypes N22 and SY63 may adopt different approaches to reduce heat damage.

Agronomic characterization and genetic analysis of the supernume-r ary spikelet in tetraploid wheat(Triticum turgidum L.)

The supernumerary spikelets（SS）characters of tetraploid wheat（Triticum turgidum L.）resulting in more spikelets and kernels per spike,thus enhancing sink capacity may contribute to potential wheat yield improvement.In order to investigate the effect of different SS types on agronomic characters and understand the genetic base of SS phenotype in tetraploid wheat,near isogenic lines（NILs）,bh-50 with normal spikelets（NS）,bh-51 with four-rowed spikelets（FRS）,bh-52 with short-ramified spikelets（SRS）,and bh-53 with long-ramified spikelets（LRS）in a Triticum durum cv.ZY1286 genetic background were developed by continuous backcrossing.Agronomic characters showed that the SS phenotype lines,bh-51,bh-52and bh-53 have significant increase in the number of spikelets and grains per spike compared with the NS phenotype line bh-50（P〈0.05）,and bh-53 line showed much more increase than those of bh-51 and bh-52.However,bh-53 had the lowest grain weight and the longest spike development stage than those of other spike phenotypes.These results indicated that the different SS types have different effects on the agronomic and spike characters.Genetic analysis through bh-50/bh-51and bh-51/bh-53 F2 populations showed that a recessive major gene controlled the spike architecture to transform from NS to FRS,and a dominant major gene determined the change of spike phenotype from FRS to RS.DNA sequences of Tt BH/WFZP ortholog on chromosome 2AS revealed that a single nucleotide polymorphism（SNP）substitution happened in the open reading frame（ORF）region of all the SS tetraploid wheat accessions,which may lead to the generation of lateral meristems between glume and lemma during the immature spike development.While the fates of the lateral meristems,developing into lateral spikelets or branched spikelets,may be determined by another major gene.Our results presented here may advance our understanding and knowledge of the genes and genetic pathways determining the spike architecture development in wheat.

Dissection of environmental and physiological effects on the temperature difference between superior and inferior spikelets within a rice panicle

Temperature of a plant organ constitutes an integrative index to its eco-physiological properties and status.However,little attempt has been made to dissect the combined effect of ecological and physiological factors on the surface temperature of a plant organ such as the rice spikelet.In this study,using a deactivated plant as reference,we developed a novel comparison method to dissect the environmental and physiological effects on temperature of rice spikelet.Three japonica rice cultivars with contrasting canopy features were used as testing materials.Temperatures of flag leaf,superior and inferior spikelets and their diurnal rhythm during grain filling stage were precisely measured by a hand-held infrared thermometer.The results showed that the variation of environmental conditions within a panicle was relatively minor,posing a limited influence on temperature difference between the superior and inferior spikelet.On the other hand,it was the intrinsic physiological properties that considerably affected the spatial variations of spikelet temperature within a panicle.Chemical analysis of sucrose and starch in grains and bracts indicated that the superior spikelet is more physiologically active at photosynthetic assimilation and starch biosynthesis.Interestingly,sugar in bracts exhibited a pattern of diurnal changes similar to the source leaf but different from the sink grain,confirming that bracts are source organs for grain filling.Our findings yield penetrating insight into the eco-physiological foundation of spikelet temperature,thus being helpful for the application of physiological approaches in crop breeding for cooler canopy.

Physiological mechanism underlying spikelet degeneration in rice

The phenomenon of degenerated spikelets is very common in cereals, and considered as a serious physiological defect and a main constraint to grain production. Understanding the physiological mechanism in which spikelet degeneration occurs would have great significance in enhancing yield potential in grain crops. Taking rice as an example, the paper reviewed the physiological mechanism underlying spikelet degeneration, with focus on the roles of phytohormones in regulating the process. There are several hypotheses for the spikelet degeneration, such as resource limitation, self-organization, and primigenic dominance. However, convincing evidences are not enough to support the assumptions. Phytohormones including auxins, cytokinins, gibberellins, abscisic acid, and ethylene are involved in regulating spikelet degeneration in cereals. The new phytohormones of brassinosteroids and polyamines have been observed to suppress spikelet degeneration in rice. The interactions among or between plant hormones may play a more important role in regulating spikelet degeneration. However, the information on such interactions is very limited. Some agronomic practices, especially proper water and nitrogen management, could reduce spikelet degeneration but the mechanism underlying remains unclear. Further research is needed to understand the cross-talk among/between phytohormones on spikelet degeneration, to reveal the physiological and molecular mechanism in which phytohormones and their interactions regulate the degeneration of spikelets, to exploit approaches to decrease spikelet degeneration and to elucidate their mechanism.

Decrement of Sugar Consumption in Rice Young Panicle Under High Temperature Aggravates Spikelet Number Reduction

Two rice genotypes Huanghuazhan(HHZ, heat-resistant) and IR36(heat-susceptible) were subjected to high-temperature(HT, 40℃) and normal-temperature(NT, 32℃) treatments at the spikelet differentiation stage. HT treatment inhibited spikelet differentiation, aggravated spikelet degeneration, reduced spikelet size, and disordered carbohydrate allocation. Meanwhile, HT treatment increased nonstructural carbohydrate content in leaves, but decreased that in stems and young panicles, and the same tendencies of sucrose and starch contents were observed in leaves and stem. However, HT treatment significantly increased the sucrose content and sharply decreased the glucose and fructose contents in young panicles. Lower activity levels of soluble acid invertase(EC3.2.1.26) and sucrose synthase(EC2.4.1.13) were observed under HT treatment. Moreover, HT treatment reduced the activities of key enzymes associated with glycolysis and the tricarboxylic acid cycle, which indicated sucrose consumption was inhibited in young panicles under HT treatment. Exogenous glucose and fructose applied under HT treatment increased the spikelet number more than exogenous sucrose. In conclusion, the results demonstrated that the reduction of spikelet number under high temperature was more affected by the decrease in sugar consumption than the blocking of sucrose transport. The impairment of sucrose hydrolysis was the main reason for the inhibition of sugar utilization.

Screening for Spikelet Fertility and Validation of Heat Tolerance in a Large Rice Mutant Population

A total of 10 000 M4 individuals in Jao Hom Nil (JHN) mutant population was treated with high temperature (40 ℃ to 45 ℃) during the day time (6 h) from the booting to the harvesting stages, and ambient temperature (33 ℃ to 35℃) was used as the control. The results of screening and yield trials found that the mutant line M9962 had a high spikelet fertility of 78% under heat stress. In addition, the other mutant lines, including M3181 and M7988, had a spikelet fertility of approximately 70%. However, the JHN wild type, Sin Lek, RD15 and RD33 had very low spikelet fertility of 34%, 14%, 9% and 4%, respectively. The lower spikelet fertility at an elevated temperature resulted in a dramatic decrease of filled grain and contributed to a loss in 100-grain weight. M9962 is a potential genetic stock for use in a heat tolerance breeding programme. In addition, spikelet fertility at high temperature was representative of heat tolerance and can be used as a screening trait for heat tolerance during the reproductive phase on a large scale.

Enclosed stigma contributes to higher spikelet fertility for rice(Oryza sativa L.) subjected to heat stress

With global warming, rice plants may be subjected to heat stress more regularly during the heatsensitive flowering stage, causing spikelet sterility and grain yield loss.Stigma exsertion is considered to increase pollen reception and promote female reproductive success.The aim of this study was to investigate the role of stigma exsertion on spikelet fertility at high temperatures.Five rice cultivars(Liangyoupeijiu, Shanyou 63, Huanghuazhan, Nagina 22, and IR64) with differing degrees of stigma exsertion were cultivated and exposed to high temperature at anthesis.Heat-tolerant cultivars did not always show a high percentage of spikelets with exserted stigmas, and vice versa.Irrespective of the presence of more pollen grains on exserted stigmas, spikelets with exserted stigmas did not show greater spikelet fertility than spikelets with fewer exserted stigmas or hidden stigmas under heat stress.GA3 application augmented the percentage of spikelets with exserted stigmas;however, it did not increase spikelet fertility under heat stress.Spikelet fertility of whole panicles was negatively correlated with the percentage of spikelets with exserted stigmas, but positively with that with hidden stigmas.Viability of the hidden stigmas was less reduced than that of exserted stigmas under heat stress, suggesting that hidden stigmas have an advantage in maintaining viability.Heat stress delayed anther dehiscence and reduced the viabilities of both exserted stigmas and pollens, thereby causing low spikelet fertility.Together, these results suggest that high spikelet fertility does not depend on stigma exsertion and that enclosed stigma generally contributes to higher spikelet fertility and heat tolerance under high-temperature conditions during flowering in rice.

Temperature Difference Between the Air and Organs of Rice Plant and Its Relation to Spikelet Fertility

Based on the experiment of measuring panicles and leaves, air temperature, and humidity above the canopy of rice cultivars after heading in 2005 and 2006, we investigated the temperature difference （TD） between the air and organs of rice plant and its relationship with spikelet fertility. The results showed that TDs between the air and organs of rice varied with air temperature, air humidity, and plant type. For similar air humidity, TDs were lower at the air temperature of 28.5℃ than at higher temperature of 35.5℃, whereas for the same air temperature, the TDs decreased as the air humidity increased. TDs were also affected by plant type of the cultivars. Erect panicle cultivars showed higher TDs than those with droopy panicles under similar climatic conditions, and cultivars with panicles above flag leaf （PAFL） had higher TDs than those with panicles below the flag leaf （PBFL）. Cultivars grown in a location with lower air humidity and higher temperature, such as Taoyuan, China, had higher spikelet fertility than those in higher humidity under the similar air temperature during the grain filling stage. This is partially attributed to the larger TDs under the lower humidity. Rowspacing and the ratio of basal-tillering to panicle-spikelet fertilizer showed a significant influence on TD and subsequently on spikelet fertility, suggesting the possibility of increasing spikelet fertility by agronomic management.

Avoidance of Linkage Drag Between Blast Resistance Gene and the QTL Conditioning Spikelet Fertility Based on Genotype Selection Against Heading Date in Rice

Previous study showed that a linkage drag between a blast resistance gene Pi25（t） and QTLs conditioning spikelet fertility （qSF-6） and number of filled grains per panicle （qNFGP-6） was detected on the short arm of chromosome 6. A larger population was used for further verification, and the results confirmed the linkage drag between the blast resistance gene and QTL conditioning spikelet fertility, other than QTL conditioning number of filled grains per panicle. Breakdown or avoidance of the linkage drag could be achieved by selection against the genotype background of a heading-date gene （qHD-7） that resided in the region between RM2 and RM214 on chromosome 7. For further validation, two lines with almost identical genotypes on all chromosomal regions except the Pi25（t） region on chromosome 6 were chosen to develop a new population The results showed that qSF-6 could be further subdivided into qSF-6-1 and qSF-6-2. When the genotype of the region between RM2 and RM214 was from rice variety Zhong 156, the linkage drag between Pi25（t） and qSF-6-2 was detected and the allele of qSF-6-2 from rice variety Gumei 2 reduced the spikelet fertility. When the genotype of the region between RM2 and RM214 was from Gumei 2, no linkage drag was detected. This indicates that the linkage drag between the blast resistance gene and the QTL conditioning spikelet fertility could be broken down or avoided under a certain background genotype selection against heading-date and provides a marker aided solution for high level of blast resistance and yield breeding in rice and other crops as well.

Alterations of Panicle Antioxidant Metabolism and Carbohydrate Content and Pistil Water Potential Involved in Spikelet Sterility in Rice under Water-Deficit Stress

Two rice genotypes with different drought tolerance, namely Jin 23B （drought tolerant） and Zhenshan 97B （drought sensitive）, were used to study the antioxidant enzyme activities, soluble sugar and starch contents in spikelets, pistil water potential and pollen number on a stigma under water-deficit stress at the flowering stage, which were involved in the spikelet sterility. Compared with respective controls, drought stress induced more serious decreases of superoxide dismutase （SOD）, peroxidase （POD） and catalase （CAT） activities and more significant increase of malonaldehyde （MDA） content in spikekets of Zhenshan 97B than in Jin 23B on 9 and 12 days after water stress （DAWS）. The soluble sugar and starch contents increased significantly in spikelets of Jin 23B, but decreased significantly in spikelets of Zhenshan 97B during 9-12 DAWS. The pistil maintained higher water potential in Jin 23B than in Zhenshan 97B during 3-6 DAWS and 9-12 DAWS. In addition, water stress induced more significant decrease in the pollen number on a stigma as well as the percentage of unfilled grains in Zhenshan 97B than in Jin 23B. Thus, it is suggested that water stress induced spikelet sterility by damaging antioxidant enzyme activities, reducing carbohydrate content in spikelets and decreasing pistil water potential at the flowering stage in rice.

Gene mapping and candidate gene analysis of aberrant-floral spikelet 1(afs1) in rice(Oryza sativa L.)

The spikelet is a unique inflorescence structure in grasses. However, the molecular mechanism that regulates its development remains unclear, and we therefore characterize a spikelet mutant of rice(Oryza sativa L.), aberrant-floral spikelet 1(afs1), which was derived from treatment of Xinong 1 B with ethyl methanesulfonate. In the afs1 mutant, the spikelet developed an additional lemma-like organ alongside the other normally developed floral organs, and the paleae were degenerated to differing degrees with or without normally developed inner floral organs. Genetic analysis revealed that the afs1 phenotype was controlled by a single recessive gene. The AFS1 gene was mapped between the insertion/deletion(In Del) marker Indel19 and the simple sequence repeat marker RM16893, with a physical distance of 128.5 kb on chromosome 4. Using sequence analysis, we identified the deletion of a 5-bp fragment and a transversion from G to A within LOC_Os04 g32510/LAX2, which caused early termination of translation in the afs1 mutant. These findings suggest that AFS1 may be a new allele of LAX2, and is involved in the development of floral organs by regulating the expression of genes related to their development. The above results provide a new view on the function of LAX2, which may also regulate the development of spikelets.

Effects of Heat Stress at Vegetative and Reproductive Stages on Spikelet Fertility

Seed-setting rate, yield components and grain quality traits of 169 accessions from an exotic rice germplasm were tested under high temperatures from 40 oC to 45 oC for 6 h during the daytime at the vegetative and reproductive stages, respectively. The results showed that heat stress significantly decreased the seed-setting rate of all the accessions, but the heat stress effects varied among accessions. Based on the decreases in seed-setting rate at high temperatures, N22 was the most tolerant, followed by AUS17, M9962, SONALEE and AUS16. Moreover, the reductions in seed-setting rate and yield under heat stress were more serious at the vegetative stage （45 d before heading） than at the booting stage （15 d before heading）. In addition, heat stress also affected grain quality, especially by conferring chalkiness to most of the accessions, but SONALEE did not change much. The heat-tolerant accessions identified here and the phenotype protocols developed could be used in future genetic studies and breeding programmes focused on heat tolerance.

QTL mapping for grain number per spikelet in wheat using a high-density genetic map

Grain number per spikelet (GNS) is a key determinant of grain yield in wheat.A recombinant inbred line population comprising 300 lines was developed from a cross between a high GNS variety H461 and Chinese Spring from which the reference genome assembly of bread wheat was obtained.Both parents and the recombinant inbred lines were genotyped using the wheat 55K single nucleotide polymorphism(SNP) array.A high-density genetic map containing 21,197 SNPs was obtained.These markers covered each of the 21 chromosomes with a total linkage distance of 3792.71 c M.Locations of these markers in this linkage map were highly consistent with their physical locations in the genome assembly of Chinese Spring.The two parents and the whole RIL population were assessed for GNS in two consecutive years at two different locations.Based on multi-environment phenotype data and best liner unbiased prediction values,three quantitative trait loci (QTL) for GNS were identified.One of them located on chromosomes 2B and the other two on 2D.Phenotypic variation explained by these loci varied from 3.07%to26.57%.One of these QTL,QGns.sicau-2D-2,was identified in each of all trials conducted.Based on the best linear unbiased prediction values,this locus explained 19.59%–26.57%of phenotypic variation.A KASP(Kompetitive Allele-Specific PCR) marker closely linked with this locus was generated and used to validate the effects of this locus in three different genetic backgrounds.The identified QTL and the KASP marker developed for it will be highly valuable in fine-mapping the locus and in exploiting it for markerassisted selection in wheat breeding programs.

Infection and Colonization of Pathogenic Fungus Fusarium proliferatum in Rice Spikelet Rot Disease

Rice spikelet rot disease(RSRD), caused by Fusarium proliferatum, is an emerging disease. So far, the effects of diseased rice floral organs as well as the primary infection sites and stages of this pathogen are not determined. We investigated changes in the floral organs, along with the infection processes of the pathogen in plants inoculated with F. proliferatum and labelled with a green fluorescent protein during different growth stages of rice. The results showed that RSRD is not a systemic infectious disease, which has negative effects on the fertility of the infected rice. F. proliferatum caused brown colored anthers, crinkled pistils and ovaries, pollen grain deformities and anther indehiscence. The number of pollen grains on the stigmas decreased significantly in the infected spikelets, and the anther dehiscence and seed-setting rate successively declined by 69% and 73%, respectively, as a result of the infection. The initial infection stage occurred at the pollen cell maturity stage, and the primary invasion sites were determined to be the anthers of rice. It was noted that the pathogen mainly damaged the pollen cells, and with the exception of the filaments, proceeded to colonize the pistils and endosperm.

Gene mapping and candidate gene analysis of multi-floret spikelet 3(mfs3) in rice(Oryza sativa L.)

Rice(Oryza sativa L.) is one of the most important food crops worldwide and a model monocot plant for gene function analysis, so it is an ideal system for studying flower development. This study reports a mutant, named multi-floret spikelet 3(mfs3), which is related to the spikelet development in rice and derived from the ethylmethane sulfonate(EMS)-treated rice cultivar XIDA 1 B. In mfs3, the main body of palea(bop) was degenerated severely and only glume-like marginal regions of palea(mrp) remained, while other floral organs developed normally, indicating that the palea identity was seriously influenced by the mutation. It was also observed that the number of floral organs was increased in some spikelets, including 2 lemmas, 4 mrp, 4 lodicules, 8–10 stamens, and 2 pistils, which meant that the spikelet determinacy was lost to some degree in mfs3. Furthermore, genetic analysis demonstrated that the mfs3 trait was controlled by a single recessive gene. Using 426 F2 mutants derived from the cross between sterile line 56 S and mfs3, the MULTI-FLORET SPIKELET 3(MFS3) gene was mapped between the molecular markers RM19347 and RM19352 on Chr.6, with a physical distance of 106.3 kb. Sequencing of candidate genes revealed that an 83-bp fragment loss and a base substitution occurred in the LOC_Os06 g04540 gene in the mutant, confirming preliminarily that the LOC_Os06 g04540 gene was the MFS3 candidate gene. Subsequent q PCR analysis showed that the mutation caused the down-regulation of Os MADS1 and FON1 genes, and the up-regulation of Os IDS1 and SNB genes, which are all involved in the regulation of spikelet development. The MFS3 mutation also significantly reduced the transcription of the REP gene, which is involved in palea development. These results indicated that the MFS3 gene might be involved in the spikelet meristem determinacy and palea identity by regulating the expression of these related genes.

Identification and Gene Mapping of a multi-floret spikelet 1 (mfs1) Mutant Associated with Spikelet Development in Rice

In this study, a rice spikelet mutant, multi-floret spikelet 1 （mfsl）, which was derived from ethylmethane sulfonate （EMS）- treated Jinhui 10 （Oryza sativa L. ssp. indica） exhibited pleiotropic defects in spikelet development. The mfsl spikelet displayed degenerated the empty glume, elongated the rachilla, the extra lemma-like organ and degraded the palea. Additionally, mfsl flowers produced varied numbers of inner floral organs. The genetic analysis revealed that the mutational trait was controlled by a single recessive gene. With 401 recessive individuals from the F2 segregation population, the MFS1 gene was finally mapped on chromosome 5, an approximate 350 kb region. The present study will be useful for cloning and functional analysis of MFS1, which would facilitate understanding of the molecular mechanism involved in spikelet development in rice.