Fine Mapping and Candidate Gene Analysis of qSTL3, a Stigma Length-Conditioning Locus in Rice (Oryza sativa L.)

The efficiency of hybrid seed production can be improved by increasing the percentage of exserted stigma, which is closely related to the stigma length in rice. In the chromosome segment substitute line (CSSL) population derived from Nipponbare (recipient) and Kasalath (donor), a single CSSL (SSSL14) was found to show a longer stigma length than that of Nipponbare. The difference in stigma length between Nipponbare and SSSL14 was controlled by one locus (qSTL3). Using 7,917 individuals from the SSSL14/Nipponbare F₂ population, the qSTL3 locus was delimited to a 19.8-kb region in the middle of the short arm of chromosome 3. Within the 19.8-kb chromosome region, three annotated genes (LOC_Os03g14850, LOC_Os03g14860 and LOC_Os03g14880) were found in the rice genome annotation database. According to gene sequence alignments in LOC_Os03g14850, a transition of G (Nipponbare) to A (Kasalath) was detected at the 474-bp site in CDS. The transition created a stop codon, leading to a deletion of 28 amino acids in the deduced peptide sequence in Kasalath. A T-DNA insertion mutant (05Z11CN28) of LOC_Os03g14850 showed a longer stigma length than that of wild type (Zhonghua 11), validating that LOC_Os03g14850 is the gene controlling stigma length. However, the Kasalath allele of LOC_Os03g14850 is unique because all of the alleles were the same as that of Nipponbare at the 474-bp site in the CDS of LOC_Os03g14850 among the investigated accessions with different stigma lengths. A gene-specific InDel marker LQ30 was developed for improving stigma length during rice hybrid breeding by marker-assisted selection.     

Fine mapping and candidate gene analysis of LM3, a novel lesion mimic gene in rice

A rice lesion mimic mutant, lm3, was obtained by the mutagenesis of an indica cultivar, 93-11, using γ-ray radiation. Brownish lesions appeared on the leaves of lm3 at the young seedling stage and persisted until the ripening stage. The lm3 mutant was characterised by a shorter plant height and delayed heading compared with the wild-type 93-11. A genetic analysis indicated that the lesion mimic phenotype was controlled by a single recessive gene. Using simple sequence repeat (SSR) markers, the target gene LM3 was first located between marker RM5748 and RM14906 on chromosome 3. We then developed Insertion-Deletion (InDel) markers to fine-map LM3, and the locus was localised to a 29 kb region defined by two InDel markers, In12571 and In12600. Five ORFs were predicted in the candidate region, and DNA sequencing detected a single-nucleotide polymorphism (SNP) in the coding region of LOC Os03g21900. The SNP in the fourth exon (C in 93-11; T in lm3) of LOC_Os03g21900 results in the substitution of a proline (P) with a serine (S) at the 140^th amino acid of the deduced uroporphyrinogen decarboxylase protein. We did not detect polymorphisms in the other predicted ORF regions between lm3 and 93-11. These results suggest that LOC_Os03g21900 is the most likely candidate gene for LM3.     

Fine mapping of grain length QTLs on chromosomes 1 and 7 in Basmati rice (Oryza sativa L.)

Grain dimensions (length, breadth and length/breadth ratio) are important quality attributes of Basmati rice for its high consumer acceptance. Earlier we identified two significant quantitative trait loci (QTL) intervals on chromosomes 1 and 7 for grain dimensions in Basmati rice using a population of recombinant inbred lines (RILs) from cross between Basmati variety Pusa 1121 and a short grain non-aromatic variety Pusa 1342. For fine mapping of these QTLs, 184 F₆ RILs were grown and phenotyped in the normal rice growing season at two different locations. Forty-nine new SSR markers targeting these QTL intervals were tested and nine were found polymorphic between the parents. Using revised genetic maps adding new markers, the grain length QTL qGRL1.1 on chromosome 1 was narrowed down to 108?kbp from the earlier reported 6,133?kbp. There were total 13 predicted gene models in this interval which includes the probable candidate gene for the exceptionally high grain length of Basmati variety Pusa 1121. Similarly, two tandem QTL intervals qGRL7.1 and qGRL7.2 on chromosome 7 were merged into a single one narrowed down to 2,390?kbp from the earlier reported length of 5,269?kbp. This region of chromosome 7 also has co-localized QTLs for grain breadth and length to breadth ratio. SSR markers tightly linked to the QTL at a map distance of ??0.2?cM were developed for the qGRL1.1 and qGRL7.1 loci that could be used for marker-assisted breeding. Validation of earlier published markers for the grain length gene GS3 on chromosome 3 showed no difference between Pusa 1121 and Pusa 1342, highlighting the significance of qGRL1.1 and qGRL7.1 for the extra grain length of Basmati variety Pusa 1121.     

Fine mapping and candidate gene analysis of dense and erect panicle 3, DEP3, which confers high grain yield in rice (Oryza sativa L.)

Architecture of the rice inflorescence, which is determined mainly by the morphology, number and length of primary and secondary inflorescence branches, is an important agronomical trait. In the current study, we characterized a novel dense and erect panicle (EP) mutant, dep3, derived from the Oryza sativa ssp. japonica cultivar Hwacheong treated with N-methyl-N-nitrosourea. The panicle of the dep3 mutant remained erect from flowering to full maturation, whereas the panicle of the wild type plant began to droop after flowering. The dep3 mutation also regulated other panicle characteristics, including panicle length, grain shape and grain number per panicle. Anatomical observations revealed that the dep3 mutant had more small vascular bundles and a thicker culm than wild type plants, explaining the EP phenotype. Genetic analysis indicated that the phenotype with the dense and EP was controlled by a single recessive gene, termed dep3. The DEP3 gene was identified as the candidate via a map-based cloning approach and was predicted to encode a patatin-like phospholipase A2 (PLA2) superfamily domain-containing protein. The mutant allele gene carried a 408?bp genomic deletion within LOC_Os06g46350, which included the last 47?bp coding region of the third exon and the first 361?bp of the 3??-untranslated region. Taken together, our results indicated that the patatin-like PLA2 might play a significant role in the formation of vascular bundles, and that the dep3 mutant may provide another EP resource for rice breeding programs.     

New PCR-based sequence-tagged site marker for bacterial blight resistance gene Xa38 of rice

Bacterial blight (BB), caused by Xanthomonas oryzae pv. oryzae, is a major disease of rice managed largely through the deployment of resistance genes. Xa38, a BB resistance gene identified from Oryza nivara acc. IRGC 81825, was mapped on chromosome 4L in a 38.4-kb region. The closely linked markers for this gene, identified earlier, were simple sequence repeat marker RM17499 and sequence-tagged site markers developed from loci Os04g53060 and Os04g53120. Marker Os04g53060 is dominant while the other two markers show smaller size differences difficult to resolve accurately on agarose gel. Based on gene annotation, three nucleotide binding site?Cleucine-rich repeat genes present in the target region were cloned from O. nivara and sequenced. One of the loci, LOC_Os04g53050, had a 48-base-pair deletion in O. nivara acc. IRGC 81825 compared to the cultivated rice. Primers were designed around the deletion and the resulting marker is codominant and easy to score in agarose gel. The newly designed marker co-segregated with Xa38, amplifying products of 269?bp in O. nivara and 317?bp in cultivated rice. This marker could be more useful for marker-assisted selection than ones reported earlier.     

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。     

Map-based cloning of the ERECT PANICLE 3 gene in rice

Panicle architecture in rice can have a strong influence on yield. Using N-methyl-N-nitrosourea mutagenesis, we isolated an erect panicle mutant, Hep, from Hwasunchalbyeo, a glutinous japonica rice cultivar. Genetic analysis revealed that the erect panicle phenotype was controlled by a single recessive mutation designated erect panicle 3 (ep3). Genetic mapping revealed that the ep3 mutation was located on the short arm of chromosome 2 in a 0.1 cM region delimited by the STS markers STS5803-5 and STS5803-7. The ep3 locus corresponded to 46.8 kb region and contained six candidate genes. Comparison of the DNA sequences of the candidate genes from wild-type and erect panicle plants revealed a single base-pair change in the second exon of LOC_Os02g15950, which is predicted to result in a nonsense mutation. LOC_Os02g15950 encodes a putative F-box protein containing 515 amino acids and is expressed throughout the plant during all growth stages. A line carrying a T-DNA insertion in LOC_ Os02g15950 was obtained and shown to have the same phenotype as the ep3 mutant, thus confirming the identification of LOC_Os02g15950 as the ERECT PANICLE 3 (EP3) gene. The ep3 mutation causes a significant increase in the number of small vascular bundles as well as the thickness of parenchyma in the peduncle, which results in the erect panicle phenotype.     

Identification and Characterization of a Novel Yellow Leaf Mutant <i>yl1</i> in Rice

Leaf-color mutants play an important role in the study of chlorophyll metabolism, chloroplast development, and photosynthesis system. In this study, the yellow leaf 1 (yl1) rice mutant was identified from the ethyl methane sulfonate-treated mutant progeny of Lailong, a glutinous japonica rice landrace cultivated in Guizhou Province, China. Results showed that yl1 exhibited yellow leaves with decreased chlorophyll content throughout the growth period. Chloroplast development in the yl1 mutant was disrupted, and the grana lamellae was loosely packed and disordered. RNA sequencing and real-time quantitative polymerase chain reaction (qRT-PCR) analysis revealed that the chlorophyll synthesis-related genes OsCHLH, OsCHLM, OsCHLG, PORB, and YGL8, as well as the chloroplast development-related genes FtsZ, OsRpoTp, and RbcL, were down-regulated in the yl1 mutant. Genetic analysis revealed that the yellow leaf phenotype of yl1 was controlled by recessive nuclear gene. By employing the MutMap method, the mutation responsible for the phenotype was mapped to a 6.17 Mb region between 17.34 and 23.51 Mb on chromosome 3. Two non-synonymous single-nucleotide polymorphisms (SNPs) located in the gene locus LOC_Os03g31210 and LOC_Os03g36760 were detected in this region. The two SNPs were further confirmed by PCR and Sanger sequencing. The expression patterns of the two candidate genes indicated that LOC_Os03g36760 showed greater potential for functional verification. Subcellular protein localization revealed that the encoded product of LOC_Os03g36760 was localized in the nucleus, cytoplasm, and plasma membrane. These results will be useful for further characterization and cloning of the yl1 gene, and for research on the molecular mechanisms controlling biogenesis and chloroplast biochemical processes.     

Candidate genes and molecular markers associated with brown planthopper (<Emphasis Type="Italic">Nilaparvata lugens</Emphasis> Stål) resistance in rice cultivar Rathu Heenati

Brown planthopper (BPH) is a destructive insect pest of rice and causes severe yield loss. In attempts to develop a BPH-resistant rice variety, Rathu Heenati (RH), a rice cultivar with a strong BPH resistance, has been used as the donor in breeding programs. Quantitative trait loci analysis was conducted for the area under the curve of BPH damage scores of a backcross (BC₃F₅) population infested by six different BPH populations. Single nucleotide polymorphism (SNP) markers on chromosome 4, i.e., LecRK2-SNP and LecRK3-SNP, and markers on chromosome 6, i.e., Bph32-SNP and SSR23, were identified to be associated with resistance against five BPH populations. To identify genes on chromosome 6 that are involved in BPH resistance, expression analysis was conducted for genes located in the genomic region of Bph32-SNP and SSR23. Genes that showed differential expression ofRH at 24 h after BPH infestation, when compared to an RH control, were identified. Those that encode proteins putatively involved in the BPH resistance mechanism are LOC_Os06g03240, LOC_Os06g03380, LOC_Os06g03486, LOC_Os06g03514, LOC_Os06g03520, LOC_Os06g03610, LOC_Os06g03676, and LOC_Os06g03890. SNP markers were developed from several differentially expressed genes and were validated by genotyping in the backcross population. The SNP marker developed from LOC_Os06g03514 showed the highest association with BPH resistance and the gene may be involved in the BPH resistance mechanism. This SNP marker will be useful in breeding programs for BPH resistance.     

Genetic and Cytological Analysis of a Novel Type of Low Temperature-Dependent Intrasubspecific Hybrid Weakness in Rice

Hybrid weakness (HW) is an important postzygotic isolation which occurs in both intra- and inter-specific crosses. In this study, we described a novel low temperature-dependent intrasubspecific hybrid weakness in the F₁ plants derived from the cross between two indica rice varieties Taifeng A and V1134. HW plants showed growth retardation, reduced panicle number and pale green leaves with chlorotic spots. Cytological assay showed that there were reduced cell numbers, larger intercellular spaces, thicker cell walls, and abnormal development of chloroplast and mitochondria in the mature leaves from HW F₁ plants in comparison with that from both of the parental lines. Genetic analysis revealed that HW was controlled by two complementary dominant genes Hw3 from V1134 and Hw4 from Taifeng A. Hw3 was mapped in a 136 kb interval between the markers Indel1118 and Indel1117 on chromosome 11, and Hw4 was mapped in the region of about 15 cM between RM182 and RM505 on chromosome 7, respectively. RT-PCR analysis revealed that only LOC_Os11g44310, encoding a putative calmodulin-binding protein (OsCaMBP), differentially expressed among Taifeng A, V1134 and their HW F₁. No recombinant was detected using the markers designed based on the sequence of LOC_Os11g44310 in the BC₁F₂ (Taifeng A//Taifeng A/V1134) population. Hence, LOC_Os11g44310 was probably the candidate gene of Hw3. Gene amplification suggested that LOC_Os11g44310 was present in V1134 and absent in Taifeng A. BLAST search revealed that LOC_Os11g44310 had one copy in the japonica genomic sequence of Nipponbare, and no homologous sequence in the indica reference sequence of 9311. Our results indicate that Hw3 is a novel gene for inducing hybrid weakness in rice.     

Subcellular localization of proteins of Oryza sativa L. in the model tobacco and tomato plants

The cellular localization and molecular interactions are indicative of functions of a protein. The development of a simple and efficient method for subcellular localization of a protein is indispensable to elucidate gene function in plants. In this study, we assessed the feasibility of Agrobacterium-mediated transformation (agroinfiltration) of tobacco and tomato leaf tissue to follow intracellular targeting of proteins from rice fused to green fluorescent protein (GFP). For this, a simple in planta assay for subcellular localization of rice proteins in the heterologous host systems of tobacco and tomato leaf via transient transformation was developed. We have tested the applicability of this method by expressing GFP fusions of the putative antiphagocytic protein 1 (APP1) (OsAPP, LOC_Os03g56930) and ZOS3-18-C2H2 zinc-finger protein (OsZF1, LOC_Os03g55540) from Oryza sativa L. subsp. japonica in tobacco and tomato leaf tissues. Our results demonstrate the suitability of GFP as a reporter in gene expression studies in tomato cv. MicroTom. The use of GFP-fused proteins from rice for subcellular targeting in the heterologous hosts of tobacco and tomato plant systems has been confirmed.Key words: agroinfiltration, confocal microscopy, GFP fusion protein, tomato cv, microtom     

A novel QTL <Emphasis Type="Italic">qSPP2.2</Emphasis> controlling spikelet per panicle identified from <Emphasis Type="Italic">Oryza longistaminata</Emphasis> (A. Chev. et Roehr.), mapped and transferred to <Emphasis Type="Italic">Oryza sativa</Emphasis> (L.)

Increasing the rice productivity from the current 10 to 12 tons/ha to meet the demand of estimated 8.8 billion people in 2035 is posing a major challenge. Wild relatives of rice contain some novel genes which can help in improving rice yield. Spikelet per panicle (SPP) is a valuable trait for determining yield potential in rice. In this study, a major QTL for increasing SPP has been identified, mapped, and transferred from African wild rice O. longistaminata to O. sativa (L.). The QTL was mapped on the long arm of chromosome 2 in a 167.1 kb region flanked by SSR markers RM13743 and RM13750, which are 1.0 cM apart, and is designated as qSPP2.2. The QTL explained up to 30% of phenotypic variance in different generations/seasons and showed positive additive effect of allele contributed by O. longistaminata. In addition, O. longistaminata allele in qSPP2.2 contributed to increase in grains per panicle, but decrease in the tillers per plant. The 167.1 kb region contains 23 predicted genes. Based on the functional annotation, three genes, LOC_Os02g44860, LOC_Os02g44990, and LOC_Os02g45010, were selected as putative candidates for characterization. Sequence analysis of the three genes revealed functional variations between the parental lines for LOC_Os02g44990 and a variation in 5′UTR for LOC_Os02g45010 which will help further to identify putative candidate gene(s). This is the first yield component QTL to be identified, mapped, and transferred from O. longistaminata.     

水稻蒸煮品质相关QTL定位及候选基因分析

挖掘与稻米蒸煮品质相关的数量性状基因座(quantitative trait locus, QTL)，分析候选基因，并通过遗传育种手段改良稻米蒸煮品质相关性状，可有效提升稻米的口感。以籼稻华占(Huazhan, HZ)、粳稻热研2号(Nekken2)及由其构建的120个重组自交系(recombinant inbred lines, RILs)群体为实验材料，测定成熟期稻米的糊化温度(gelatinization temperature, GT)、胶稠度(gel consistency, GC)和直链淀粉含量(amylose content, AC)。结合高密度分子遗传图谱进行QTL定位，共检测到26个与稻米蒸煮品质相关的QTLs (糊化温度相关位点1个、胶稠度相关位点13个、直链淀粉含量相关位点12个)，其中最高奇数的可能性(likelihood of odd, LOD)值达30.24。通过实时荧光定量PCR (quantitative real-time polymerase chain reaction, qRT-PCR)分析定位区间内候选基因的表达量，发现6个基因在双亲间的表达量差异显著，推测LOC_Os04g20270和LOC_Os11g40100的高表达可能会极大地提高稻米的胶稠度，而LOC_Os01g04920和LOC_Os02g17500的高表达以及LOC_Os03g02650和LOC_Os05g25840的低表达有助于降低直链淀粉含量。这些结果为培育优质水稻新品种奠定了分子基础，并为揭示稻米蒸煮品质的分子调控机制提供了重要的遗传资源。     

Fine mapping and identification of a novel locus <Emphasis Type="Italic">qGL12.2</Emphasis> control grain length in wild rice (<Emphasis Type="Italic">Oryza rufipogon</Emphasis> Griff.)

Key message

A wild rice QTL qGL12.2 for grain length was fine mapped to an 82-kb interval in chromosome 12 containing six candidate genes and none was reported previously.

Abstract

Grain length is an important trait for yield and commercial value in rice. Wild rice seeds have a very slender shape and have many desirable genes that have been lost in cultivated rice during domestication. In this study, we identified a quantitative trait locus, qGL12.2, which controls grain length in wild rice. First, a wild rice chromosome segment substitution line, CSSL41, was selected that has longer glume and grains than does the Oryza sativa indica cultivar, 9311. Next, an F₂ population was constructed from a cross between CSSL41 and 9311. Using the next-generation sequencing combined with bulked-segregant analysis and F₃ recombinants analysis, qGL12.2 was finally fine mapped to an 82-kb interval in chromosome 12. Six candidate genes were found, and no reported grain length genes were found in this interval. Using scanning electron microscopy, we found that CSSL41 cells are significantly longer than those of 9311, but there is no difference in cell widths. These data suggest that qGL12.2 is a novel gene that controls grain cell length in wild rice. Our study provides a new genetic resource for rice breeding and a starting point for functional characterization of the wild rice GL gene.

     

Rice ORMDL Controls Sphingolipid Homeostasis Affecting Fertility Resulting from Abnormal Pollen Development

The orosomucoids (ORM) are ER-resisdent polypeptides encoded by ORM and ORMDL (ORM-like) genes. In humans, ORMDL3 was reported as genetic risk factor associated to asthma. In yeast, ORM proteins act as negative regulators of sphingolipid synthesis. Sphingolipids are important molecules regulating several processes including stress responses and apoptosis. However, the function of ORM/ORMDL genes in plants has not yet been reported. Previously, we found that temperature sensitive genetic male sterility (TGMS) rice lines controlled by tms2 contain a deletion of about 70 kb in chromosome 7. We identified four genes expressed in panicles, including an ORMDL ortholog, as candidates for tms2. In this report, we quantified expression of the only two candidate genes normally expressed in anthers of wild type plants grown in controlled growth rooms for fertile and sterile conditions. We found that only the ORMDL gene (LOC_Os07g26940) showed differential expression under these conditions. To better understand the function of rice ORMDL genes, we generated RNAi transgenic rice plants suppressing either LOC_Os07g26940, or all three ORMDL genes present in rice. We found that the RNAi transgenic plants with low expression of either LOC_Os07g26940 alone or all three ORMDL genes were sterile, having abnormal pollen morphology and staining. In addition, we found that both sphingolipid metabolism and expression of genes involved in sphingolipid synthesis were perturbed in the tms2 mutant, analogous to the role of ORMs in yeast. Our results indicated that plant ORMDL proteins influence sphingolipid homeostasis, and deletion of this gene affected fertility resulting from abnormal pollen development.     

Phenotypic and Candidate Gene Analysis of a New Floury Endosperm Mutant (osagpl2-3) in Rice

A floury endosperm mutant, osagpl2-3, was isolated from the M₂ generation of japonica rice cultivar Nipponbare following ethyl methane sulfonate mutagenesis. The osagpl2-3 mutant produced a white-core endosperm compared to the transparent endosperm of the wild type (WT). The results from scanning electron microscope showed that the osagpl2-3 mutant grains comprised of round and loosely packed starch granules, some of which were compounded. The analysis for cooking and nutrition quality traits indicated that the values of gel consistency, gelatinization temperature, and rapid viscosity analysis profile of osagpl2-3 grains were lower than those of the WT. Besides, the protein content, the contents of nine different amino acids, and the thermodynamic parameters of T _p and ??T _1/2 in osagpl2-3 were also different from those of the WT. Genetic analysis revealed that osagpl2-3 mutation was controlled by a single recessive gene. The osagpl2-3 gene was mapped between InDel markers R1M30 and ID1-12 on rice chromosome 1. In the candidate region of the Nipponbare genome, an annotated gene, LOC_Os01g44220 which encodes a large subunit of putative ADP-glucose pyrophosphrylase named OsAPL2 was considered the optimal candidate. Cloning and sequencing of LOC_Os01g44220 in different plants of the osagpl2-3 mutants revealed a single nucleotide mutation (G??A) in the open reading frame region, which led to a substitution of an acidic amino acid Glu (E) by a basic amino acid Lys (K) accordingly. Furthermore, the mutant site is close to the functional domain which interacts with the ADP-Glc. In brief, these results suggested that the osagpl2-3 is a new mutant of OsAPL2.