Yuanlin Duan

Characterization of Osmads6-5, a null allele, reveals that OsMADS6 is a critical regulator for early flower development in rice (Oryza sativa L.)

AGL6-clade genes are a subfamily of MADS-box genes and preferentially expressed in floral organs. OsMADS6 and OsMADS17 are two AGL6-like genes in rice. OsMADS17 has been shown to play a minor role in floral development and appears to result from a duplication of OsMADS6. OsMADS6 was initially named as MFO1 for mosaic floral organs based on its moderate mutant phenotypes. So far, four moderate or weak mutant alleles of OsMADS6 have been described, providing valuable insights into its role in flower development. Here, we report a null allele of OsMADS6 (Osmads6-5), which exhibited a strong mutant phenotype in spikelet without affecting vegetative traits, causing all floral organs except lemma homeotically transformed into lemma-like organs (LLOs) as well as an indeterminate floral meristem, thus resulting in a mutant floret consisting of reiterating whorls of lemma and LLOs. In consistently, over-expression of OsMADS6 led to additional lodicule-, stamen- and carpel-like organs. Expression analysis showed that OsMADS6 controls the formation of the incipient primordia of lodicule, stamen and carpel via regulating the expression of class B, C and SEP-like MADS-box genes. Taken together, our results revealed that OsMADS6 acts as a critical regulator for early flower development in rice and provide novel insights into the molecular mechanism of OsMADS6.

Genetic analysis and mapping of gene fzp(t) controlling spikelet differentiation in rice

A mutant of spikelet differentiation in rice called frizzle panicle (fzp) was discovered in the progeny of a cross between Oryza sativa ssp. indica cv. V20B and cv. Hua1B. The mutant exhibits normal plant morphology but has apparently fewer tillers. The most striking change in fzp is that its spikelet differentiation is completely blocked, with unlimited subsequent rachis branches generated from the positions where spikelets normally develop in wild-type plants. Genetic analysis suggests that fzp is controlled by a single recessive gene, which is temporarily named fzp(t). Based on its mutant phenotype, fzp(t) represents a key gene controlling spikelet differentiation. Some F2 mutant plants derived from various genetic background appeared as the “middle type”, suggesting that the action of fzp(t) is influenced by the presence of redundant, modifier or interactive genes. By using simple sequence repeat (SSR) markers and bulked segregant analysis (BSA) method, fzp(t) gene was mapped in the terminal region of the long arm of chromosome 7, with RM172 and RM248 on one side, 3.2 cM and 6.4 cM from fzp(t), and RM18 and RM234 on the other side, 23.1 cM and 26.3 cM from fzp(t), respectively. These results will facilitate the positional cloning and function studies of the gene.

Dwarf and deformed flower 1, encoding an F-box protein, is critical for vegetative and floral development in rice (Oryza sativa L.)

Recent studies have shown that F-box proteins constitute a large family in eukaryotes, and play pivotal roles in regulating various developmental processes in plants. However, their functions in monocots are still obscure. In this study, we characterized a recessive mutant dwarf and deformed flower 1-1 (ddf1-1) in Oryza sativa (rice). The mutant is abnormal in both vegetative and reproductive development, with significant size reduction in all organs except the spikelet. DDF1 controls organ size by regulating both cell division and cell expansion. In the ddf1-1 spikelet, the specification of floral organs in whorls 2 and 3 is altered, with most lodicules and stamens being transformed into glume-like organs and pistil-like organs, respectively, but the specification of lemma/palea and pistil in whorls 1 and 4 is not affected. DDF1 encodes an F-box protein anchored in the nucleolus, and is expressed in almost all vegetative and reproductive tissues. Consistent with the mutant floral phenotype, DDF1 positively regulates B-class genes OsMADS4 and OsMADS16, and negatively regulates pistil specification gene DL. In addition, DDF1 also negatively regulates the Arabidopsis LFY ortholog APO2, implying a functional connection between DDF1 and APO2. Collectively, these results revealed that DDF1, as a newly identified F-box gene, is a crucial genetic factor with pleiotropic functions for both vegetative growth and floral organ specification in rice. These findings provide additional insights into the molecular mechanism controlling monocot vegetative and reproductive development.

Molecular cloning and functional characterization of OsJAG gene based on a complete-deletion mutant in rice (Oryza sativa L.).

In this article, we report an independent work of positional cloning and functional characterization of OsJAG gene in rice. The merit of our work is that we used a genuine null mutant, in which the wild-type allele was completely deleted. This allowed us to identify the mutant phenotypes accurately without the interference of residual function of the target gene. OsJAG is an important gene with pleiotropy, expressing almost throughout the plant and acting in both vegetative phase and reproductive phase. But its main and crucial roles are in regulating the development of all floral organs, especially in specifying the identity of stamens. Interestingly, OsJAG does not affect the number of floral organ primordial and so of floral organs in each whorl, suggesting that OsJAG does not influence the initiation of floral organ primordia, but affect the developmental fate of all floral organs after their primordia have initiated. Loss of OsJAG function results in maldevelopment of all floral organs, such as degenerated lemma and palea, elongated lodicules and deformed and sterile pistil. The stamen appears to be more sensitive to the mutation. All the six stamens in a mutant floret were thoroughly transformed into six pistil-like organs developed at the presumptive positions of the stamens in whorl 3.

Genetic analysis and gene mapping ofleafy head (lhd), a mutant blocking the differentiation of rachis branches in rice (Oryza sativa L.)

A rice mutant calledleafy head (lhd), in which the differentiation of rachis branches is blocked, was identified in a doubled haploid (DH) population derived through F1 anther culture from a cross between rice (Oryza sativa L.) indica cultivar Gui-630 and japonica cultivar Taiwanjing. The mutant is shorter in plant height, possessing smaller and clumpy leaves, and always stays at the vegetative growth stage. Genetic analysis suggests thatlhd is controlled by a single recessive gene, which is temporarily namedlhd(t). The phenotype of the mutant suggests thatLHD(t) is a key gene controlling the differentiation of rachis branches. In order to map the gene, two F2 populations were constructed by crossing thelhd heterozygote with varieties Minghui-77 (indica) and Jinghua-8 (japonica). In the F2 oflhd heterozygote × Jinghua-8, some mutant plants appeared as the “medium type”, suggesting that the lhd phenotype could be influenced by genetic backgrounds. With the published SSR markers of RM series and additional SSR markers developed by ourselves and using the methods of bulked segregant analysis (BSA) and mutant analysis (with 498 mutant plants in total),LHD(t) gene was mapped onto the distal region of the long arm of chromosome 10. Markers SSR1, RM269, RM258, RM304 and RM171 were located on one side with distances of 6.4, 16.6, 18.4, 22.2 and 26.3 cM toLHD(t); whereas markers SSR4 and SSR5 were on the other side with distances of 0.6 and 2.2 cM toLHD(t). The results will facilitate the positional cloning and functional study of theLHD(t) gene.

Fine Mapping of qBlsr5a, a QTL Controlling Resistance to Bacterial Leaf Streak in Rice

The quantitative trait locus (QTL) qBlsr5a on the short arm of rice (Oryza sativa L.) chromosome 5 has been proved to have the largest effect on the resistance to rice bacterial leaf streak (Xanthomonas oryzae pv. oryzicola, BLS). Using Acc8558 (highly resistant to BLS) as the donor and H359 (highly susceptible to BLS) as the recipient, a near-isogenic line (NIL) H359-BLSR5a was developed through backcross and only qBlsr5a was introgressed from the donor parent. A big F2 population (2,265 individuals) was constructed by hybridizing the NILs with H359 and 120 individuals with extreme phenotypes (lesion length < 2 cm) were selected. Eighty-five out of the 120 individuals were identified as homozygous resistant genotypes at the target QTL after examining their progeny lines (F2:3). By genotyping these homozygous individuals with SSR markers and performing linkage analysis, qBlsr5a was mapped to an interval between SSR markers RM153 and RM159, which covered a range of 2.4 cM or 290 kb.

Toward the positional cloning of qBlsr5a, a QTL underlying resistance to bacterial leaf streak, using overlapping sub-CSSLs in rice.

Bacterial leaf steak (BLS) is one of the most destructive diseases in rice. Studies have shown that BLS resistance in rice is quantitatively inherited, controlled by multiple quantitative trait loci (QTLs). A QTL with relatively large effect, qBlsr5a, was previously mapped in a region of ∼380 kb on chromosome 5. To fine map qBlsr5a further, a set of overlapping sub-chromosome segment substitution lines (sub-CSSLs) were developed from a large secondary F2 population (containing more than 7000 plants), in which only the chromosomal region harboring qBlsr5a was segregated. By genotyping the sub-CSSLs with molecular markers covering the target region and phenotyping the sub-CSSLs with artificial inoculation, qBlsr5a was delimited to a 30.0-kb interval, in which only three genes were predicted. qRT-PCR analysis indicated that the three putative genes did not show significant response to the infection of BLS pathogen in both resistant and susceptible parental lines. However, two nucleotide substitutions were found in the coding sequence of gene LOC_Os05g01710, which encodes the gamma chain of transcription initiation factor IIA (TFIIAγ). The nucleotide substitutions resulted in a change of the 39th amino acid from valine (in the susceptible parent) to glutamic acid (in the resistant parent). Interestingly, the resistant parent allele of LOC_Os05g01710 is identical to xa5, a major gene resistant to bacterial leaf blight (another bacterial disease of rice). These results suggest that LOC_Os05g01710 is very possibly the candidate gene of qBlsr5a.

Knockdown of NtMed8, a Med8-like gene, causes abnormal development of vegetative and floral organs in tobacco (Nicotiana tabacum L.)

Med8, a subunit of mediator complex, has proved to possess crucial functions in many organisms from yeast to human. In plant, the med8 mutant of Arabidopsis thaliana displayed delayed anthesis and increased number of leaves during the vegetative period. However, the roles of Med8 in other flowering plants are still unknown. To investigate the function of Med8 ortholog in tobacco (Nicotiana tabacum L.; named as NtMed8), we created transgenic tobacco plants with repressed NtMed8 expression mediated by RNA interference (RNAi). Compared with the wild type, the NtMed8-RNAi plants exhibited: more leaves with smaller but thicker blades; larger cells and vascular bundles with lower stomata density in leaves; swelled chloroplasts with thicker and lumen-enlarged thylakoids; weaker root system with fewer lateral roots; larger flowers and floral organs; flowering earlier under long day, but later under short day conditions; and male sterile with larger but less germinable pollens. In addition, quantitative RT-PCR indicated that NtMed8 is expressed in both vegetative and floral tissues. Subcellular localization analysis by transient expression of fusion protein in Nicotiana benthamiana leaves showed that NtMed8 was located in both plasma membrane and nucleus. These results suggest that NtMed8 plays important roles in both vegetative and reproductive development, and the function of Med8 appears to be, at least partially, conserved in flowering plants.

Genetic Analysis and Mapping of an Enclosed Panicle Mutant Locus esp1 in Rice (Oryza sativa L.)

Abstract A mutant was isolated from the M2 of 60Co-γ ray mutagenized male-fertility restorer line Zao-R974 in rice. The mutant showed pleiotropic phenotypes including dwarfism, delayed heading time, short and partially enclosed panicles, short uppermost internode, decreased grain and secondary branch numbers per panicle, and partially degenerated spikelets. The mutant was named as esp1 (enclosed shorter panicle 1). Genetic analysis indicated that the mutant phenotype was controlled by a recessive locus. Spraying exogenous GA3 did not rescue the panicle enclosure. Using an F2 and a BC1 population of the cross between esp1 and a japonica cultivar Nipponbare, we mapped the ESP1 locus to a region of ∼260 kb on chromosome 11. This result provides a basis for further map-based cloning of the ESP1 locus.

Fine mapping and candidate identification of SST, a gene controlling seedling salt tolerance in rice (Oryza sativa L.)

Using a recessive mutant with enhanced salt tolerance at seedling stage obtained from an indica rice cultivar R401 by gamma-ray irradiation, a novel gene controlling salt tolerance in rice was previously mapped to a 406-kb region on chromosome 6. We named the gene Seedling Salt Tolerance (SST). In this study, with a large F2 population derived from a cross between mutant sst and a japonica cultivar Nipponbare (salt sensitive), SST was further fine mapped to a 17-kb interval between InDel markers ID27101 and ID27118, in which only one gene (OsSPL10) was predicted. Sequencing analysis indicated that the 232nd base of the coding sequence of OsSPL10 was deleted in the sst allele, resulting in a frameshift mutation. The result strongly suggested that OsSPL10 should be the candidate of SST. OsSPL10 is a member of the SBP-box gene family. This is the first time that the SBP-box gene family is found to be probably involved in the regulation of seedling salt tolerance in plant.