Huazhong Guan

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.

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.

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.

Genetic analysis and gene mapping for a salt tolerant mutant at seedling stage in rice: Genetic analysis and gene mapping for a salt tolerant mutant at seedling stage in rice

A salt tolerant mutant at seedling stage was obtained from an M2 population of radiation mutagenesis of an indica rice cultivar R401. The mutant seedlings could survive under the treatment of sodium chloride solution at the concentration of 150 mmol/L, while the wild-type control seedlings withered and died. An F2 population was developed from a cross between a japonica cultivar Nipponbare and the salt tolerant mutant. By investigating the performance of the F2 population under the stress of 150 mmol/L NaCl solution, we found that the mutant phenotype was caused by the recessive mutation of a single gene, temporarily designated SST(t). Bulked segregant analysis (BSA) based on the F2 mapping population revealed that SST(t) is located on chromosome 6. By analyzing 137 typical salt-tolerant F2 plants using molecular markers, SST(t) was mapped in a 2.3 cM (or 406 kb) interval between InDel markers ID26847 and ID27253, with genetic distances of 1.2 cM and 1.1 cM to the two markers, respectively.

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.

Genetic analysis and gene mapping of DDF1 , a pleiotropic gene involving in both vegetable and reproductive growth in rice: Genetic analysis and gene mapping of DDF1 , a pleiotropic gene involving in both vegetable and reproductive growth in rice

There are many pleiotropic genes playing key roles in regulating both vegetative growth and reproductive development in plants. A dwarf mutant of rice with deformed flowers, named as ddf1, was identified from indica rice breeding lines. Genetic analysis indicated that ddf1 was resulted from the recessive mutation of a single gene, temporarily named as DDF1. This result suggested that DDF1 is a pleiotropic gene, which controls both vegetative growth and reproductive development in rice. To map this gene, an F2 population was developed by crossing the ddf1 heterozygote with the tropical japonica rice variety DZ60. By means of bulked segregant analysis and small population-based linkage analysis using the published RM-series rice SSR markers, DDF1 was preliminarily mapped in a region between markers RM588 and RM587 on chromosome 6 with the genetic distances of 3.8 cM and 2.4 cM to the two markers, respectively. By developing new SSR markers in this interval according to the published rice genome sequence, we further mapped DDF1 in a 165 kb interval. The results will facilitate cloning of DDF1.