Huqu Zhai

Isolation and initial characterization of GW5, a major QTL associated with rice grain width and weight

Grain weight is a major determinant of crop grain yield and is controlled by naturally occurring quantitative trait loci (QTLs). We earlier identified a major QTL that controls rice grain width and weight, GW5, which was mapped to a recombination hotspot on rice chromosome 5. To gain a better understanding of how GW5 controls rice grain width, we conducted fine mapping of this locus and uncovered a 1 212-bp deletion associated with the increased grain width in the rice cultivar Asominori, in comparison with the slender grain rice IR24. In addition, genotyping analyses of 46 rice cultivars revealed that this deletion is highly correlated with the grain-width phenotype, suggesting that the GW5 deletion might have been selected during rice domestication. GW5 encodes a novel nuclear protein of 144 amino acids that is localized to the nucleus. Furthermore, we show that GW5 physically interacts with polyubiquitin in a yeast two-hybrid assay. Together, our results suggest that GW5 represents a major QTL underlying rice width and weight, and that it likely acts in the ubiquitin-proteasome pathway to regulate cell division during seed development. This study provides novel insights into the molecular mechanisms controlling rice grain development and suggests that GW5 could serve as a potential tool for high-yield breeding of crops.

D14–SCF D3 -dependent degradation of D53 regulates strigolactone signalling

Strigolactones (SLs), a newly discovered class of carotenoid-derived phytohormones, are essential for developmental processes that shape plant architecture and interactions with parasitic weeds and symbiotic arbuscular mycorrhizal fungi. Despite the rapid progress in elucidating the SL biosynthetic pathway, the perception and signalling mechanisms of SL remain poorly understood. Here we show that DWARF 53 (D53) acts as a repressor of SL signalling and that SLs induce its degradation. We find that the rice (Oryza sativa) d53 mutant, which produces an exaggerated number of tillers compared to wild-type plants, is caused by a gain-of-function mutation and is insensitive to exogenous SL treatment. The D53 gene product shares predicted features with the class I Clp ATPase proteins and can form a complex with the α/β hydrolase protein DWARF 14 (D14) and the F-box protein DWARF 3 (D3), two previously identified signalling components potentially responsible for SL perception. We demonstrate that, in a D14- and D3-dependent manner, SLs induce D53 degradation by the proteasome and abrogate its activity in promoting axillary bud outgrowth. Our combined genetic and biochemical data reveal that D53 acts as a repressor of the SL signalling pathway, whose hormone-induced degradation represents a key molecular link between SL perception and responses.

DTH8 Suppresses Flowering in Rice, Influencing Plant Height and Yield Potential Simultaneously

The three most important agronomic traits of rice (Oryza sativa), yield, plant height, and flowering time, are controlled by many quantitative trait loci (QTLs). In this study, a newly identified QTL, DTH8 (QTL for days to heading on chromosome 8), was found to regulate these three traits in rice. Map-based cloning reveals that DTH8 encodes a putative HAP3 subunit of the CCAAT-box-binding transcription factor and the complementary experiment increased significantly days to heading, plant height, and number of grains per panicle in CSSL61 (a chromosome segment substitution line that carries the nonfunctional DTH8 allele) with the Asominori functional DTH8 allele under long-day conditions. DTH8 is expressed in most tissues and its protein is localized to the nucleus exclusively. The quantitative real-time PCR assay revealed that DTH8 could down-regulate the transcriptions of Ehd1 (for Early heading date1) and Hd3a (for Heading date3a; a rice ortholog of FLOWERING LOCUS T) under long-day conditions. Ehd1 and Hd3a can also be down-regulated by the photoperiodic flowering genes Ghd7 and Hd1 (a rice ortholog of CONSTANS). Meanwhile, the transcription of DTH8 has been proved to be independent of Ghd7 and Hd1, and the natural mutation of this gene caused weak photoperiod sensitivity and shorter plant height. Taken together, these data indicate that DTH8 probably plays an important role in the signal network of photoperiodic flowering as a novel suppressor as well as in the regulation of plant height and yield potential.

Brassinosteroids Regulate Grain Filling in Rice

Genes controlling hormone levels have been used to increase grain yields in wheat (Triticum aestivum) and rice (Oryza sativa). We created transgenic rice plants expressing maize (Zea mays), rice, or Arabidopsis thaliana genes encoding sterol C-22 hydroxylases that control brassinosteroid (BR) hormone levels using a promoter that is active in only the stems, leaves, and roots. The transgenic plants produced more tillers and more seed than wild-type plants. The seed were heavier as well, especially the seed at the bases of the spikes that fill the least. These phenotypic changes brought about 15 to 44% increases in grain yield per plant relative to wild-type plants in greenhouse and field trials. Expression of the Arabidopsis C-22 hydroxylase in the embryos or endosperms themselves had no apparent effect on seed weight. These results suggested that BRs stimulate the flow of assimilate from the source to the sink. Microarray and photosynthesis analysis of transgenic plants revealed evidence of enhanced CO2 assimilation, enlarged glucose pools in the flag leaves, and increased assimilation of glucose to starch in the seed. These results further suggested that BRs stimulate the flow of assimilate. Plants have not been bred directly for seed filling traits, suggesting that genes that control seed filling could be used to further increase grain yield in crop plants.

A Chlorophyll-Deficient Rice Mutant with Impaired Chlorophyllide Esterification in Chlorophyll Biosynthesis

Chlorophyll (Chl) synthase catalyzes esterification of chlorophyllide to complete the last step of Chl biosynthesis. Although the Chl synthases and the corresponding genes from various organisms have been well characterized, Chl synthase mutants have not yet been reported in higher plants. In this study, a rice (Oryza Sativa) Chl-deficient mutant, yellow-green leaf1 (ygl1), was isolated, which showed yellow-green leaves in young plants with decreased Chl synthesis, increased level of tetrapyrrole intermediates, and delayed chloroplast development. Genetic analysis demonstrated that the phenotype of ygl1 was caused by a recessive mutation in a nuclear gene. The ygl1 locus was mapped to chromosome 5 and isolated by map-based cloning. Sequence analysis revealed that it encodes the Chl synthase and its identity was verified by transgenic complementation. A missense mutation was found in a highly conserved residue of YGL1 in the ygl1 mutant, resulting in reduction of the enzymatic activity. YGL1 is constitutively expressed in all tissues, and its expression is not significantly affected in the ygl1 mutant. Interestingly, the mRNA expression of the cab1R gene encoding the Chl a/b-binding protein was severely suppressed in the ygl1 mutant. Moreover, the expression of some nuclear genes associated with Chl biosynthesis or chloroplast development was also affected in ygl1 seedlings. These results indicate that the expression of nuclear genes encoding various chloroplast proteins might be feedback regulated by the level of Chl or Chl precursors.

Quantitative Trait Loci (QTL) Analysis For Rice Grain Width and Fine Mapping of an Identified QTL Allele gw-5 in a Recombination Hotspot Region on Chromosome 5

Rice grain width and shape play a crucial role in determining grain quality and yield. The genetic basis of rice grain width was dissected into six additive quantitative trait loci (QTL) and 11 pairs of epistatic QTL using an F7 recombinant inbred line (RIL) population derived from a single cross between Asominori (japonica) and IR24 (indica). QTL by environment interactions were evaluated in four environments. Chromosome segment substitution lines (CSSLs) harboring the six additive effect QTL were used to evaluate gene action across eight environments. A major, stable QTL, qGW-5, consistently decreased rice grain width in both the Asominori/IR24 RIL and CSSL populations with the genetic background Asominori. By investigating the distorted segregation of phenotypic values of rice grain width and genotypes of molecular markers in BC4F2 and BC4F3 populations, qGW-5 was dissected into a single recessive gene, gw-5, which controlled both grain width and length–width ratio. gw-5 was narrowed down to a 49.7-kb genomic region with high recombination frequencies on chromosome 5 using 6781 BC4F2 individuals and 10 newly developed simple sequence repeat markers. Our results provide a basis for map-based cloning of the gw-5 gene and for marker-aided gene/QTL pyramiding in rice quality breeding.

Mapping segregation distortion loci and quantitative trait loci for spikelet sterility in rice ( Oryza sativa L.)

Markers with segregation ratio distortion are commonly observed in data sets used for quantitative trait locus (QTL) mapping. In this study, a multipoint method of maximum likelihood (ML) was newly developed to estimate the positions and effects of the segregation distortion loci (SDLs) in two F2 populations of rice (Oryza sativa L.), i.e. Taichung65/Bhadua (TB; japonica-indica cross) and CPSLO17/W207-2 (CW; japonica-japonica). Of the four parents, W207-2 and Bhadua were found to be spikelet semi-sterile and stably inherited through selfing, and spikelet fertility segregated in the two populations. Therefore, recombination frequencies were recalculated after mapping the SDLs by using the multipoint method, and the molecular linkage maps of the two F2 populations were constructed to detect QTLs underlying spikelet fertility. As a result, five SDLs in the TB population were mapped on chromosomes 1, 3, 8 and 9, respectively. Two major QTLs underlying spikelet fertility, namely qSS-6a and qSS-8a, were detected on chromosomes 6 and 8, respectively. In the CW population, a total of 12 SDLs were detected on all 12 chromosomes except 1, 5, 7 and 11. Three QTLs underlying spikelet sterility, namely qSS-2, qSS-6b and qSS-8b on chromosomes 2, 6 and 8, were determined on the whole genome scale. Interestingly, both qSS-6a and qSS-6b, detected in the two F2 populations respectively, were located on a similar position as the S5 gene on chromosome 6; while qSS-8a and qSS-8b were also simultaneously detected on similar positions of the short arm of chromosome 8 in the two populations, which should be a new sterility gene showing the same type of zygotic selection.

Transcriptome analysis of grain-filling caryopses reveals involvement of multiple regulatory pathways in chalky grain formation in rice

BackgroundGrain endosperm chalkiness of rice is a varietal characteristic that negatively affects not only the appearance and milling properties but also the cooking texture and palatability of cooked rice. However, grain chalkiness is a complex quantitative genetic trait and the molecular mechanisms underlying its formation are poorly understood.ResultsA near-isogenic line CSSL50-1 with high chalkiness was compared with its normal parental line Asominori for grain endosperm chalkiness. Physico-biochemical analyses of ripened grains showed that, compared with Asominori, CSSL50-1 contains higher levels of amylose and 8 DP (degree of polymerization) short-chain amylopectin, but lower medium length 12 DP amylopectin. Transcriptome analysis of 15 DAF (day after flowering) caryopses of the isogenic lines identified 623 differential expressed genes (P < 0.01), among which 324 genes are up-regulated and 299 down-regulated. These genes were classified into 18 major categories, with 65.3% of them belong to six major functional groups: signal transduction, cell rescue/defense, transcription, protein degradation, carbohydrate metabolism and redox homeostasis. Detailed pathway dissection demonstrated that genes involved in sucrose and starch synthesis are up-regulated, whereas those involved in non-starch polysaccharides are down regulated. Several genes involved in oxidoreductive homeostasis were found to have higher expression levels in CSSL50-1 as well, suggesting potential roles of ROS in grain chalkiness formation.ConclusionExtensive gene expression changes were detected during rice grain chalkiness formation. Over half of these differentially expressed genes are implicated in several important categories of genes, including signal transduction, transcription, carbohydrate metabolism and redox homeostasis, suggesting that chalkiness formation involves multiple metabolic and regulatory pathways.

Pi64, Encoding a Novel CC-NBS-LRR Protein, Confers Resistance to Leaf and Neck Blast in Rice

Rice blast caused by Magnaporthe oryzae poses a major threat to rice production worldwide. The utilization of host resistance (R) genes is considered to be the most effective and economic means to control rice blast. Here, we show that the japonica landrace Yangmaogu (YMG) displays a broader spectrum of resistance to blast isolates than other previously reported broad-spectrum resistant (BSR) cultivars. Genetic analysis suggested that YMG contains at least three major R genes. One gene, Pi64, which exhibits resistance to indica-sourced isolate CH43 and several other isolates, was mapped to a 43-kb interval on chromosome 1 of YMG. Two open reading frames (NBS-1 and NBS-2) encoding nucleotide-binding site and leucine-rich repeat proteins were short-listed as candidate genes for Pi64. Constructs containing each candidate gene were transformed into three susceptible japonica cultivars. Only transformants with NBS-2 conferred resistance to leaf and neck blast, validating the idea that NBS-2 represents the functional Pi64 gene. Pi64 is constitutively expressed at all development stages and in all tissues examined. Pi64 protein is localized in both the cytoplasm and nucleus. Furthermore, introgression of Pi64 into susceptible cultivars via gene transformation and marker-assisted selection conferred high-level and broad-spectrum leaf and neck blast resistance to indica-sourced isolates, demonstrating its potential utility in breeding BSR rice cultivars.

SSR Mapping of Brown Planthopper Resistance Gene Bph9 in Kaharamana, an Indica Rice (Oryza sativa L.)

The brown planthopper (BPH) is one of the most serious insects pests of rice, and the host resistance has been recognized as one of the most economic and effective measures for BPH management. In this study, we conducted a molecular-based genetic analysis of Bph9 in Kaharamana, a Sri Lanka rice variety resistant to BPH insects of East and Southeast Asia. An F2 segregating population composed of 180 plants was constructed from the cross between Kaharamana and 02428, and each F2 plant was self-crossed to obtain F2:3 family. The bulked seedling test method was used to evaluate the resistance of F2:3 families, and the genotype of each F2 plant was inferred from the phenotype of corresponding F2:3 family. Linkage analysis indicated that the resistant gene Bph9 in Kaharamana was located between SSR markers RM463 and RM5341 on chromosome 12 with linkage distances of 6.8 cM and 9.7 cM, respectively. The time- and money-saving SSR markers would be helpful in the application of Bph9 in breeding program via marker-assisted selection.