Wenzhu Jiang

High-resolution mapping of two rice brown planthopper resistance genes, Bph20(t) and Bph21(t), originating from Oryza minuta

Brown planthopper (BPH) is one of the most destructive insect pests of rice. Wild species of rice are a valuable source of resistance genes for developing resistant cultivars. A molecular marker-based genetic analysis of BPH resistance was conducted using an F2 population derived from a cross between an introgression line, ‘IR71033-121-15’, from Oryza minuta (Accession number 101141) and a susceptible Korean japonica variety, ‘Junambyeo’. Resistance to BPH (biotype 1) was evaluated using 190 F3 families. Two major quantitative trait loci (QTLs) and two significant digenic epistatic interactions between marker intervals were identified for BPH resistance. One QTL was mapped to 193.4-kb region located on the short arm of chromosome 4, and the other QTL was mapped to a 194.0-kb region on the long arm of chromosome 12. The two QTLs additively increased the resistance to BPH. Markers co-segregating with the two resistance QTLs were developed at each locus. Comparing the physical map positions of the two QTLs with previously reported BPH resistance genes, we conclude that these major QTLs are new BPH resistance loci and have designated them as Bph20(t) on chromosome 4 and Bph21(t) on chromosome 12. This is the first report of BPH resistance genes from the wild species O. minuta. These two new genes and markers reported here will be useful to rice breeding programs interested in new sources of BPH resistance.

SPL28 encodes a clathrin‐associated adaptor protein complex 1, medium subunit μ1 (AP1M1) and is responsible for spotted leaf and early senescence in rice (Oryza sativa)

To expand our understanding of cell death in plant defense responses, we isolated a novel rice (Oryza sativa) spotted leaf mutant (spl28) that displays a lesion mimic phenotype in the absence of pathogen attack through treatment of Hwacheongbyeo (an elite Korean japonica cultivar) with N-methyl-N-nitrosourea (MNU). Early stage development of the spl28 mutant was normal. However, after flowering, spl28 mutants exhibited a significant decrease in chlorophyll content, soluble protein content, and photosystem II efficiency, and high concentrations of reactive oxygen species (ROS), phytoalexin, callose, and autofluorescent phenolic compounds that localized in or around the lesions. The spl28 mutant also exhibited significantly enhanced resistance to rice blast and bacterial blight. Using a map-based cloning approach, we determined that SPL28 encodes a clathrin-associated adaptor protein complex 1, medium subunit micro 1 (AP1M1), which is involved in the post-Golgi trafficking pathway. A green fluorescent protein (GFP) fusion protein of SPL28 (SPL28::GFP) localized to the Golgi apparatus, and expression of SPL28 complemented the membrane trafficking defect of apm1-1 Delta yeast mutants. SPL28 was ubiquitously expressed and contained a highly conserved adaptor complex medium subunit (ACMS) family domain. SPL28 appears to be involved in the regulation of vesicular trafficking, and SPL28 dysfunction causes the formation of hypersensitive response (HR)-like lesions, leading to the initiation of leaf senescence.

Inactivation of the UGPase1 gene causes genic male sterility and endosperm chalkiness in rice (Oryza sativa L.).

A rice genic male-sterility gene ms-h is recessive and has a pleiotropic effect on the chalky endosperm. After fine mapping, nucleotide sequencing analysis of the ms-h gene revealed a single nucleotide substitution at the 3′-splice junction of the 14th intron of the UDP-glucose pyrophosphorylase 1 (UGPase1; EC2.7.7.9) gene, which causes the expression of two mature transcripts with abnormal sizes caused by the aberrant splicing. An in vitro functional assay showed that both proteins encoded by the two abnormal transcripts have no UGPase activity. The suppression of UGPase by the introduction of a UGPase1-RNAi construct in wild-type plants nearly eliminated seed set because of the male defect, with developmental retardation similar to the ms-h mutant phenotype, whereas overexpression of UGPase1 in ms-h mutant plants restored male fertility and the transformants produced T1 seeds that segregated into normal and chalky endosperms. In addition, both phenotypes were co-segregated with the UGPase1 transgene in segregating T1 plants, which demonstrates that UGPase1 has functional roles in both male sterility and the development of a chalky endosperm. Our results suggest that UGPase1 plays a key role in pollen development as well as seed carbohydrate metabolism.

Characterization and Mapping of a Shattering Mutant in Rice That Corresponds to a Block of Domestication Genes

Easy shattering reduces yield due to grain loss during harvest in cereals. Shattering is also a hindrance in breeding programs that use wild accessions because the shattering habit is often linked to desirable traits. We characterized a shattering mutant line of rice, Hsh, which was derived from a nonshattering japonica variety, Hwacheong, by N-methyl-N-nitrosourea (MNU) treatment. The breaking tensile strength (BTS) of the grain pedicel was measured using a digital force gauge to evaluate the degree of shattering of rice varieties at 5, 10, 15, 20, 25, 30, 35, and 40 days after heading (DAH). The BTS of Hwacheong did not decrease with increasing DAH, maintaining a level of 180–240 gf, while that of Hsh decreased greatly during 10–20 DAH and finally stabilized at 50 gf. Optical microscopy revealed that Hsh had a well-developed abscission layer similar to the wild rice Oryza nivara (accession IRGC105706), while Hwacheong did not produce an abscission layer, indicating that the shattering of Hsh was caused by differentiation of the abscission layer. On the basis of the BTS value and morphology of the abscission layer of F1 plants and segregation data in F2 populations, it was concluded that the easy shattering of Hsh was controlled by the single recessive gene sh-h. The gene sh-h was determined to be located on rice chromosome 7 by bulked segregant analysis. Using 14 SSR markers on rice chromosome 7, the gene sh-h was mapped between the flanking markers RM8262 and RM7161 at distances of 1.6 and 2.0 cM, respectively. An SSR marker Rc17 cosegregated with the gene sh-h. The locus sh-h for shattering was tightly linked to the Rc locus conferring red pericarp, as well as a QTL qSDs-7-1 for seed dormancy, implying that this region might represent a domestication block in the evolutionary pathway of rice.

PCR Marker-Based Evaluation of the Eating Quality of Japonica Rice (Oryza sativa L.)

Evaluation of eating quality in early breeding generations of rice is critical to developing varieties with better palatability. This paper reports DNA markers associated with eating quality of temperate japonica rice and an evaluation method aided by multiple regression analysis. A total of 30 markers comprising STSs, SNPs, and SSRs were tested for their association with palatability using 22 temperate japonica varieties with different palatability values. Eating quality-related traits of the 22 varieties were also measured. Of the 30 markers, 18 were found to be significantly associated with palatability and, consequently, a model regression equation with an R2 value of 0.99 was formulated to estimate the palatability by the marker data set. Validation of the model equation using selected breeding lines indicated that the marker set and the equation are highly applicable to evaluation of the palatability of cooked rice in temperate japonica varieties.

Fine mapping and candidate gene analysis of hwh1 and hwh2 , a set of complementary genes controlling hybrid breakdown in rice

Hybrid breakdown (HB), a phenomenon of reduced viability or fertility accompanied with retarded growth in hybrid progenies, often arises in the offspring of intersubspecific hybrids between indica and japonica in rice. We detected HB plants in F8 recombinant inbred lines derived from the cross between an indica variety, Milyang 23, and a japonica variety, Tong 88-7. HB plants showed retarded growth, with fewer tillers and spikelets. Genetic analysis revealed that HB was controlled by the complementary action of two recessive genes, hwh1 and hwh2, originating from each of both parents, which were fine-mapped on the short arm of chromosome 2 and on the near centromere region of the long arm of chromosome 11, respectively. A comparison of the sequences of candidate genes among both parents and HB plants revealed that hwh1 encoded a putative glucose-methanol-choline oxidoreductase with one amino acid change compared to Hwh1 and that hwh2 probably encoded a putative hexose transporter with a six amino acid insertion compared to Hwh2. Investigation of the distribution of these alleles among 54 japonica and indica cultivars using candidate gene-based markers suggested that the two loci might be involved in developing reproductive barriers between two subspecies.

Fine mapping and candidate gene analysis of the floury endosperm gene, FLO(a), in rice

In addition to its role as an energy source for plants, animals and humans, starch is also an environmentally friendly alternative to fossil fuels. In rice, the eating and cooking quality of the grain is determined by its starch properties. The floury endosperm of rice has been explored as an agronomical trait in breeding and genetics studies. In the present study, we characterized a floury endosperm mutant, flo(a), derived from treatment of Oryza sativa ssp. japonica cultivar Hwacheong with MNU. The innermost endosperm of the flo(a) mutant exhibited floury characteristics while the outer layer of the endosperm appeared normal. Starch granules in the flo(a) mutant formed a loosely-packed crystalline structure and X-ray diffraction revealed that the overall crystallinity of the starch was decreased compared to wild-type. The FLO(a) gene was isolated via a map-based cloning approach and predicted to encode the tetratricopeptide repeat domaincontaining protein, OsTPR. Three mutant alleles contain a nucleotide substitution that generated one stop codon or one splice site, respectively, which presumably disrupts the interaction of the functionally conserved TPR motifs. Taken together, our map-based cloning approach pinpointed an OsTPR as a strong candidate of FLO(a), and the proteins that contain TPR motifs might play a significant role in rice starch biosynthetic pathways.

Shotgun proteomic analysis for detecting differentially expressed proteins in the reduced culm number rice

To survey protein expression patterns in the reduced culm number (RCN) rice, a comparative shotgun proteomic analysis was conducted. For large‐scale protein identification, multidimensional protein identification technology (MudPIT) coupled with pre‐fractionation of plant shoot proteins led to the identification of 3004 non‐redundant rice proteins. By statistically comparing relative amounts of 1353 reproducibly identified proteins between the RCN rice and the wild‐type rice, 44 differentially expressed proteins were detected, where 42 proteins were increased and 2 proteins were decreased in the RCN rice. These proteins appear to have roles in glycolysis, trichloroacetic acid cycle, secondary metabolism, nutrient recycling, and nucleotide metabolism and repair. Consequently, we hypothesized that the RCN rice might fail to maintain sugar nutrient homeostasis. This was confirmed with the observation that the sucrose concentration was increased significantly in the RCN rice compared with the wild‐type rice. Also, the RCN rice showed a hypersensitive response to exogenous sucrose treatment.

Identification of QTLs for hybrid fertility in inter-subspecific crosses of rice (Oryza sativa L.)

Two subspecies in rice, japonica and indica, have their own ecotypic traits. However, reproductive barriers such as spikelet sterility in hybrid progenies between subspecies have been an obstacle in breeding programs for combining desirable traits from both subspecies through inter-subspecific hybridization. The 166 F9 RILs and two BC1F1s’ were analyzed for spikelet and pollen fertility with the parents and F1 between Dasanbyeo (DS, indica) / TR22183 (TR, japonica). A frame map was constructed using a total of 218 polymorphic STS and SSR markers. In both BC1F1s’ of DS//DS/TR and TR//DS/TR, clusters of significant QTLs for spikelet and pollen fertility were identified on the short arm of chromosome 5 and chromosome 8. Nine and ten digenic epistatic interactions for DS//DS/TR and TR//DS//TR were identified, respectively. HF-QTLs were detected at the similar position with subspecies-specific markers and segregation distortion loci, implying that HF-QTLs might be associated with the differentiation of indica and japonica. Hybrid fertility/sterility and its relationship with other traits are discussed in relation to the reproductive barriers between subspecies of rice.

QTL analyses of heterosis for grain yield and yield-related traits in indica-japonica crosses of rice (Oryza sativa L.)

Two sets of rice materials, 166 RILs derived from a cross between Milyang 23 (Korean indica-type rice) and Tong 88-7 (japonica Rice), and BC1F1 hybrids derived from crosses between the RILs and the female parent, Milyang 23, were produced to identify QTLs for heterosis of yield and yield-related traits. The QTLs were detected from three different phenotype data sets including the RILs, BC1F1 hybrids, and mid-parental heterosis data set acquired from the definition of mid-parental heterosis. A total of 57 QTLs were identified for nine traits. Of eight QTLs detected for yield heterosis, five overlapped with other heterosis QTLs for yield-related traits such as spikelet number per panicle, days to heading, and spikelet fertility. Four QTLs for yield heterosis, gy1.1, py6, gy10, and py11, were newly identified in this study. We identified a total of 17 EpQTLs for yield heterosis that explain 21.4 ∼ 59.0 % of total phenotypic variation, indicating that epistatic interactions may play an important role in heterosis.