Identification of the rice D-genome chromosomes by genomic in situ hybridisation

 The 24 rice D-genome chromosomes were identified among the 48 chromosomes of O. latifolia, which comprise the C- and D-genomes, using genomic in situ hybridisation (GISH). The B-genome chromosomes were also discriminated from the C-genome chromosomes in O. minuta (BBCC) by GISH. A comparison of the differences in the fluorescence intensity between the C and D genomes within O. latifolia (CCDD), and between the B and C genomes within O. minuta, indicated that the overall nucleotide-sequence homology between the B and C genomes is less than that between the C and D genomes. The origin of the D genome and the phylogenetic relationship of the D genome among the rice genomes are discussed, based on the results obtained. Received: 5 June 1997 / Accepted: 19 June 1997     

Identification and characterization of two rice autophagy associated genes, OsAtg8 and OsAtg4

Autophagy is an intracellular process for vacuolar degradation of cytoplasmic components. The molecular machinery responsible for yeast and mammalian autophagy has begun to be elucidated at the cellular level. A genome-wide search revealed significant conservation among autophagy genes in yeast and Arabidopsis. Up till now, however, there is no report about rice autophagy associated genes. Here we cloned OsAtg8 and OsAtg4 from Oryza sativa and detected their expression patterns in various tissues. Immunoblotting analysis showed that carboxyl terminus of OsAtg8 can be cleaved in yeast cell. Mutation analysis revealed that the conserved Gly117 residue of OsAtg8 was essential for its characteristic C-terminal cleavage as similar to that found in mammalian and yeast Atg8. We further proved that OsAtg8 interacted with OsAtg4, and this interaction was not affected by the conserved Gly117 mutation. Our results demonstrate that Atg8 conjugation pathway is conserved in rice and may play important roles in rice autophagy.     

Identification of genomic regions that affect grain-mould incidence and other traits of agronomic importance in sorghum

Grain-mould is a major problem in grain sorghum utilization as mouldy grain has a reduced quality due to the deterioration of the endosperm and reduced embryo viability. Here, our objective was to use genome mapping to improve knowledge of genetic variation and co-variation for grain-mould incidence and other inter-related agronomic traits. Grain-mould incidence, kernel-milling hardness, grain density, plant height, panicle peduncle length, foliar-disease incidence, and plant color were measured on 125 F₅ genotypes derived from a cross of Sureño and RTx430. Quantitative trait loci were detected by means of 130 mapped markers (44 microsatellites, 85 AFLPs, one morphological-trait locus) distributed among ten linkage groups covering 970 cM. One to five QTLs affected each trait, with the exception of grain density for which no QTLs were detected. Grain-mould incidence was affected by five QTLs each accounting for between 10 and 23% of the phenotypic variance. The effects and relative positions of QTLs for grain-mould incidence were in accordance with the QTL distribution of several inter-related agronomic traits (e.g., plant height, peduncle length) and with the correlation between these phenotypic traits and grain-mould incidence. The detection of QTLs for grain-mould incidence was dependent on the environment, which is consistent with heritibility estimates that show strong environmental and genotype × environment effects. Several genomic regions affected multiple traits including one region that affected grain-mould incidence, plant height, panicle peduncle length, and grain-milling hardness, and a second region that influenced grain-mould (in four environments) and plant height. One genomic region, which harbors loci for plant color, influenced the severity of foliar disease symptoms and the incidence of grain-mould in one environment. Collectively QTLs detected in the present population explained between 10% and 55% of the phenotypic variance observed for a given trait.     

Dorsoventral asymmetry of photosynthesis and photoinhibition in flag leaves of two rice cultivars that differ in nitrogen response and leaf angle

Rice is believed to show photosynthetic symmetry between adaxial and abaxial leaf sides. To verify this, we re‐examined dorsoventral asymmetry in photosynthesis, chlorophyll fluorescence and anatomical traits in flag leaves of two Oryza sativa cultivars that differ in nitrogen (N) response and in leaf angle: ‘Akenohoshi’, a cultivar that can adapt to low‐N (LN), with low leaf angle (more erect leaves), and ‘Shirobeniya’, a cultivar that is unable to adapt to LN, with higher leaf angle. Plants were grown under standard‐N (SN) and LN conditions. LN leaves of both cultivars became more erect than SN, but LN Akenohoshi still had more erect ones than Shirobeniya. Contrary to results of previous studies, leaves of both cultivars showed an asymmetry in photosynthetic rate between adaxial and abaxial sides (higher on the adaxial side) under SN. SN leaves of both cultivars showed lower susceptibility to photoinhibition on the adaxial side than on the abaxial side. However, leaves of Akenohoshi showed less asymmetry in these traits under LN than under SN, whereas leaves of Shirobeniya had similar degrees of asymmetry in these traits under both SN and LN. Both cultivars also showed dorsoventral asymmetry in anatomical traits of mesophyll tissue regardless of N level, but the degree of asymmetry was lower in LN Akenohoshi. These data reveal that rice leaves exhibit dorsoventral asymmetry in photosynthetic and anatomical features, and that the degree of asymmetry varies with cultivar and N level. It is suggested that lower leaf angles (particularly in Akenohoshi) in the presence of LN represent a light acclimation to prevent photoinhibition.     

Identification and characterization of regions of the rice genome associated with broad-spectrum, quantitative disease resistance

Much research has been devoted to understanding the biology of plant-pathogen interactions. The extensive genetic analysis of disease resistance in rice, coupled with the sequenced genome and genomic resources, provides the opportunity to seek convergent evidence implicating specific chromosomal segments and genes in the control of resistance. Published data on quantitative and qualitative disease resistance in rice were synthesized to evaluate the distributions of and associations among resistance loci. Quantitative trait loci (QTL) for resistance to multiple diseases and qualitative resistance loci (R genes) were clustered in the rice genome. R genes and their analogs of the nucleotide binding site-leucine-rich repeat class and genes identified on the basis of differential representation in disease-related EST libraries were significantly associated with QTL. Chromosomal segments associated with broad-spectrum quantitative disease resistance (BS-QDR) were identified. These segments contained numerous positional candidate genes identified on the basis of a range of criteria, and groups of genes belonging to two defense-associated biochemical pathways were found to underlie one BS-QDR region. Genetic dissection of disease QTL confidence intervals is needed to reduce the number of positional candidate genes for further functional analysis. This study provides a framework for future investigations of disease resistance in rice and related crop species.     

Identification and mapping of expressed genes, simple sequence repeats and transposable elements in centromeric regions of rice chromosomes.

The genomic sequences derived from rice centromeric regions were analyzed to facilitate the comprehensive understanding of the rice genome. A rice centromere-specific satellite sequence, RCS2/TrsD/CentO, was used to screen P1-derived artificial chromosome (PAC) and bacterial artificial chromosome (BAC) genomic libraries derived from Oryza sativa L. ssp. japonica cultivar Nipponbare. Physical maps of the centromeric regions were constructed by DNA fingerprinting methods and the aligned clones were analyzed by end sequencing. BLAST analysis revealed the composition of genes, centromeric satellites and other repetitive elements, such as RIRE7/CRR, RIRE8, Squiq, Anaconda, CACTA and miniature inverted-repeat transposable elements. Fiber-fluorescent in situ hybridization analysis also indicated the presence of distinct clusters of RCS2/TrsD/CentO satellite interspersed with other elements, instead of a long homogeneous region. Several expressed genes, sequences representative of ancestral organellar insertions, relatively long simple sequence repeats (SSRs), and sequences corresponding to 5S and 45S ribosomal RNA genes were also identified. Thirty-one gene sequences showed high-similarity to rice full-length cDNA sequences that had not been matched to the published rice genome sequence in silico. These results suggest the presence of expressed genes within and around the clusters of RCS2/TrsD/CentO satellites in unsequenced centromeric regions of the rice chromosomes.     

Identification of genomic regions that interact with a viable allele of the Drosophila protein tyrosine phosphatase corkscrew

Signaling by receptor tyrosine kinases (RTKs) is critical for a multitude of developmental decisions and processes. Among the molecules known to transduce the RTK-generated signal is the nonreceptor protein tyrosine phosphatase Corkscrew (Csw). Previously, Csw has been demonstrated to function throughout the Drosophila life cycle and, among the RTKs tested, Csw is essential in the Torso, Sevenless, EGF, and Breathless/FGF RTK pathways. While the biochemical function of Csw remains to be unambiguously elucidated, current evidence suggests that Csw plays more than one role during transduction of the RTK signal and, further, the molecular mechanism of Csw function differs depending upon the RTK in question. The isolation and characterization of a new, spontaneously arising, viable allele of csw, csw(lf), has allowed us to undertake a genetic approach to identify loci required for Csw function. The rough eye and wing vein gap phenotypes exhibited by adult flies homo- or hemizygous for csw(lf) has provided a sensitized background from which we have screened a collection of second and third chromosome deficiencies to identify 33 intervals that enhance and 21 intervals that suppress these phenotypes. We have identified intervals encoding known positive mediators of RTK signaling, e.g., drk, dos, Egfr, E(Egfr)B56, pnt, Ras1, rolled/MAPK, sina, spen, Src64B, Star, Su(Raf)3C, and vein, as well as known negative mediators of RTK signaling, e.g., aos, ed, net, Src42A, sty, and su(ve). Of particular interest are the 5 lethal enhancing intervals and 14 suppressing intervals for which no candidate genes have been identified.     

The flexible interrelation between AOX respiratory pathway and photosynthesis in rice leaves.

Alternative respiratory pathway was investigated in rice seedlings grown under total darkness, light/dark cycle, or continuous light. The capacity of the alternative pathway was relatively higher in leaves that had longer light exposure. An analysis of rice AOX1 multigene family revealed that AOX1c, but not AOX1a and AOX1b, had a light-independent expression. The alternative oxidase (AOX) inhibitor, salicylhydroxamic acid (SHAM, 1mM), inhibited nearly 68% of the capacity of the alternative pathway in leaves grown under different light conditions. The plants grown under different light periods were treated with SHAM and then were exposed to illumination for 4h. The transition from dark to 4h of light stimulated the capacity of alternative pathway in etiolated rice seedlings and in those grown under light/dark cycle, whereas the capacity of the alternative pathway was constant in seedlings grown under continuous light with additional 4h of illumination. Etiolated leaves did not show any CO(2) fixation after 4h of illumination, and the increase in chlorophyll content was delayed by the SHAM pretreatment. When seedlings grown under light/dark cycle were moved from dark and exposed to 4h of light, increases in chlorophyll content and CO(2) fixation rate were reduced by SHAM. Although these parameters were stable in plants grown under continuous light, SHAM decreased CO(2) fixation rate but not the chlorophyll content. These results indicate that the role and regulation of AOX in light are determined by the developmental stage of plant photosynthetic apparatus.     

RFLP analysis of genomic regions associated with cooked-kernel elongation in rice

As a result of earlier breeding efforts, portions of the genome of Basmati 370 have been introgressed into a rice breeding line, B8462T3-710. Cooked-kernel elongation was increased in this breeding line to a level equal to that of Basmati 370. The objective of this study was to identify and locate quantitative trait loci (QTLs) associated with cooked-kernel elongation in an F3 population derived from a cross between B8462T3710 and the reduced-elongation recurrent parent variety, Dellmont. DNA from the parental lines and Basmati 370 as a control, were screened for RFLPs using 170 clones chosen to cover the rice genome at intervals of 8 cM on average. Eighteen markers identified RFLPs common to Basmati 370 and B8462T3-710, but different from Dellmont, suggesting possible associations with kernel elongation. The B8462T3-710/Dellmont F3 population was analyzed for segregation of those RFLPs and for kernel elongation. Analysis of variance of the kernel elongation ratio revealed that two markers, 14.6 cM apart on chromosome 8, are significantly associated with this trait (RZ323 P 0.005, RZ562 P 0.05). Interval mapping suggests a single QTL with a close proximity to RZ323. This QTL was tested in F6 lines derived from the same cross and the presence of the B8462T3-710 segment detected by RZ323 caused a highly significant increase of the kernel elongation ratio (P 0.04). In addition, the QTL for kernel elongation and a gene for aroma, which are major components of the grain quality characteristics of Basmati-type rices, showed linkage. The availability of linked markers to the QTL may facilitate early selection for kernel elongation in rice breeding programs.     

Identification of genomic regions affecting plant height in sorghum and maize

The objective of this study was to use restriction fragment length polymorphisms (RFLPs) to determine the genetic location and effects of genomic regions controlling plant height in sorghum. F₂ plants (152) from the cross CK60 x PI229828 were used. Genomic and cDNA clones (106) identified 111 loci distributed among ten linkage groups covering 1299 cM. Interval mapping identified four regions, each in a separate linkage group. These regions may correspond to loci (dw) previously identified by alleles with qualitative effects. Also, these regions identified in sorghum may be orthologous to those previously reported for plant height in maize. Gene effects and gene action varied among genomic regions. In each region, PI229828 alleles resulted in increased plant height. Each region accounted for 9.2–28.7% of the phenotypic variation. Positive, additive effects ranged from 15 to 32cm. Tallness was dominant or overdominant and conferred by alleles from PI229828 for three quantitative trait loci (QTL). At the fourth QTL, PI229828 contributed to increased plant height, but short stature was partially dominant. One digenic interaction was significant. The presence of a PI229828 allele at one region diminished the effects of the other region. A multiple model indicated that these four regions collectively accounted for 63.4% of the total phenotypic variation. The utility of this information for germplasm conversion through backcross breeding is discussed.Journal Paper No. J. 15649 of the Iowa Agriculture and Home Economic Experiment Station, Ames, Iowa. Project No. 3134     

Low humidity effects on photosynthesis in single leaves of C4 plants

Summary It was hypothesized that since sub-stomatal carbon dioxide concentrations are often saturating to photosynthesis at ambient external concentrations in C₄ plants at high light, photosynthesis might be insensitive to partial stomatal closure caused by large leaf-air water vapor pressure difference. The response of stomatal conductance and photosynthesis at high irradiance to vapor pressure difference was determined under uniform conditions in C₄ plants grown under controlled conditions, and outdoors. In several cases, photosynthesis was less sensitive to stomatal closure than it would have been if photosynthesis had a linear response to sub-stomatal carbon dioxide concentration. No change in photosynthesis at up to 25 mbar vapor pressure difference was demonstrated in the C₄ species Portulaca oleracea and Amaranthus hypochondriacus, despite reductions in stomatal conductance of 32 and 17%, respectively. Sensitivity of photosynthesis to leaf-air vapor pressure difference was found to depend on the species and on the growth conditions.     

Stomatal control of photosynthesis in detached leaves of woody and herbaceous plants

Photosynthesis and transpiration were assayed in leaves and needles of some woody species (Pinus sibirica Du Tour, P. sylvestris L., Larix sibirica Ledeb., Betula pendula Roth., Quercus robur L.), annual herbaceous plants (Amaranthus cruentus L. cv. Tampala, Celosia argentea L. f. cristata (L.), Gomphrena dispersa Standl., Solanum tuberosum L., Helianthus annuus L., H. tuberosus L.), and perennial herbs (Inula helenium L., Poligonum weyrichii F. Schmidt, Polymnia sonchifolia Poepp. & Endl.). A high-precision portable gas-analyzing system GFS-3000 with a climate-controlled chamber was used for measurements on leaves both before and after leaf detachment from the shoot under conditions optimal for photosynthesis: photosynthetically active radiation of 2000 μE/(m² s), 22–25°C, and a relative humidity of 65–70%. The steady-state gas exchange in illuminated leaves of all plant species examined was characterized by a directly proportional relationship between photosynthesis and transpiration (R ₂ = 0.87). This means that the temporal course of H₂O and CO₂ gas exchange in detached leaves suffices to characterize the status of stomatal control of photosynthesis. The general trend in the effect of leaf detachment, observed in herbaceous and woody plants, is that the stomatal control of photosynthesis was retained within first 3–5 min after leaf excision. By contrast, the increase in transpiration after leaf detachment was species-specific. Because of this circumstance, the measurements of transpiration by rapid weighing method may result in overestimation of transpiration rates by 10–15% for some plant species, compared to steady-state rates of gas exchange in undetached leaves.     

Identification of early senescence-associated genes in rice flag leaves

Leaf senescence is one of the key stages of plant leaf development. It is a highly complex but ordered process involving expression of large scale senescence associated genes, and its molecular mechanisms still remain unclear. By using suppression subtractive hybridization, 815 ESTs that are up-regulated at the onset of rice flag leaf senescence have been isolated. A total of 533 unigenes have been confirmed by macroarray detection and sequencing. 183 of these unigenes have GO annotations, involved in macromolecule metabolism, protein biosynthesis regulation, energy metabolism, gene expression regulations, detoxification, pathogenicity and stress, cytoskeleton organization and flower development. Another 121 unigenes co-localized with previously reported known stay-green QTLS. RT-PCR analysis on the other novel genes indicated that they can be up-regulated in natural early senescence and induced by hormone. Our results indicate that senescence is closely related to various metabolic pathways, thus providing new insight into the onset of leaf senescence mechanism. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Polymorphisms and genomic organization of repetitive DNA from centromeric regions of Arabidopsis chromosomes.

A highly abundant repetitive DNA sequence family of Arabidopsis, AtCon, is composed of 178-bp tandemly repeated units and is located at the centromeres of all five chromosome pairs. Analysis of multiple copies of AtCon showed 95% conservation of nucleotides, with some alternative bases, and revealed two boxes, 30 and 24 bp long, that are 99% conserved. Sequences at the 3' end of these boxes showed similarity to yeast CDEI and human CENP-B DNA-protein binding motifs. When oligonucleotides from less conserved regions of AtCon were hybridized in situ and visualized by using primer extension, they were detected on specific chromosomes. When used for polymerase chain reaction with genomic DNA, single primers or primer pairs oriented in the same direction showed negligible amplification, indicating a head-to-tail repeat unit organization. Most primer pairs facing in opposite directions gave several strong bands corresponding to their positions within AtCon. However, consistent with the primer extension results, some primer pairs showed no amplification, indicating that there are chromosome-specific variants of AtCon. The results are significant because they elucidate the organization, mode of amplification, dispersion, and evolution of one of the major repeated sequence families of Arabidopsis. The evidence presented here suggests that AtCon, like human alpha satellites, plays a role in Arabidopsis centromere organization and function.     

Sequencing and characterization of telomere and subtelomere regions on rice chromosomes 1S, 2S, 2L, 6L, 7S, 7L and 8S

Telomeres, which are important for chromosome maintenance, are composed of long, repetitive DNA sequences associated with a variety of telomere-binding proteins. We characterized the organization and structure of rice telomeres and adjacent subtelomere regions on the basis of cytogenetic and sequence analyses. The length of the rice telomeres ranged from 5.1 to 10.8 kb, as revealed by both fibre-fluorescent in situ hybridization and terminal restriction-fragment assay. Physical maps of the chromosomal ends were constructed from a fosmid library. This facilitated sequencing of the telomere regions of chromosomes 1S, 2S, 2L, 6L, 7S, 7L and 8S. The resulting sequences contained conserved TTTAGGG telomere repeats, which indicates that the physical maps partly covered the telomere regions of the respective chromosome arms. These repeats were organized in the order of 5'-TTTAGGG-3' from the chromosome-specific region, except in chromosome 7S, in which seven inverted copies also existed in tandem array. Analysis of the telomere-flanking regions revealed the occurrence of deletions, insertions, or chromosome-specific substitutions of single nucleotides within the repeat sequences at the junction between the telomere and subtelomere. The sequences of the 500-kb regions of the seven chromosome ends were analysed in detail. A total of 598 genes were predicted in the telomeric regions. In addition, repetitive sequences derived from various kinds of retrotransposon were identified. No significant evidence for segmental duplication could be detected within or among the subtelomere regions. These results indicate that the rice chromosome ends are heterogeneous in both sequence and characterization.