Development of 12 sets of chromosome segment substitution lines that enhance allele mining in Asian cultivated rice.

Many agronomic traits that are important in rice breeding are controlled by multiple genes. The extensive time and effort devoted so far to identifying and selecting such genes are still not enough to target multiple agronomic traits in practical breeding in Japan because of a lack of suitable plant materials in which to efficiently detect and validate beneficial alleles from diverse genetic resources. To facilitate the comprehensive analysis of genetic variation in agronomic traits among Asian cultivated rice, we developed 12 sets of chromosome segment substitution lines (CSSLs) with the japonica background, 11 of them in the same genetic background, using donors representing the genetic diversity of Asian cultivated rice. Using these materials, we overviewed the chromosomal locations of 1079 putative QTLs for seven agronomic traits and their allelic distribution in Asian cultivated rice through multiple linear regression analysis. The CSSLs will allow the effects of putative QTLs in the highly homogeneous japonica background to be validated.

Genetic architecture of leaf photosynthesis in rice revealed by different types of reciprocal mapping populations.

The improvement of leaf net photosynthetic rate (An) is a major challenge in enhancing crop productivity. However, the genetic control of An among natural genetic accessions is still poorly understood. The high-yielding indica cultivar Takanari has the highest An of all rice cultivars, 20-30% higher than that of the high-quality japonica cultivar Koshihikari. By using reciprocal backcross inbred lines and chromosome segment substitution lines derived from a cross between Takanari and Koshihikari, we identified three quantitative trait loci (QTLs) where the Takanari alleles enhanced An in plants with a Koshihikari genetic background and five QTLs where the Koshihikari alleles enhanced An in plants with a Takanari genetic background. Two QTLs were expressed in plants with both backgrounds (type I QTL). The expression of other QTLs depended strongly on genetic background (type II QTL). These beneficial alleles increased stomatal conductance, the initial slope of An versus intercellular CO2 concentration, or An at CO2 saturation. Pyramiding of these alleles consistently increased An. Some alleles positively affected biomass production and grain yield. These alleles associated with photosynthesis and yield can be a valuable tool in rice breeding programs via DNA marker-assisted selection.

Detection of QTLs for cold tolerance of rice cultivar 'Kuchum' and effect of QTL pyramiding.

KEY MESSAGE: A QTL for cold tolerance at the booting stage of rice cultivar 'Kuchum' was detected and delimited into a 1.36 Mb region, and a cold-tolerant line was developed by QTL pyramiding. Low temperature in summer causes pollen sterility in rice, resulting in a serious loss of yield. The second most widely grown rice cultivar in Japan, 'Hitomebore', has been developed as a cultivar highly tolerant to low temperature at the booting stage. However, even 'Hitomebore' exhibits sterility at a temperature lower than 18.5 °C. Further improvement of cold tolerance of rice is required. In the present study, QTLs for cold tolerance in a Bhutanese rice variety, 'Kuchum', were analyzed using backcrossed progenies and a major QTL, named qCT-4, was detected on chromosome 4. Evaluating cold tolerance of seven types of near isogenic lines having 'Kuchum' alleles around qCT-4 with a 'Hitomebore' genetic background, qCT-4 was delimited to a region of ca. 1.36 Mb between DNA markers 9_1 and 10_13. Homozygous 'Kuchum' alleles at qCT-4 showed an effect of increasing seed fertility by ca. 10 % under cold-water treatment. Near isogenic lines of 'Hitomebore' having 'Silewah' alleles of Ctb1 and Ctb2 and a 'Hokkai PL9' allele of qCTB8 did not exhibit higher cold tolerance than that of 'Hitomebore'. On the other hand, a qLTB3 allele derived from a Chinese cultivar 'Lijiangxintuanheigu' increased cold tolerance of 'Hitomebore', and pyramiding of the qCT-4 allele and the qLTB3 allele further increased seed fertility under cold-water treatment. Since NILs of 'Hitomebore' with the 'Kuchum' allele of qCT-4 were highly similar to 'Hitomebore' in other agronomic traits, the qCT-4 allele is considered to be useful for developing a cold-tolerant cultivar.

Genetic architecture of variation in heading date among Asian rice accessions.

BACKGROUND: Heading date, a crucial factor determining regional and seasonal adaptation in rice (Oryza sativa L.), has been a major selection target in breeding programs. Although considerable progress has been made in our understanding of the molecular regulation of heading date in rice during last two decades, the previously isolated genes and identified quantitative trait loci (QTLs) cannot fully explain the natural variation for heading date in diverse rice accessions. RESULTS: To genetically dissect naturally occurring variation in rice heading date, we collected QTLs in advanced-backcross populations derived from multiple crosses of the japonica rice accession Koshihikari (as a common parental line) with 11 diverse rice accessions (5 indica, 3 aus, and 3 japonica) that originate from various regions of Asia. QTL analyses of over 14,000 backcrossed individuals revealed 255 QTLs distributed widely across the rice genome. Among the detected QTLs, 128 QTLs corresponded to genomic positions of heading date genes identified by previous studies, such as Hd1, Hd6, Hd3a, Ghd7, DTH8, and RFT1. The other 127 QTLs were detected in different chromosomal regions than heading date genes. CONCLUSIONS: Our results indicate that advanced-backcross progeny allowed us to detect and confirm QTLs with relatively small additive effects, and the natural variation in rice heading date could result from combinations of large- and small-effect QTLs. We also found differences in the genetic architecture of heading date (flowering time) among maize, Arabidopsis, and rice.

Next generation long-culm rice with superior lodging resistance and high grain yield, Monster Rice 1.

During late 1960s Green Revolution, researchers utilized semidwarf 1 (sd1) to improve the yield and lodging resistance in rice (Oryza sativa L.). However, sd1 has a negative effect to culm strength and biomass production. To increase yield dramatically in 21th century, development of next generation long-culm rice for non-lodging and high grain yield independent of sd1 has been needed. The present study developed Monster Rice 1, a long-culm and heavy-panicle type of rice line and compared it with Takanari, a high-yielding semidwarf rice variety about yield and lodging resistance associated traits. Brown rice yield and bending moment at breaking of the basal elongated internode were higher in Monster Rice 1 than those in Takanari due to a large number of spikelets per panicle and thicker culm. Furthermore, to identify QTLs with superior alleles for these traits, QTL and haplotype analyses were performed using F2 population and recombinant inbred lines derived from a cross between Monster Rice 1 and Takanari. The results from this study suggest that long-culm and heavy-panicle type of rice with a superior lodging resistance by culm strength can perform its high yield potential by using these identified QTLs contributing yield and lodging resistance.

Finding the superior allele of japonica-type for increasing stem lodging resistance in indica rice varieties using chromosome segment substitution lines.

BACKGROUND: In cereal crops, stem lodging can be classified into two types: stem-breaking type and stem-bending type. To improve stem-lodging resistance, the strong culm traits of superior lodging-resistant varieties must be characterized. The identification of quantitative trait loci (QTLs) and the corresponding genes associated with the parameters for bending moment at breaking (M) and flexural rigidity (FR) is expected to enable the efficient development of lodging-resistant varieties. A set of Chromosome Segment Substitution Lines (CSSLs) derived from the cross between Takanari and Koshihikari were used in this study to identify QTLs associated with lodging resistance. RESULTS: The indica variety Takanari possesses large M due to its large section modulus (SM) despite its small bending stress (BS), whereas Takanari also has large FR due to its large secondary moment of inertia (SMI) and Young's modulus (YM). The QTLs for BS were assigned to chromosomes 3, 5, 6, 8, 9, 10, 11, and 12. Koshihikari alleles increased BS in these QTLs. The YM was increased by substitution of the Koshihikari chromosomal segments on chromosomes 2, 10, and 11. Other QTLs mapped to chromosomes 7 and 12, such that the Koshihikari alleles contributed to the decrease of YM. QTLs for cellulose density were assigned to chromosomes 1, 3, and 5, which were replaced by substitutions of Koshihikari segments. The QTLs for hemicellulose, cellulose, and holocellulose densities identified on chromosome 5 overlapped with those for BS, indicating the positive effect of the Koshihikari segment on increasing BS. CONCLUSIONS: These results suggested that the QTLs for the densities of cell wall materials in japonica varieties contributed to increased BS and might be utilized for improving lodging resistance in indica varieties of rice.

qUVR-10, a major quantitative trait locus for ultraviolet-B resistance in rice, encodes cyclobutane pyrimidine dimer photolyase.

Rice qUVR-10, a quantitative trait locus (QTL) for ultraviolet-B (UVB) resistance on chromosome 10, was cloned by map-based strategy. It was detected in backcross inbred lines (BILs) derived from a cross between the japonica variety Nipponbare (UV resistant) and the indica variety Kasalath (UV sensitive). Plants homozygous for the Nipponbare allele at the qUVR-10 locus were more resistant to UVB compared with the Kasalath allele. High-resolution mapping using 1850 F(2) plants enabled us to delimit qUVR-10 to a <27-kb genomic region. We identified a gene encoding the cyclobutane pyrimidine dimer (CPD) photolyase in this region. Activity of CPD photorepair in Nipponbare was higher than that of Kasalath and nearly isogenic with qUVR-10 [NIL(qUVR-10)], suggesting that the CPD photolyase of Kasalath was defective. We introduced a genomic fragment containing the CPD photolyase gene of Nipponbare to NIL(qUVR-10). Transgenic plants showed the same level of resistance as Nipponbare did, indicating that the qUVR-10 encoded the CPD photolyase. Comparison of the qUVR-10 sequence in the Nipponbare and Kasalath alleles revealed one probable candidate for the functional nucleotide polymorphism. It was indicated that single-base substitution in the CPD photolyase gene caused the alteration of activity of CPD photorepair and UVB resistance. Furthermore, we were able to develop a UV-hyperresistant plant by overexpression of the photolyase gene.

A novel DNA polymerase homologous to Escherichia coli DNA polymerase I from a higher plant, rice (Oryza sativa L.).

A novel DNA polymerase, designated as OsPolI-like, has been identified from the higher plant, rice (Oryza sativa L. cv. Nipponbare). The OsPolI-like cDNA was 3765 bp in length, and the open reading frame encoded a predicted product of 977 amino acid residues with a molecular weight of 100 kDa. The OsPolI-like gene has been mapped to chromosome 8 and contains 12 exons and 11 introns. The encoded protein showed a high degree of sequence and structural homology to Escherichia coli pol I protein, but differed from DNA polymerase gamma and theta. The DNA polymerase domain of OsPolI-like showed DNA polymerase activity. Subcellular fractionation analysis suggested that the protein is localized in the plastid. Northern and western blotting, and in situ hybridization analyses demonstrated preferential expression of OsPolI-like in meristematic tissues such as shoot apical meristem, root apical meristem, leaf primordia and the marginal meristem. Interestingly, no expression was detected in mature leaves, although they have a high chloroplast content. These properties indicated that OsPolI-like is a novel plant DNA polymerase. The function of OsPolI-like is discussed in relation to plastid maturation.

Precise estimation of genomic regions controlling lodging resistance using a set of reciprocal chromosome segment substitution lines in rice.

Severe lodging has occurred in many improved rice varieties after the recent strong typhoons in East and Southeast Asian countries. The indica variety Takanari possesses strong culm characteristics due to its large section modulus, which indicates culm thickness, whereas the japonica variety Koshihikari is subject to substantial bending stress due to its thick cortical fibre tissue. To detect quantitative trait loci (QTLs) for lodging resistance and to eliminate the effects of genetic background, we used reciprocal chromosome segment substitution lines (CSSLs) derived from a cross between Koshihikari and Takanari. The oppositional effects of QTLs for section modulus were confirmed in both genetic backgrounds on chromosomes 1, 5 and 6, suggesting that these QTLs are not affected by the genetic background and are controlled independently by a single factor. The candidate region of a QTL for section modulus included SD1. The section modulus of NIL-sd1 was lower than that of Koshihikari, whereas the section modulus of NIL-SD1 was higher than that of Takanari. This result indicated that those regions regulate the culm thickness. The reciprocal effects of the QTLs for cortical fibre tissue thickness were confirmed in both genetic backgrounds on chromosome 9 using CSSLs.

Characterization of DNA polymerase delta from a higher plant, rice (Oryza sativa L.).

DNA polymerase delta (pol delta), which is comprised of at least two essential subunits, is an important enzyme involved in DNA replication and repair. We have cloned and characterized both the catalytic and small subunits of pol delta from rice (Oryza sativa L. cv. Nipponbare). The open reading frames of OsPoldelta1 and delta2 encoded a predicted product of 1105 amino acid residues with a molecular weight of 124 kDa for OsPoldelta1, and of 429 residues with a molecular weight of 48 kDa for OsPoldelta2. Northern blotting analysis indicated that OsPoldelta1 and delta2 transcripts were expressed strongly in proliferating tissues such as shoot apical meristem. The expression patterns of both subunits in the organs were slightly different. Therefore, we analyzed the spatial distribution pattern of OsPoldelta1 transcripts by in situ hybridization. In the shoot apex, OsPoldelta1 mRNA was abundant in the shoot apical meristem. In the roots, the OsPoldelta1 transcript accumulated at high levels in the root apical meristem. In mature leaves, OsPoldelta1 was induced after UV irradiation, but OsPoldelta2 was not. The amounts of the OsPoldelta1 and delta2 mRNAs in the rice cells changed rapidly during cell proliferation. These results indicated that the levels of OsPoldelta expression are markedly correlated with cell proliferation, and that some of OsPoldelta might have special roles in the leaves.

Increased lodging resistance in long-culm, low-lignin gh2 rice for improved feed and bioenergy production.

Lignin modification has been a breeding target for the improvements of forage digestibility and energy yields in forage and bioenergy crops, but decreased lignin levels are often accompanied by reduced lodging resistance. The rice mutant gold hull and internode2 (gh2) has been identified to be lignin deficient. GH2 has been mapped to the short arm of chromosome 2 and encodes cinnamyl-alcohol dehydrogenase (CAD). We developed a long-culm variety, 'Leaf Star', with superior lodging resistance and a gh phenotype similar to one of its parents, 'Chugoku 117'. The gh loci in Leaf Star and Chugoku 117 were localized to the same region of chromosome 2 as the gh2 mutant. Leaf Star had culms with low lignin concentrations due to a natural mutation in OsCAD2 that was not present in Chugoku 117. However, this variety had high culm strength due to its strong, thick culms. Additionally, this variety had a thick layer of cortical fiber tissue with well-developed secondary cell walls. Our results suggest that rice can be improved for forage and bioenergy production by combining superior lodging resistance, which can be obtained by introducing thick and stiff culm traits, with low lignin concentrations, which can be obtained using the gh2 variety.

Interaction between proliferating cell nuclear antigen (PCNA) and a DnaJ induced by DNA damage.

Proliferating cell nuclear antigen (PCNA) is an essential protein for both DNA replication and DNA repair. In the present study using two-hybrid analysis with PCNA from rice, Oryza sativa L. cv. Nipponbare (OsPCNA), we found that OsPCNA interacted with rice DnaJ protein. We have identified DnaJ and designated it as OsDnaJ. OsDnaJ was able to bind to OsPCNA in vitro. Transcripts of OsDnaJ were found to be strongly expressed in the proliferating cells. mRNA of DnaJ was induced by UV and DNA-damaging agents such as H2O2. The expression patterns of OsPCNA were almost the same as OsDnaJ. The relationship between OsPCNA and OsDnaJ is discussed.

Spontaneously occurring mutations in the cyclobutane pyrimidine dimer photolyase gene cause different sensitivities to ultraviolet-B in rice.

Sensitivity to ultraviolet-B (UVB) radiation (280-320 nm) varies widely among rice cultivars. We previously indicated that UV-resistant rice cultivars are better able to repair cyclobutane pyrimidine dimers (CPDs) through photorepair than are UV-sensitive cultivars. In this paper, we report that UVB sensitivity in rice, in part, is the result of defective CPD photolyase alleles. Surjamkhi (indica) exhibited greater sensitivity to UVB radiation and was more deficient in CPD photorepair ability compared with UV-resistant Sasanishiki (japonica). The deficiency in CPD photorepair in Surjamkhi resulted from changes in two nucleotides at positions 377 and 888 in the photolyase gene, causing alterations of two deduced amino acids at positions 126 and 296 in the photolyase enzyme. A linkage analysis in populations derived from Surjamkhi and Sasanishiki showed that UVB sensitivity is a quantitative inherited trait and that the CPD photolyase locus is tightly linked with a quantitative trait locus that explains a major portion of the genetic variation for this trait. These results suggest that spontaneously occurring mutations in the CPD photolyase gene cause different degrees of sensitivity to UVB in rice, and that the resistance of rice to UVB radiation could be increased by increasing the photolyase function through conventional breeding or bioengineering.