Inheritance of plant elongation ability at seedling stage in rice

Summary In a comparison of methods to study inheritance of plant elongation ability, 15-, 20-, 25-, 30-, and 35-day-old F₂ populations of a cross between Baisbish (floating variety) and IR42 (nonelongating semidwarf modern variety) of rice, (Oryza sativa L.) were subjected to 65 cm water depth for 7 days. Frequency distribution of plant height before and after submergence was obtained. Bimodal curves in 15-, 20- and 25-day-old populations gave good fits to 9:7 elongating: nonelongating plants, suggesting that elongation was due to two dominant complementary genes. Segregation in the 30-day-old population was not clear-cut. A seedling age of 20 days was subsequently chosen for further studies.Two F₂'s involving floating rice and a nonelongating semidwarf; four F₂'s involving floating rice and an elongating semidwarf; and two F₂'s involving elongating and nonelongating semidwarf parents were studied with 20-day-old seedlings in the same way. Floating rice combinations with nonelongating semidwarf parents as well as with elongating semidwarf parents segregated into 9:7 elongating: nonelongating ratio. It is possible that because elongating and nonelongating dwarf parents did not differ much in elongation ability at seedling age, their combination with floating rice parents provided similar segregation. The F₂ distributions for height in elongating and nonelongating dwarf cross combinations were continuous with one peak.Genetic constitution of parents proposed are Sd1 Sd1 El El for floating parents, sd1 sd1 El El for semidwarf elongating, and sd1 sd1 el el for dwarf nonelongating.     

Radiation induced variations in photoperiod-sensitivity,thermo-sensitivity and the number of days to heading in rice

Summary Radiation induced semi-dwarf mutants derived from five japonica type varieties of rice were studied with regard to their photoperiod-sensitivity, thermo-sensitivity and the number of days to heading. The experiment was carried out under the natural conditions at Taipei. The coefficient of photoperiod-sensitivity (T.D.M.) and thermo-sensitivity (byx. z and byz. x) as developed by Oka (1954) were estimated for the mutants in comparison with their original varieties.It was observed that these various physiological characters could be altered rather easily by mutations. Mutants showed wider ranges in both positive and negative directions than their original varieties in all the physiological characters studied. Even though heading date depends on both photoperiod-sensitivity and thermo-sensitivity, it was estimated which of the two contributed more to the induced earliness in each mutant. This offers a basis for selecting early maturing lines of rice.Taiwan Agricultural Research Institute, Taipei, Taiwan, Republic of China.     

SSR analysis of chromosome 8 regions associated with aroma and cooked kernel elongation in Basmati rice

Aroma and cooked kernel elongation (CKE) are the two most important quality traits, which differentiate the highly valued Basmati rice from other rice types. Previous studies on genetic analysis have shown that genes/QTLs for these two traits are linked and present on chromosome number 8. We have evaluated the genetic diversity in 33 rice genotypes representative of the traditional Basmati (TB), cross-bred Basmati derived from indica × Basmati rice crosses and non-Basmati (indica and japonica) rice varieties for chromosome number 8 using 26 SSR markers including a specific marker (SCU-SSR1) for RG28 locus; the results have been compared with whole genome based SSR allelic data. The 26 SSR markers (24 polymorphic and 2 monomorphic) amplified a total of 106 alleles; 21 of these alleles were detected to be unique, present in only one genotype. The number and size of the alleles, and polymorphism information content (PIC) values ranged between 1–8, 87–312 and 0–0.736 bp, respectively. SCU-SSR1 marker amplified a total of three alleles (128, 129 and 130 bp). All the TB varieties except Basmati 217 (129 bp) and 7/13 cross-bred Basmati varieties had the 130 bp allele. Alleles of 129 and 128 bp were present in majority of the indica and japonica varieties, respectively. The average pair-wise Jaccard similarity coefficients for TB, indica and japonica varieties were 0.512, 0.483 and 0.251, respectively. Average similarity coefficient between TB and japonica was higher (0.236) compared to that between TB and indicas (0.150). Genetic relationships as determined by Principal Component Analysis (PCA, NTSYS-pc), PowerMarker tree, and Structure analyses, clearly showed high-level differentiation between TB and indica rice varieties, which formed two distinct clusters. The cross-bred Basmati and japonica rice genotypes were placed between these two clusters. Basmati 217 and Ranbir Basmati were quite divergent from rest of the TB varieties. Some of cross-bred Basmati varieties including Super, CSR30 and kernel were closer to TB. Indica rice varieties, CSR10 (salt tolerant variety) and Pokkali (salt tolerant landrace) formed a separate distinct cluster. The Pritchard structure analysis divided the rice genotypes in four major sub-populations of TB, cross-bred Basmati, indica and japonica (including Ranbir Basmati and Basmati 217) rice varieties. Chromosome 8 data-set showed a positive correlation (Mantel test, r = 0.739) with the allelic data-set for 30 SSR markers well-distributed on 12 rice chromosomes indicating a higher level of similarity between the two. The study demonstrates the distinctness of TB from other rice types (indica and japonica) and also provides several novel markers for differentiation between TB rice supplies from cheaper cross-bred Basmati and long-grain non-Basmati varieties at commercial level.     

Genetic analysis of semidwarf mutants induced in indica rice (Oryza sativa L.)

The mode of inheritance of five semidwarf (SD) mutants and allelic relationship with DGWG (sd1) locus was studied. The five SD mutants viz., Basmati 370 (SD1), Basmati 370 (SD2), Basmati 370 (SD3), TCA 2 (SD) and TCA P2-5 (SD) when crossed with their tall parents exhibited monogenic inheritance of 3 tall: 1 semidwarf in the F2 progenies. The SD mutants were also crossed with semidwarf varieties (DGWG and Bala) possessing sd1 gene. Crosses between Basmati 370 (SD1) × Bala, Basmati 370 (SD3) × Bala and TCA 2 (SD) × DGWG produced tall F1 hybrids and in F2 generation, modified dihybrid ratios (9 : 6 : 1 and 9 : 7) were observed indicating that the mutants Basmati 370 (SD1), Basmati 370 (SD3) and TCA 2 (SD) are non-allelic to sd1 gene. Whereas, the mutants Basmati 370 (SD2) and TCA P2-5 (SD) when crossed with Bala produced semidwarf F1 hybrids and in F2 generation, segregation for plant height was not observed indicating that these mutants are allelic to sd1 gene. The three non-allelic SD mutants identified in the present study can be used in rice breeding as alternative gene sources for semidwarfism. This revised version was published online in July 2006 with corrections to the Cover Date.     

Screening of Rice Varieties for Endosperm Storage Proteins

A total of 118 rice varieties/lines (Oryza sativa L.) from Bangladesh were analyzed for endosperm storage proteins on a single seed basis. The screening was done by observing the profiles of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with standard japonica variety ‘Kinmaze’. Six groups were classified on the basis of the poly-peptide banding patterns. Two-dimensional gel electrophoresis was performed to ensure the SDS-PAGE results in selected varieties. The analysis of extracted protein fractions revealed that one variety had low prolamin and one variety had high glutelin content.     

Inheritance of morphological traits of periwinkle mutants with modified contents and yields of leaf and root alkaloids

Periwinkle Catharanthus roseus is a medicinally important plant producing anticancer and antihypertension alkaloids. Two mutants, one dwarf and one semidwarf and both, with a high alkaloid content in the roots and one mutant with a wavy leaf margin and a high alkaloid content in its leaves were obtained after induced chemical mutagenesis with ethyl methane sulphonate and N-nitroso-N-ethylurea in the variety ‘Nirmal’, which is resistant to dieback disease. These mutants were evaluated in the M₃ and M₄ generations. The dwarf and semidwarf mutants differed from the parental variety for many morphological characters, while the mutant with the wavy leaf margin differed mainly in leaf size and leaf thickness. Although both dwarf and semidwarf mutants showed a significantly higher alkaloid content in their roots in both generations, only the semidwarf mutant gave a significantly higher (23%) root alkaloid yield than the parental variety. The mutant with the wavy leaf margin showed a significantly higher alkaloid content in the leaves in both the M₃ and M₄ generations and also had a significantly higher (21%) leaf alkaloid yield than the parental variety. All three mutants were found to be controlled by monogenic recessive genes; the genes for ‘dwarfness’ and ‘semidwarfness’ were allelic to each other, with the allele for semidwarfness being dominant over the allele for dwarfness. The gene for the wavy leaf margin was inherited independently of the genes for dwarfness and semidwarfness.     

Rice (Oryza sativa L.) germplasm suitable for direct sowing under flooded soil surface

Summary Screening for rice germplasm which can establish seedling from flooded soil was conducted using 256 accessions of conserved germplasm from the International Rice Germplasm Center (IRGC) and 404 accessions from the International Network for Genetic Evaluation for Rice (INGER), IRRI. IRGC germplasm represented broad genetic diversity while INGER germplasm involved desirable agronomic characters. Seeds germinated for 2 d were planted at 25 mm depth in seedling trays. The trays were then submerged to a depth of 30–50 mm. Seedling establishment was evaluated by analyzing leaf development, seedling height, and percentage establishment 15 d after planting. Eight percent and 2% of IRGC and INGER germplasm, respectively, were identified statistically as superior to the control semidwarf IR varieties. Among the superior germplasm were those from Northeast India and Bangladesh which were adapted to deepwater and early summer rainfed lowland cultures. These could be utilized as parents in breeding programs which aim to develop varieties suitable for direct seeding technology (i.e., germinated seeds are sown under the surface of flooded soil).     

Analysis on the spectra and level of wide-compatibility conferred by three neutral alleles in inter-subspecific hybrids of rice (Oryza sativa L.) using near isogenic lines

Wide‐compatibility varieties are a special class of rice germplasm that is able to produce fertile hybrids when crossed to either indica or japonica subspecies. Previous studies determined the f5 allele from ‘Dular’ (f5‐Du), f6 allele from ‘Dular’ (f6‐Du) and S5 allele from ‘02428’ (S5‐08) as neutral alleles conferring wide‐compatibility. However, the possible extent of the effect of the three neutral alleles has not been fully characterized because of the narrow range of the tester varieties used and the highly complex differentiation in Asian cultivated rice. In this study, we further developed the five near‐isogenic lines with higher recovery rates of the recurrent parent genome, and testcrossed to 14 japonica varieties, which have been widely used in rice breeding programmes in China. The results clearly revealed that all three neutral alleles exhibited substantial effects on spikelet fertility in most of the indica–japonica testcrosses, which indicated that these hybrid sterility loci have been extensively differentiated between indica and japonica varieties. The magnitudes of effects on spikelet fertility averaged over various crosses seem to be similar among the three neutral alleles, with f5‐Du, f6‐Du and S5‐08 alleles increasing spikelet fertility by 15.09%, 13.99% and 14.25%, respectively. The testcrosses involving f5‐Du allele generally showed much smaller variation in pollen fertility than others. The pyramiding lines with two neutral alleles showed a wider spectrum and a higher level of wide compatibility than others, whereas most of the increases in hybrid fertility couldn’t be simply explained by additive effects, suggesting the very complexity of wide compatibility and hybrid sterility. The indica–japonica hybrids involving restorer lines as one of their parents showed much higher pollen fertility (almost normal) and also higher spikelet fertility. The implications of the findings in rice breeding programmes are also discussed.     

Complexity of indica-japonica varietal differentiation in Bangladesh rice landraces revealed by microsatellite markers

To understand the genetic diversity and indica-japonica differentiation in Bangladesh rice varieties, a total of 151 accessions of rice varieties mostly Bangladesh traditional varieties including Aus, Boro, broadcast Aman, transplant Aman and Rayada varietal groups were genotyped using 47 rice nuclear SSRs. As a result, three distinct groups were detected by cluster analysis, corresponding to indica, Aus and japonica rice. Among deepwater rice varieties analyzed some having particular morphological features that mainly corresponded to the japonica varietal group. Some small seeded and aromatic varieties from Bangladesh also corresponded to the japonica varietal group. This research for the first time establishes that the japonica varietal group is a prominent component of traditional varieties in Bangladesh, particularly in deepwater areas.     

Characterization of near-isogenic lines carrying QTL for high spikelet number with the genetic background of an indica rice variety IR64 (Oryza sativa L.)

Total spikelet number per panicle (TSN) is one of the most important traits associated with rice yield potential. This trait was assessed in a set of 334 chromosomal segment introgression lines (ILs: BC₃-derived lines), developed from new plant type (NPT) varieties as donor parents and having the genetic background of an indica-type rice variety IR64. Among the 334 ILs, five lines which had different donor parents and showed significantly higher TSN than IR64 were used for genetic analysis. Quantitative trait locus (QTL) analysis was conducted using F₂ populations derived from crosses between IR64 and these ILs. As a result, a QTL for high TSN (one from each NPT donor variety) was detected on common region of the long arm of chromosome 4. The effect of the QTL was confirmed by an increase in TSN of five near-isogenic lines (NILs) developed in the present study. The variation in TSN was found among these NILs, attributing to the panicle architecture in the numbers of primary, secondary and tertiary branches. The NILs for TSN and the SSR markers linked to the TSN QTLs are expected to be useful materials for research and breeding to enhance the yield potential of rice varieties.     

Cytoplasm and cytoplasm-nucleus interactions affect agronomic traits in japonica rice

Some sources of cytoplasm have been favoredin rice breeding for unknown reasons. Tostudy cytoplasmic effects on agronomictraits, five core japonica parents inOryza sativa, Xinan 175, Reimei,Keqing No. 3, Todorokiwase, and Toride No.1, which are ancestors of 75 % of cultivarsbred in Yunnan, China, were used as femaleparents in crosses with 3 distinctjaponica rice cultivars, 8-126,Lijiangxintuanheigu, and Norinmochi No.20as males. These nuclear genomes weresubstituted into the five cytoplasms viaseven backcrosses using the original maleas recurrent parent. Fifteen combinationsof BC₇F₂ and their parents weresown in Jinghong during the late season(July-October) for agronomic evaluation in1999. Meanwhile, all materials werescreened for low temperature tolerancebased upon two methods in Kunming (1916 mabove sea level): natural field and lowtemperature water (19 °C) cyclingirrigation at booting stage. Spikeletfertility was used as an indication of lowtemperature tolerance. Effects of cytoplasmon yield, width of flag leaf, and lowtemperature tolerance were significant. Significant cytoplasm-nucleus interactionon yield, plant height, and low temperaturetolerance were also observed. These resultsindicated that cytoplasm andcytoplasm-nucleus interaction playedimportant roles in yield, low temperaturetolerance, and some important agronomictraits in japonica rice. The role ofcytoplasm and cytoplasm-nucleus interactionshould be considered in future ricebreeding and resources work.     

高能重离子辐射对水稻的生物学效应

本文观察了数种高能重离子辐射处理水稻干种子的生物学效应，结果表明；与具低传能线密度的＾Ｃｏ６０－γ射线相比，这些具高ＬＥＴ的重离子能更有效地抑制受照射种子萌发生出的幼苗的生长，诱导根尖细胞和花粉母细胞的染色体畸变和微核形成，降低当代植株的结实率，并发后代出现形态性状及农艺性状的变异，在一定剂量范围内，辐射诱导的上述数种生物学效应与吸收剂量间呈线性关系，根据剂一－效应关系曲线，估算了重离子辐射的上述     

A new gene controlling hybrid sterility in rice (Oryza sativa L.)

Hybrids between rice subspecies indica and japonica display strong heterosis. However, semi-sterility of inter-subspecific hybrids between indica and japonica varieties is a major obstacle for application of hybrid vigor in rice production. Semi-sterility was previously ascribed to allelic interaction at a number of different loci, whereas, wide-compatibility varieties can overcome hybrid sterility. Variety Nekken 2, which is a source of wide compatibility genes, showed sterility when crossed to the Korean variety Yeong Pung. Genetic and cytological analyses revealed that the semi-sterility was caused by partial abortion of the embryo sac. Genome-wide analysis of the backcross population, Nekken 2/Yeong Pung//Nekken 2 identified two independent loci for hybrid sterility on chromosomes 1 and 12, explaining 18.99 and 18.03% of the phenotypic variance, respectively. To confirm this result, another population of the same backcross containing 216 individuals was tested at a different site in a different year. The locus on chromosome 12 was detected again. Based on the study, the stable QTL on chromosome 12 appeared to be different from previously reported genes for this trait, and was designated as S35(t).     

Seedling emergence,coleoptile length,and plant height relationships in crosses of dwarf and standard-height wheats

Summary Seedling emergence was closely correlated with coleoptile length and plant height among parents, F₂ and F₃, populations of crosses involving dwarf wheats Olesen Dwarf (CI 14497), Norin 10 derivative D6301, Tom Thumb derivative D6899, and the standard-height varieties Ramona 50 and Nainari 60. Genetic mechanisms that governed plant height also influenced coleoptile length, but the relative effects of genes showing dominant or epistatic effects appeared to be different. With respect to the two parents involved in each of 15 crosses, mean F₂ coleoptile lengths were consistently closer to the low parent value than were corresponding mean F₂ plant heights. A slight curvilinear relationship was also found between coleoptile length and plant height of F₃ lines. The results suggest that selection of semidwarf wheats with long coleoptiles and improved emergence properties from crosses involving the dwarf wheats of this study would be unlikely.     

Genetic analysis of a novel dominant rice dwarf mutant 986083D

This study was to determine the agronomic and genetic characteristics of a novel rice dominant dwarf mutant 986083D (japonica) and its potential in breeding. 986083D derived from the anther culture of an autotetraploid indica/japonica hybrid and its progeny segregated into normal and dwarf plants. Homozygous and heterozygous 986083D plants looked similar phenotypically, showing shortened stature, erect leaves, more tillers and poor fertility. The segregation ratio of dwarf to normal plants fit the expected 3:1 by χ²-test in 77 out of 88 tested lines. Crosses between homozygous 986083D and eight other rice varieties had uniform semi-dwarf F₁ plants. The F₁ plants from crosses between heterozygous 986083D and five other varieties had normal and semi-dwarf plants close to the expected ratio of 1:1. The reduction of plant height in F₁ plants ranged from 40.0 to 53.5% in a subtropical environment and from 37.5 to 48.2% in a temperate environment. 986083D showed moderate sensitivity to exogenously applied GA₃ in terms of elongation of shoots and induction of α-amylase activity in the endosperm. Linkage analysis showed that the dominant dwarf gene (designated as Dx) in 986083D was not allelic to D53. Dx was roughly mapped to the short arm of chromosome 8. All results showed that 986083D was a novel mutant controlled by single dominant gene, providing a valuable material in rice breeding. Ruizhen Qin, Yang Qiu contributed equally to this work.     

Development of cleaved amplified polymorphic sequence (CAPS) markers linked to a green rice leafhopper resistance gene, Grh3(t)

The chromosomal location of the resistance gene for green rice leafhopper (GRLH), an injurious insect for rice, has been determined and RFLP markers closely linked to this gene have been identified. The susceptible japonica rice variety ‘Nipponbare’ was crossed with a resistant japonica rice line ‘Aichi42’, in which green rice leaf hopper resistance had been introduced from an indica variety ‘Rantaj‐emas2’, and the 100 F₂ plants obtained were used for linkage analysis. The green rice leafhopper resistance gene, Grh3(t), was mapped between RFLP markers C288B and C133A on chromosome 6 and co‐segregated with C81. Of the RFLP markers tightly linked to Grh3(t), C81 was converted to a SCAR marker and C133A to a cleaved amplified polymorphic sequence marker that could distinguish the heterozygous genotype to establish an effective marker‐aided selection system for the GRLH resistance gene.     

Assessment of genetic diversity within and among Basmati and non-Basmati rice varieties using AFLP,ISSR and SSR markers

Molecular markers provide novel tools to differentiate between the various grades of Basmati rice, maintain fair-trade practices and to determine its relationship with other rice groups in Oryza sativa. We have evaluated the genetic diversity and patterns of relationships among the 18 rice genotypes representative of the traditional Basmati, cross-bred Basmati and non-Basmati (indica and japonica) rice varieties using AFLP, ISSR and SSR markers. All the three marker systems generated higher levels of polymorphism and could distinguish between all the 18 rice cultivars. The minimum number of assay-units per system needed to distinguish between all the cultivars was one for AFLP, two for ISSR and five for SSR. A total of 171 (110 polymorphic), 240 (188 polymorphic) and 160 (159 polymorphic) bands were detected using five primer combinations of AFLP, 25 UBC ISSR primers and 30 well distributed, mapped SSR markers, respectively. The salient features of AFLP, ISSR and SSR marker data analyzed using clustering algorithms, principal component analysis, Mantel test and AMOVA analysis are as given below: (i) the two traditional Basmati rice varieties were genetically distinct from indica and japonica rice varieties and invariably formed a separate cluster, (ii) the six Basmati varieties developed from various indica × Basmati rice crosses and backcrosses were grouped variably depending upon the marker system employed; CSR30 and Super being more closer to traditional Basmati followed by HKR228, Kasturi, Pusa Basmati 1 and Sabarmati, (iii) AFLP, ISSR and SSR marker data-sets showed moderate levels of positive correlation (Mantel test, r = 0.42–0.50), and (iv) the partitioning of the variance among and within rice groups (traditional Basmati, cross-bred Basmati, indica and japonica) using AMOVA showed greater variation among than within groups using SSR data-set, while reverse was true for both ISSR and AFLP data-sets. The study emphasizes the need for using a combination of different marker systems for a comprehensive genetic analysis of Basmati rice germplasm. The high-level polymorphism generated by SSR, ISSR and AFLP assays described in this study shall provide novel markers to differentiate between traditional Basmati rice supplies from cheaper cross-bred Basmati and long-grain non-Basmati varieties at commercial level.The first two authors have equal contribution     

Susceptibility of rice to sheath blight: an assessment of the diversity of rice germplasm according to genetic groups and morphological traits

A sample of 200 accessions was selected from IRRI’s rice (Oryza sativa) germplasm bank, that represents rice diversity in terms of genetic groups (aus, indica, tropical japonica, temperate japonica, and aromatic) and crop duration cycles of the accessions. The sample also captured the diversity of rice germplasm in terms of morphological traits. The accessions were assessed for their level of susceptibility to sheath blight in a field experiment, by inoculating hills at the centre of microfields and measuring disease intensity. Morphological traits of all accessions were also measured. Analyses of variance indicated significant effects of genetic groups and genotypes on sheath blight intensity, measured by different variables (lesion height, relative lesion height, leaf severity, sheath severity, and disease incidence on tillers). Results from multivariate analyses (canonical correlation, hierarchical cluster analyses, correspondence analysis, and discriminant analysis), and logistic regressions indicated that morphological traits were strongly associated with disease intensity, and that their effect was larger than that of genetic groups. In particular, plant height was associated with low sheath blight intensity. Effects of genetic groups could however also be detected, and the combination of results suggested a ranking of genetic groups with increasing level of susceptibility to sheath blight as: aus < indica < japonica.     

Selection of Semidwarf, Early Maturing and Blast Resistant Mutants after Mutagenic Seed Treatment in Two Locally Adapted Indian Rice Cultivars

The objective of this study was to induce semidwarf, early maturing and blast resistant mutants in two adapted Indian rice cultivars. Seeds of the rice cultivars ‘Madhu Malti’ and ‘Phul Patas 72’ were treated with gamma rays and ethyl methane sulfonate (EMS) as well as a combination of gamma rays and EMS. A total of 96 dwarf (< 75 cm) and semidwarf (< 100 cm) and 104 early flowering/maturing M₂ plants were identified among the 18,060 M₂ plants observed. Sixty-eight blast resistant M₂ plants were also identified. The true mutant status of these plants has yet to be determined in progeny tests. The induction of desired mutant types in locally adapted cultivars could prove to be superior over their introduction from non-adapted foreign materials via backcrossing in that the important agronomic characteristics of adaptation to the local environment are retained in the mutants.     

A simple method to control the seed purity of japonica hybrid rice varieties using PCR-based markers

Cytoplasmic male sterility (CMS) by the cms‐bo cytoplasm and its restoration by the nuclear restorer gene, Rf‐1, are used for seed production of japonica hybrid rice varieties. To produce pure hybrid seeds, a prerequisite is to properly manage the seed purity of parental lines, especially CMS lines. In this study, three dominant polymerase chain reaction (PCR)‐based markers (M1, M2 and M3) were developed to detect mutual contamination in seed batches of CMS lines, maintainer lines, restorer lines and hybrids. M1 detected the mitochondrial sequence that was present in the cytoplasm of common japonica varieties and absent in the cms‐bo cytoplasm. M2 and M3 detected the chromosomal sequence related to the Rf‐1 allele in restorer lines and the rf‐1 allele in common japonica varieties, respectively. By the strategic use of these markers, japonica hybrids and their parental lines could be efficiently distinguished from each other. Furthermore, sensitivity tests for the three markers with a series of crude DNA samples prepared from polished grains demonstrated that these markers could detect one contaminating grain among 500 or 1000 grains. Therefore, the bulk PCR analyses with the markers developed here probably make it possible to control the seed purity of japonica hybrids properly by selecting appropriate seed batches of their parental lines quickly and efficiently.