RAPD variability in rice (Oryza sativa L.) plants derived from desiccation-tolerant calli

Genetic modification from selfed progenies of 18 rice (Oryza sativa L.) plants regenerated from callus tissues which survived desiccation, were investigated at the DNA level using the random amplified polymorphic DNA (RAPD) method. Twelve 10-mer random primers were used to amplify DNA of progenies from the regenerated plants, and a total of 228 PCR products and 1780 DNA fragments were obtained by primers, generating between four to thirteen major bands. The size of the amplified fragments ranged from 0.2 to 2.55 kb. The results showed that 10 out of 12 primers produced polymorphic bands, two primers (RA31 and RA185) showed no polymorphism among plants tested. A dendrogram of the genetic distance was constructed based on their polymorphism, demonstrating that somaclonal variation exists in rice plants regenerated from callus which survived the desiccation treatment. Part of this variation can be useful in rice breeding. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genetical and morphological characterization of cold tolerance at fertilization stage in rice

Cold temperature during the reproductive phase leads to seed sterility, which reduces yield and decreases the grain quality of rice. The fertilization stage, ranging from pollen maturation to the completion of fertilization, is sensitive to unsuitable temperature. Improving cold tolerance at the fertilization stage (CTF) is an important objective of rice breeding program in cold temperature areas. In this study, we characterized fertilization behavior under cold temperature to define the phenotype of CTF and identified quantitative trait loci (QTLs) for CTF. A wide variation in CTF levels has been identified among local cultivars in Hokkaido, which is one of the most northern regions for rice cultivation in the world. Clear varietal differences in pollen germination, and pollen tube elongation due to cold temperature have been observed. These differences may confer a degree of CTF among this population. We conducted QTL analysis for CTF using 120 backcrossed inbred lines derived from a cross between Eikei88223 (vigorous CTF) and Suisei (very weak CTF). Three QTLs for CTF were identified. A clear effect by QTL, qCTF7, for increasing the level of CTF was validated using advanced progeny. These results will facilitate marker-assist selection for desirable QTLs for CTF in rice breeding program.     

Heterogeneous plastid genomes in anther culture-derived albino rice plants

Large-scale deletions in the plastid genome of albino plants derived fromanther culture of japonica rice have in previous studies been detected bySouthern blot analysis. In the present study, PCR was applied to detectlarge-scale deletions in the plastid genome of albino and green plantsgenerated from the anther culture of japonica-indica hybrid-derived DHlines. The rice plastid genome (134.5 kbp) was separated into sevenregions and each one (approximately 20 kbp) was amplified by long PCR. The plastid sequences of all 22 albino and 19 green plants were amplifiedat all the seven regions, indicating that large-scale deletions in the plastidgenome were not detected by long PCR. The concentrations of two plastidgenome regions were measured by competitive PCR. Four of 29 albinoplants analyzed showed different concentrations between the two regions,indicating that both deleted and complete plastid molecules were presentin each of the four albino plants. Such plastid genome heterogeneity wasnot detected in 17 green plants.     

Characterization of rice (Oryza sativa L.) F3 populations selected for salt resistance. I. Physiological behaviour during vegetative growth

Plants belonging to a somaclonal family (R₃-1-23, obtained from a salt-resistant callus of the salt-sensitive cultivar I Kong Pao (IKP, salt-sensitive)) were crossed with this intital cultivar IKP or with plants of the elite breeding line IR31785 (IR31, extremely salt-sensitive). Two other crosses were also performed: IR31 × IKP and IKP × Aiwu, where Aiwu is a moderatly salt-resistant cultivar. The physiological behaviour of F₃ populations was analysed after 21 and 42 days of exposure to 0 or 30 mM NaCl and compared to the behaviour of plants selected within parental populations. A clear improvement in terms of survival percentages was demonstrated for plants issued from IR31 × 1-23 and IR31 × IKP. These F₃ populations exhibited lower Na accumulation in shoots, higher Ca accumulation in roots, higher ratios of shoot P/root P than the parents after 42 days of salt exposure. Salt stress induced a decrease of shoot K, an increase of shoot Cl and proline and a decrease of osmotic potential in all populations. Correlation analysis indicated that no physiological parameter recorded in the absence of salt stress may be used as a reliable selection marker to predict the plant behaviour in salt stress conditions. K/Na ratio recorded in the stressed plants was significantly correlated with surviving percentages recorded after 90 days of salt stress, suggesting that this physiological parameter in stress conditions appears to be the most reliable selection criterion for salt resistance in rice. This revised version was published online in August 2006 with corrections to the Cover Date.     

Molecular mapping of S-c, an F1 pollen sterility gene in cultivated rice

Hybrids between indica and japonica rice varieties usually show partial sterility, and are a major limiting factor in the utilization of heterosis at subspecific level. When studying male-gamete (pollen) abortion, a possibly important cause for sterility, six loci (S-a, S-b, S-c, S-d, S-e and S-f) for F₁ pollen sterility were identified. Here we report genetic and linkage analysis of S-c locus using molecular markers in a cross between Taichung 65, a japonica variety carrying allele S-c ^j, and its isogenic line TISL5, carrying alleleS-c ^j. Our results show that pollen sterility occurring in the hybrids is controlled by one locus. We used 208 RFLP markers, as well as 500 RAPD primers, to survey the polymorphism between Taichung 65 and TISL5. Six RFLP markers located on a small region of chromosome 3, detected different RFLP patterns. Co-segregation analysis of fertility and RFLP patterns with 123 F₂ plants confirmed that the markers RG227, RG391, R1420 were completely linked with the S-c locus. The genetic distances between the markers C730, RG166 and RG369 and the S-c locus were 0.5 cM, 3.4 cM, and 3.4 cM respectively. Distorted F₂ ratios were also observed for these 4 RFLP markers in the cross. This result suggests that the `one locus sporo-gametophytic' model could explain F₁ hybrid pollen sterility in cultivated rice. RG227, the completely linked marker, has been converted to STS marker for marker-assisted selection. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

Genetics of stem elongation ability in rice (Oryza sativa L.)

Summary The genetics of stem elongation ability in rice was studied in parents, F₁, F₂ and backcross generations of six crosses. Segregation analysis indicated dominance for stem elongation ability. Estimation of genetic parameters under epistatic model indicated more than one locus control stem elongation ability and both additive and nonadditive gene effects were important. Epistatic effects were predominant over additive and dominance effects with an important role of duplicate type of epistasis. The occurrence of significant additive and additive x additive types of genetic variation and the moderately high broad sense heritability indicated the possibility of selection for an increased manifestation of stem elongation ability.     

Identification of near-isogenic lines: an innovative approach,validated for root and shoot morphological characters in a mapping population of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Near-isogenic lines (NILs) constitute valuable tools in genetic investigations and plant breeding programs. Conventional methods for developing these are time consuming and tedious. An innovative method for identifying NILs is proposed and validated. The method involves computation of simple correlation coefficients of all possible pairs of genotypes within a mapping population using molecular marker data, and phenotypic characterization of those pairs with very high positive correlation. The pairs having both genomic and phenotypic similarity except for a single trait are considered as NILs. This strategy was tested with a doubled haploid mapping population involving CT9993 and IR62266. This population was saturated with 315 markers and comprised 154 lines. The pairs showing very high correlation coefficients (0.70–0.97) and differing for less than 10% of the markers were considered as Genotypically Closely Related Pairs (GCRPs). Graphical genotyping was employed to visualize the genome of the closely related lines. A total of 39 such pairs were subjected to rigorous evaluation for root and shoot morphological traits in two contrasting moisture regimes. Four GCRPs under well-watered condition and ten GCRPs under low moisture stress condition are statistically significant for a single phenotypic trait and are considered as NILs for their respective traits and would be the valuable materials for genetic studies. Mapped QTLs and candidate genes were employed to explain the probable cause of phenotypic difference in NILs.     

The embryogenic potential of rice cell suspensions affects their recovery following cryogenic storage

Summary The rates of recovery from cryogenic storage of suspension cultures of the Japonica rice cultivar Taipei 309, as determined by the reduction of triphenyl tetrazolium chloride and cell regrowth, were significantly influenced by the embryogenic potential of the non-frozen cultures.     

Inheritance of aroma in rice

Summary Inheritance of an aroma was worked out in crosses between Brimful from Nepal as an aromatic rice and leading Japanese varieties Koshihikari and Nipponbare as non-aromatic ones. The F₂ pattern of segregation for aroma to non-aroma was 3:13 indicating one dose gene for aroma and one dose inhibitor gene in two crosses. This ratio was confirmed by genetic behavior of F₃ populations.     

Inheritance of scent in rice

Summary Inheritance of scent in rice and its association with pigmented characters were studied in T-141 × K-44-1. Four complementary genes appeared to control scent, one of them showing linkage with a complementary gene for glume colour and a supplementary gene for red pigment in apiculus. This group formed part of V (Prp)linkage group of Misro et al. (1966) in which case Pg and P were additions.Pl.480 Project on linkage maps in rice.     

A comparison of somaclonal variation in rice plants derived and not derived from protoplasts

To clarify the effect of the type of in vitro culture on the generation of somaclonal variation, protoplast-derived rice plants were compared with rice plants derived from suspension culture or primary calli (not derived from protoplasts). Regenerated plants showing polyploid-like phenotypes appeared at a higher frequency (33–70%) in plants derived from protoplasts than in those not derived from protoplasts (3–6%). In the first progeny (R₁) generation of all regenerated plants, 120 of 368 lines (33%) segregated plants with mutated characters such as albino, dwarf and sterile. In quantitative traits, 62 (21%) and 144 (50%) of 290 Rj lines showed shorter culm and lower seed-fertility, respectively, compared with the control line derived from the selfed seeds of the original cultivar. The frequency of the mutant-possessing R₁ lines among lines derived from protoplasts was not significantly different from those not derived from protoplasts. These results indicate that isolation and culture of protoplasts does not enhance genetic changes other than cytogenetical changes.     

Variations of the vascular bundle system in Asian rice cultivars

Summary The capacity of vascular bundle system transporting assimilates from source to sink is one of the limiting factors for rice (Oryza sativa L.) yield. Genetic variation in vascular bundle system should be investigated for improvement of transporting efficiency. A total of 531 Asian rice cultivars were studied for vascular bundles in the peduncle and its relation to spike morphology. A few difference was detected for the number of primary rachis branches of the spike among the cultivars from various countries. However, the number of vascular bundles significantly differed among the cultivars; those from Nepal, Bangladesh and India bore more vascular bundles than those from Japan. Therefore, the ratio of vascular bundles to rachis branches (V/R ratio) was nearly 1.0 in cultivars from Japan, while those from Nepal, Bangladesh and India ranged from 1.3 to 2.2. Chinese and Indonesian cultivars incuded two types of high and low V/R ratios. Cultivars with high V/R ratio showed positive reactions to the phenol solution, while those with low V/R ratio were negative reactions, suggesting that the former cultivars are of Indica type and the latter of Japonica type. It was concluded that the relationship between vascular bundles and rachis branches differentiated among the ecotypes of Asian cultivated rice.     

Inheritance of grain threshability in rice

Summary Inheritance of grain threshability was studied in a cross between two indica rice varieties. Hard threshability was found to be completely dominant over easy threshability. The segregation pattern indicated threshability to be monogenically controlled.     

Inheritance of salt tolerance in rice

Summary The genetic behavior of salt tolerance was studied in artificially salinized conditions at the International Rice Research Institute.Divergent selection, carried out at a salinity level where the ECe was 15.2 mmhos/cm at 25 C in F₃ lines from two crosses confirmed the effects of salt tolerance on F₄ progeny with realized heritability values of 0.39 and 0.62, respectively.In a cross between two tolerant cultivars there was clear over-dominance for tolerance, despite the high environmental fluctuation which resulted in a low genetic response as indicated by a low but significant repeatability of 0.20–0.25, and many progeny lines more tolerant than the parents were recovered. The superior tolerance of these progenies compared to the parents was confirmed subsequently at 3 different salt levels. In the same experiment a cross between tolerant and susceptible cultivars produced some progeny of comparable tolerance with tolerant sources.In a 6×6 diallel cross experiment with two tolerant, moderate, and susceptible varieties each, both general and specific combining ability were significant.The findings indicate the possibility of breeding rices more tolerant than existing tolerant cultivars through cumulative crosses of tolerant cultivars. Further improvement can be attained by crossing highly tolerant lines with donors of good agronomic traits and pest and disease resistance.     

QTL-based analysis of genotype-by-environment interaction for grain yield of rice in stress and non-stress environments

Use of DNA-based markers can accelerate cultivar development in variable cultivation environments since, in contrast to phenotype, DNA markers are environment-independent. In an effort to elucidate the genetic basis of genotype-by-environment interaction (G × E) for yield of rice (Oryza sativa L.), the associations between 139 AFLP markers and grain yield were determined for rice grown in fresh water (EC of 0.65 dS m⁻¹) and saline conditions (EC of 4–8 dS m⁻¹) with 0 kg ha⁻¹ or 100 kg ha⁻¹ nitrogen fertilizer in the years 2000 and 2001. A population of recombinant inbred lines of rice, developed from an IR29 × Pokkali cross, was used in the study. Both genotype × salinity and genotype × nitrogen level interactions were significant, with the genotype × salinity interaction being stronger. Through multiple regression analysis using a stepwise procedure for selecting markers, 36 markers were detected for grain yield in the four test conditions and of these 28 were detected in only one test condition implying strong environmental specificity for yield QTL expression. However, the fact that eight QTLs were detected in more than one test condition points to the existence of wide-adaptability genes in this cross. Markers with significant associations with yield explained between 37% and 48% of the yield variation in each test condition. Superior genotypes of rice were identified in all four test conditions based on their marker signatures. Furthermore, across N fertilizer regimes, yield predicted from summed additive effects of QTLs were significantly correlated with observed yield in the same year and across years. Thus marker-assisted selection can help breeders overcome the problem of low selection efficiency encountered during phenotypic selection for yield in stress environments.     

Crosses between cultivars and tissue culture-selected plants for salt resistance improvement in rice, Oryza sativa

Several R₂ somaclonal families were derived from plants regenerated from a salt‐resistant callus of the salt‐sensitive rice cultivar ‘I Kong Pao’ (IKP). The family R₂‐1‐23, in the presence of NaCl exhibited higher yield performances than the initial cultivar. This improvement in salinity resistance, however, was not transmitted to following generations; despite a higher number of spikelets per plant, family R₃‐1‐23 did not perform better than the initial cultivar because of a very low seed set. This somaclonal family, its initial being the cultivar IKP, the breeding line IR₃1785 (extremely salt‐sensitive) and the cultivar ‘Aiwu’ (moderately salt‐resistant), were used as parents for production ofhybrids. Four crosses, IKP×R3‐1‐23, IR31785 ×R3‐1‐23, IR31785× IKP and IKP בAiwu’, were performed. Most of the F₁ hybrids cultivated in the absence of salt exhibited increased performances compared with the mid‐parent, suggesting an heterosis effect for yield‐related parameters. F₂ populations were screened for salinity resistance and a clear improvement for yield in stress conditions was recorded for populations derived from IK×R3‐1‐23, IR31785×R3‐1‐23 and IR31785×IKP, although the mean level of increase over the mid‐parent (RIMP) varied depending on the population, the presence or absence of stress, and the quantified parameters. The results are discussed in relation to the usefulness of in vitro selection for obtaining interesting somaclonal variants useful to be integrated in classical breeding programmes for salinity resistance in rice.     

Photoperiod sensitivity gene controlling heading date in rice cultivars in the northernmost region of Japan

Rice is grown in diverse environments at latitudes ranging from 53^°N to 40^°S. In Japan, Hokkaido is the northernmost rice cultivation region(42–45^°N latitude). Only extremely early maturing (heading) varieties that have extremely low photoperiod sensitivity are adapted to this area. Heading date is the most important trait in adaptation to this particular environment. Also, the efficient manipulation of heading date is a crucial component of rice improvement. To determine the genetic basis for heading date among cultivars grown in Hokkaido, the heading behaviour was analyzed. Clear segregations were observed. To estimate the photoperiod sensitivity of the genes involved, the cultivars and F₁ plants from crosses between the cultivars were grown under different daylength conditions. The results indicated that the genes controlling heading date are photoperiod sensitive, suggesting they play important roles in the northernmost rice cultivation regions in Japan, to which only cultivars with extremely low photoperiod sensitivity are adapted. This revised version was published online in August 2006 with corrections to the Cover Date.     

Isolation and characterization of a rice mutant insensitive to cool temperatures on amylose synthesis

The Wx ^b gene, one of the alleles at the rice waxy(wx) locus, is activated at cool temperatures during seed development, andas a result, a large amount of amylose is accumulated causing a reductionin rice grain quality. We found that the seeds of a du mutant couldbe visibly distinguished depending on whether they matured at cool ornormal temperatures. Using these characteristics, we isolated a mutantcandidate insensitive to cool temperatures. While the amylose content inthe original line was about 2% at a normal temperature (28 °C)and 12% at a cool temperature (21 °C), in the mutant candidate(coi) the amylose content was not affected by temperatures, i.e. theamylose content was about 3% at both temperatures. This finding incombination with the results of an immunoblot analysis indicated that theabsence of an increase in the amylose content in this mutant was caused bya constant level of Wx gene expression at normal and cooltemperature. Genetic analysis revealed that this insensitivity to cooltemperatures was caused by a single recessive mutation. This mutantshould be useful in breeding programs designed to produce rice of desiredquality at cool temperatures and in understanding genetic and molecularmechanisms that respond to slight changes in temperature.