QTL-based analysis of leaf senescence in an indica/japonica hybrid in rice (Oryza sativa L.)

In order to identify quantitative trait loci (QTLs) for leaf senescence and related traits in rice (Oryza sativa L.), we developed two backcross populations, indica/japonica// japonica and indica/japonica//indica, using IR36 as the indica parent and Nekken-2 as the japonica parent. The QTLs were mapped using a set of simple sequence-repeat markers (SSRs) in the BC₁F₁ population. Senescence was characterized in these plants by measuring the leaf chlorophyll content 25 days after flowering (DAF), the reduction in chlorophyll content (the difference between the chlorophyll content at flowering and at 25 DAF), and the number of late-discoloring leaves per panicle at 25 DAF in five plants from each BC₁F₂ line. These plants were moved into a temperature-controlled growth cabinet at the time of flowering and allowed to mature under identical conditions. Eleven QTLs were detected in the two populations. The major of QTLs for senescence were found on the short arm of chromosome 6 and on the long arm of chromosome 9. Of these, one QTL on chromosome 6 and two on chromosome 9 were verified by confirming the effects of the genotypes on the phenotypes of the BC₁F₃ lines. The japonica parent was found to contribute to late senescence at all but one QTL. Based on a comparison of the effects of heterozygotes and homozygotes on the phenotypic values of each QTL genotype, we concluded that the differential senescence observed in the indica-japonica hybrid was not due to over-dominance; rather, it was the result of partial-dominance genes that were donated from either of the parents.     

Three-dimensional analysis of the senescence program in rice (Oryza sativa L.) coleoptiles

The cytological sequence of senescence-related changes in coleoptiles of rice (Oryza sativa L. cv. Nippon-bare) was studied using fluorescence and electron microscopy. The coleoptiles reach full size 3 d after sowing, then rapidly senesce and wither completely by day 7. The interveinal region in cross-sections taken 1 mm from the tip of the coleoptile was selected for this analysis. Fluorescence microscopy using samples embedded in Technovit 7100 resin, electron microscopy and immunoelectron microscopy using DNA-specific antibodies were used to elucidate the sequence of senescence-related events. These occur in the following order: (i) degradation of the chloroplast DNA (cpDNA); (ii) condensation of the nucleus in conjunction with a decrease in the size of the dense-chromatin region, shrinkage of the chloroplast, degradation of ribulose-1, 5-bisphosphate carboxylase/oxygenase, dilation of the thylakoid membranes, increase in size and number of osmiophilic globules, condensation of the cytoplasm; (iii) disorganization of the nucleus, degeneration of the tonoplast; (iv) complete loss of the cytoplasmic components, distortion of the cell wall, invasion of microorganisms into the intercellular spaces and ultimately into the cell itself. The mitochondria maintain their ultrastructural integrity and a constant level of mitochondrial DNA throughout senescence. In young mesophyll cells, invagination of the tonoplast into the vacuole frequently occurs. This occasionally includes cytoplasmic material, which is digested in the vacuole as senescence proceeds. Immunoelectron microscopy suggests that cpDNA degradation involves rough digestion first, rather than rapid, direct decomposition of the DNA into nucleotides. The fragmented cpDNA is then dispersed throughout the chloroplast and cytoplasm. Received: 9 April 1998 / Accepted: 11 June 1998     

Three-dimensional analysis of the senescence program in rice (Oryza sativa L.) coleoptiles

The coleoptile of rice (Oryza sativa L. cv. Nippon-bare) emerges from an imbibed seed on day 2 after sowing. Then, it matures and senesces rapidly. For analysis of the senescence pattern within individual coleoptiles, we monitored the distribution of chlorophyll (Chl) in entire coleoptiles and in cross-sections of coleoptiles by recording the autofluorescence of Chl. Degradation of Chl was apparent at the tip of the margins of opened-out coleoptiles on day 4, when the overall levels of soluble protein and Chl per coleoptile had reached maximum values. Then, senescence proceeded from the tip to the base and from the inner mesophyll cells towards the outer epidermis, excluding tissues along vascular bundles. Further analysis of cellular senescence using samples embedded in Technovit 7100 resin revealed that the senescence of each green mesophyll cell followed an identical program, which consisted of the following steps: (i) degradation of chloroplast DNA; (ii) condensation of the nucleus, decrease in the size of chloroplasts, degradation of ribulose-1,5-bisphosphate carboxylase/oxygenase and chloroplast inner membranes; (iii) disorganization of the nucleus; (iv) complete loss of cellular components, distortion of the cell wall. Although the timing of each step and the rate at which each step was completed differed among cells of different locations within the coleoptile, this sequence was observed in all mesophyll cells in the coleoptile. Received: 31 July 1997 / Accepted: 28 October 1997     

RFLP analysis of rice (Oryza sativa L.) introgression lines

Summary Fifty-two introgression lines (BC₂F₈) from crosses between two Oryza sativa parents and five accessions of O. officinalis were analyzed for the introgression of O. officinalis chromosome segments. DNA from the parents and introgression lines was analyzed with 177 RFLP markers located at approximately 10-cM intervals over the rice chromosomes. Most probe/enzyme combinations detected RFLPs between the parents. Of the 174 informative markers, 28 identified putative O. officinalis introgressed chromosome segments in 1 or more of the introgression lines. Introgressed segments were found on 11 of the 12 rice chromosomes. In most cases of introgression, O. sativa RFLP alleles were replaced by O. officinalis alleles. Introgressed segments were very small in size and similar in plants derived from early and later generations. Some nonconventional recombination mechanism may be involved in the transfer of such small chromosomal segments from O. officinalis chromosomes to those of O. sativa. Some of the introgressed segments show association with genes for brown planthopper (BPH) resistance in some introgressed lines, but not in others. Thus, none of the RFLP markers could be unambiguously associated with BPH resistance.     

Diallel analysis of heading date in rice (Oryza sativa L.)

Summary The genetics of heading date was investigated in an 8×8 diallel set of crosses involving diverse rice cultivars. Wr, Vr graph analysis revealed the presence of a complementary type of non-allelic interaction which apparently affected the position and slope of the regression line such as if there were overdominance. Omission of two interacting parents resulted in a 6×6 subset of diallel crosses from which, as observed in the Wr, Vr graph, the non-allelic interaction had disappeared and the regression line exhibited partial dominance. Estimates of the genetic components of variation were in close conformity with the results obtained from the Wr, Vr graph: the average degree of dominance, as measured by (H₁/D)^1/2, was in overdominance range in the interacting 8×8 set of diallel crosses whereas it was reduced to partial dominance in the non-interacting 6×6 set of crosses. Further analysis by a standardized deviations graph indicated that earliness was controlled, on the average, by an excess of dominant alleles.     

Genetic analysis of plant regeneration in rice (Oryza sativa L.)

Summary A diallel study involving reciprocal crosses of four genotypes (IR8, 36, 54, and 64) was carried out to understand the genetic mechanism of plant regeneration from immature embryo-calli in rice. Regeneration frequency (% of calli that produced plants) varied from a high of 86% for IR54 to a low of 0% for IR36, while regenerated plants per embryo numbered from 0 to 7 when these same IR lines and the F₁ hybrids were compared. Combining ability analysis revealed that both nuclear (with both additive and dominant effects) and cytoplasmic genes are important in controlling regeneration in rice. Parental lines and F1 hybrids with high ability to regenerate were identified.     

Genetic analysis of salinity tolerance in rice (Oryza sativa L.)

Summary The genetics of salinity tolerance in rice was investigated by a nine-parent complete diallel including reciprocals. Test materials involved susceptible (IR28, IR29, and MI-48), moderately tolerant (IR4595-4-1-13, IR9884-54-3-1E-P1, and IR10206-29-2-1), and tolerant (Nona Bokra, Pokkali, and SR26B) parents. Twoweek-old seedlings were grown in a salinized (EC = 12 dS/m) culture solution for 19 days under controlled conditions in the IRRI phytotron. Typical characteristics of salinity tolerance in rice were found to be Na⁺ exclusion and an increased absorption of K⁺ to maintain a good Na-K balance in the shoot. Genetic component analysis (GCA) revealed that a low Na-K ratio is governed by both additive and dominance gene effects. The trait exhibited overdominance, and two groups of genes were detected. Environmental effects were large, and the heritability of the trait was low. Our findings suggest that when breeding for salt tolerance, selection must be done in a later generation and under controlled conditions in order to minimize environmental effects. Modified bulk and single-seed descent would be the suitable breeding methods. Combining ability analysis revealed that both GCA and specific combining ability (SCA) effects were important in the genetics of salt tolerance. Moderately tolerant parents — e.g., IR4595-4-1-13 and IR9884-54-3-1E-P1 — were the best general combiners. Most of the best combinations had susceptible parents crossed either to moderate or tolerant parents. The presence of reciprocal effects among crosses necessitates the use of susceptible parents as males in hybridization programs. Large heterotic effects suggest the potential of hybrid rice for salt-affected lands.     

Genetic analysis of waxy locus in rice (Oryza sativa L.)

Summary Inheritance of waxy locus was studied in crosses of a waxy variety with four non-waxy parents having high-, intermediate-, low- or very low-amylose content. The analysis for amylose content was done on a single grain basis in parents, F₁, F₂, B₁F₁, and B₂F₁ seeds. The waxy parent lacking synthesis of amylose content was found to differ from the ones having high-, intermediate-, low- or very low-amylose content by one gene with major effect. Dosage effects for amylose content were observed to have great influence on segregation pattern and efficiency of selection. Selection efficiency for amylose content can be enhanced by selecting for endosperm appearance in early segregating generations.     

Heterosis breeding in rice (Oryza sativa L.)

Summary Studies conducted at the International Rice Research Institute (IRRI) during 1980 and 1981 have shown up to 73% heterosis, 59% heterobeltiosis and 34% standard heterosis for yield in rice. The latter was estimated in comparison to commercial varieties: IR36 and IR42 (yield 4–5 t/ha in wet season trials and 7–8 t/ha in dry season trials). Generally speaking, absolute yield was lower and extent of standard heterosis was higher in wet season than in dry season with some exception. Yields up to 5.9 t/ha (22% standard heterosis) in the wet season and 10.4 t/ha (34% standard heterosis) in the dry season were obtained. Most of the hybrids performed better in some season while some performed better in both seasons. Hybrids showed better lodging resistance although they were 5–10 cm taller. F₁ hybrids had significant positive correlations with the parental traits viz., yield (r = 0.446), tillering (r = 0.746), height (r = 0.810) and flowering (r = 0.843). Selection of parents among elite breeding lines on the basis of their per se yield performance, diverse origin and resistance to insects and diseases should give heterotic combination. Yield advantage of hybrids was due primarily to increase in number of spikelets per unit area even though tiller number was reduced. Grain weight was either the same or slightly higher. High yielding hybrids also showed significant heterosis and heterobeltiosis for total dry matter and harvest index. For commercial utilization of heterosis in rice, effective male sterility and fertility restoration systems are available and up to 45% natural outcrossing on male sterile lines has been observed. Consequently, F₁ rice hybrid have been successfully developed and used in China. Prospects of developing hybrid rice varieties elsewhere appear bright especially in countries that have organized seed production, certification and distribution programs and where hybrid seed can be produced at a reasonable cost.     

Potentiality of leaf sheath cells for regeneration of rice (Oryza sativa L.) plants

Summary Regeneration of rice plantlets (Oryza sativa L.) from calli originated from leaf sheath cells was made possible. This was possible in tissues initially grown in media containing 2.4-D (2.4-dichlorophenoxyacetic acid) at low temperature and illumination. The slow growing tissues were subsequently subjected to growth conditions at an elevated temperature and higher illumination with addition of kinetin and IAA and without 2.4-D. The suitability of leaf sheath cells for protoplast technology is indicated by this success.     

Genetics of gel consistency in rice (Oryza sativa L.)

Inheritance of gel consistency in rice was studied in crossés involving highamylose, low-gelatinizalion temperature parents with hard, medium, and soft gel consistency. The results of single-grain analysis of parents, F₁, F₂, B₁F₁, B₂F₂, and their reciprocal crosses from a single-season harvest showed that the differences between hard and soft, hard and medium, and medium and soft gel consistency are under monogenic control and that modifiers affect the expression of the trait. Multiple alleles at the same locus, hereby designated asgec ^a for medium gel consistency andgec ^b for soft gel consistency, were recessive to the wild type allele for hard gel consistency andgec ^a was dominant overgec ^b. The results indicate that selection for desired gel consistency can effectively be done in early segregating generations.     

Genomic analysis of polycarpellary rice (Oryza sativa L.) through whole genome resequencing

There is a natural floral organ mutant of rice (var. Jugal) where the florets, popularly known as spikelet bear multiple carpels and produce multiple kernels in most of its grain. In our earlier work a detailed study has been done on its morpho-anatomical structure with allelic diversity and expression study of the major genetic loci associated with floral organ development. In present study high throughput whole genome sequencing was done which generated about of 3.7 million base pair genomic data for downstream analysis. The reads were about 101 bases long and mapped to the Oryza sativa var. Nipponbare as reference genome. Genome wide variant analysis detected 1,096,419 variants which included 943,033 SNPs and 153,386 InDels. A total of 24,920 non-synonymous SNPs were identified for 11,529 identified genes. Chromosome-wise distribution of uniquely mapped reads onto reference genome showed that maximum reads were mapped to 1st chromosome and least to 9th chromosome. 10th chromosome showed highest density of variations (about 325.6 per 100 kb genome sequence). Detailed sequence analysis of 23 floral organ developmental genes detected 419 potent variants where DL (Drooping Leaf) and OSH1 (Oryza sativa Homeobox1) genes showed highest number (32) of variants; whereas, MADS21 (Minichromosome Agamous Deficient Serum Factor 21) gene have lowest number (5) of variants. The information generated in this study will enrich the genomics of floral organ development in indica rice and cereal crops in general.

     

Clustered QTL for source leaf size and yield traits in rice (Oryza sativa L.)

Improvement of plant type plays an important role in super-high yield breeding in rice (Oryza sativa L.). In the present study, a set of backcross recombinant inbred lines derived from a cross of 9311 and Zhenshan97, both elite indica hybrid parents, were developed to identify quantitative trait loci (QTL) for flag leaf size, panicle and yield traits. Forty-seven QTL for 14 traits were detected in common in the two environmental trials, of which nine genomic regions contained clustered QTL affecting plant type traits and yield traits. Four co-localized QTL on chromosomes 1, 6, 7 and 8 involving QTL for flag leaf size (flag leaf length, width and area) contained the QTL for yield traits such as panicle weight (PW) and secondary branch number (SBN), and in all cases alleles from 9311 increased source leaf size and were associated with increased sink size and yield (SBN and PW). Using a subset of overlapping substitution lines for the QTL region on chromosome 1, the QTL were validated and narrowed into a 990?kbp interval (RM3746?CRM10435) with pleiotropic effects on flag leaf size, PW, SBN and spikelet number per panicle. These QTL clusters with large effects on source leaf size and yield-related traits provide good targets for marker-assisted breeding for plant type improvement and high-yield potential in rice.     

Cytogenetical analysis on aneuploids obtained from pollenclones of rice (Oryza sativa L.)

Summary Rice aneuploids were obtained from 1,715 pollenclones with a mean frequency of 10.2% in anther culture (1983 to 1985). Among the aneuploids obtained, the frequency of primary trisomics ranged from 5.4% to 6,7%, tetrasomics from 1.1% to 1.7% monosomics from 0.9% to 1.3%, nullisomics from 0.5% to 1% and double trisomics from 0.5% to 0.7%. The chromosome complements of those aneuploids were identified by pachytene analysis on the absolute length of the extra chromosomes. Pollen clonal aneuploids showed a different range of variation in agronomic characters from dihaploids of the same origin but the phenotypic variations ressembled those found in aneuploids created by conventional breeding methods. The meiotic chromosome behavior of PMC revealed various chromosomal aberrations of aneuploids: loose pairing, trivalents, univalents, straggling chromosomes, bridges and laggards.     

Gene mapping related to yellow green leaf in a mutant line in rice (Oryza sativa L.)

A mutant, which derived from the restorer line Jinhui10 treated with EMS, showed completely yellow green leaves, and it had low chlorophyll content and poor agronomic characteristics during the growing stage. The F1 plants from the cross between normal × the mutant showed normal green leaves, and the segregation ratio of normal to yellow green leaves was 3 : 1 in F₂ population. It indicated that the trait was controlled by a single recessive nuclear gene, temporarily designated asygl3. The geneygl3 was mapped between RM468 and RM3684 with genetic distances 8.4 cM and 1.8 cM on chromosome 3. This result would be used as genetic information for fine mapping and map-based cloning ofygl3 gene.