Genetic variation detected by DNA fingerprinting with a rice minisatellite probe in Oryza sativa L.

A rice minisatellite probe detecting DNA fingerprints was used to assess genetic variation in cultivated rice (Oryza sativa L.). Fifty-seven cultivars of rice, including 40 closely related cultivars released in the US, were studied. Rice DNA fingerprinting revealed high levels of polymorphism among distantly related cultivars. The variability of fingerprinting pattern was reduced in the closely related cultivars. A genetic similarity index (S) was computed based on shared fragments between each pair of cultivars, and genetic distance (D) was used to construct the dendrograms depicting genetic relationships among rice cultivars. Cluster analysis of genetic distance tended to group rice cultivars into different units corresponding with their varietal types and breeding pedigrees. However, by comparison with the coefficients of parentage, the criterion of relatedness based on DNA fingerprints appeared to overestimate the genetic relationships between some of the closely related US cultivars. Although this may reduce the power of fingerprints for genetic analysis, we were able to demonstrate that DNA fingerprinting with minisatellite sequences is simpler and more sensitive than most other types of marker systems in detecting genetic variation in rice.This paper reports the results of research only. Mention of a proprietary product does not consititute an endorsement or a recommendation for its use by the USDA or the University of Missouri. Contribution from the US Department of Agriculture, Agricultural Research Service, Plant Genetics Research Unit, and the University of Missouri Agricultural Experiment Station Journal Series No. 12178.     

Mapping and genome organization of microsatellite sequences in rice (Oryza sativa L.)

In order to enhance the resolution of an existing genetic map of rice, and to obtain a comprehensive picture of marker utility and genomic distribution of microsatellites in this important grain species, rice DNA sequences containing simple sequence repeats (SSRs) were extracted from several small-insert genomic libraries and from the database. One hundred and eighty eight new microsatellite markers were developed and evaluated for allelic diversity. The new simple sequence length polymorphisms (SSLPs) were incorporated into the existing map previously containing 124 SSR loci. The 312 microsatellite markers reported here provide whole-genome coverage with an average density of one SSLP per 6 cM. In this study, 26 SSLP markers were identified in published sequences of known genes, 65 were developed based on partial cDNA sequences available in GenBank, and 97 were isolated from genomic libraries. Microsatellite markers with different SSR motifs are relatively uniformly distributed along rice chromosomes regardless of whether they were derived from genomic clones or cDNA sequences. However, the distribution of polymorphism detected by these markers varies between different regions of the genome. Received: 5 May 1999 / Accepted: 16 August 1999     

Molecular mapping of genes conferring aluminum tolerance in rice (Oryza sativa L.)

Crop productivity on acid soil is restricted by multiple abiotic stress factors. Aluminum (Al) tolerance seems to be a key to productivity on soil with a pH below 5.0, but other factors such as Mn toxicity and the deficiency of P, Ca and Mg also play a role. The development of Al-tolerant genotypes of rice is an urgent necessity for improving crop productivity in developing countries. Inhibition of root growth is a primary and early symptom of Al toxicity. The present study was conducted to identify genetic factors controlling the aluminum tolerance of rice. Several parameters related to Al tolerance, most importantly the relative root growth under Al stress versus non-stress conditions, were scored in 188 F₃ selfed families from a cross between an Al-tolerant Vietnamese local variety, Chiembau, and an Al-susceptible improved variety, Omon269–65. The two varieties are both Oryza sativa ssp. indica, but showed a relatively high level of DNA polymorphism, permitting the assembly of an RFLP map consisting of 164 loci spanning 1,715.8 cM, and covering most of the rice genome. A total of nine different genomic regions on eight chromosomes have been implicated in the genetic control of root and shoot growth under aluminum stress. By far the greatest effects on aluminum tolerance were associated with the region near WG110 on chromosome 1. This region does not seem to correspond to most of the genes that have been mapped for aluminum tolerance in other species, nor do they correspond closely to one another. Most results, both from physiological studies and from molecular mapping studies, tend to suggest that aluminum tolerance is a complex multi-genic trait. The identification of DNA markers (such as WG110) that are diagnostic for aluminum tolerance in particular gene pools provides an important starting point for transferring and pyramiding genes that may contribute to the sustainable improvement of crop productivity in aluminum-rich soils. The isolation of genes responsible for aluminum tolerance is likely to be necessary to gain a comprehensive understanding of this complex trait. Received: 29 March 2000 / Accepted: 16 August 2000     

Mapping of QTLs for phosphorus-deficiency tolerance in rice (Oryza sativa L.)

 Phosphorus (P) deficiency of soils is a major yield-limiting factor in rice production. Increasing the P-deficiency tolerance of rice cultivars may represent a more cost-effective solution than relying on fertilizer application. The objective of this study was to identify putative QTLs for P-deficiency tolerance in rice, using 98 backcross inbred lines derived from a japonica×indica cross and genotyped at 245 RFLP marker loci. Lines were grown on P-deficient soil and P uptake, internal P-use efficiency, dry weight, and tiller number were determined. Three QTLs were identified for dry weight and four QTLs for P uptake, together explaining 45.4% and 54.5% of the variation for the respective traits. Peaks for both traits were in good agreement which was to be expected considering the tight correlation of r=0.96 between dry weight and P uptake. For both traits the QTL linked to marker C443 on chromosome 12 had a major effect. Two of the three QTLs detected for internal P-use efficiency, including the major one on chromosome 12, coincided with QTLs for P uptake; however, whereas indica alleles increased P uptake they reduced P-use efficiency. We concluded that this was not due to the tight linkage of two genes in repulsion but rather due to an indirect effect of P uptake on P-use efficiency. Most lines with high use efficiency were characterized by very low P uptake and dry weight and apparently experienced extreme P-deficiency stress. Their higher P-use efficiency was thus the result of highly sub-optimal tissue-P concentrations and did not represent a positive adaptation to low P availability. The number of tillers produced under P deficiency is viewed as an indirect indicator of P-deficiency tolerance in rice. In addition to the major QTL on chromosome 12 already identified for all other traits, two QTLs on chromosome 4 and 12 were identified for tiller number. Their position, however, coincided with QTLs for tiller number reported elsewhere under P-sufficient conditions and therefore appear to be not related to P-deficiency tolerance. In this study P-deficiency tolerance was mainly caused by differences in P uptake and not in P-use efficiency. Using a trait indirectly related to P-deficiency tolerance such as tiller number, we detected a major QTL but none of the minor QTLs detected for P uptake or dry weight. Received: 9 February 1998 / Accepted: 29 April 1998     

RFLP mapping of the genes controlling hybrid breakdown in rice (Oryza sativa L.)

 Complementary recessive genes hwd1 and hwd2 controlling hybrid breakdown (weakness of F₂ and later generations) were mapped in rice using RFLP markers. These genes produce a plant that is shorter and has fewer tillers than normal plants when the two loci have only one or no dominant allele at both loci. A cultivar with two dominant alleles at the hwd1 locus and a cultivar with two dominant alleles at the hwd2 locus were crossed with a double recessive tester line. Linkage analysis was carried out for each gene independently in two F₂ populations derived from these crosses. hwd1 was mapped on the distal region of rice genetic linkage map for chromosome 10, flanked by RFLP markers C701 and R2309 at a distance of 0.9 centiMorgans (cM) and 0.6 cM, respectively. hwd2 was mapped in the central region of rice genetic linkage map for chromosome 7, tightly linked with 4 RFLP markers without detectable recombination. The usefulness of RFLP mapping and map information for the genes controlling reproductive barriers are discussed in the context of breeding using diverse rice germplasm, especially gene introduction by marker-aided selection.     

QTL mapping for the paste viscosity characteristics in rice (Oryza sativa L.)

In order to understand the genetic basis of the paste viscosity characteristics (RVA profile, which is tested on the Rapid Visco Analyser) of the rice grain, we mapped QTLs for RVA profile parameters using a DH population derived from a cross between an indica variety, Zai-Ye-Qing 8 (ZYQ8), and a japonica variety, Jing-Xi 17 (JX17). Evidence of genotype-by-environment interaction was found by comparing the mapped QTLs between two locations, Hainan (HN) and Hangzhou (HZ). A total of 20 QTLs for six parameters of the RVA profiles were identified at least one location. Only the waxy locus (wx) located on chromosome 6 was detected significantly at both environments for five traits, i.e. hot paste viscosity (HPV), cool paste viscosity (CPV), breakdown viscosity (BDV), consistency viscosity (CSV) and setback viscosity (SBV). This locus explained 19.5%–63.7% of the total variations at both environments, suggesting that the RVA profiles were mainly controlled by the wx gene. HPV, CPV, BDV, CSV and SBV were also controlled by other QTLs whose effects on the respective parameter were detected only in one environment, while for the peak viscosity (PKV), only 2 QTLs, 1 at HN,the other at HZ, were identified. These results indicate that RVA profiles are obviously affected by environment. Received: 18 July 1999 / Accepted: 27 August 1999     

PCR-based molecular markers for the fragrance gene in rice (Oryza sativa. L.)

The genomic DNA clone RG28, linked to the major fragrance gene of rice (fgr), was assessed for polymorphism in order to produce a PCR-based marker for fragrance. A small mono-nucleotide repeat, that was polymorphic between a pair of fragrant and non-fragrant cultivars, was identified and developed into a co-dominant PCR-based marker. The polymorphism-information-content determinations for three microsatellite markers, that have been genetically mapped near RG28, are also presented. These PCR-based markers will be highly useful in distinguishing fragrance-producing alleles from non-fragrance-producing alleles at the fgr locus. Received: 19 October 1999 / Accepted: 16 December 1999     

Geographic distribution and multilocus organization of isozyme variation of rice (Oryza sativa L.)

Genetic organization of isozyme variation in rice (Oryza sativa L.) was investigated based on 17 polymorphic isozyme loci using a sample of 511 accessions of worldwide origin. The genetic diversity within the species was very high (H=0.36 with 4.82 alleles per locus), as compared with most selfing plant species. Three diversity centers were detected for isozyme variation including South Asia, China and Southeast Asia. The accessions were classified into three well-differentiated cultivar groups corresponding to the indica and japonica subspecies, and a new unnamed group. Variation within the cultivar groups accounted for 80% of the total isozyme variation. Within-country variation accounted for 58% of the total variation while among-region and among-country variation within the cultivar groups accounted for only 14% and 8% of the total variation. Analyses using log-linear models revealed that pronounced non-random associations between and among alleles at many unlinked isozyme loci were organized in a non-hierarchical pattern, and subspecific and macro-geographic differentiation was much more pronounced in multilocus phenotype frequencies than in allelic frequencies at individual loci. These results suggest that selection on multilocus gene complexes was largely responsible for the maintenance of the extensive isozyme variation within the species and the indica-japonica differentiation. Our results further suggest the independent domestication of indica and japonica, the dual origins of the indica rice from China and South Asia (India), and the differentiation of the ecotypes ’javanica’ and the ’temperate japonica’ within the japonica subspecies. Received: 5 August 1999 / Accepted: 13 December 1999     

Inter-simple sequence repeat (ISSR) amplification for analysis of microsatellite motif frequency and fingerprinting in rice (Oryza sativa L.)

 Inter-simple sequence repeat (ISSR) amplification was used to analyze microsatellite motif frequency in the rice genome and to evaluate genetic diversity among rice cultivars. A total of 32 primers, containing different simple sequence repeat (SSR) motifs, were tested for amplification on a panel of 59 varieties, representative of the diversity of cultivated rice (Oryza sativa L.). The ISSR analysis provided insights into the organization, frequency and levels of polymorphism of different simple sequence repeats in rice. The more common dinucleotide motifs were more amenable to ISSR analysis than the more infrequent tri-, tetra- and penta-nucleotide motifs. The ISSR results suggested that within the dinucleotide class, the poly(GA) motif was more common than the poly(GT) motif and that the frequency and clustering of specific tri- and tetra-nucleotide simple sequence repeats was variable and motif-specific. Furthermore, trinucleotide ISSR markers were found to be less polymorphic than either dinucleotide or certain tetranucleotide ISSR markers, suggesting which motifs would be better targets for microsatellite marker development. The ISSR amplification pattern was used to group the rice genotypes by cluster analysis. These results were compared to surveys of the same varieties for amplified fragment length polymorphism (AFLP), restriction fragment length polymorphism (RFLP) and isozyme markers. The ISSR fingerprint could be used to differentiate the genotypes belonging to either Japonica or Indica sub species of cultivated rice and to dissect finer levels of diversity within each subspecies. A higher percentage of polymorphic bands was produced with the ISSR technique than the AFLP method, based on a similar PCR reaction. Therefore, ISSR amplification proved to be a valuable method for determining genetic variability among rice varieties and for rapidly identifying cultivars. This efficient genetic fingerprinting technique would be useful for characterizing the large numbers of rice accessions held in national and international germplasm centers. Received: 25 May 1998 / Accepted: 17 September 1998     

Comparison of the genetic diversity of common wild rice (Oryza rufipogon Griff.) and cultivated rice (O. sativa L.) using RFLP markers

Forty fourth single-copy RFLP markers were used to evaluate the genetic diversity of 122 accessions of common wild rice (CWR, Oryza rufipogon Griff.) and 75 entries of cultivated rice (Oryza sativa L. ) from more than ten Asian countries. A comparison of the parameters showing genetic diversity, including the percentage of polymorphic loci (P), the average number of alleles per locus (A), the number of genotypes (Ng), the average heterozygosity (Ho) and the average genetic multiplicity (Hs) of CWR and indica and japonica subspecies of cultivated rice from different countries and regions, indicated that CWR from China possesses the highest genetic diversity, followed by CWR from South Asia and Southeast Asia. The genetic diversity of CWR from India is the second highest. Although the average gene diversity (Hs)of the South Asian CWR is higher than that of the Southeast Asian CWR, its percentage of polymorphic loci (P), number of alleles (Na) and number of genotypes (Ng) are all smaller. It was also found that the genetic diversity of cultivated rice is obviously lower than that of CWR. At the 44 loci investigated, the number of polymorphic loci of cultivated rice is only 3/4 that of CWR, while the number of alleles, 60%, and the number of genotypes is about 1/2 that of CWR. Of the two subspecies studied, the genetic diversity of indica is higher than that of japonica. The average heterozygosity of the Chinese CWR is the highest among all the entries studied. The average heterozygosity of CWR is about two-times that of cultivated rice. It is suggested that during the course of evolution from wild rice to cultivated rice, many alleles were lost through natural and human selection, leading to the lower heterozygosity and genetic diversity of the cultivated rice. Received: 19 May 1999 / Accepted: 26 April 2000     

QTLs for cell-membrane stability mapped in rice (Oryza sativa L.) under drought stress

Cell-membrane stability (CMS) is considered to be one of the major selection indices of drought tolerance in cereals. In order to determine which genomic region is responsible for CMS, 104 rice (Oryza sativa L.) doubled haploid (DH) lines derived from a cross between CT9993–5-10–1-M and IR62266-42–6-2 were studied in the greenhouse in a slowly developed drought stress environment. Drought stress was induced on 50-day-old plants by withholding water. The intensity of stress was assessed daily by visual scoring of leaf wilting and by measuring leaf relative water content (RWC). The leaf samples were collected from both control (well-watered) and stressed plants (at 60–65% of RWC), and the standard test for CMS was carried out in the laboratory. There was no significant difference (P>0.05) in RWC between the two parental lines as well as among the 104 lines, indicating that all the plants were sampled at a uniform stress level. However, a significant difference (P<0.05) in CMS was observed between the two parental lines and among the population. No significant correlation was found between CMS and RWC, indicating that the variation in CMS was genotypic in nature. The continuous distribution of CMS and its broad-sense heritability (34%) indicates that CMS should be polygenic in nature. A linkage map of this population comprising of 145 RFLPs, 153 AFLPs and 17 microsatellite markers was used for QTL analysis. Composite interval mapping identified nine putative QTLs for CMS located on chromosomes 1, 3, 7, 8, 9, 11 and 12. The amount of phenotypic variation that was explained by individual QTLs ranged from 13.4% to 42.1%. Four significant (P<0.05) pairs of digenic interactions between the detected QTLs for CMS were observed. The identification of QTLs for this important trait will be useful in breeding for the improvement of drought tolerance in rice. This is the first report of mapping QTLs associated with CMS under a natural water stress condition in any crop plants. Received: 8 September 1999 / Accepted: 13 October 1999     

Development of a microsatellite framework map providing genome-wide coverage in rice (Oryza sativa L.)

 Ninety-four newly developed microsatellite markers were integrated into existing RFLP framework maps of four rice populations, including two doubled haploid, a recombinant inbred, and an interspecific backcross population. These simple sequence repeats (SSR) were predominantly poly(GA) motifs, targetted because of their abundance in rice. They were isolated from a previously described sheared library and a newly constructed enzyme-digested library. Differences in the average length of poly(GA) tracts were observed for clones isolated from the two libraries. The length of GA motifs averaged 21 repeat units for clones isolated from the Tsp-509-digested library, while motifs averaged 17 units for clones from the sheared library. There was no evidence of clustering of microsatellite markers near centromeres or telomeres. Mapping of the 94 newly developed markers as well as of 27 previously reported microsatellites provided genome-wide coverage of the 12 chromosomes, with an average distance of 1 SSLP (simple sequence repeat polymorphism) per 16–20 cM. Received: 13 February 1997/Accepted: 28 February 1997     

Genetic control of paste viscosity characteristics in indica rice (Oryza sativa L.)

 Paste viscosity parameters play an important role in estimating the eating, cooking and processing quality of rice. Four cytoplasmic male-sterile (CMS) lines and eight restorer (R) lines were employed in an incomplete diallel cross to analyze seed effects, cytoplasmic effects and maternal gene effects on the viscosity profiles of indica rice. The results indicated that the viscosity profiles of rice were controlled by the direct effects of the seed, by the cytoplasm and by maternal plant. The seed-direct effects (V _A +V _D ) accounted for over 51% of the total genetic variances (V _A +V _D +V _C +V _Am +V _Dm ) for all the traits, suggesting that seed direct effects were more important than maternal effects and cytoplasmic effects. The additive variances (V _A +V _Am ) were much larger than the dominance variances (V _D +V _Dm ), which revealed that additive genetic effects were the major contributors of genetic variation for the paste viscosity profiles, and that selection could be applied for viscosity traits in the early generations. Significant cytoplasmic variance (V _C ) was detected for hot paste viscosity (HPV), cool paste viscosity (CPV) and consistency viscosity (CSV). The cytoplasmic effects for these three traits can, therefore, not be neglected in rice breeding. It was also shown that seed heritabilities (h ² _o ) tended to be larger than maternal heritabilities (h ² _m ) and cytoplasmic heritabilities (h ² _c ). Prediction of the main genetic effects for 12 parents showed that CMS lines had highly positive effects on all the traits except for the breakdown viscosity (BDV), and that R lines had both positive and negative effects on the paste viscosity characteristics. Received: 3 August 1998 / Accepted: 28 November 1998     

Mapping quantitative trait loci controlling sheath blight resistance in two rice cultivars (Oryza sativa L.)

Rice sheath blight, caused by Rhizoctonia solani Kühn, is one of the three major diseases of rice. The present study was conducted with an F₂ clonal population of Jasmine 85/Lemont. The F₂ population, including 128 clonal families, was inoculated by short toothpicks incubated with a strain, RH-9 of the fungus. Based on field disease evaluations in 2 years and a genetic map with 118 evenly distributed molecular markers, we identified six quantitative trait loci (QTLs) contributing to sheath blight resistance. These QTLs, qSB-2, qSB-3, qSB-7, qSB-9-1, qSB-9-2 and qSB-11, were located on chromosomes 2, 3, 7, 9 and 11, respectively. The respective alleles of qSB-2, qSB-3, qSB-7, and qSB-9-2 from Jasmine 85 could explain 21.2%, 26.5%, 22.2% and 10.1% of the total phenotypic variation, respectively; while the alleles of qSB-9-1 and qSB-11 from Lemont could explain 9.8% and 31.2% of the total phenotypic variation. Of these qSB-2 and qSB-11 could be detected in both years, while remaining loci were detected only in a single year. Furthermore, four QTLs (qHD-2, qHD-3, qHD-5 and qHD-7) controlling heading date and three QTLs (qPH-3, qPH-4 and qPH-11) controlling plant height were also identified. Though rice sheath blight resistance may be influenced by morphological traits, such as heading date and plant height, in the present study most detected resistance loci were not linked to the loci for heading date or plant height. Received: 1 September 1999 / Accepted: 24 January 2000     

Genetic dissection of root growth in rice (Oryza sativa L.) I: a hydrophonic screen

 Root growth is an important component of the adaptation of rice to drought-prone environments. A hydroponic screen was used to study root growth of 28 rice varieties. Both maximum root length and adventitious root thickness varied widely between varieties. In general, japonica varieties had larger root systems than indica varieties. Two F₂ populations involving the thick- and long-rooted upland japonica variety ‘Azucena’ and two poor-rooting varieties, namely the upland indica‘Bala’ and the Italian japonica‘Maratelli’, were made and screened in hydroponics. Generation means analysis revealed significant additive and dominance main effects for the root length traits with a prevalence of dominance gene effects in both crosses. The dominance×dominance type of non-allelic interactions were important for maximum root length from day 7 to day 28, root volume, root thickness and root cell length in the cross ‘Bala’בAzucena’. The heritability (broad-sense) estimates varied from low to high for the traits and displayed differences between populations. This suggested that recombinant lines with improved root traits can be developed from the two crosses with selection methods that involve some form of progeny evaluation. In a companion paper, we report the mapping of quantitative trait loci (QTLs) for root growth traits in the ‘Bala’בAzucena’ population using restriction fragment length polymorphisms (RFLPs). Received: 5 May 1996 / Accepted: 14 February 1997     

Molecular mapping of the cnx2 locus involved in molybdenum cofactor biosynthesis in rice (Oryza sativa L.)

Molybdenum cofactor (Moco) is essential for nitrate reductase (NR), xanthine dehydrogenase (XDH), and aldehyde oxidase to perform their catalytic functions in plants. Moco biosynthesis is a complex process involving many genes. Little is known about the genetics and molecular aspects of Moco biosynthesis in plants and other eukaryotes. In rice, we previously isolated a Moco mutant C25 with a mutation in the CNX2 gene from a mutagenized indica cultivar IR30 and characterized its biochemical properties. This mutant was crossed with a japonica cultivar, Norin 8, to investigate the linkage of cnx2 to restriction fragment length polymorphism (RFLP) and cleaved amplified polymorphic sequence (CAPS) markers. Chlorate resistance was used to trace the cnx2 mutation because of its cosegregation with the loss of NR and XDH activities observed earlier. RFLP and CAPS analyses show the location of the cnx2 locus on the long arm of chromosome 4. It is mapped between RFLP markers C513 and C377 with a distance of 9.5 and 13.1 cM, respectively. It is also linked with CAPS marker RA0738 at a distance of 30.3 cM. Received: 25 June 2000 / Accepted: 31 August 2000     

A low-temperature method for maintaining plant regeneration activity in embryogenic callus of rice (Oryza sativa L.)

 A method was developed to maintain plant regeneration activity of rice cells (Oryza sativa L.) using embryogenic callus. Calluses were cultured in suspension, then on solid medium, to form compact globular callus resistant to low-temperature stress and with high plant regeneration activity. Callus preserved at 5  °C for 5 months regenerated plants from protoplasts at a frequency higher than from non-preserved callus from cv. Nipponbare, and cv. Koshihikari, but at lower rates from cv. Akitakomachi. Similar results were obtained from protoplasts of the three cultivars. Callus preserved at 5  °C for 8 months incurred cell damage, yet some surviving cells divided in suspension culture and eventually regenerated whole plants. Preserved and non-preserved regenerated plants showed similar levels of somaclonal variation. Received: 7 January 1999 / Revision received: 28 April 1999 / Accepted: 26 May 1999     

Comparison of two indices for evaluating regeneration ability in rice (Oryza sativa L.) through a diallel analysis

 A full diallel analysis was performed among seven rice cultivars, all of which showed different abilities of regeneration from seed-derived calli. Number of regenerated shoots and regeneration frequency were used as indices of regeneration ability. In both cases, additive effects were significant at the 0.1% level, and dominant genes had a positive effect, that is, they increased regeneration ability. Non-allelic interaction (epistasis) and maternal effects were not detected. Dominance effects were significant at the 1% and the 0.1% level when the number of regenerated shoots and regeneration frequency were used as indices, respectively. Average degree of dominance was 0.531 for the shoot regeneration index and 0.990 for the regeneration frequency index. Since broad-sense heritability was 0.919 for number of regenerated shoots and 0.736 for regeneration frequency, the former was considered to be a better index of regeneration ability than the latter. Received: 10 June 1996 / Accepted: 23 August 1996     

Direct amplification of minisatellite-region DNA with VNTR core sequences in the genus Oryza

 A polymerase chain reaction (PCR) application, involving the directed amplification of minisatellite-region DNA (DAMD) with several minisatellite core sequences as primers, was used to detect genetic variation in 17 species of the genus Oryza and several rice cultivars (O. sativa L.). The electrophoretic analysis of DAMD-PCR products showed high levels of variation between different species and little variation between different cultivars of O. sativa. Polymorphisms were also found between accessions within a species, and between individual plants within an accession of several wild species. The DAMD-PCR yielded genome-specific banding patterns for the species studied. Several DAMD-PCR-generated DNA fragments were cloned and characterized. One clone was capable of detecting multiple fragments and revealed individual-specific hybridization banding patterns using genomic DNA from wild species as well as rice cultivars. A second clone detected only a single polymorphic locus, while a third clone expressed a strong genome specificity by Southern analysis. The results demonstrated that DAMD-PCR is potentially useful for species and genome identification in Oryza. The DAMD-PCR technique also allows for the isolation of informative molecular probes to be utilized in DNA fingerprinting and genome identification in rice. Received: 1 October 1996 / Accepted: 25 April 1997     

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.