Identification of a set of RFLP probes for subspecies differentiation in Oryza sativa L.

Sixty-eight indica-japonica tester-differentiating RFLP probes were tested in seven indica and seven japonica varieties of rice (Oryza sativa L.) with four enzyme digestions (EcoRI, EcoRV, HindIII and DraI). Twenty-one DNA clones were isolated as indica-japonica subspecies-differentiating probes. A set of 13 probes was established as core probes for subspecies differentiation and a pooled blotting analysis was carried out to facilitate the application of RFLP in rice genetics and breeding practice. A dendrogram of 12 wide-compatibility varieties was constructed based on RFLPs detected by 13 core probes with single enzyme digestions. It was speculated that most RFLPs of indica-japonica differentiating probes were generated by insertions/deletions, which may be of great significance for the origin and differentiation of subspecies in Oryza sativa L.     

A major photoperiod-sensitivity gene tagged with RFLP and isozyme markers in rice

Summary Photoperiod-sensitive rice (Oryza sativa L.) cultivars are widely grown in rainfed lowland areas with unfavorable water regimes. A molecular marker for the trait would be useful in genetic and physiological studies and in developing improved photoperiod-sensitive cultivars. Previous genetic studies identified a major gene for photoperiod sensitivity on chromosome 6. We have tested an isozyme marker and several RFLP probes mapping to chromosome 6 in an attempt to identify marker(s) tightly linked to photoperiod sensitivity in tropical rice cultivars. We report here that the isozyme gene Pgi-2 is linked (23.2±4.7 cM) to the photoperiod-sensitivity gene in the cultivar GEB-24. Although association of duration with Pgi-2 alleles can be used to detect segregation of the photoperiod sensitivity gene in crosses, it will probably not be useful as a marker in selection because of its loose linkage. In contrast, a gene for photoperiod sensitivity in the cultivar Puang Rai 2 was found to be closely linked to the rice genomic clone RG64. Among 15 F₃ lines homozygous for photoperiod insensitivity, no recombinants were detected with RG64. This clone is thus an excellent probe to follow segregation of the major photoperiod-sensitivity gene in rice crosses.     

Molecular mapping of genes for resistance to rice blast (Pyricularia grisea Sacc.)

Two dominant genes conferring complete resistance to specific isolates of the rice blast fungus, Pyricularia grisea Sacc., were located on the molecular map of rice in this study. Pi-l(t) is a blast resistance gene derived from the cultivar LAC23. Its map location was determined using a pair of nearly isogenic lines (NILs) and a B₆F₃ segregating population from which the isoline was derived. RFLP analysis showed that Pi-l(t) is located near the end of chromosome 11, linked to RZ536 at a distance of 14.0±4.5 centiMorgans (cM). A second gene, derived from the cultivar Apura, was mapped using a rice doubled-haploid (DH) population. This gene was located on chromosome 12, flanked by RG457 and RG869, at a distance of 13.5+-4.3 cM and 17.7+-4.5 cM, respectively. The newly mapped gene on chromosome 12 may be allelic or closely linked toPi-ta. (=Pi-4(t)), a gene derived from Tetep that was previously reported to be linked to RG869 at a distance of 15.4±4.7 cM. The usefulness of markers linked to blast resistance genes will be discussed in the context of breeding for durable blast resistance.     

Identification of the double genome donor in spontaneous triploid tomato plants by RFLP analysis

Independent spontaneous triploid tomato plants (Lycopersicon esculentum Mill.) were collected among diploid hybrids growing in commercial greenhouses. Ploidy levels were verified by counting chromosomes, and the donor of the double genome dose was determined by restriction fragment length polymorphism (RFLP) analysis. The TG101 probe, which is tightly linked to the Tm-2^a locus, revealed different restriction patterns between TMV-resistant and TMV-susceptible parent lines. The parent donor which provided two genomes to the triploid was identified by comparing the relative intensity of alleles in the triploid with that in the diploid. The results indicate that both parents can serve as a double genome donor.     

Molecular marker heterozygosity and hybrid performance in indica and japonica rice

An essential assumption underlying markerbased prediction of hybrid performance is a strong linear correlation between molecular marker heterozygosity and hybrid performance or heterosis. This study was intended to investigate the extent of the correlations between molecular marker heterozygosity and hybrid performance in crosses involving two sets of rice materials, 9 indica and 11 japonica varieties. These materials represent a broad spectrum of the cultivated rice gene pool including landraces, primitive cultivars, historically important cultivars, modern elite cultivars and parents of superior hybrids. Varieties within each set were intermated in all possible nonreciprocal pairs resulting in 36 crosses in the indica set and 55 in the japonica set. The F₁s and their parents, 111 entries in total, were examined for performance of seven traits in a replicated field trial. The parents were surveyed for polymorphisms using 96 RFLP and ten SSR markers selected at regular intervals from a published molecular marker linkage map. Molecular marker genotypes of the F₁ hybrids were deduced from the parental genotypes. The analysis showed that, with very few exceptions, correlations in the indica dataset were higher than in that of their japonica counterparts. Among the seven traits analyzed, plant height showed the highest correlation between heterozygosity and hybrid performance and heteorsis in both indica and japonica datasets. Correlations were low to intermediate between hybrid performance and heterozygosity (both general and specific) in yield and yield component traits in both indica and japonica sets, and also low to intermediate between specific heterozygosity and heterosis in the indica set, whereas very little correlation was detected between heterosis and heterozygosity (either general or specific) in the japonica set. In comparison to the results from our previous studies, we concluded that the relationship between molecular marker heterozygosity and heterosis is variable, depending on the genetic materials used in the study, the diversity of rice germplasms and the complexity of the genetic basis of heterosis.     

Restriction fragment length polymorphism differences among Illinois long-term selection oil strains

Restriction fragment length polymorphism (RFLP) analysis was used to characterize variability in the Illinois Long-Term Selection Experiment oil strains. Considerable polymorphism was detected within each oil strain and among oil strains. Fifty-two individual plants from each of the Illinois High Oil (IHO), Illinois Low Oil (ILO), Reverse High Oil (RHO) and Reverse Low Oil (RLO) strains were sampled to determine RFLP allele/variant frequencies. Generation 90 was sampled for IHO, RHO, and RLO whereas generation 87 was sampled for ILO. Forty-nine RFLP probes distributed throughout the maize genome were used. Chi-square analysis was performed to determine if RFLP genotypes at each of the 49 RFLP loci were significantly different among strains. Oil strains that have been separated for 90 generations showed high levels of significantly-different RFLP genotypic frequencies. The comparison of ILO vs RHO gave only significant chi-square values while the comparisons of IHO vs RLO and RHO vs RLO had 111 ratios of significant to non-significant chi-square values. Strains that have been separated for only 42 generations showed a lower level of significantly-different RFLP genotypic frequencies. The comparisons of IHO vs RHO and ILO vs RLO both had only a 32 ratio of significant to non-significant chi-squares values. Detection of multiple RFLP alleles/variants among the oil strains was common with 59% of the RFLP loci examined exhibiting multiple variants. A number of RFLP loci in RHO (3) and RLO (11) were associated with a trend in RFLP allele/variant frequencies consistent with a response to reverse selection for oil concentration.     

Isolation and identification of a non-specific tandemly repeated DNA sequence in Oryza species

A tandemly repeated DNA sequence (RRS7) was isolated from Oryza alta (CCDD). RRS7-related sequences were also found tandemly arrayed in genomes AA, BB, BBCC, CC, and EE, and a small amount of RRS7-related sequences were detected in genome FF and the Oryza species with unknown genomes. DNA sequence analysis of the 1844-bp insert of RRS7 revealed that it contained six tandemly repeated units, of which five were 155 bp in length and one was 194 bp in length and contained an imperfect internal 39-bp duplication. Southern blot analysis showed that the boundary sequence contained in RRS7 is a single-copy sequence. A 155-bp consensus sequence derived from the six monomeric repeats contained no internal repeat and showed no significant homology to other currently known sequences. The results of Southern blot and sequence analysis revealed that there are at least two subfamilies present in the RRS7 family; these are represented by the DraI site and the MspI site, respectively. Restriction digestion with two pairs of isoschizomers MboI/Sau3A and MspI/HpaII demonstrated that most of the C residues in the GATC sites and the internal C in the CCGG sites of the RRS7 family in O. Alta were methylated. The usefulness of the RRS7 family in determining the evolutionary relationship of the genome DD and other Oryza genomes is discussed.     

Molecular divergence and hybrid performance in rice

This study was undertaken to determine the relationship between genetic distance of the parents based on molecular markers and F₁ performance in a set of diallel crosses involving eight commonly used parental lines in hybrid rice production. The F₁s and their parents were measured for five traits including heading date, plant height, straw weight, grain yield and biomass. The parental lines were assayed for DNA polymorphisms using two classes of markers: 140 probes for restriction fragment length polymorphisms (RFLPs) and 12 simple sequence repeats (SSRs), resulting in a total of 105 polymorphic markers well spaced along the 12 rice chromosomes. SSRs detected more polymorphism than RFLPs among the eight lines. A cluster analysis based on marker genotypes separated these eight lines into three groups which agree essentially with the available pedigree information. Correlations were mostly low between general heterozygosity based on all the markers and F₁ performance and heterosis. In contrast, very high correlations were detected between midparent heterosis and specific heterozygosity based on the markers that detected significant effects for all the five traits; these correlations may have practical utility in predicting heterosis. The analyses also suggest the existence of two likely heterotic groups in the rice germplasm represented by these eight lines.     

小鼠DNA的限制性片段长度多态性分析

用人类肌红蛋白基因内含子中３３ｂｐ的小卫星ＤＮＡ重复序列为探针,经ｐＢＲ３２２的ＥｃｏＲ Ｉ正向测序引物和α－３２Ｐ－ｄＣＴＰ在Ｔａｑ ＤＮＡ聚合酶作用下做合成标记,与６种小鼠的ＤＮＡ限制性片段杂交。结果显示,每个品系平均出现可分辨带纹在１３条以上,不同品系小鼠间的杂交带纹表现出高度的多态性。多态性带纹主要分布在６￣２３ｋｂ区段;同一近交系的不同个体间的带纹特点及分布完全一致;ＣＳ７与ＡＫＲ及二者     

Production and characterization of a complete set of individual chromosome additions from Oryza officinalis to Oryza sativa using RFLP and GISH analyses

Monosomic alien addition lines (MAALs) are valuable materials for comparative analyses of two distinct genomes, for elucidating introgression mechanisms, and for dissecting genes controlling complex traits. In the study reported here, MAALs of rice containing the complete genome of Oryza sativa and individual chromosomes of Oryza officinalis were produced. Interspecific hybridizations were made between O. sativa L. ssp. Japonica (CV, Hejiang 19, 2 n=24, AA) and O. officinalis (Acc. HY018, 2 n=24, CC). Two backcrosses were made to the cultivated rice to obtain BC₂F₁ plants. Through RFLP and GISH analyses, 25 MAALs (2 n=25, AA+1C) were identified and divided into 12 syntenic groups, designated MAALs 1–12. MAALs 1, 2, 3, 5, 7 and 10 were each represented by one plant, MAALs 8, 11 and 12 by two plants, MAALs 6 and 9 by four plants, and MAAL 4 by five plants. An ideogram of the C-genome of O. officinalis was constructed, based on GISH analysis of the interspecific hybrid and the MAALs. Comparative RFLP maps showed strong syntenic associations between the A-genomes and C-genomes. Chromosomal arrangements such as translocations and duplications were detected in different alien chromosomes of the MAALs. The complete set of O. officinalis MAALs generated here provides a novel manipulation platform for exploiting and utilizing the O. officinalis genome and carrying out genetic studies.     

Tagging genes for blast resistance in rice via linkage to RFLP markers

Summary Both Pi-2(t) and Pi-4(t) genes of rice confer complete resistance to the blast fungal pathogen Pyricularia oryzae Cav. As economically important plant genes, they have been recently characterized phenotypically, yet nothing is known about their classical linkage associations and gene products. We report here the isolation of DNA markers closely linked to these blast resistance genes in rice. The DNA markers were identified by testing 142 mapped rice genomic clones as hybridization probes against Southern blots, consisting of DNA from pairs of nearly isogenic lines (NILs) with or without the target genes. Chromosomal segments introgressed from donor genomes were distinguished by restriction fragment length polymorphisms (RFLPs) between the NILs. Linkage associations of the clones with Pi-2(t) and Pi4(t) were verified using F₃ segregating populations of known blast reaction. Cosegregation of the resistant genotype and donor-derived allele indicated the presence of linkage between the DNA marker and a blast resistance gene. RFLP analysis showed that Pi-2(t) is closely linked to a single-copy DNA clone RG64 on chromosome 6, with a distance of 2.8+1.4(SE) cMorgans. Another blast resistance gene, Pi-4(t), is 15.3+4.2(SE) cMorgans away from a DNA clone RG869 on chromosome 12. These chromosomal regions can now be examined with additional markers to define the precise locations of Pi-2(t) and Pi-4(t). Tightly linked DNA markers may facilitate early selection for blast resistance genes in breeding programs. These markers may also be useful to map new genes for resistance to blast isolates. They may ultimately lead to the cloning of those genes via chromosome walking. The gene tagging approach demonstrated in this paper may apply to other genes of interest for both monogenic and polygenic traits.     

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.     

Evaluation of restriction fragment length polymorphism in Cucumis melo

Summary The objectives of this study were to assess the degree of restriction fragment length polymorphism (RFLP) in Cucumis melo and to determine interrelationships among cultivated varieties. Initial screening of a genomic PstI library revealed that approximately 40% of the clones were repetitive. A total of 162 unique and low-copy sequence clones were hybridized to seven diverse accesions of C. melo and a C. sativus cultivar Pacer to evaluate RFLP variation. Of these, 130 probes (80%) detected a polymorphism between C. melo accessions and C. sativus, and the majority were polymorphic with more than one enzyme digest. In contrast, only 53 probes (33%) were useful in differentiating at least one of the seven accessions. Of those, only 9% were informative with more than one enzyme digest. This indicates that within C. melo, the differences among accessions are due to infrequent base substitutions, whereas between the two species, differences are mainly due to genome rearrangements such as insertions and deletions or numerous base substitutions. Of the informative probes, 34 were used in analyzing 44 C. melo lines to establish a data base of RFLP hybridization patterns. Percent similarity based on RFLP profiles was computed among lines and analyzed by principal component analysis, to visualize relationships among lines. There were clear demarcations among, but not within, muskmelon and honeydew groups.     

Telomere-homologous sequences occur near the centromeres of many tomato chromosomes

Several bacteriophage lambda clones containinginterstitialtelomererepeats (ITR) were isolated from a library of tomato genomic DNA by plaque hybridization with the clonedArabidopsis thaliana telomere repeat. Restriction fragments lacking highly repetitive DNA were identified and used as probes to map 14 of the 20 lambda clones. All of these markers mapped near the centromere on eight of the twelve tomato chromosomes. The exact centromere location of chromosomes 7 and 9 has recently been determined, and all ITR clones that localize to these two chromosomes map to the marker clusters known to contain the centromere. High-resolution mapping of one of these markers showed cosegregation of the telomere repeat with the marker cluster closest to the centromere in over 9000 meiotic products. We propose that the map location of interstitial telomere clones may reflect specific sequence interchanges between telomeric and centromeric regions and may provide an expedient means of localizing centromere positions.     

RFLP mapping of quantitative trait loci controlling seed aliphatic-glucosinolate content in oilseed rape (Brassica napus L)

We report the RFLP mapping of quantitative trait loci (QTLs) which regulate the total seed aliphaticglucosinolate content in Brassica napus L. A population of 99 F₁-derived doubled-haploid (DH) recombinant lines from a cross between the cultivars Stellar (low-glucosinolate) and Major (high-glucosinolate) was used for singlemarker analysis and the interval mapping of QTLs associated with total seed glucosinolates. Two major loci, GSL-1 and GSL-2, with the largest influence on total seed aliphatic-glucosinolates, were mapped onto LG 20 and LG 1, respectively. Three loci with smaller effects, GSL-3, GSL-4 and GSL-5, were tentatively mapped to LG 18, LG 4 and LG 13, respectively. The QTLs acted in an additive manner and accounted for 71 % of the variation in total seed glucosinolates, with GSL-1 and GSL-2 accounting for 33% and 17%, respectively. The recombinant population had aliphatic-glucosinolate levels of between 6 and 160 moles per g^-1 dry wt of seed. Transgressive segregation for high seed glucosinolate content was apparent in 25 individuals. These phenotypes possessed Stellar alleles at GSL-3 and Major alleles at the four other GSL loci demonstrating that low-glucosinolate genotypes (i.e. Stellar) may possess alleles for high glucosinolates which are only expressed in particular genetic backgrounds. Gsl-elong and Gsl-alk, loci which regulate the ratio of individual aliphatic glucosinolates, were also mapped. Gsl-elong-1 and Gsl-elong-2, which control elongation of the -amino-acid precursors, mapped to LG 18 and LG 20 and were coincident with GSL loci which regulate total seed aliphatic glucosinolates. A third tentative QTL, which regulates side-chain elongation, was tentatively mapped to LG 12. Gsl-alk, which regulates H₃CS-removal and side-chain de-saturation, mapped to LG 20.     

The suitability of restriction fragment length polymorphisms as genetic markers in maize

Summary Strain identification in Zea mays by restriction fragment length polymorphism should be feasible due to the high degree of polymorphism found at many loci. The polymorphism in maize is apparently higher than that currently known for any other organism. Five randomly selected maize inbred lines were examined by Southern filter hybridization with probes of cloned low copy sequences. Typically, several alleles could be distinguished among the inbred lines with any one probe and an appropriately selected restriction enzyme. Despite considerable polymorphism at the DNA level, 16 RFLP markers in three inbred lines of maize were examined for six to 11 generations and found be stable. Mapping of RFLP markers in maize can be accelerated by the use of B-A translocation stocks, which enable localization of a marker to chromosome arm in one generation. The use of recombinant inbred lines in further refinement of the map is discussed.     

Restriction fragment length polymorphism analysis of loci associated with disease resistance genes and developmental traits in Pisum sativum L.

An F₂ population of pea (Pisum sativum L.) consisting of 174 plants was analysed by restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) techniques. Ascochyta pisi race C resistance, plant height, flowering earliness and number of nodes were measured in order to map the genes responsible for their variation. We have constructed a partial linkage map including 3 morphological character genes, 4 disease resistance genes, 56 RFLP loci, 4 microsatellite loci and 2 RAPD loci. Molecular markers linked to each resistance gene were found: Fusarium wilt (6 cM from Fw), powdery mildew (11 cM from er) and pea common Mosaic virus (15 cM from mo). QTLs (quantitative traits loci) for Ascochyta pisi race C resistance were mapped, with most of the variation explained by only three chromosomal regions. The QTL with the largest effect, on chromosome 4, was also mapped using a qualitative, Mendelian approach. Another QTL displayed a transgressive segregation, i.e. the parental line that was susceptible to Ascochyta blight had a resistance allele at this QTL. Analysis of correlations between developmental traits in terms of QTL effects and positions suggested a common genetic control of the number of nodes and earliness, and a loose relationship between these traits and height.     

Molecular marker analysis of Helianthus annuus L. 2. Construction of an RFLP linkage map for cultivated sunflower

A detailed linkage map of Helianthus annuus was constructed based on segregation at 234 RFLP loci, detected by 213 probes, in an F₂ population of 289 individuals (derived from a cross between the inbred lines HA89 and ZENB8). The genetic markers covered 1380 centiMorgans (cM) of the sunflower genome and were aranged in 17 linkage groups, corresponding to the haploid number of chromosomes in this species. One locus was found to be unlinked. Although the average interval size was 5.9 cM, there were a number of regions larger than 20 cM that were devoid of markers. Genotypic classes at 23 loci deviated significantly from the expected ratios (121 or 31), all showing a reduction in the ZENB8 homozygous class. The majority of these loci were found to map to four regions on linkage groups G, L and P.