Characterization and genetic mapping of a short, highly repeated, interspersed DNA sequence from rice (Oryza sativa L.).

Summary A short, highly repeated, interspersed DNA sequence from rice was characterized using a combination of techniques and genetically mapped to rice chromosomes by restriction fragment length polymorphism (RFLP) analysis. A consensus sequence (GGC)_n, where n varies from 13–16, for the repeated sequence family was deduced from sequence analysis. Southern blot analysis, restriction mapping of repeat element-containing genomic clones, and DNA sequence analysis indicated that the repeated sequence is interspersed in the rice genome, and is heterogeneous and divergent. About 200000 copies are present in the rice genome. Single copy sequences flanking the repeat element were used as RFLP markers to map individual repeat elements. Eleven such repeat elements were mapped to seven different chromosomes. The strategy for characterization of highly dispersed repeated DNA and its uses in genetic mapping, DNA fingerprinting, and evolutionary studies are discussed.     

Mapping of RFLP and qualitative trait loci in Brassica rapa and comparison to the linkage maps of B. napus,B. oleracea,and Arabidopsis thaliana

A linkage map of restriction fragment length polymorphisms (RFLPs) was constructed for oilseed, Brassica rapa, using anonymous genomic DNA and cDNA clones from Brassica and cloned genes from the crucifer Arabidopsis thaliana. We also mapped genes controlling the simply inherited traits, yellow seeds, low seed erucic acid, and pubescence. The map included 139 RFLP loci organized into ten linkage groups (LGs) and one small group covering 1785 cM. Each of the three traits mapped to a single locus on three different LGs. Many of the RFLP loci were detected with the same set of probes used to construct maps in the diploid B. oleracea and the amphidiploid B. napus. Comparisons of the linkage arrangements between the diploid species B. rapa and B. oleracea revealed six LGs with at least two loci in common. Nine of the B. rapa LGs had conserved linkage arrangements with B. napus LGs. The majority of loci in common were in the same order among the three species, although the distances between loci were largest on the B. rapa map. We also compared the genome organization between B. rapa and A. thaliana using RFLP loci detected with 12 cloned genes in the two species and found some evidence for a conservation of the linkage arrangements. This B. rapa map will be used to test for associations between segregation of RFLPs, detected by cloned genes of known function, and traits of interest.     

RFLP tagging of a gene for aroma in rice

Summary We report here the identification of a DNA marker closely linked to a gene for aroma in rice. The DNA marker was identified by testing 126 mapped rice genomic, cDNA, and oat cDNA, clones as hybridization probes against Southern blots, consisting of DNA from a pair of nearly isogenic lines (NILs) with or without the aroma gene. Chromosomal segments introgressed from the donor genome were distinguished by RFLPs between the NILs. Linkage association of the clone with the gene was verified using an F₃ segregating for aroma. Cosegregation of the scented phenotype and donor-derived allele indicated the presence of linkage between the DNA marker and the gene. RFLP analysis showed that the gene is linked to a single-copy DNA clone, RG28, on chromosome 8, at a distance of 4.5 cM. The availability of a linked DNA marker may facilitate early selection for the aroma gene in rice breeding programs.     

Comparative RFLP mapping of an allotetraploid wild rice species (Oryza latifolia) and cultivated rice (O. sativa)

The purpose of this study was to construct a comparative RFLP map of an allotetraploid wild rice species, Oryza latifolia, and to study the relationship between the CCDD genome of O. latifolia and the AA genome of O. sativa. A set of RFLP markers, which had been previously mapped to the AA genome of cultivated rice, were used to construct the comparative map. Fifty-eight F₂ progeny, which were derived from a single F₁ plant, were used for segregation analysis. The comparative RFLP map contains 149 DNA markers, including 145 genomic DNA markers from cultivated rice, 3 cDNA markers from oat, and one known gene (waxy, from maize). Segregation patterns reflected the allotetraploid ancestry of O. latifolia, and the CC and DD genomes were readily distinguished by most probes tested. There is a high degree of conservation between the CCDD genome of O. latifolia and the AA genome of O. sativa based on our data, but some inversions and translocations were noted.     

A linkage map of rye

A linkage map of rye (Secale cereale L.) is presented which comprises 60 loci including RFLPs, RAPDs, isozyme, morphological and physiological markers. The genetics and linkage relationships of these markers were investigated in several inbred lines of rye. For the RFLP mapping a genomic library of PstI-digested DNA was constructed from which 50 size-selected clones were analysed. The portion of single-copy and multi-copy DNA and the frequency of polymorphic DNA was determined. The markers are unequally distributed over the seven chromosomes of rye. Many of them exhibit a distorted segregation. The main region of deviating segregation ratios could be localized near the self-incompatibility loci.     

Restriction fragment length polymorphism analysis of CCDD genome species of the genus Oryza L.

Restriction fragment length polymorphisms (RFLPs) were studied in fourteen accessions of CCDD genome allotetraploid wild rice species (Oryza latifolia, O. alta and O. grandiglumis). Fourteen nuclear RFLP markers previously mapped in AA genome-cultivated rice were used as probes. A phylogenetic tree, constructed by parsimony analysis based on RFLPs, grouped the accessions according to their geographic origin from Central or South America. Oryza alta, O. grandiglumis and one accession of O. latifolia grouped together as a subgroup, and our results suggested that the three taxa should be considered as populations of a single complex species. Duplicate loci, representing the two constituent genomes of the allotetraploid, were observed for most RFLP markers. By comparing RFLPs from the allotetraploids with those from a CC genome diploid wild species (O. officinalis), it was possible to detect RFLPs specific for both the CC and DD genomes of the allotetraploid. In inter-accession F2 populations, independent segregation of RFLP markers for CC and DD genomes was observed.     

An RFLP species-specific DNA sequence for the A genome of rice.

A DNA sequence, pOs6.20, was cloned from the nuclear genome of cultivated rice (Oryza sativa L.) by hybridization with a human minisatellite sequence. At high hybridization stringency, a subfragment of the rice sequence, pOs6.20.3, detected low-copy restriction fragment length polymorphisms (RFLPs), which behaved as Mendelian genetic markers. This subfragment detected multicopy RFLPs between an indica and a javanica cultivar at medium hybridization stringency. The sequences detected by pOs6.20.3 at high hybridization stringency did not occur in rice species possessing the B, C, D, E, and F genomes. These results suggest that the RFLPs detected by this sequence can be placed on a genetic map of rice and that this sequence can be used as a species-specific probe for the A genome of rice.     

Restriction fragment length polymorphism and allozyme linkage map of Cuphea lanceolata

Summary Cuphea lanceolata Ait. has had a significant role in the domestication of Cuphea and is a useful experimental organism for investigating how medium-chain lipids are synthesized in developing seeds. To expand the genetics of this species, a linkage map of the C. lanceolata genome was constructed using five allozyme and 32 restriction-fragment-length-polymorphism (RFLP) marker loci. These loci were assigned to six linkage groups that correspond to the six chromosomes of this species. Map length is 288 cM. Levels of polymorphism were estimated for three inbred lines of C. lanceolata and an inbred line of C. viscosissima using 84 random genomic clones and two restriction enzymes, EcoRI and HindIII. Of the probes 29% detected RFLPs between C. lanceolata and C. viscosissima lines. Crosses between these species can be exploited to expand the map.     

Comparison of RAPD and RFLP genetic markers in determining genetic similarity among Brassica oleracea L. genotypes

Genetic similarity among 45 Brassica Oleracea genotypes was compared using two molecular markers, random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphisms (RFLPs). The genotypes included 37 broccolis (var. italica), five cauliflowers (var. botrytis) and three cabbages (var. capitata) which represented a wide range of commercially-available germplasm, and included open-pollinated cultivars, commercial hybrids, and inbred parents of hybrid cultivars. Fifty-six polymorphic RFLP bands and 181 polymorphic RAPD bands were generated using 15 random cDNA probes and 62 10-mer primers, respectively. The objectives were to compare RFLP and RAPD markers with regard to their (1) sampling variance, (2) rank correlations of genetic distance among sub-samples, and (3) inheritance. A bootstrap procedure was used to generate 200 random samples of size n (n=2,3,5,... 55) independently from the RAPD and RFLP data sets. The coefficient of variance (CV) was estimated for each sample. Pooled regressions of the coefficient of variance on bootstrap sample size indicated that the rate of decrease in CV with increasing sample size was the same for RFLPs and RAPDs. The rank correlation between the Nei-Li genetic similarity values for all pairs of genotypes (990) based on RFLP and RAPD data was 0.745. Differences were observed between the RFLP and RAPD dendrograms of the 45 genotypes. Overlap in the distributions of rank correlations between independent sub-samples from the RAPD data set, compared to correlations between RFLP and RAPD sub-samples, suggest that observed differences in estimation of genetic similarity between RAPDs and RFLPs is largely due to sampling error rather than due to DNA-based differences in how RAPDs and RFLPs reveal polymorphisms. A crossing algorithm was used to generate hypothetical banding patterns of hybrids based on the genotypes of the parents. The results of this study indicate that RAPDs provide a level of resolution equivalent to RFLPs for detemination of the genetic relationships among genotypes.     

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.