Genetic diversity and differentiation of indica and japonica rice detected by RFLP analysis

Summary Genetic diversity and differentiation in indica and japonica groups of the cultivated rice (Oryza sativa L.) were studied by assaying DNA restriction fragment length polymorphisms of 12 indica and 14 japonica rice lines digested with three restriction endonucleases. A total of 49 probes were selected to represent the entire RFLP map at intervals of 20–30 cM. It was shown that 95 of the 145 possible probe/enzyme combinations, involving 43 probes and all three enzymes, detected restriction fragment length variation, and the degree of polymorphism varied greatly from one probe/enzyme combination to another. These results demonstrate that indica rice is genetically more diverse than japonica type. Significant differentiation between the two rice groups was detected by 33 probes representing 11 of the 12 rice chromosomes. It was deduced that the processes leading to differentiation involved a combination of molecular events that include base substitutions and insertion/deletions.     

A genetic linkage map for tef [Eragrostis tef (Zucc.) Trotter]

Tef [Eragrostis tef (Zucc.) Trotter] is the major cereal crop in Ethiopia. Tef is an allotetraploid with a base chromosome number of 10 (2n = 4x = 40) and a genome size of 730 Mbp. Ninety-four F₉ recombinant inbred lines (RIL) derived from the interspecific cross, Eragrostis tef cv. Kaye Murri × Eragrostis pilosa (accession 30-5), were mapped using restriction fragment length polymorphisms (RFLP), simple sequence repeats derived from expressed sequence tags (EST–SSR), single nucleotide polymorphism/insertion and deletion (SNP/INDEL), intron fragment length polymorphism (IFLP) and inter-simple sequence repeat amplification (ISSR). A total of 156 loci from 121 markers was grouped into 21 linkage groups at LOD 4, and the map covered 2,081.5 cM with a mean density of 12.3 cM per locus. Three putative homoeologous groups were identified based on multi-locus markers. Sixteen percent of the loci deviated from normal segregation with a predominance of E. tef alleles, and a majority of the distorted loci were clustered on three linkage groups. This map will be useful for further genetic studies in tef including mapping of loci controlling quantitative traits (QTL), and comparative analysis with other cereal crops.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Linkage Arrangement of RFLP loci in progenies from crosses between doubled haploid Asparagus officinalis L. clones

A preliminary genetic map of the dioecious species Asparagus officinalis L. (2n = 20) has been constructed on the basis of restriction fragment length polymorphism (RFLP) and isozyme marker data. With DNA samples digested with either EcoRI or HindIII 61 out of 148 probes (41%) identified RFLPs in six families of doubled haploid lines obtained through anther culture. A higher level of polymorphism (65%) was observed when a single family was screened for RFLPs using six distinct restriction enzymes. Segregation analysis of the BC progenies (40–80 individuals) resulted in a 418-cM extended map comprising 43 markers: 39 RFLPs, three isozymes and one morphological (sex). These markers are clustered in 12 linkage groups and four of them exhibited significant deviations from the expected 11 ratio. One isozyme and three RFLP markers were assigned to the sex chromosome.     

DNA polymorphisms in grain sorghum (Sorghum bicolor (L.) Moench)

Molecular markers [random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP)] were used to determine the frequency of DNA polymorphism in grain sorghum (Sorghum bicolor (L.) Moench). Twenty-nine oligonucleotide primers were employed for RAPDs, generating a total of 262 DNA fragments, of which 145 were polymorphic in at least one pairwise comparison between 36 genotypes. Individual primers differed significantly in their ability to detect genetic polymorphism in the species. The overall frequency of polymorphisms was low with a mean frequency of 0.117 polymorphisms per RAPD band being obtained from all pairwise comparisons between genotypes, with maximum and minimum values of 0.212 and 0.039, respectively. Results from phenetic analysis of bandsharing data were consistent with current sub-specific groupings of the species, with clusters of Durra, Zerazera, Caud-Nig, Caud-Kaura and Caffrorum being discernible. The results also indicated that individuals of a similar taxonomic grouping but different geographic origin may be genetically less identical than previously considered. Similar frequencies of polymorphism to that obtained with RAPDs were obtained with RFLPs. Results from these experiments indicated that a high level of genetic uniformity exists within S. bicolor.     

Sheep linkage mapping: restriction fragment length polymorphism detection with heterologous cDNA probes

Summary. A selection of cattle, human and sheep cDNA probes were screened against sheep genomic DNA, cut with 10 different restriction enzymes, to assess the usefulness of these probes for restriction fragment length polymorphism (RFLP) linkage studies in sheep. Two-thirds of the cattle cDNA probes showed moderate to strong homology with sheep DNA samples, compared with less than half of the human cDNA probes at the final washing stringency chosen for the experiments. The set of probes tested detected a useful frequency of RFLPs. Fifty-seven per cent of probes showing moderate to strong homology identified RFLPs with one or more restriction enzymes. Restriction enzymes that detected RFLPs most frequently in sheep were Taq I and Msp I. The results show that sheep and cattle cDNA probes, including candidate genes for production traits, identified a high frequency of RFLPs suitable for genetic mapping in sheep.     

Toward closing rice telomere gaps: mapping and sequence characterization of rice subtelomere regions

Despite the collective efforts of the international community to sequence the complete rice genome, telomeric regions of most chromosome arms remain uncharacterized. In this report we present sequence data from subtelomere regions obtained by analyzing telomeric clones from two 8.8 × genome equivalent 10-kb libraries derived from partial restriction digestion with HaeIII or Sau3AI (OSJNPb HaeIII and OSJNPc Sau3AI). Seven telomere clones were identified and contain 25–100 copies of the telomere repeat (CCCTAAA)_n on one end and unique sequences on the opposite end. Polymorphic sequence-tagged site markers from five clones and one additional PCR product were genetically mapped on the ends of chromosome arms 2S, 5L, 10S, 10L, 7L, and 7S. We found distinct chromosome-specific telomere-associated tandem repeats (TATR) on chromosome 7 (TATR7) and on the short arm of chromosome 10 (TATR10s) that showed no significant homology to any International Rice Genome Sequencing Project (IRGSP) genomic sequence. The TATR7, a degenerate tandem repeat which is interrupted by transposable elements, appeared on both ends of chromosome 7. The TATR10s was found to contain an inverted array of three tandem repeats displaying an interesting secondary folding pattern that resembles a telomere loop (t-loop) and which may be involved in a protective function against chromosomal end degradation.Electronic Supplementary Material Supplementary material is available for this article at     

The comparison of RFLP,RAPD, AFLP and SSR (microsatellite) markers for germplasm analysis

The utility of RFLP (restriction fragment length polymorphism), RAPD (random-amplified polymorphic DNA), AFLP (amplified fragment length polymorphism) and SSR (simple sequence repeat, microsatellite) markers in soybean germplasm analysis was determined by evaluating information content (expected heterozygosity), number of loci simultaneously analyzed per experiment (multiplex ratio) and effectiveness in assessing relationships between accessions. SSR markers have the highest expected heterozygosity (0.60), while AFLP markers have the highest effective multiplex ratio (19). A single parameter, defined as the marker index, which is the product of expected heterozygosity and multiplex ratio, may be used to evaluate overall utility of a marker system. A comparison of genetic similarity matrices revealed that, if the comparison involved both cultivated (Glycine max) and wild soybean (Glycine soja) accessions, estimates based on RFLPs, AFLPs and SSRs are highly correlated, indicating congruence between these assays. However, correlations of RAPD marker data with those obtained using other marker systems were lower. This is because RAPDs produce higher estimates of interspecific similarities. If the comparisons involvedG. max only, then overall correlations between marker systems are significantly lower. WithinG. max, RAPD and AFLP similarity estimates are more closely correlated than those involving other marker systems.Abbreviations RFLP restriction fragment length plymorphism - RAPD random-amplified polymorphic DNA - AFLP amplified fragment length polymorphism - SSR simple sequence repeat - PCR polymerase chain reaction - TBE Tris-borate-EDTA buffer - MI marker index - SENA sum of effective numbers of alleles     

5S ribosomal gene clusters in wheat: pulsed field gel electrophoresis reveals a high degree of polymorphism

Summary The long-range structure of 5S rRNA gene clusters has been investigated in wheat (Triticum aestivum L.) by means of pulsed field gel electrophoresis. Using aneuploid stocks, 5S rRNA gene clusters were assigned to sites on chromosomes 1B, 1D, 513 and 5D. Cluster sizes were evaluated and the copy number of 5S DNA repeats was estimated at 4700-5200 copies for the short repeating unit (410 bp) and about 3100 copies for the long repeat (500 bp) per haploid genome. A comparison of wheat cultivars revealed extremely high levels of polymorphism in the 5S rRNA gene clusters. With one restriction enzyme digest all varieties tested gave unique banding patterns and, on a per fragment basis, 21-fold more polymorphism was detected among cultivars for 5S DNA compared to standard restriction fragment length polymorphisms (RFLPs) detected with single copy clones. Experiments with aneuploid stocks suggest that the 5S rRNA gene clusters at several chromosomal sites contribute to this polymorphism. A number of previous reports have shown that wheat cultivars are not easily distinguished by isozymes or RFLPs. The high level of variation detected in 5S rRNA gene clusters therefore offers the possibility of a sensitive fingerprinting method for wheat. 5S DNA and other macro-satellite sequences may also serve as hypervariable Mendelian markers for genetic and breeding experiments in wheat.     

Unigene derived microsatellite markers for the cereal genomes

Unigene derived microsatellite (UGMS) markers have the advantage of assaying variation in the expressed component of the genome with unique identity and positions. We characterized the microsatellite motifs present in the unigenes of five cereal species namely, rice, wheat, maize, Sorghum and barley and compared with those in Arabidopsis. The overall UGMS frequency in the five cereal species was 1/7.6 kb. The maximum UGMS frequency was in rice (1/3.6 kb) and the lowest in wheat (1/10.6 kb). GC-rich trinucleotide repeat motifs coding for alanine followed by arginine and the dinucleotide repeat motif GA were found to be abundant UGMS classes across all the five cereal species. Primers could be designed for 95% (wheat and barley) to 97% (rice) of the identified microsatellites. The proportion and frequency of occurrence of long hypervariable class I (≥20 nucleotides) and potentially variable class II (12–20 nucleotides) UGMS across five cereal species were characterized. The class I UGMS markers were physically mapped in silico on to the finished rice genome and bin-mapped in wheat. Comparative mapping based on class I UGMS markers in rice and wheat revealed syntenic relationships between the two genomes. High degree of conservation and cross-transferability of the class I UGMS markers were evident among the five cereal species, which was validated experimentally. The class I UGMS-conserved orthologous set (COS) markers identified in this study would be useful for understanding the evolution of genes and genomes in cereals. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

A PvuII polymorphism in the 5′ flanking region of the apolipoprotein AIV gene: its use to study genetic variation determining serum lipid and apolipoprotein concentration

Summary We have used a 1.05-kb unique genomic fragment from the 5 end of the apolipoprotein (apo) CIII gene to identify a restriction fragment length polymorphism (RFLP) detected with the restriction enzyme PvuII, in the apoCIII-apoAIV intergenic region. In a sample of 220 normolipidaemic individuals from the UK population, the frequency of the rare allele, VB2 is 0.054. The PvuII polymorphism is in apparent linkage equilibrium with three other RFLPs of this gene cluster, detected with the restriction enzymes XmnI, PstI and SstI, but in linkage disequilibrium with an RFLP in the apoCIII gene also detected with PvuII. Taken together, these five RFLPs have a PIC (polymorphism information content) value of 0.8, and therefore are informative for genetic studies. Individuals with the genotype VB1VB2 had lower mean concentrations of apoAI, and HDL-cholesterol than individuals with the genotype VB1VB1. However these differences were not statistically significant.     

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     

Molecular mapping of rice chromosomes

Summary We report the construction of an RFLP genetic map of rice (Oryza sativa) chromosomes. The map is comprised of 135 loci corresponding to clones selected from a PstI genomic library. This molecular map covers 1,389 cM of the rice genome and exceeds the current classical maps by more than 20%. The map was generated from F₂ segregation data (50 individuals) from a cross between an indica and javanica rice cultivar. Primary trisomics were used to assign linkage groups to each of the 12 rice chromosomes. Seventy-eight percent of the clones assayed revealed RFLPs between the two parental cultivars, indicating that rice contains a significant amount of RFLP variation. Strong correlations between size of hybridizing restriction fragments and level of polymorphism indicate that a significant proportion of the RFLPs in rice are generated by insertions/delections. This conclusion is supported by the occurrence of null alleles for some clones (presumably created by insertion or deletion events). One clone, RG229, hybridized to sequences in both the indica and javanica genomes, which have apparently transposed since the divergence of the two cultivars from their last common ancestor, providing evidence for sequence movement in rice. As a by product of this mapping project, we have discovered that rice DNA is less C-methylated than tomato or maize DNA. Our results also suggest the notion that a large fraction of the rice genome (approximately 50%) is single copy.     

DNA restriction fragment length polymorphisms correlate with isozyme diversity in Phaseolus vulgaris L.

Summary Genetic variation in Phaseolus vulgaris L. (P. vulgaris) was investigated at the isozyme and DNA levels. We constructed a library of size-selected Pst I clones of P. vulgaris nuclear DNA. Clones from this library were used to examine 14 P. vulgaris accessions for restriction fragment length polymorphisms (RFLPs). DNAs from each accession were analyzed with three restriction enzymes and 18 single copy probes. The same accessions were also examined for variability at 16 isozyme loci. Accessions included four representatives of the T phaseolin group and five representatives each of the C and S phaseolin groups. One member of the S group (the breeding line XR-235-1-1) was derived from a cross between P. vulgaris and P. coccineus. Isozymes and RFLPs revealed very similar patterns of genetic variation. Little variation was observed among accessions with C and T phaseolin types or among those with the S phaseolin type. However, both isozyme and RFLP data grouped accessions with S phaseolin separately from those accessions with C or T phaseolin. The highest degree of polymorphism was observed between XR-235-1-1 and members of the C/T group. RFLP markers will supplement isozymes, increasing the number of polymorphic loci that can be analyzed in breeding, genetic, and evolutionary studies of Phaseolus.     

The X chromosome shows less genetic variation at restriction sites than the autosomes.

Using a standard technique, 122 single-copy probes were screened for their ability to detect restriction fragment length polymorphisms (RFLPs) in the human genome. The use of a standardized RFLP screening enables the introduction of statistical methods in the analysis of differences in RFLP content between chromosomes and enzymes. RFLPs were detected from panels containing at least 17 unrelated chromosomes, digested with TaqI, MspI, BglII, HindIII, EcoRI, and PstI. Forty autosomal probes, representing a sample of 2,710 base pairs (bp) per haploid genome, were tested, and 24 RFLPs were found. With 82 X-chromosomal probes, 17 RFLPs were found in 6,228 bp per haploid genome. The frequency of X-chromosomal RFLPs is three times less than that of the autosomes; this difference is highly significant (P = less than .001). The frequency of RFLPs revealed by various restriction enzymes and the possibility that the X chromosome is a "low mutation" niche in the human genome are discussed.     

The human fumarylacetoacetase gene: characterisation of restriction fragment length polymorphisms and identification of haplotypes in tyrosinemia type 1 and pseudodeficiency

Summary Deficiency of human fumarylacetoacetase (FAH) activity results in hereditary tyrosinemia type I. Using the restriction enzymes BglII, KpnI and StuI and a 1.3-kb cDNA probe for the FAH gene, we have found 6 restriction fragment length polymorphisms (RFLPs). These RFLPs were utilised in 3 tyrosinemia families in which one or both parents are carriers of both a tyrosinemia and a pseudodeficiency gene for FAH. Full information was obtained in two of these families. The polymorphisms identified 6 haplotypes. The haplotype distribution was significantly different in 32 unrelated tyrosinemia patients compared with a reference population of 100 individuals. The combined polymorphism information content was 0.77.     

The pattern of insertion/deletion polymorphism in Arabidopsis thaliana

Little is known about variation of nucleotide insertion/deletions (indels) within species. In Arabidopsis thaliana, we investigated indel polymorphism patterns between two genome sequences and among 96 accessions at 1215 loci. Our study identified patterns in the variation of indel density, size, GC content and distribution, and a correlation between indels and substitutions. We found that the GC content in indel sequences was lower than that in non-indel sequences and that indels typically occur in regions with lower GC content. Patterns of indel frequency distribution among populations were more consistent with neutral expectation than substitution patterns. We also found that the local level of substitutions is positively correlated with indel density and negatively correlated with their distance to the closed indel, suggesting that indels play an important role in nucleotide variation.     

Towards an integrated linkage map of common bean

Summary Two genomic libraries were established to provide markers to develop an integrated map combining molecular markers and genes for qualitative and quantitative morpho-agronomic traits in common bean. Contrasting characteristics were observed for the two libraries. While 89% of the PstI clones were classified as single-copy sequences, only 21% of the EcoRIBamHI clones belonged in that category. Clones of these two libraries were hybridized against genomic DNA of nine genotypes chosen according to their divergent evolutionary origin and contrasting agronomic traits. Eight restriction enzymes were used in this study. PstI clones revealed 80–90% polymorphism between the Andean and Middle American gene pools and 50–60% polymorphism within these gene pools. However, under the same conditions only 30% of the EcoRI-BamHI clones showed polymorphism between the Middle American and Andean gene pools. Hybridization with PstI clones to EcoRI-, EcoRV-, or HindIII-digested genomic DNA resulted in a cumulative frequency of polymorphism of approximately 80%. Hybridizations to BamHI-, HaeIII-, HinfI-, PstI-, and XbaI-digested genomic DNA detected no additional polymorphisms not revealed by the former three enzymes. In the PstI library, a positive correlation was observed between the average size of hybridizing restriction fragments and the frequency of polymorphism detected by each restriction enzyme. This relationship is consistent with the higher proportion of insertion/deletion events compared with the frequency of nucleotide substitutions observed in that library.     

Simple-sequence repeat markers used in merging linkage maps of melon (Cucumis melo L.)

A set of 118 simple sequence repeat (SSR) markers has been developed in melon from two different sources: genomic libraries (gSSR) and expressed sequence-tag (EST) databases (EST-SSR). Forty-nine percent of the markers showed polymorphism between the Piel de Sapo (PS) and PI161375 melon genotypes used as parents for the mapping populations. Similar polymorphism levels were found in gSSR (51.2%) and EST-SSR (45.5%). Two populations, F₂ and a set of double haploid lines (DHLs), developed from the same parent genotypes were used for map construction. Twenty-three SSRs and 79 restriction fragment length polymorphisms (RFLPs), evenly distributed through the melon genome, were used to anchor the maps of both populations. Ten cucumber SSRs, 41 gSSRs, 16 EST-SSR, three single nucleotide polymorphism (SNP) markers, and the Nsv locus were added in the DHL population. The maps developed in the F₂ and DHL populations were co-linear, with similar lengths, except in linkage groups G1, G9, and G10. There was segregation distortion in a higher proportion of markers in the DHL population compared with the F₂, probably caused by selection during the construction of DHLs through in vitro culture. After map merging, a composite genetic map was obtained including 327 transferable markers: 226 RFLPs, 97 SSRs, three SNPs, and the Nsv locus. The map length is 1,021 cM, distributed in 12 linkage groups, and map density is 3.11 cM/marker. SSR markers alone cover nearly 80% of the map length. This map is proposed as a basis for a framework melon map to be merged with other maps and as an anchor point for map comparison between species of the Cucurbitaceae family.Electronic Supplementary Material Supplementary material is available for this article at     

影响籼粳稻DNA限制性片段长度多态性检测的因素分析

对窄叶青(籼稻)和京系17(梗稻)的RFLP进行了系统分析。结果表明,某种酶多态性检测能力与同时检测到此多态性的其余酶的数目之间存在极显著的正相关(r=0.962**)。由此推论,籼粳稻大部分RFLP可能来自大范围DNA的结构变化而不是碱基取代。cDNA克隆虽然具有高度的保守性,但却具有比基因组克隆更高的多态性检测能力。在实验使用的探针范围内,探针长度和检测到的多态性无相关性。由探针检测到的多态性位点均匀地散布在水稻的12对染色体上,这可能是这两个品种所属的亚种之间的遗传距离较大的原因。     

Ribosomal DNA variation and its phylogenetic implication in the genusBrachypodium (Poaceae)

The structure of ribosomal DNA ofBrachypodium and several other grass species was investigated using a heterologous rDNA probe from wheat. Several different rDNA families were present among perennial and annual species within the genus. In contrast to the annual species the perennial species exhibited a very low degree of repeat length variation. An extra Eco RI site and a Hin dIII site were observed in the IGS, which distinguishedBrachypodium from other grass genera. The restriction fragment length polymorphism and length variation of the repeat units have taxonomic value withinBrachypodium and are correlated with the classification ofBrachypodium derived from other data.