Structural diversity and evolution of the Rf-1 locus in the genus Oryza

The Rf-1 locus in rice is agriculturally important as it restores fertility in plants with BT-type cytoplasmic male sterility (CMS). The Rf-1 locus contains several duplicated copies of the gene responsible for restoration of fertility. We analyzed the genomic structure of the Rf-1 locus in the genus Oryza to clarify the structural diversity and evolution of the locus. We identified six genes (Rf-1A to Rf-1F) with homology to Rf-1 at this locus in Oryza species with an AA genome. The Rf-1 locus structures in the rice accessions examined were very complex and fell into at least six classification types. The nucleotide sequences of the duplicated genes and their flanking regions were highly conserved suggesting that the complex Rf-1 locus structures were produced by homologous recombination between the duplicated genes. The fact that complex Rf-1 locus structures were common to Oryza species that have evolved independently indicates that a duplication of the ancestral Rf-1 gene occurred early in rice evolution and that homologous recombination resulted in the diversification of Rf-1 locus structures. Additionally, the amino acid sequences of each duplicated gene were conserved between species. This suggests that the duplicated genes in the Rf-1 locus may have divergent functions and may act by controlling mitochondrial gene expression in rice as occurs in the restoration of CMS.     

New in silico insight into the synteny between rice (Oryza sativa L.) and maize (Zea mays L.) highlights reshuffling and identifies new duplications in the rice genome

A unigene set of 1411 contigs was constructed from 2629 redundant maize expressed sequence tags (ESTs) mapped on the maizeDB genetic map. Rice orthologous sequences were identified by blast alignment against the rice genomic sequence. A total of 1046 (74%) maize contigs were associated with their corresponding homologues in the rice genome and 656 (47%) defined as potential orthologous relationships. One hundred and seventeen (8%) maize EST contigs mapped to two distinct loci on the maize genetic map, reflecting the tetraploid nature of the maize genome. Among 492 mono-locus contigs, 344 (484 redundant ESTs) identify collinear blocks between maize chromosomes 2 and 4 and a single rice chromosome, defining six new collinear regions. Fine-scale analysis of collinearity between rice chromosomes 1 and 5 with maize chromosomes 3, 6 and 8 shows the presence of internal rearrangements within collinear regions. Mapping of maize contigs to two distinct loci on the rice sequence identifies five new duplication events in rice. Detailed analysis of a duplication between rice chromosomes 1 and 5 shows that 11% of the annotated genes from the chromosome 1 locus are found duplicated on the chromosome 5 paralogous counterpart, indicating a high degree of re-organisations. The implications of these findings for map-based cloning in collinear regions are discussed.     

Meiotic Recombination between Duplicated Genetic Elements in SACCHAROMYCES CEREVISIAE

We have studied the meiotic recombination behavior of strains carrying two types of duplications of an 18.6-kilobase HIS4 Bam HI fragment. The first type is a direct duplication of the HIS4 Bam HI fragment in which the repeated sequences are separated by Escherichia coli plasmid sequences. The second type, a tandem duplication, has no sequences intervening between the repeated yeast DNA. The HIS4 genes in each region were marked genetically so that recombination events between the duplicated segments could be identified. Meiotic progeny of the strains carrying the duplication were analyzed genetically and biochemically to determine the types of recombination events that had occurred. Analysis of the direct vs. tandem duplication suggests that the E. coli plasmid sequences are recombinogenic in yeast when homozygous. In both types of duplications recombination between the duplicated HIS4 regions occurs at high frequency and involves predominantly interchromosomal reciprocal exchanges (equal and unequal crossovers). The striking observation is that intrachromosomal reciprocal recombination is very rare in comparison with interchromosomal reciprocal recombination. However, intrachromosomal gene conversion occurs at about the same frequency as interchromosomal gene conversion. Reciprocal recombination events between regions on the same chromatid are the most infrequent exchanges. These data suggest that intrachromosomal reciprocal exchanges are suppressed.     

Distance-Independence of Mitotic Intrachromosomal Recombination in Saccharomyces Cerevisiae

Many genetic studies have shown that the frequency of homologous recombination depends largely on the distance in which recombination can occur. We have studied the effect of varying the length of duplicated sequences on the frequency of mitotic intrachromosomal recombination in Saccharomyces cerevisiae. We find that the frequency of recombination resulting in the loss of one of the repeats and the intervening sequences reaches a plateau when the repeats are short. In addition, the frequency of recombination to correct a point mutation contained in one of these repeats is not proportional to the size of the duplication but rather depends dramatically on the location of the mutation within the repeated sequences. However, the frequency of mitotic interchromosomal reciprocal recombination is dependent on the distance separating the markers. The difference in the response of intrachromosomal and interchromosomal mitotic recombination to increasing lengths of homology may indicate there are different rate-limiting steps for recombination in these two cases. These findings have important implications for the maintenance and evolution of duplicated sequences.     

Genetic and Segregation Analysis of Escherichia coli Strains Containing a Tandem Duplication of the trpD-purB Region of the Chromosome

Genetic and segregation analysis of Escherichia coli strains containing a partial duplication of the trp operon reveal that the 2.5-min-long region trpD-purB is duplicated in tandem in the chromosome. The adjacent loci cysB and fabD are not duplicated. Although one copy of the duplicated region is longer than the maximum size of bacteriophage P1kc transducing fragments, the frequency at which the duplicated segment trpDCBA is transferred by transduction to tonB-trp deletion strains is equal to that observed for transfer of the normal trp operon. This suggests that three-point recombination events believed to account for transduction of long duplications occur as frequently as two-point recombination events believed to account for normal transduction. Cotransduction frequencies of trpDCBA with the duplicated loci tonB, galU, tyrT, and hemA are very similar to those for the trp operon with the same loci. This indicates that normal genetic linkage is maintained during the three-point recombination event. However, purB, which is normally unlinked to trp by transduction, is closely linked to trpDCBA and thus must be near the repeat point of the duplication. Transduction tests with point mutations in the trp operon indicated that the repeat point occurs near the normal boundary between trpE and trpD. Segregation analysis of heterogenotes constructed from tonB-trp deletion strains shows that the frequency at which a marker is lost is approximately proportional to its distance from the repeat point. This finding is consistent with a random, singlesite crossover event during segregation. Several observations indicate that non-reciprocal genetic exchange also occurs between copies of the duplication. Analysis of heterogenotes containing dadR1 and dadR(+) demonstrate that the mutant allele is transdominant.     

Analysis of IS1236-mediated gene amplification events in Acinetobacter baylyi ADP1

Recombination between insertion sequence copies can cause genetic deletion, inversion, or duplication. However, it is difficult to assess the fraction of all genomic rearrangements that involve insertion sequences. In previous gene duplication and amplification studies of Acinetobacter baylyi ADP1, an insertion sequence was evident in approximately 2% of the characterized duplication sites. Gene amplification occurs frequently in all organisms and has a significant impact on evolution, adaptation, drug resistance, cancer, and various disorders. To understand the molecular details of this important process, a previously developed system was used to analyze gene amplification in selected mutants. The current study focused on amplification events in two chromosomal regions that are near one of six copies of the only transposable element in ADP1, IS1236 (an IS3 family member). Twenty-one independent mutants were analyzed, and in contrast to previous studies of a different chromosomal region, IS1236 was involved in 86% of these events. IS1236-mediated amplification could occur through homologous recombination between insertion sequences on both sides of a duplicated region. However, this mechanism presupposes that transposition generates an appropriately positioned additional copy of IS1236. To evaluate this possibility, PCR and Southern hybridization were used to determine the chromosomal configurations of amplification mutants involving IS1236. Surprisingly, the genomic patterns were inconsistent with the hypothesis that intramolecular homologous recombination occurred between insertion sequences following an initial transposition event. These results raise a novel possibility that the gene amplification events near the IS1236 elements arise from illegitimate recombination involving transposase-mediated DNA cleavage.     

Genomic background predicts the fate of duplicated genes: evidence from the yeast genome

Gene duplication with subsequent divergence plays a central role in the acquisition of genes with novel function and complexity during the course of evolution. With reduced functional constraints or through positive selection, these duplicated genes may experience accelerated evolution. Under the model of subfunctionalization, loss of subfunctions leads to complementary acceleration at sites with two copies, and the difference in average rate between the sequences may not be obvious. On the other hand, the classical model of neofunctionalization predicts that the evolutionary rate in one of the two duplicates is accelerated. However, the classical model does not tell which of the duplicates experiences the acceleration in evolutionary rate. Here, we present evidence from the Saccharomyces cerevisiae genome that a duplicate located in a genomic region with a low-recombination rate is likely to evolve faster than a duplicate in an area of high recombination. This observation is consistent with population genetics theory that predicts that purifying selection is less effective in genomic regions of low recombination (Hill-Robertson effect). Together with previous studies, our results suggest the genomic background (e.g., local recombination rate) as a potential force to drive the divergence between nontandemly duplicated genes. This implies the importance of structure and complexity of genomes in the diversification of organisms via gene duplications.     

Development of microsatellite markers and characterization of simple sequence length polymorphism (SSLP) in rice (Oryza sativa L.)

Microsatellite markers containing simple sequence repeats (SSR) are a valuable tool for genetic analysis. Our objective is to augment the existing RFLP map of rice with simple sequence length polymorphisms (SSLP). In this study, we describe 20 new microsatellite markers that have been assigned to positions along the rice chromosomes, characterized for their allelic diversity in cultivated and wild rice, and tested for amplification in distantly related species. Our results indicate that the genomic distribution of microsatellites in rice appears to be random, with no obvious bias for, or clustering in particular regions, that mapping results are identical in intersubspecific and interspecific populations, and that amplification in wild relatives ofOryza sativa is reliable in species most closely related to cultivated rice but becomes less successful as the genetic distance increases. Sequence analysis of SSLP alleles in three relatedindica varieties demonstrated the clustering of complex arrays of SSR motifs in a single 300-bp region with independent variation in each. Two microsatellite markers amplified multiple loci that were mapped onto independent rice chromosomes, suggesting the presence of duplicated regions within the rice genome. The availability of increasing numbers of mapped SSLP markers can be expected to increase the power and resolution of genome analysis in rice.     

Development of microsatellite markers and characterization of simple sequence length polymorphism (SSLP) in rice (Oryza sativa L.)

Microsatellite markers containing simple sequence repeats (SSR) are a valuable tool for genetic analysis. Our objective is to augment the existing RFLP map of rice with simple sequence length polymorphisms (SSLP). In this study, we describe 20 new microsatellite markers that have been assigned to positions along the rice chromosomes, characterized for their allelic diversity in cultivated and wild rice, and tested for amplification in distantly related species. Our results indicate that the genomic distribution of microsatellites in rice appears to be random, with no obvious bias for, or clustering in particular regions, that mapping results are identical in intersubspecific and interspecific populations, and that amplification in wild relatives ofOryza sativa is reliable in species most closely related to cultivated rice but becomes less successful as the genetic distance increases. Sequence analysis of SSLP alleles in three relatedindica varieties demonstrated the clustering of complex arrays of SSR motifs in a single 300-bp region with independent variation in each. Two microsatellite markers amplified multiple loci that were mapped onto independent rice chromosomes, suggesting the presence of duplicated regions within the rice genome. The availability of increasing numbers of mapped SSLP markers can be expected to increase the power and resolution of genome analysis in rice.     

Identification and High-Density Mapping of Gene-Rich Regions in Chromosome Group 5 of Wheat

The distribution of genes and recombination in the wheat genome was studied by comparing physical maps with the genetic linkage maps. The physical maps were generated by mapping 80 DNA and two phenotypic markers on an array of 65 deletion lines for homoeologous group 5 chromosomes. The genetic maps were constructed for chromosome 5B in wheat and 5D in Triticum tauschii. No marker mapped in the proximal 20% chromosome region surrounding the centromere. More than 60% of the long arm markers were present in three major clusters that physically encompassed <18% of the arm. Because 48% of the markers were cDNA clones and the distributions of the cDNA and genomic clones were similar, the marker distribution may represent the distribution of genes. The gene clusters were identified and allocated to very small chromosome regions because of a higher number of deletions in their surrounding regions. The recombination was suppressed in the centromeric regions and mainly occurred in the gene-rich regions. The bp/cM estimates varied from 118 kb for gene-rich regions to 22 Mb for gene-poor regions. The wheat genes present in these clusters are, therefore, amenable to molecular manipulations parallel to the plants with smaller genomes like rice.     

Detection of tandem duplications and implications for linkage analysis.

The first demonstration of an autosomal dominant human disease caused by segmental trisomy came in 1991 for Charcot-Marie-Tooth disease type 1A (CMT1A). For this disorder, the segmental trisomy is due to a large tandem duplication of 1.5 Mb of DNA located on chromosome 17p11.2-p12. The search for the CMT1A disease gene was misdirected and impeded because some chromosome 17 genetic markers that are linked to CMT1A lie within this duplication. To better understand how such a duplication might affect genetic analyses in the context of disease gene mapping, we studied the effects of marker duplication on transmission probabilities of marker alleles, on linkage analysis of an autosomal dominant disease, and on tests of linkage homogeneity. We demonstrate that the undetected presence of a duplication distorts transmission ratios, hampers fine localization of the disease gene, and increases false evidence of linkage heterogeneity. In addition, we devised a likelihood-based method for detecting the presence of a tandemly duplicated marker when one is suspected. We tested our methods through computer simulations and on CMT1A pedigrees genotyped at several chromosome 17 markers. On the simulated data, our method detected 96% of duplicated markers (with a false-positive rate of 5%). On the CMT1A data our method successfully identified two of three loci that are duplicated (with no false positives). This method could be used to identify duplicated markers in other regions of the genome and could be used to delineate the extent of duplications similar to that involved in CMT1A.     

The structures of integration sites in transgenic rice

Extensive genomic sequencing and sequence motif analysis have been conducted over the integration sites of two transgenic rice plants, #478 and #559, carrying the luciferase gene and/or hygromycin phosphotransferase gene. The transgenes reside in a region with inverted structure and a large duplication of rice genome over 2 kb. Integration was found at the AT-rich region and/or at the repetitive sequence region, including a SAR-like structure, retrotransposon and telomere repeats. The presence of a patch of sequence homology between plasmid and target DNA, and a small region of duplication involving the target DNA around the recombination site, implicated illegitimate recombination in the process of gene integration. Massive rearrangement of genomic DNA including deletion or translocation was also observed at the integration site and the flanking region of the transgene. The recognition sites of DNA topoisomerases I or II were observed in the rearranged sequences. Since only three junctions of transgenic rice were implicated in the illegitimate recombination and extensive rearrangement of the rice genome, rice protoplasts may be active in this process.     

Mapping 49 quantitative trait loci at high resolution through sequencing-based genotyping of rice recombinant inbred lines

Mapping chromosome regions responsible for quantitative phenotypic variation in recombinant populations provides an effective means to characterize the genetic basis of complex traits. We conducted a quantitative trait loci (QTL) analysis of 150 rice recombinant inbred lines (RILs) derived from a cross between two cultivars, Oryza sativa ssp. indica cv. 93-11 and Oryza sativa ssp. japonica cv. Nipponbare. The RILs were genotyped through next-generation sequencing, which accurately determined the recombination breakpoints and provided a new type of genetic markers, recombination bins, for QTL analysis. We detected 49 QTL with phenotypic effect ranging from 3.2 to 46.0% for 14 agronomics traits. Five QTL of relatively large effect (14.6–46.0%) were located on small genomic regions, where strong candidate genes were found. The analysis using sequencing-based genotyping thus offers a powerful solution to map QTL with high resolution. Moreover, the RILs developed in this study serve as an excellent system for mapping and studying genetic basis of agricultural and biological traits of rice.     

Duplication and Gene Conversion in the Drosophila melanogaster Genome

Using the genomic sequences of Drosophila melanogaster subgroup, the pattern of gene duplications was investigated with special attention to interlocus gene conversion. Our fine-scale analysis with careful visual inspections enabled accurate identification of a number of duplicated blocks (genomic regions). The orthologous parts of those duplicated blocks were also identified in the D. simulans and D. sechellia genomes, by which we were able to clearly classify the duplicated blocks into post- and pre-speciation blocks. We found 31 post-speciation duplicated genes, from which the rate of gene duplication (from one copy to two copies) is estimated to be 1.0×10⁻⁹ per single-copy gene per year. The role of interlocus gene conversion was observed in several respects in our data: (1) synonymous divergence between a duplicated pair is overall very low. Consequently, the gene duplication rate would be seriously overestimated by counting duplicated genes with low divergence; (2) the sizes of young duplicated blocks are generally large. We postulate that the degeneration of gene conversion around the edges could explain the shrinkage of “identifiable” duplicated regions; and (3) elevated paralogous divergence is observed around the edges in many duplicated blocks, supporting our gene conversion–degeneration model. Our analysis demonstrated that gene conversion between duplicated regions is a common and genome-wide phenomenon in the Drosophila genomes, and that its role should be especially significant in the early stages of duplicated genes. Based on a population genetic prediction, we applied a new genome-scan method to test for signatures of selection for neofunctionalization and found a strong signature in a pair of transporter genes.     

Rapid evolution in a pair of recent duplicate segments of rice

Gene duplication has been considered the most important way of generating genetic novelties. The subsequent evolution right after gene duplication is critical for new function to occur. Here we analyzed the evolutionary pattern for a recently duplicated segment between rice chromosomes 11 and 12. This duplication event was estimated to occur about 6 million years ago, during the divergence of the B- and C-genome rice species. The duplicate segment in chromosome 12 has significantly higher frequency of sequence rearrangement rate than non-duplicated regions. The rearrangement rate is approximately 6.5 breakages/Mb per million years, about six times higher than the fastest rate ever reported in eukaryotes. The genes within both segments experienced accelerated nucleotide substitution rates revealed by synonymous (Ks) and non-synonymous divergence (Ka) between Oryza sativa indica and O. sativa japonica. Analysis using EST data also implicates rapid divergence in expression between these segmental duplicate genes. These overall rapid changes from different perspective for the first time provide evidence that relaxation of selection also occurs in large-scale duplications.     

2R or not 2R: Testing hypotheses of genome duplication in early vertebrates

The widely popular hypothesis that there were two rounds of genome duplication by polyploidization early in vertebrate history (the 2R hypothesis) has been difficult to test until recently. Among the lines of evidence adduced in support of this hypothesis are relative genome size, relative gene number, and the existence of genomic regions putatively duplicated during polyploidization. The availability of sequence for a substantial portion of the human genome makes possible the first rigorous tests of this hypothesis. Comparison of gene family size in the human genome and in invertebrate genomes shows no evidence of a 4:1 ratio between vertebrates and invertebrates. Furthermore, explicit phylogenetic tests for the topology expected from two rounds of polyploidization have revealed alternative topologies in a substantial majority of human gene families. Likewise, phylogenetic analyses have shown that putatively duplicated genomic regions often include genes duplicated at widely different times over the evolution of life. The 2R hypothesis thus can be decisively rejected. Rather, current evidence favors a model of genome evolution in which tandem duplication, whether of genomic segments or of individual genes, predominates.     

High Frequency Repeat-Induced Point Mutation (Rip) Is Not Associated with Efficient Recombination in Neurospora

Duplicated DNA sequences in Neurospora crassa are efficiently detected and mutated during the sexual cycle by a process named repeat-induced point mutation (RIP). Linked, direct duplications have previously been shown to undergo both RIP and deletion at high frequency during premeiosis, suggesting a relationship between RIP and homologous recombination. We have investigated the relationship between RIP and recombination for an unlinked duplication and for both inverted and direct, linked duplications. RIP occurred at high frequency (42-100%) with all three types of duplications used in this study, yet recombination was infrequent. For both inverted and direct, linked duplications, recombination was observed, but at frequencies one to two orders of magnitude lower than RIP. For the unlinked duplication, no recombinants were seen in 900 progeny, indicating, at most, a recombination frequency nearly three orders of magnitude lower than the frequency of RIP. In a direct duplication, RIP and recombination were correlated, suggesting that these two processes are mechanistically associated or that one process provokes the other. Mutations due to RIP have previously been shown to occur outside the boundary of a linked, direct duplication, indicating that RIP might be able to inactivate genes located in single-copy sequences adjacent to a duplicated sequence. In this study, a single-copy gene located between elements of linked duplications was inactivated at moderate frequencies (12-14%). Sequence analysis demonstrated that RIP mutations had spread into these single-copy sequences at least 930 base pairs from the boundary of the duplication, and Southern analysis indicated that mutations had occurred at least 4 kilobases from the duplication boundary.     

Inherited Interstitial Duplications of Proximal 15q: Genotype-Phenotype Correlations

We present the cytogenetic, molecular cytogenetic, and molecular genetic results on 20 unrelated patients with an interstitial duplication of the proximal long arm of chromosome 15. Multiple probes showed that the Prader-Willi/Angelman critical region (PWACR) was included in the duplication in 4/20 patients, each ascertained with developmental delay. The duplication was also found in two affected but not in three unaffected sibs of one of these patients. All four probands had inherited their duplication from their mothers, three of whom were also affected. Two of the affected mothers also carried a maternally inherited duplication, whereas the duplication in the unaffected mother and in an unaffected grandmother was paternal in origin, raising the possibility of a parental-origin effect. The PWACR was not duplicated in the remaining 16 patients, of whom 4 were referred with developmental delay. In the 14 families for which parental samples were available, the duplication was inherited with equal frequency from a phenotypically normal parent, mother or father. Comparative genomic hybridization undertaken on two patients suggested that proximal 15q outside the PWACR was the origin of the duplicated material. The use of PWACR probes discriminates between a large group of duplications of no apparent clinical significance and a smaller group, in which a maternally derived PWACR duplication is consistently associated with developmental delay and speech difficulties but not with overt features of either Prader-Willi syndrome or Angelman syndrome.     

Coevolution of PERB11 (MIC) and HLA Class I Genes with HERV-16 and Retroelements by Extended Genomic Duplication

The recent availability of genomic sequence information for the class I region of the MHC has provided an opportunity to examine the genomic organization of HLA class I (HLAcI) and PERB11/MIC genes with a view to explaining their evolution from the perspective of extended genomic duplications rather than by simple gene duplications and/or gene conversion events. Analysis of genomic sequence from two regions of the MHC (the alpha- and beta-blocks) revealed that at least 6 PERB11 and 14 HLAcI genes, pseudogenes, and gene fragments are contained within extended duplicated segments. Each segment was searched for the presence of shared (paralogous) retroelements by RepeatMasker in order to use them as markers of evolution, genetic rearrangements, and evidence of segmental duplications. Shared Alu elements and other retroelements allowed the duplicated segments to be classified into five distinct groups (A to E) that could be further distilled down to an ancient preduplication segment containing a HLA and PERB11 gene, an endogenous retrovirus (HERV-16), and distinctive retroelements. The breakpoints within and between the different HLAcI segments were found mainly within the PERB11 and HLA genes, HERV-16, and other retroelements, suggesting that the latter have played a major role in duplication and indel events leading to the present organization of PERB11 and HLAcI genes. On the basis of the features contained within the segments, a coevolutionary model premised on tandem duplication of single and multipartite genomic segments is proposed. The model is used to explain the origins and genomic organization of retroelements, HERV-16, DNA transposons, PERB11, and HLAcI genes as distinct segmental combinations within the alpha- and beta-blocks of the human MHC. Received: 5 December 1998 / Accepted: 27 January 1999