Recombination events generating a novel Rp1 race specificity

Genes at the maize Rp1 rust resistance complex often mispair in meiosis, which allows genes to recombine unequally, creating recombinant haplotypes. Four recombinant haplotypes were identified from progeny of an Rp1-D/Rp1-I heterozygote that conferred a nonparental resistance specificity designated Rp1-I*. Sequence comparisons of paralogs in the recombinant and parental haplotypes demonstrated that all four recombinants were derived from intergenic (between gene) recombination events. The sequence of paralogs in the HRp1-I parental haplotype indicated this haplotype includes 41 or more rp1 genes, at least 31 of which are transcribed. The results indicate that most of the novel resistance specificities that have arisen spontaneously at Rp1 are the result of reassort ment of existing Rp1 genes.     

The maize rp1 rust resistance gene identifies homologues in barley that have been subjected to diversifying selection

A number of agronomically important grasses (sorghum, wheat, panicum, sugar cane, oats, rice and barley) are shown to contain sequences homologous to rp1, a maize gene that confers race-specific resistance to the rust fungus Puccinia sorghi. Mapping of rp1-related sequences in barley identified three unlinked loci on chromosomes 1HL, 3HL and 7HS. The locus located on chromosome 7HS comprises a small gene family of at least four members, two of which were isolated and are predicted to encode nucleotide binding site-leucine-rich repeat (NBS-LRR) proteins that are respectively 58% and 60% identical to the maize rp1 protein. Evidence of positive selection for sequence diversification acting upon these two barley genes was observed; however, diversifying selection was restricted to the carboxy terminal half of the LRR domain. One of these rp1 homologous genes cosegregated with the barley Rpg1 stem rust resistance gene amongst 148 members of the Steptoe × Morex double haploid mapping family. Three other unrelated resistance gene-like sequences, potentially encoding NBS-LRR proteins, are also shown to be linked to the Rpg1 locus but not cosegregating with the gene. Received: 2 August 1999 / Accepted: 28 September 1999     

Disease Lesion Mimicry Caused by Mutations in the Rust Resistance Gene rp1

The rp1 locus of maize controls race-specific resistance to the common rust fungus Puccinia sorghi. Four mutant or recombinant Rp1 alleles (rp1-NC3, Rp1-D21, Rp1-MD19, and Rp1-Kr1N) were identified. They condition necrotic phenotypes in the absence of the rust pathogen. These Rp1 lesion mimics fall into three different phenotypic classes: (1) The rp1-NC3 and Rp1-D21 alleles require rust infection or other biotic stimulus to initiate necrotic lesions. These alleles react strongly to all maize rust biotypes tested and also to nonhost rusts. (2) The Rp1-MD19 allele, which has a similar phenotype, also requires a biotic stimulus to initiate lesions. However, Rp1-MD19 shows the race specificity of the Rp1-D gene. (3) The Rp1-Kr1N allele specifies a diffuse necrotic phenotype in the absence of any biotic stimulus and a race-specific reaction when inoculated with maize rust.     

Recombination between paralogues at the Rp1 rust resistance locus in maize

Rp1 is a complex rust resistance locus of maize. The HRp1-D haplotype is composed of Rp1-D and eight paralogues, seven of which also code for predicted nucleotide binding site-leucine rich repeat (NBS-LRR) proteins similar to the Rp1-D gene. The paralogues are polymorphic (DNA identities 91-97%), especially in the C-terminal LRR domain. The remaining family member encodes a truncated protein that has no LRR domain. Seven of the nine family members, including the truncated gene, are transcribed. Sequence comparisons between paralogues provide evidence for past recombination events between paralogues and diversifying selection, particularly in the C-terminal half of the LRR domain. Variants selected for complete or partial loss of Rp1-D resistance can be explained by unequal crossing over that occurred mostly within coding regions. The Rp1-D gene is altered or lost in all variants, the recombination breakpoints occur throughout the genes, and most recombinant events (9/14 examined) involved the same untranscribed paralogue with the Rp1-D gene. One recombinant with a complete LRR from Rp1-D, but the amino-terminal portion from another homologue, conferred the Rp1-D specificity but with a reduced level of resistance.     

Evolution of the rat kallikrein gene family: Gene conversion leads to functional diversity

Summary Kallikrein-like simple serine proteases are encoded by closely related members of a gene family in several mammalian species. Molecular cloning and genomic Southern blot analysis after conventional and pulsed-field gel electrophoresis indicate that the rat kallikrein gene family comprises 15–20 members, probably closely linked at a single locus. Determination of the nucleotide sequences of the rGK-3,-4, and-6 genes here completes sequence data for a total of nine rat kallikrein family members. Comparison of the rat gene sequences to each other and to those of human and mouse kallikrein family genes reveals patterns of relatedness indicative of concerted evolution. Analysis of nucleotide sequence variants in kallikrein family members shows that most sequence variants are shared by multiple family members; the patterns of shared variants are complex and indicate multiple short gene conversions between family members. Sequence exchanges between family members generate novel assortments of variants in amino acid coding regions that may affect substrate specificity and thereby contribute to the diversity of enzyme activity. Furthermore, small sequence exchanges also may play a role in generating the diverse patterns of tissue-specific expression of rat family members. These analyses indicate an important role for gene conversion in the evolution of the functional diversity of these duplicated genes.     

Allelic and haplotypic diversity at the rp1 rust resistance locus of maize

The maize Rp1 rust resistance locus is a complex consisting of a family of closely related resistance genes. The number of Rp1 paralogs in different maize lines (haplotypes) varied from a single gene in some stocks of the inbred A188 to >50 genes in haplotypes carrying the Rp1-A and Rp1-H specificities. The sequences of paralogs in unrelated haplotypes differ, indicating that the genetic diversity of Rp1-related genes is extremely broad in maize. Two unrelated haplotypes with five or nine paralogs had identical resistance phenotypes (Rp1-D) encoded in genes that differed by three nucleotides resulting in a single amino acid substitution. Genes in some haplotypes are more similar to each other than to any of the genes in other haplotypes indicating that they are evolving in a concerted fashion.     

Structural analysis of the maize rp1 complex reveals numerous sites and unexpected mechanisms of local rearrangement

Rp1 is a complex disease resistance locus in maize that is exceptional in both allelic variability and meiotic instability. Genomic sequence analysis of three maize BACs from the Rp1 region of the B73 inbred line revealed 4 Rp1 homologs and 18 other gene-homologous sequences, of which at least 16 are truncated. Thirteen of the truncated genes are found in three clusters, suggesting that they arose from multiple illegitimate break repairs at the same sites or from complex repairs of each of these sites with multiple unlinked DNA templates. A 43-kb region that contains an Rp1 homolog, six truncated genes, and three Opie retrotransposons was found to be duplicated in this region. This duplication is relatively recent, occurring after the insertion of the three Opie elements. The breakpoints of the duplication are outside of any genes or identified repeat sequence, suggesting a duplication mechanism that did not involve unequal recombination. A physical map and partial sequencing of the Rp1 complex indicate the presence of 15 Rp1 homologs in regions of approximately 250 and 300 kb in the B73 inbred line. Comparison of fully sequenced Rp1-homologous sequences in the region demonstrates a history of unequal recombination and diversifying selection within the Leu-rich repeat 2 region, resulting in chimeric gene structures.     

Comparative sequence analysis of the sorghum Rph region and the maize Rp1 resistance gene complex

A 268-kb chromosomal segment containing sorghum (Sorghum bicolor) genes that are orthologous to the maize (Zea mays) Rp1 disease resistance (R) gene complex was sequenced. A region of approximately 27 kb in sorghum was found to contain five Rp1 homologs, but most have structures indicating that they are not functional. In contrast, maize inbred B73 has 15 Rp1 homologs in two nearby clusters of 250 and 300 kb. As at maize Rp1, the cluster of R gene homologs is interrupted by the presence of several genes that appear to have no resistance role, but these genes were different from the ones found within the maize Rp1 complex. More than 200 kb of DNA downstream from the sorghum Rp1-orthologous R gene cluster was sequenced and found to contain many duplicated and/or truncated genes. None of the duplications currently exist as simple tandem events, suggesting that numerous rearrangements were required to generate the current genomic structure. Four truncated genes were observed, including one gene that appears to have both 5' and 3' deletions. The maize Rp1 region is also unusually enriched in truncated genes. Hence, the orthologous maize and sorghum regions share numerous structural features, but all involve events that occurred independently in each species. The data suggest that complex R gene clusters are unusually prone to frequent internal and adjacent chromosomal rearrangements of several types.     

Homologues of the maize rust resistance gene Rp1-D are genetically associated with a major rust resistance QTL in sorghum

As part of a comparative mapping study between sugarcane and sorghum, a sugarcane cDNA clone with homology to the maize Rp1-D rust resistance gene was mapped in sorghum. The cDNA probe hybridised to multiple loci, including one on sorghum linkage group (LG) E in a region where a major rust resistance QTL had been previously mapped. Partial sorghum Rp1-D homologues were isolated from genomic DNA of rust-resistant and -susceptible progeny selected from a sorghum mapping population. Sequencing of the Rp1-D homologues revealed five discrete sequence classes: three from resistant progeny and two from susceptible progeny. PCR primers specific to each sequence class were used to amplify products from the progeny and confirmed that the five sequence classes mapped to the same locus on LG E. Cluster analysis of these sorghum sequences and available sugarcane, maize and sorghum Rp1-D homologue sequences showed that the maize Rp1-D sequence and the partial sugarcane Rp1-D homologue were clustered with one of the sorghum resistant progeny sequence classes, while previously published sorghum Rp1-D homologue sequences clustered with the susceptible progeny sequence classes. Full-length sequence information was obtained for one member of a resistant progeny sequence class ( Rp1-SO) and compared with the maize Rp1-D sequence and a previously identified sorghum Rp1 homologue ( Rph1-2). There was considerable similarity between the two sorghum sequences and less similarity between the sorghum and maize sequences. These results suggest a conservation of function and gene sequence homology at the Rp1 loci of maize and sorghum and provide a basis for convenient PCR-based screening tools for putative rust resistance alleles in sorghum.     

Recombinant Rp1 genes confer necrotic or nonspecific resistance phenotypes

Genes at the Rp1 rust resistance locus of maize confer race-specific resistance to the common rust fungus Puccinia sorghi. Three variant genes with nonspecific effects (HRp1 -Kr1N, -D*21 and -MD*19) were found to be generated by intragenic crossing over within the LRR region. The LRR region of most NBS-LRR encoding genes is quite variable and codes for one of the regions in resistance gene proteins that controls specificity. Sequence comparisons demonstrated that the Rp1-Kr1N recombinant gene was identical to the N-terminus of the rp1-kp2 gene and C-terminus of another gene from its HRp1-K grandparent. The Rp1-D*21 recombinant gene consists of the N-terminus of the rp1-dp2 gene and C-terminus of the Rp1-D gene from the parental haplotype. Similarly, a recombinant gene from the Rp1-MD*19 haplotype has the N-terminus of an rp1 gene from the HRp1-M parent and C-terminus of the rp1-D19 gene from the HRp1-D parent. The recombinant Rp1 -Kr1N, -D*21 and -MD*19 genes activated defense responses in the absence of their AVR proteins triggering HR (hypersensitive response) in the absence of the pathogen. The results indicate that the frequent intragenic recombination events that occur in the Rp1 gene cluster not only recombine the genes into novel haplotypes, but also create genes with nonspecific effects. Some of these may contribute to nonspecific quantitative resistance but others have severe consequences for the fitness of the plant.     

Pronounced intraspecific haplotype divergence at the RPP5 complex disease resistance locus of Arabidopsis

In Arabidopsis ecotype Landsberg erecta (Ler), RPP5 confers resistance to the pathogen Peronospora parasitica. RPP5 is part of a clustered multigene family encoding nucleotide binding-leucine-rich repeat (LRR) proteins. We compared 95 kb of DNA sequence carrying the Ler RPP5 haplotype with the corresponding 90 kb of Arabidopsis ecotype Columbia (Col-0). Relative to the remainder of the genome, the Ler and Col-0 RPP5 haplotypes exhibit remarkable intraspecific polymorphism. The RPP5 gene family probably evolved by extensive recombination between LRRs from an RPP5-like progenitor that carried only eight LRRs. Most members have variable LRR configurations and encode different numbers of LRRs. Although many members carry retroelement insertions or frameshift mutations, codon usage analysis suggests that regions of the genes have been subject to purifying or diversifying selection, indicating that these genes were, or are, functional. The RPP5 haplotypes thus carry dynamic gene clusters with the potential to adapt rapidly to novel pathogen variants by gene duplication and modification of recognition capacity. We propose that the extremely high level of polymorphism at this complex resistance locus is maintained by frequency-dependent selection.     

Translation of some maize small heat shock proteins is initiated from internal in-frame AUGs.

Etiolated maize radicles (inbred Oh43) subjected to a brief heat shock synthesize a family of small heat shock proteins (approximately 18 kD) that is composed of at least 12 members. We previously described the cDNA-derived sequence of three maize shsp mRNAs (cMHSP18-1, cMHSP18-3, and cMHSP18-9). In this report, we demonstrate that the mRNA transcribed in vitro from one of these cDNAs (cMHSP18-9) is responsible for the synthesis of three members of the shsp family, and we suggest that cMHSP18-3 may be responsible for the synthesis of three additional members and cMHSP18-1 for the synthesis of two other members of this family. The fact that these genes do not contain introns, coupled with the observations reported herein, suggest that maize may have established another method of using a single gene to produce a number of different proteins.     

Small auxin upregulated RNA (SAUR) gene family in maize: Identification,evolution, and its phylogenetic comparison with Arabidopsis,rice, and sorghum

Small auxin-up RNAs(SAURs)are the early auxin-responsive genes represented by a large multigene family in plants.Here,we identified 79 SAUR gene family members from maize(Zea mays subsp.mays)by a reiterative database search and manual annotation.Phylogenetic analysis indicated that the SAUR proteins from Arabidopsis,rice,sorghum,and maize had divided into 16 groups.These genes were non-randomly distributed across the maize chromosomes,and segmental duplication and tandem duplication contributed to the expansion of the maize SAUR gene family.Synteny analysis established orthology relationships and functional linkages between SAUR genes in maize and sorghum genomes.We also found that the auxin-responsive elements were conserved in the upstream sequences of maize SAUR members.Selection analyses identified some significant site-specific constraints acted on most SAUR paralogs.Expression profiles based on microarray data have provided insights into the possible functional divergence among members of the SAUR gene family.Quantitative real-time PCR analysis indicated that some of the 10 randomly selected ZmSAUR genes could be induced at least in maize shoot or root tissue tested.The results reveal a comprehensive overview of the maize SAUR gene family and may pave the way for deciphering their function during plant development.     

Microsatellite marker-based genetic diversity assessment among exotic and native maize inbred lines of Bangladesh

Hybrid development is basically dependent on the variability among available genetic resources. Polymorphism among the maize inbreds is essentially needed for maize hybridization. This study aimed at the assessment of diversity among 22 maize inbreds by 18 microsatellite markers. The study identified 187 alleles at 18 SSR loci. The amplified allele frequency per microsatellite locus was 10.4 and the highest allele per locus was 17 in SSR primer pair phi026. SSR primer set p-umc1292, phi074 and phi090 showed the lowest 6 alleles per genotype per locus. The locus phi026 showed the highest degree of gene diversity (0.92), and the locus p-umc1292 had the lowest of gene diversity (0.77) with a mean value of 0.862 among the microsatellites. At each site, the most prevalent allele varied between 0.14 (bnlg371) and 0.36. (p-umc1292). At any given locus, an average of 0.22 out of the 22 selected maize inbred lines had a common major allele. The average value of the polymorphic information content (PIC) was 0.85, within the range of 0.74 at the lowest to 0.92 at the highest. The higher PIC values of phi026 and nc013 established them to be the best markers for maize inbred lines. The UPGMA clustering generated seven distinct groups having 12.5% of similarity coefficient. The results revealed that inbred lines E10, E27, E19, E34, E35, E4, E43, E28, E11, E21, E17, E38, E25, E34, E14, E16, E39 and E3 were more diversified. These lines are promising to be used as parent materials for hybrid maize development in the future.     

Small auxin upregulated RNA （SAUR） gene family in maize： Identification,evolution, and its phylogenetic comparison with Arabidopsis,rice, and sorghum

Small auxin-up RNAs （.SAURs） are the early auxin- responsive genes represented by a large multigene family in plants. Here, we identified 79 SAUR gene family members from maize （Zea mays subsp, mays） by a reiterative database search and manual annotation. Phylogenetic analysis indicated that the SAUR proteins from Arabidopsis, rice, sorghum, and maize had divided into 16 groups. These genes were non-randomly distributed across the maize chromosomes, and segmental duplication and tandem duplication contributed to the expansion of the maize .SAUR gene family. Synteny analysis established ortholos~J relationships and functional linkages between SAUR genes in maize and sorghum genomes. We also found that the auxin-responsive elements were conserved in the upstream sequences of maize SAUR members. Selection analyses identified some significant site-specific constraints acted on most SAUR paralogs. Expression profiles based on microarray data have provided insights into the possible functional divergence among members of the .SAUR gene family. Quantitative real-time PCR analysis indicated that some of the 10 randomly selected ZmSAUR genes could be induced at least in maize shoot or root tissue tested. The results reveal a comprehensive overview of the maize .SAUR gene family and may pave the way for deciphering their function during pJant development.     

The Rp3 disease resistance gene of maize: mapping and characterization of introgressed alleles

The Rp3 locus of maize conditions race-specific resistance to a fungal rust pathogen, Puccinia sorghi. Both morphological and DNA markers were employed to characterize alleles of Rp3 and to accurately position Rp3 on the maize genetic map. DNA marker polymorphisms distinctive to each Rp3 allele were identified, allowing the identification of specific Rp3 alleles in cases where rust races that differentiate particular alleles are not available. In a population of 427 progeny, Rp3 and Rg1 were found to be completely linked, while Lg3 was approximately 3 cM proximal on the long arm of chromosome 3. In this same population, 12 RFLP markers were mapped relative to Rp3; the closest markers were UMC102 (about 1cM distal to Rp1) and NPI114 (1–2 cM proximal). These and additional DNA probes were used to characterize the nature and extent of flanking DNA that was carried along when six different Rp3 alleles were backcrossed into a single background. Depending upon the allele investigated, a minimum of 2–10cM of polymorphic DNA flanking the Rp3 locus was retained through the introgression process. In addition, many of the probes that map near Rp3 were found to detect an additional fragment in the Rp3 region, indicating that portions of this chromosomal segment have been tendemly duplicated. The materials and results generated will permit marker-assisted entry of Rp3 into different maize backgrounds and lay the foundation for the eventual map-based cloning of Rp3.