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Analysis of non-TIR NBS-LRR resistance gene analogs in Musa acuminata Colla: isolation, RFLP marker development, and physical mapping.

Miller RN, Bertioli DJ, Baurens FC, Santos CM, Alves PC, Martins NF, Togawa RC, Souza MT, Pappas GJ - BMC Plant Biol. (2008)

Bottom Line: This is the first large scale analysis of NBS-LRR RGAs in M. acuminata Calcutta 4.Contig sequences were deposited in GenBank and assigned numbers ER935972 - ER936023.RGA sequences and isolated BACs are a valuable resource for R-gene discovery, and in future applications will provide insight into the organization and evolution of NBS-LRR R-genes in the Musa A and B genome.

View Article: PubMed Central - HTML - PubMed

Affiliation: Postgraduate program in Genomic Science and Biotechnology, Universidade Católica de Brasília, SGAN 916, Módulo B, CEP 70,790-160, Brasília, DF, Brazil. rngmiller@gmail.com.

ABSTRACT

Background: Many commercial banana varieties lack sources of resistance to pests and diseases, as a consequence of sterility and narrow genetic background. Fertile wild relatives, by contrast, possess greater variability and represent potential sources of disease resistance genes (R-genes). The largest known family of plant R-genes encode proteins with nucleotide-binding site (NBS) and C-terminal leucine-rich repeat (LRR) domains. Conserved motifs in such genes in diverse plant species offer a means for isolation of candidate genes in banana which may be involved in plant defence.

Results: A computational strategy was developed for unbiased conserved motif discovery in NBS and LRR domains in R-genes and homologues in monocotyledonous plant species. Degenerate PCR primers targeting conserved motifs were tested on the wild cultivar Musa acuminata subsp. burmannicoides, var. Calcutta 4, which is resistant to a number of fungal pathogens and nematodes. One hundred and seventy four resistance gene analogs (RGAs) were amplified and assembled into 52 contiguous sequences. Motifs present were typical of the non-TIR NBS-LRR RGA subfamily. A phylogenetic analysis of deduced amino-acid sequences for 33 RGAs with contiguous open reading frames (ORFs), together with RGAs from Arabidopsis thaliana and Oryza sativa, grouped most Musa RGAs within monocotyledon-specific clades. RFLP-RGA markers were developed, with 12 displaying distinct polymorphisms in parentals and F1 progeny of a diploid M. acuminata mapping population. Eighty eight BAC clones were identified in M. acuminata Calcutta 4, M. acuminata Grande Naine, and M. balbisiana Pisang Klutuk Wulung BAC libraries when hybridized to two RGA probes. Multiple copy RGAs were common within BAC clones, potentially representing variation reservoirs for evolution of new R-gene specificities.

Conclusion: This is the first large scale analysis of NBS-LRR RGAs in M. acuminata Calcutta 4. Contig sequences were deposited in GenBank and assigned numbers ER935972 - ER936023. RGA sequences and isolated BACs are a valuable resource for R-gene discovery, and in future applications will provide insight into the organization and evolution of NBS-LRR R-genes in the Musa A and B genome. The developed RFLP-RGA markers are applicable for genetic map development and marker assisted selection for defined traits such as pest and disease resistance.

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Multiple loci polymorphisms observed in M. acuminata parentals with RGA genetic markers. Polymorphisms were observed in DraI, HindIII, and EcoRV-digested genomic DNA from M. acuminata spp. microcarpa genetic map parentals Borneo and Pisang Lilin, following hybridization of Southern blots with RGA probes MaRGA08 (panel A) and MaRGA37 (panel B).
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Figure 3: Multiple loci polymorphisms observed in M. acuminata parentals with RGA genetic markers. Polymorphisms were observed in DraI, HindIII, and EcoRV-digested genomic DNA from M. acuminata spp. microcarpa genetic map parentals Borneo and Pisang Lilin, following hybridization of Southern blots with RGA probes MaRGA08 (panel A) and MaRGA37 (panel B).

Mentions: From a total of 33 Musa RGAs evaluated as RFLP markers with restricted genomic DNA from mapping population parentals M. acuminata Borneo and Pisang Lilin, 30 displayed single locus or multiple loci polymorphisms on parentals, with at least one restriction enzyme (Table 3). Across the polymorphisms observed, 12 distinct fingerprint types were observed, when using enzymes DraI and HindIII. RGA probes MaRGA04, MaRGA07, MaRGA08, MaRGA12, MaRGA13, MaRGA14, MaRGA16, MaRGA22, MaRGA37, MaRGA41, MaRGA43, and MaRGA46 represented each polymorphism pattern. Figure 3 shows examples of multiple loci polymorphisms observed on Southern blots of restricted parental DNA hybridized with probes MaRGA08 and MaRGA37. Segregation of selected polymorphic bands according to Mendelian ratios in a subset of F1 progeny for this mapping population is depicted in Figure 4.


Analysis of non-TIR NBS-LRR resistance gene analogs in Musa acuminata Colla: isolation, RFLP marker development, and physical mapping.

Miller RN, Bertioli DJ, Baurens FC, Santos CM, Alves PC, Martins NF, Togawa RC, Souza MT, Pappas GJ - BMC Plant Biol. (2008)

Multiple loci polymorphisms observed in M. acuminata parentals with RGA genetic markers. Polymorphisms were observed in DraI, HindIII, and EcoRV-digested genomic DNA from M. acuminata spp. microcarpa genetic map parentals Borneo and Pisang Lilin, following hybridization of Southern blots with RGA probes MaRGA08 (panel A) and MaRGA37 (panel B).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2262081&req=5

Figure 3: Multiple loci polymorphisms observed in M. acuminata parentals with RGA genetic markers. Polymorphisms were observed in DraI, HindIII, and EcoRV-digested genomic DNA from M. acuminata spp. microcarpa genetic map parentals Borneo and Pisang Lilin, following hybridization of Southern blots with RGA probes MaRGA08 (panel A) and MaRGA37 (panel B).
Mentions: From a total of 33 Musa RGAs evaluated as RFLP markers with restricted genomic DNA from mapping population parentals M. acuminata Borneo and Pisang Lilin, 30 displayed single locus or multiple loci polymorphisms on parentals, with at least one restriction enzyme (Table 3). Across the polymorphisms observed, 12 distinct fingerprint types were observed, when using enzymes DraI and HindIII. RGA probes MaRGA04, MaRGA07, MaRGA08, MaRGA12, MaRGA13, MaRGA14, MaRGA16, MaRGA22, MaRGA37, MaRGA41, MaRGA43, and MaRGA46 represented each polymorphism pattern. Figure 3 shows examples of multiple loci polymorphisms observed on Southern blots of restricted parental DNA hybridized with probes MaRGA08 and MaRGA37. Segregation of selected polymorphic bands according to Mendelian ratios in a subset of F1 progeny for this mapping population is depicted in Figure 4.

Bottom Line: This is the first large scale analysis of NBS-LRR RGAs in M. acuminata Calcutta 4.Contig sequences were deposited in GenBank and assigned numbers ER935972 - ER936023.RGA sequences and isolated BACs are a valuable resource for R-gene discovery, and in future applications will provide insight into the organization and evolution of NBS-LRR R-genes in the Musa A and B genome.

View Article: PubMed Central - HTML - PubMed

Affiliation: Postgraduate program in Genomic Science and Biotechnology, Universidade Católica de Brasília, SGAN 916, Módulo B, CEP 70,790-160, Brasília, DF, Brazil. rngmiller@gmail.com.

ABSTRACT

Background: Many commercial banana varieties lack sources of resistance to pests and diseases, as a consequence of sterility and narrow genetic background. Fertile wild relatives, by contrast, possess greater variability and represent potential sources of disease resistance genes (R-genes). The largest known family of plant R-genes encode proteins with nucleotide-binding site (NBS) and C-terminal leucine-rich repeat (LRR) domains. Conserved motifs in such genes in diverse plant species offer a means for isolation of candidate genes in banana which may be involved in plant defence.

Results: A computational strategy was developed for unbiased conserved motif discovery in NBS and LRR domains in R-genes and homologues in monocotyledonous plant species. Degenerate PCR primers targeting conserved motifs were tested on the wild cultivar Musa acuminata subsp. burmannicoides, var. Calcutta 4, which is resistant to a number of fungal pathogens and nematodes. One hundred and seventy four resistance gene analogs (RGAs) were amplified and assembled into 52 contiguous sequences. Motifs present were typical of the non-TIR NBS-LRR RGA subfamily. A phylogenetic analysis of deduced amino-acid sequences for 33 RGAs with contiguous open reading frames (ORFs), together with RGAs from Arabidopsis thaliana and Oryza sativa, grouped most Musa RGAs within monocotyledon-specific clades. RFLP-RGA markers were developed, with 12 displaying distinct polymorphisms in parentals and F1 progeny of a diploid M. acuminata mapping population. Eighty eight BAC clones were identified in M. acuminata Calcutta 4, M. acuminata Grande Naine, and M. balbisiana Pisang Klutuk Wulung BAC libraries when hybridized to two RGA probes. Multiple copy RGAs were common within BAC clones, potentially representing variation reservoirs for evolution of new R-gene specificities.

Conclusion: This is the first large scale analysis of NBS-LRR RGAs in M. acuminata Calcutta 4. Contig sequences were deposited in GenBank and assigned numbers ER935972 - ER936023. RGA sequences and isolated BACs are a valuable resource for R-gene discovery, and in future applications will provide insight into the organization and evolution of NBS-LRR R-genes in the Musa A and B genome. The developed RFLP-RGA markers are applicable for genetic map development and marker assisted selection for defined traits such as pest and disease resistance.

Show MeSH
Related in: MedlinePlus