Cloning and characterization of NBS-LRR class resistance-gene candidate sequences in citrus

• Published in: Theoretical and applied genetics Vol. 101; no. 5-6; pp. 814 - 822
• Main Authors: DENG, Z, HUANG, S, LING, P, CHEN, C, YU, C, WEBER, C. A, MOORE, G. A, GMITTER, F. G
• Format: Journal Article
• Language: English
• Published: Heidelberg Springer 01.10.2000; Berlin Springer Nature B.V
• Subjects: Agronomy. Soil science and plant productions; Binding sites; Biological and medical sciences; Citrus; Citrus fruits; Citrus tristeza virus; Cloning; Comparative analysis; Ctv gene; Cultivars; DNA sequencing; Flowers & plants; Fundamental and applied biological sciences. Psychology; Generalities; Genes; Genetic aspects; Genetics and breeding of economic plants; Nematoda; Nucleotide sequencing; Pest resistance; Physiological aspects; Plant diseases; Plant genetics; Plant immunology; Plant resistance; Poncirus; Signal transduction; Tyr1 gene; Varietal selection. Specialized plant breeding, plant breeding aims; Viruses; United States; United States > US; Genetic marker; Plant pathogen; Citrus fruit; Rutaceae; Homology; Binding site; Pest; Gene; Dicotyledones; Angiospermae; Nematoda; Genetic mapping; Nucleotide sequence; Phylogenetic tree; Chromosome; Citrus; Resistance; Virus; Aminoacid; Helmintha; Nucleotide; Nemathelminthia; Spermatophyta; Invertebrata; Polymorphism
• ISSN: 0040-5752; 1432-2242
• DOI: 10.1007/s001220051548

Numerous disease resistance gene-like DNA sequences were cloned from an intergeneric hybrid of Poncirus and Citrus, using a PCR approach with degenerate primers designed from conserved NBS (nucleotide-binding site) motifs found in a number of plant resistance genes. Most of the cloned genomic sequences could be translated into polypeptides without stop codons, and the sequences contained the characteristic motifs found in the NBS-LRR class of plant disease resistance genes. Pairwise comparisons of these polypeptide sequences indicated that they shared various degrees of amino-acid identity and could be grouped into ten classes (RGC1-RGC10). When the sequences of each class were compared with known resistance-gene sequences, the percentage of amino-acid identity ranged from 18.6% to 48%. To facilitate genetic mapping of these sequences and to assess their potential linkage relationship with disease resistance genes in Poncirus, we developed CAPS markers by designing specific primers based on the cloned DNA sequences and subsequently identifying restriction enzymes that revealed genetic polymorphisms. Three of the amplified DNA fragment markers (designated as 18P33a, Pt9a, and Pt8a) were associated with the citrus tristeza virus resistance gene (Ctv), and one fragment (Pt8a) was associated with the major gene responsible for the citrus nematode resistance (Tyr1); both genes are from Poncirus and of importance to citrus survival and production. These polymorphic fragments were located on two local genetic linkage maps of the chromosome region from Ctv to Tyr1. These results indicate that resistance-gene candidate sequences amplified with the NBS-derived degenerate primers are valuable sources for developing markers in disease resistance-gene tagging, mapping, and cloning.