Full-genome analysis of resistance gene homologues in rice

The availability of the rice genome sequence enabled the global characterization of nucleotide-binding site (NBS)–leucine-rich repeat (LRR) genes, the largest class of plant disease resistance genes. The rice genome carries approximately 500 NBS–LRR genes that are very similar to the non-Toll/interleukin-1 receptor homology region (TIR) class (class 2) genes of Arabidopsis but none that are homologous to the TIR class genes. Over 100 of these genes were predicted to be pseudogenes in the rice cultivar Nipponbare, but some of these are functional in other rice lines. Over 80 other NBS-encoding genes were identified that belonged to four different classes, only two of which are present in dicotyledonous plant sequences present in databases. Map positions of the identified genes show that these genes occur in clusters, many of which included members from distantly related groups. Members of phylogenetic subgroups of the class 2 NBS–LRR genes mapped to as many as ten different chromosomes. The patterns of duplication of the NBS–LRR genes indicate that they were duplicated by many independent genetic events that have occurred continuously through the expansion of the NBS–LRR superfamily and the evolution of the modern rice genome. Genetic events, such as inversions, that inhibit the ability of recently duplicated genes to recombine promote the divergence of their sequences by inhibiting concerted evolution.Electronic Supplementary Material Supplementary material is available for this article at     

Isolation of TIR and nonTIR NBS–LRR resistance gene analogues and identification of molecular markers linked to a powdery mildew resistance locus in chestnut rose (Rosa roxburghii Tratt)

Toll and interleukin-1 receptor (TIR) and nonTIR nucleotide binding site–leucine rich repeat (NBS–LRR) resistance gene analogues (RGAs) were obtained from chestnut rose (Rosa roxburghii Tratt) by two PCR-based amplification strategies (direct amplification and overlap extension amplification) with degenerate primers designed to the conserved P-loop, kinase-2, and Gly-Leu-Pro-Leu (GLPL) motifs within the NBS domain of plant resistance gene (R gene) products. Thirty-four of 65 cloned PCR fragments contained a continuous open reading frame (ORF) and their predicted protein products showed homology to the NBS–LRR class R proteins in the GenBank database. These 34 predicted protein sequences exhibited a wide range (19.5–99.4%) of sequence identity among them and were classified into two distinct groups by phylogenetic analysis. The first group consisted of 23 sequences and seemed to belong to the nonTIR NBS–LRR RGAs, since they contained group specific motifs (RNBS-A-nonTIR motif) that are often present in the coiled-coil domain of the nonTIR NBS–LRR class R genes. The second group comprised 11 sequences that contained motifs found in the TIR domain of TIR NBS–LRR class R genes. Restriction fragment length polymorphic (RFLP) markers were developed from some of the RGAs and used for mapping powdery mildew resistance genes in chestnut rose. Three markers, RGA22C, RGA4A, and RGA7B, were identified to be linked to a resistance gene locus, designated CRPM1 for chestnut rose powdery mildew resistance 1, which accounted for 72% of the variation in powdery mildew resistance phenotype in an F1 segregating population. To our knowledge, this is the first report on isolation, phylogenetic analysis and potential utilization as genetic markers of RGAs in chestnut rose.     

Motifs specific for the ADR1 NBS–LRR protein family in <Emphasis Type="Italic">Arabidopsis</Emphasis> are conserved among NBS–LRR sequences from both dicotyledonous and monocotyledonous plants

The activated disease resistance (ADR) 1 gene encodes a protein that possesses an N-terminal coiled-coil (CC) motif, nucleotide-binding site (NBS) and C-terminal leucine-rich repeat (LRR) domains. ADR1 belongs to a small, atypical Arabidopsis thaliana sub-class containing four CC–NBS–LRR genes. The NBS region of most NBS–LRR proteins possesses numerous conserved motifs. In contrast, the LRR domain, which is subject to positive selection, is highly variable. Surprisingly, sequence analysis revealed that the LRR domain of the ADR1 sub-class was more conserved than the NBS region. Sequence analysis identified two novel conserved motifs, termed TVS and PKAE, specific for this CC–NBS–LRR sub-class. The TVS motif is adjacent to the P-loop, whereas the PKAE motif corresponded to the inter-domain region termed the NBS–LRR linker, which was conserved within the different CC–NBS–LRR classes but varied among classes. These ADR1-specific motifs were employed to identify putative ADR1 homologs in phylogenetically distant and agronomically important plant species. Putative ADR1 homologs were identified in 11 species including rice and in 3 further Poaceae species. The ADR1 sub-class of CC–NBS–LRR proteins is therefore conserved in both monocotyledonous and dicotyledonous plant species.     

Isolation of a full-length CC–NBS–LRR resistance gene analog candidate from sugar pine showing low nucleotide diversity

The nucleotide-binding-site and leucine-rich-repeat (NBS–LRR) class of R proteins is abundant and widely distributed in plants. By using degenerate primers designed on the NBS domain in lettuce, we amplified sequences in sugar pine that shared sequence identity with many of the NBS–LRR class resistance genes catalogued in GenBank. The polymerase chain reaction products were used to probe a cDNA library constructed from needle tissue of sugar pine seedlings. A full-length cDNA was obtained that demonstrated high predicted amino acid sequence similarity to the coiled coil (CC)–NBS–LRR subclass of NBS–LRR resistance proteins in GenBank. Sequence analyses of this gene in megagametophytes from two sugar pine trees segregating for the hypersensitive response to white pine blister rust revealed zero nucleotide variation. Moreover, there was no variation found in 24 unrelated sugar pine trees except for three single-nucleotide polymorphisms located in the 3′ untranslated region. Compared to other genes sequenced in Pinaceae, such a low level of sequence variation in unrelated individuals is unusual. Although, numerous studies have reported that plant R genes are under diversifying selection for specificity to evolving pathogens, the resistance gene analog discussed here appears to be under intense purifying selection.An erratum to this article can be found at     

Receptor-like genes in the major resistance locus of lettuce are subject to divergent selection.

Disease resistance genes in plants are often found in complex multigene families. The largest known cluster of disease resistance specificities in lettuce contains the RGC2 family of genes. We compared the sequences of nine full-length genomic copies of RGC2 representing the diversity in the cluster to determine the structure of genes within this family and to examine the evolution of its members. The transcribed regions range from at least 7.0 to 13.1 kb, and the cDNAs contain deduced open reading frames of approximately 5. 5 kb. The predicted RGC2 proteins contain a nucleotide binding site and irregular leucine-rich repeats (LRRs) that are characteristic of resistance genes cloned from other species. Unique features of the RGC2 gene products include a bipartite LRR region with >40 repeats. At least eight members of this family are transcribed. The level of sequence diversity between family members varied in different regions of the gene. The ratio of nonsynonymous (Ka) to synonymous (Ks) nucleotide substitutions was lowest in the region encoding the nucleotide binding site, which is the presumed effector domain of the protein. The LRR-encoding region showed an alternating pattern of conservation and hypervariability. This alternating pattern of variation was also found in all comparisons within families of resistance genes cloned from other species. The Ka /Ks ratios indicate that diversifying selection has resulted in increased variation at these codons. The patterns of variation support the predicted structure of LRR regions with solvent-exposed hypervariable residues that are potentially involved in binding pathogen-derived ligands.     

Multiple genetic processes result in heterogeneous rates of evolution within the major cluster disease resistance genes in lettuce

Resistance Gene Candidate2 (RGC2) genes belong to a large, highly duplicated family of nucleotide binding site-leucine rich repeat (NBS-LRR) encoding disease resistance genes located at a single locus in lettuce (Lactuca sativa). To investigate the genetic events occurring during the evolution of this locus, approximately 1.5- to 2-kb 3' fragments of 126 RGC2 genes from seven genotypes were sequenced from three species of Lactuca, and 107 additional RGC2 sequences were obtained from 40 wild accessions of Lactuca spp. The copy number of RGC2 genes varied from 12 to 32 per genome in the seven genotypes studied extensively. LRR number varied from 40 to 47; most of this variation had resulted from 13 events duplicating two to five LRRs because of unequal crossing-over within or between RGC2 genes at one of two recombination hot spots. Two types of RGC2 genes (Type I and Type II) were initially distinguished based on the pattern of sequence identities between their 3' regions. The existence of two types of RGC2 genes was further supported by intron similarities, the frequency of sequence exchange, and their prevalence in natural populations. Type I genes are extensive chimeras caused by frequent sequence exchanges. Frequent sequence exchanges between Type I genes homogenized intron sequences, but not coding sequences, and obscured allelic/orthologous relationships. Sequencing of Type I genes from additional wild accessions confirmed the high frequency of sequence exchange and the presence of numerous chimeric RGC2 genes in nature. Unlike Type I genes, Type II genes exhibited infrequent sequence exchange between paralogous sequences. Type II genes from different genotype/species within the genus Lactuca showed obvious allelic/orthologous relationships. Trans-specific polymorphism was observed for different groups of orthologs, suggesting balancing selection. Unequal crossover, insertion/deletion, and point mutation events were distributed unequally through the gene. Different evolutionary forces have impacted different parts of the LRR.     

Mapping and analysis of a novel candidate Fusarium wilt resistance gene FOC1 in Brassica oleracea

Background

Cabbage Fusarium wilt is a major disease worldwide that can cause severe yield loss in cabbage (Brassica olerecea). Although markers linked to the resistance gene FOC1 have been identified, no candidate gene for it has been determined so far. In this study, we report the fine mapping and analysis of a candidate gene for FOC1 using a double haploid (DH) population with 160 lines and a F₂ population of 4000 individuals derived from the same parental lines.

Results

We confirmed that the resistance to Fusarium wilt was controlled by a single dominant gene based on the resistance segregation ratio of the two populations. Using InDel primers designed from whole-genome re-sequencing data for the two parental lines (the resistant inbred-line 99–77 and the highly susceptible line 99–91) and the DH population, we mapped the resistance gene to a 382-kb genomic region on chromosome C06. Using the F₂ population, we narrowed the region to an 84-kb interval that harbored ten genes, including four probable resistance genes (R genes): Bol037156, Bol037157, Bol037158 and Bol037161 according to the gene annotations from BRAD, the genomic database for B. oleracea. After correcting the model of the these genes, we re-predicted two R genes in the target region: re-Bol037156 and re-Bol0371578. The latter was excluded after we compared the two genes’ sequences between ten resistant materials and ten susceptible materials. For re-Bol037156, we found high identity among the sequences of the resistant lines, while among the susceptible lines, there were two types of InDels (a 1-bp insertion and a 10-bp deletion), each of which caused a frameshift and terminating mutation in the cDNA sequences. Further sequence analysis of the two InDel loci from 80 lines (40 resistant and 40 susceptible) also showed that all 40 R lines had no InDel mutation while 39 out of 40 S lines matched the two types of loci. Thus re-Bol037156 was identified as a likely candidate gene for FOC1 in cabbage.

Conclusions

This work may lay the foundation for marker-assisted selection as well as for further function analysis of the FOC1 gene.     

Spatial patterns of diversity at the putative recognition domain of resistance gene candidates in wild bean populations

Leucine Rich Repeats (LRR) domains have been identified on most known plant resistance genes and appear to be involved in the specific recognition of pathogen strains. Here we explore the processes which may drive the evolution of this putative recognition domain. We developed AFLP markers specifically situated in the LRR domain of members of the PRLJ1 complex Resistance Gene Candidate (RGC) family identified in common bean (Phaseolus vulgaris). Diversity for these markers was assessed in ten wild populations of P. vulgaris and compared to locally co-occurring pathogen populations of Colletotrichum lindemuthianum. Nine PRLJ1 LRR specific markers were obtained. Marker sequences revealed that RGC diversity at PRLJ1 is similar to that at other complex R-loci. Wild bean populations showed contrasting levels of PRLJ1 LRR diversity and were all significantly differentiated. We could not detect an effect of local C. lindemuthianum population diversity on the spatial distribution of P. vulgaris PRLJ1 diversity. However, host populations have been previously assessed for neutral (RAPD) markers and for resistance phenotypes to six strains of C. lindemuthianum isolated from cultivated bean fields. A comparative analysis of PRLJ1 LRR diversity and host diversity for resistance phenotypes indicated that evolutionary processes related to the antagonistic C. lindemuthianum/P. vulgaris interaction are likely to have shaped molecular diversity of the putative recognition domains of the PRLJ1 RGC family members.     

Analyses of phylogeny, evolution, conserved sequences and genome-wide expression of the ICK/KRP family of plant CDK inhibitors

Background and Aims

The cell cycle is controlled by cyclin-dependent kinases (CDKs), and CDK inhibitors are major regulators of their activities. The ICK/KRP family of CDK inhibitors has been reported in several plants, with seven members in arabidopsis; however, the phylogenetic relationship among members in different species is unknown. Also, there is a need to understand how these genes and proteins are regulated. Furthermore, little information is available on the functional differences among ICK/KRP family members.

Methods

We searched publicly available databases and identified over 120 unique ICK/KRP protein sequences from more than 60 plant species. Phylogenetic analysis was performed using 101 full-length sequences from 40 species and intron–exon organization of ICK/KRP genes in model species. Conserved sequences and motifs were analysed using ICK/KRP protein sequences from arabidopsis (Arabidopsis thaliana), rice (Orysa sativa) and poplar (Populus trichocarpa). In addition, gene expression was examined using microarray data from arabidopsis, rice and poplar, and further analysed by RT-PCR for arabidopsis.

Key Results and Conclusions

Phylogenetic analysis showed that plant ICK/KRP proteins can be grouped into three major classes. Whereas the C-class contains sequences from dicotyledons, monocotyledons and gymnosperms, the A- and B-classes contain only sequences from dicotyledons or monocotyledons, respectively, suggesting that the A- and B-classes might have evolved from the C-class. This classification is also supported by exon–intron organization. Genes in the A- and B- classes have four exons, whereas genes in the C-class have only three exons. Analysis of sequences from arabidopsis, rice and poplar identified conserved sequence motifs, some of which had not been described previously, and putative functional sites. The presence of conserved motifs in different family members is consistent with the classification. In addition, gene expression analysis showed preferential expression of ICK/KRP genes in certain tissues. A model has been proposed for the evolution of this gene family in plants.     

Genetic diversity of NBS–LRR class disease-resistance gene analogs in cultivated and wild eggplants

The genes encoding the nucleotide-binding site (NBS) and leucine-rich repeat (LRR) motifs constitute a large gene family in plants and have attracted much interest, because most of the plant disease-resistance genes that have been cloned are from this gene family. In this study, degenerate oligonucleotide primers, designed on the basis of conserved regions of the NBS domains from known plant resistance genes, were used to isolate resistance gene analogs (RGAs) from cultivated and wild eggplants, i.e., S. melongena, S. aethiopicum gr. Gilo, S. linnaeanum, S. integrifolium, S. sisymbriifolium, and S. khasianum. Sequence analysis indicated that the cloned eggplant RGAs belong to the non-TIR–NBS–LRR type, which are very similar to the R genes or the RGAs identified in other plant species, especially Solanaceae plants, suggesting the existence of common ancestors. Wide genetic diversity of eggplant RGAs was observed both in interspecific and intraspecific sequences, and eight distinct families of eggplant RGAs were identified. Further studies revealed a high average ratio of synonymous to non-synonymous substitution and a low level of recombination. These results suggest that NBS-encoding sequences of RGAs in cultivated and wild eggplants are subject to gradual accumulation of mutations leading to purifying selection. This is the first report of NBS–LRR class RGAs in eggplants.     

The utility of PacBio circular consensus sequencing for characterizing complex gene families in non-model organisms

Background

Molecular characterization of highly diverse gene families can be time consuming, expensive, and difficult, especially when considering the potential for relatively large numbers of paralogs and/or pseudogenes. Here we investigate the utility of Pacific Biosciences single molecule real-time (SMRT) circular consensus sequencing (CCS) as an alternative to traditional cloning and Sanger sequencing PCR amplicons for gene family characterization. We target vomeronasal gene receptors, one of the most diverse gene families in mammals, with the goal of better understanding intra-specific V1R diversity of the gray mouse lemur (Microcebus murinus). Our study compares intragenomic variation for two V1R subfamilies found in the mouse lemur. Specifically, we compare gene copy variation within and between two individuals of M. murinus as characterized by different methods for nucleotide sequencing. By including the same individual animal from which the M. murinus draft genome was derived, we are able to cross-validate gene copy estimates from Sanger sequencing versus CCS methods.

Results

We generated 34,088 high quality circular consensus sequences of two diverse V1R subfamilies (here referred to as V1RI and V1RIX) from two individuals of Microcebus murinus. Using a minimum threshold of 7× coverage, we recovered approximately 90% of V1RI sequences previously identified in the draft M. murinus genome (59% being identical at all nucleotide positions). When low coverage sequences were considered (i.e. < 7× coverage) 100% of V1RI sequences identified in the draft genome were recovered. At least 13 putatively novel V1R loci were also identified using CCS technology.

Conclusions

Recent upgrades to the Pacific Biosciences RS instrument have improved the CCS technology and offer an alternative to traditional sequencing approaches. Our results suggest that the Microcebus murinus V1R repertoire has been underestimated in the draft genome. In addition to providing an improved understanding of V1R diversity in the mouse lemur, this study demonstrates the utility of CCS technology for characterizing complex regions of the genome. We anticipate that long-read sequencing technologies such as PacBio SMRT will allow for the assembly of multigene family clusters and serve to more accurately characterize patterns of gene copy variation in large gene families, thus revealing novel micro-evolutionary patterns within non-model organisms.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-720) contains supplementary material, which is available to authorized users.     

Differential spreading of HinfI satellite DNA variants during radiation in Centaureinae

Background and Aims

Subtribe Centaureinae appears to be an excellent model group in which to analyse satellite DNA and assess the influence that the biology and/or the evolution of different lineages have had on the evolution of this class of repetitive DNA. Phylogenetic analyses of Centaureinae support two main phases of radiation, leading to two major groups of genera of different ages. Furthermore, different modes of evolution are observed in different lineages, reflected by morphology and DNA sequences.

Methods

The sequences of 502 repeat units of the HinfI satellite DNA family from 38 species belonging to ten genera of Centaureinae were isolated and compared. A phylogenetic reconstruction was carried out by maximum likelihood and Bayesian inference.

Key Results

Up to eight different HinfI subfamilies were found, based on the presence of a set of diagnostic positions given by a specific mutation shared by all the sequences of one group. Subfamilies V–VIII were mostly found in older genera (first phase of radiation in the subtribe, late Oligocene–Miocene), although some copies of these types of repeats were also found in some species of the derived genera. Subfamilies I–IV spread mostly in species of the derived clade (second phase of radiation, Pliocene to Pleistocene), although repeats of these subfamilies exist in older species. Phylogenetic trees did not group the repeats by taxonomic affinity, but sequences were grouped by subfamily provenance. Concerted evolution was observed in HinfI subfamilies spread in older genera, whereas no genetic differentiation was found between species, and several subfamilies even coexist within the same species, in recently radiated groups or in groups with a history of recurrent hybridization of lineages.

Conclusions

The results suggest that the eight HinfI subfamilies were present in the common ancestor of Centaureinae and that each spread differentially in different genera during the two main phases of radiation following the library model of satellite DNA evolution. Additionally, differential speciation pathways gave rise to differential patterns of sequence evolution in different lineages. Thus, the evolutionary history of each group of Centaureinae is reflected in HinfI satellite DNA evolution. The data reinforce the value of satellite DNA sequences as markers of evolutionary processes.     

The major resistance gene cluster in lettuce is highly duplicated and spans several megabases.

At least 10 Dm genes conferring resistance to the oomycete downy mildew fungus Bremia lactucae map to the major resistance cluster in lettuce. We investigated the structure of this cluster in the lettuce cultivar Diana, which contains Dm3. A deletion breakpoint map of the chromosomal region flanking Dm3 was saturated with a variety of molecular markers. Several of these markers are components of a family of resistance gene candidates (RGC2) that encode a nucleotide binding site and a leucine-rich repeat region. These motifs are characteristic of plant disease resistance genes. Bacterial artificial chromosome clones were identified by using duplicated restriction fragment length polymorphism markers from the region, including the nucleotide binding site-encoding region of RGC2. Twenty-two distinct members of the RGC2 family were characterized from the bacterial artificial chromosomes; at least two additional family members exist. The RGC2 family is highly divergent; the nucleotide identity was as low as 53% between the most distantly related copies. These RGC2 genes span at least 3.5 Mb. Eighteen members were mapped on the deletion breakpoint map. A comparison between the phylogenetic and physical relationships of these sequences demonstrated that closely related copies are physically separated from one another and indicated that complex rearrangements have shaped this region. Analysis of low-copy genomic sequences detected no genes, including RGC2, in the Dm3 region, other than sequences related to retrotransposons and transposable elements. The related but divergent family of RGC2 genes may act as a resource for the generation of new resistance phenotypes through infrequent recombination or unequal crossing over.     

Comparative molecular cytogenetic analyses of a major tandemly repeated DNA family and retrotransposon sequences in cultivated jute Corchorus species (Malvaceae)

Background and Aims

The cultivated jute species Corchorus olitorius and Corchorus capsularis are important fibre crops. The analysis of repetitive DNA sequences, comprising a major part of plant genomes, has not been carried out in jute but is useful to investigate the long-range organization of chromosomes. The aim of this study was the identification of repetitive DNA sequences to facilitate comparative molecular and cytogenetic studies of two jute cultivars and to develop a fluorescent in situ hybridization (FISH) karyotype for chromosome identification.

Methods

A plasmid library was generated from C. olitorius and C. capsularis with genomic restriction fragments of 100–500 bp, which was complemented by targeted cloning of satellite DNA by PCR. The diversity of the repetitive DNA families was analysed comparatively. The genomic abundance and chromosomal localization of different repeat classes were investigated by Southern analysis and FISH, respectively. The cytosine methylation of satellite arrays was studied by immunolabelling.

Key Results

Major satellite repeats and retrotransposons have been identified from C. olitorius and C. capsularis. The satellite family CoSat I forms two undermethylated species-specific subfamilies, while the long terminal repeat (LTR) retrotransposons CoRetro I and CoRetro II show similarity to the Metaviridea of plant retroelements. FISH karyotypes were developed by multicolour FISH using these repetitive DNA sequences in combination with 5S and 18S–5·8S–25S rRNA genes which enable the unequivocal chromosome discrimination in both jute species.

Conclusions

The analysis of the structure and diversity of the repeated DNA is crucial for genome sequence annotation. The reference karyotypes will be useful for breeding of jute and provide the basis for karyotyping homeologous chromosomes of wild jute species to reveal the genetic and evolutionary relationship between cultivated and wild Corchorus species.     

Reproduction of Meloidogyne spp. on Resistant Peanut Genotypes from Three Breeding Programs

Three described species of root-knot nematode parasitize peanut (Arachis hypogaea): Meloidogyne arenaria race 1 (Ma), M. hapla (Mh), and M. javanica (Mj). Peanut cultivars with broad resistance to Meloidogyne spp. will be useful regardless of the species present in the field. The objective of this study was to determine whether peanut genotypes with resistance to M. arenaria originating from three different breeding programs were also resistant to M. hapla and M. javanica. The experiment used a factorial arrangement (completely randomized) with peanut genotype and nematode population as the factors. The five peanut genotypes were ''COAN'' and AT 0812 (highly resistant to Ma), C209-6-13 (moderately resistant to Ma), and ''Southern Runner'' and ''Georgia Green'' (susceptible to Ma). The four nematode populations were two isolates of Ma (Gibbs and Gop) and one isolate each of Mh and Mj. On COAN or AT 0812, both Ma and Mj produced <10% of the eggs produced on Georgia Green. On the peanut genotype C209-6-13, Ma and Mj produced about 50% of the eggs produced on Georgia Green. None of the resistant genotypes exhibited a high level of resistance to Mh. The lack of resistance to Mh in any cultivars or advanced germplasm is a concern because the identity of a Meloidogyne sp. in a particular peanut field is generally not known. Breeding efforts should focus on moving genes for resistance to M. hapla into advanced peanut germplasm, and combining genes for resistance to the major Meloidogyne spp. in a single cultivar.