Nucleotide-binding site (NBS) disease resistance genes play an important role in defending plants from a variety of pathogens and insect pests. Many R-genes have been identified in various plant species. However, little is known about the NBS-encoding genes in Brachypodium distachyon. In this study, using computational analysis of the B. distachyon genome, we identified 126 regular NBS-encoding genes and characterized them on the bases of structural diversity, conserved protein motifs, chromosomal locations, gene duplications, promoter region, and phylogenetic relationships. EST hits and full-length cDNA sequences (from Brachypodium database) of 126 R-like candidates supported their existence. Based on the occurrence of conserved protein motifs such as coiled-coil (CC), NBS, leucine-rich repeat (LRR), these regular NBS-LRR genes were classified into four subgroups: CC-NBS-LRR, NBS-LRR, CC-NBS, and X-NBS. Further expression analysis of the regular NBS-encoding genes in Brachypodium database revealed that these genes are expressed in a wide range of libraries, including those constructed from various developmental stages, tissue types, and drought challenged or nonchallenged tissue.

BACKGROUND Genetically anchored physical maps of large eukaryotic genomes have proven useful both for their intrinsic merit and as an adjunct to genome sequencing. Cultivated tetraploid cottons, Gossypium hirsutum and G. barbadense, share a common ancestor formed by a merger of the A and D genomes about 1-2 million years ago. Toward the long-term goal of characterizing the spectrum of diversity among cotton genomes, the worldwide cotton community has prioritized the D genome progenitor Gossypium raimondii for complete sequencing. RESULTS A whole genome physical map of G. raimondii, the putative D genome ancestral species of tetraploid cottons was assembled, integrating genetically-anchored overgo hybridization probes, agarose based fingerprints and 'high information content fingerprinting'(HICF). A total of 13,662 BAC-end sequences and 2,828 DNA probes were used in genetically anchoring 1585 contigs to a cotton consensus genetic map, and 370 and 438 contigs, respectively to Arabidopsis thaliana (AT) and Vitis vinifera (VV) whole genome sequences. CONCLUSION Several lines of evidence suggest that the G. raimondii genome is comprised of two qualitatively different components. Much of the gene rich component is aligned to the Arabidopsis and Vitis vinifera genomes and shows promise for utilizing translational genomic approaches in understanding this important genome and its resident genes. The integrated genetic-physical map is of value both in assembling and validating a planned reference sequence.

Genetic mapping studies have suggested that diploid cotton (Gossypium) might be an ancient polyploid. However, further evidence is lacking due to the complexity of the genome and the lack of sequence resources. Here, we used the grape (Vitis vinifera) genome as an out-group in two different approaches to further explore evidence regarding ancient genome duplication (WGD) event(s) in the diploid Gossypium lineage and its (their) effects: a genome-level alignment analysis and a local-level sequence component analysis. Both studies suggest that at least one round of genome duplication occurred in the Gossypium lineage. Also, gene densities in corresponding regions from Gossypium raimondii, V. vinifera, Arabidopsis thaliana and Carica papaya genomes are similar, despite the huge difference in their genome sizes and the different number of WGDs each genome has experienced. These observations fit the model that differences in plant genome sizes are largely explained by transposon insertions into heterochromatic regions.

Tomato (Solanum lycopersicum) is a major crop plant and a model system for fruit development. Solanum is one of the largest angiosperm genera and includes annual and perennial plants from diverse habitats. Here we present a high-quality genome sequence of domesticated tomato, a draft sequence of its closest wild relative, Solanum pimpinellifolium, and compare them to each other and to the potato genome (Solanum tuberosum). The two tomato genomes show only 0.6% nucleotide divergence and signs of recent admixture, but show more than 8% divergence from potato, with nine large and several smaller inversions. In contrast to Arabidopsis, but similar to soybean, tomato and potato small RNAs map predominantly to gene-rich chromosomal regions, including gene promoters. The Solanum lineage has experienced two consecutive genome triplications: one that is ancient and shared with rosids, and a more recent one. These triplications set the stage for the neofunctionalization of genes controlling fruit characteristics, such as colour and fleshiness.

MCScan is an algorithm able to scan multiple genomes or subgenomes in order to identify putative homologous chromosomal regions, and align these regions using genes as anchors. The MCScanX toolkit implements an adjusted MCScan algorithm for detection of synteny and collinearity that extends the original software by incorporating 14 utility programs for visualization of results and additional downstream analyses. Applications of MCScanX to several sequenced plant genomes and gene families are shown as examples. MCScanX can be used to effectively analyze chromosome structural changes, and reveal the history of gene family expansions that might contribute to the adaptation of lineages and taxa. An integrated view of various modes of gene duplication can supplement the traditional gene tree analysis in specific families. The source code and documentation of MCScanX are freely available at http://chibba.pgml.uga.edu/mcscan2/.

We report the annotation and analysis of the draft genome sequence of Brassica rapa accession Chiifu-401-42, a Chinese cabbage. We modeled 41,174 protein coding genes in the B. rapa genome, which has undergone genome triplication. We used Arabidopsis thaliana as an outgroup for investigating the consequences of genome triplication, such as structural and functional evolution. The extent of gene loss (fractionation) among triplicated genome segments varies, with one of the three copies consistently retaining a disproportionately large fraction of the genes expected to have been present in its ancestor. Variation in the number of members of gene families present in the genome may contribute to the remarkable morphological plasticity of Brassica species. The B. rapa genome sequence provides an important resource for studying the evolution of polyploid genomes and underpins the genetic improvement of Brassica oil and vegetable crops.

Genome duplication (GD) has permanently shaped the architecture and function of many higher eukaryotic genomes. The angiosperms (flowering plants) are outstanding models in which to elucidate consequences of GD for higher eukaryotes, owing to their propensity for chromosomal duplication or even triplication in a few cases. Duplicated genome structures often require both intra- and inter-genome alignments to unravel their evolutionary history, also providing the means to deduce both obvious and otherwise-cryptic orthology, paralogy and other relationships among genes. The burgeoning sets of angiosperm genome sequences provide the foundation for a host of investigations into the functional and evolutionary consequences of gene and GD. To provide genome alignments from a single resource based on uniform standards that have been validated by empirical studies, we built the Plant Genome Duplication Database (PGDD; freely available at http://chibba.agtec.uga.edu/duplication/), a web service providing synteny information in terms of colinearity between chromosomes. At present, PGDD contains data for 26 plants including bryophytes and chlorophyta, as well as angiosperms with draft genome sequences. In addition to the inclusion of new genomes as they become available, we are preparing new functions to enhance PGDD.

We present SSR-based genetic maps from a cross between Miscanthus sacchariflorus Robustus and M. sinensis, the progenitors of the promising cellulosic biofuel feedstock Miscanthus × giganteus. cDNA-derived SSR markers were mapped by the two-way pseudo-testcross model due to the high heterozygosity of each parental species. A total of 261 loci were mapped in M. sacchariflorus, spanning 40 linkage groups and 1,998.8 cM, covering an estimated 72.7% of the genome. For M. sinensis, a total of 303 loci were mapped, forming 23 linkage groups and 2,238.3 cM, covering 84.9% of the genome. The use of cDNA-derived SSR loci permitted alignment of the Miscanthus linkage groups to the sorghum chromosomes, revealing a whole genome duplication affecting the Miscanthus lineage after the divergence of subtribes Sorghinae and Saccharinae, as well as traces of the pan-cereal whole genome duplication. While the present maps provide for many early research needs in this emerging crop, additional markers are also needed to improve map density and to further characterize the structural changes of the Miscanthus genome since its divergence from sorghum and Saccharum.

BACKGROUND: Both single gene and whole genome duplications (WGD) have recurred in angiosperm evolution. However, the evolutionary effects of different modes of gene duplication, especially regarding their contributions to genetic novelty or redundancy, have been inadequately explored. RESULTS: In Arabidopsis thaliana and Oryza sativa (rice), species that deeply sample botanical diversity and for which expression data are available from a wide range of tissues and physiological conditions, we have compared expression divergence between genes duplicated by six different mechanisms (WGD, tandem, proximal, DNA based transposed, retrotransposed and dispersed), and between positional orthologs. Both neo-functionalization and genetic redundancy appear to contribute to retention of duplicate genes. Genes resulting from WGD and tandem duplications diverge slowest in both coding sequences and gene expression, and contribute most to genetic redundancy, while other duplication modes contribute more to evolutionary novelty. WGD duplicates may more frequently be retained due to dosage amplification, while inferred transposon mediated gene duplications tend to reduce gene expression levels. The extent of expression divergence between duplicates is discernibly related to duplication modes, different WGD events, amino acid divergence, and putatively neutral divergence (time), but the contribution of each factor is heterogeneous among duplication modes. Gene loss may retard inter-species expression divergence. Members of different gene families may have non-random patterns of origin that are similar in Arabidopsis and rice, suggesting the action of pan-taxon principles of molecular evolution. CONCLUSION: Gene duplication modes differ in contribution to genetic novelty and redundancy, but show some parallels in taxa separated by hundreds of millions of years of evolution.

BACKGROUND Evolution of the Brassica species has been recursively affected by polyploidy events, and comparison to their relative, Arabidopsis thaliana, provides means to explore their genomic complexity. RESULTS A genome-wide physical map of a rapid-cycling strain of B. oleracea was constructed by integrating high-information-content fingerprinting (HICF) of Bacterial Artificial Chromosome (BAC) clones with hybridization to sequence-tagged probes. Using 2907 contigs of two or more BACs, we performed several lines of comparative genomic analysis. Interspecific DNA synteny is much better preserved in euchromatin than heterochromatin, showing the qualitative difference in evolution of these respective genomic domains. About 67% of contigs can be aligned to the Arabidopsis genome, with 96.5% corresponding to euchromatic regions, and 3.5%(shown to contain repetitive sequences) to pericentromeric regions. Overgo probe hybridization data showed that contigs aligned to Arabidopsis euchromatin contain ~80% of low-copy-number genes, while genes with high copy number are much more frequently associated with pericentromeric regions. We identified 39 interchromosomal breakpoints during the diversification of B. oleracea and Arabidopsis thaliana, a relatively high level of genomic change since their divergence. Comparison of the B. oleracea physical map with Arabidopsis and other available eudicot genomes showed appreciable 'shadowing' produced by more ancient polyploidies, resulting in a web of relatedness among contigs which increased genomic complexity. CONCLUSIONS A high-resolution genetically-anchored physical map sheds light on Brassica genome organization and advances positional cloning of specific genes, and may help to validate genome sequence assembly and alignment to chromosomes.All the physical mapping data is freely shared at a WebFPC site (http://lulu.pgml.uga.edu/fpc/WebAGCoL/brassica/WebFPC/; Temporarily password-protected: account: pgml; password: 123qwe123.

Whole genome duplication ~70 million years ago provided raw material for Poaceae (grass) diversification. Comparison of rice (Oryza sativa), sorghum (Sorghum bicolor), maize (Zea mays), and Brachypodium distachyon genomes revealed that one paleo-duplicated chromosome pair has experienced very different evolution than all the others. For tens of millions of years, the two chromosomes have experienced illegitimate recombination that has been temporally restricted in a stepwise manner, producing structural stratification in the chromosomes. These strata formed independently in different grass lineages, with their similarities (low sequence divergence between paleo-duplicated genes) preserved in parallel for millions of years since the divergence of these lineages. The pericentromeric region of this homeologous chromosome pair accounts for two-thirds of the gene content differences between the modern chromosomes. Both intriguing and perplexing is a distal chromosomal region with the greatest DNA similarity between surviving duplicated genes but also with the highest concentration of lineage-specific gene pairs found anywhere in these genomes and with a significantly elevated gene evolutionary rate. Intragenomic similarity near this chromosomal terminus may be important in hom(e)ologous chromosome pairing. Chromosome structural stratification, together with enrichment of autoimmune response-related (nucleotide binding site-leucine-rich repeat) genes and accelerated DNA rearrangement and gene loss, confer a striking resemblance of this grass chromosome pair to the sex chromosomes of other taxa.

A meta-analysis was performed to understand the role of zinc finger domains in proteins of resistance (R) genes cloned from different crops. We analyzed protein sequences of seventy R genes of various crops in which twenty six proteins were found to have zinc finger domains along with nucleotide binding sites - leucine rice repeats (NBS-LRR) domains. We identified thirty four zinc finger domains in the R proteins of nine crops and were grouped into 19 types of zinc fingers. The size of individual zinc finger domain within the R genes varied from 11 to 84 amino acids, whereas the size of proteins containing these domains varied from 263 to 1305 amino acids. The biophysical analysis revealed that molecular weight of Pi54 zinc finger was lowest whereas the highest one was found in rice Pib zinc finger named as Transposes Transcription Factor (TTF). The instability (R(2) = 0.95) and the aliphatic (R(2) = 0.94) indices profile of zinc finger domains follows the polynomial distribution pattern. The pairwise identity analysis showed that the Lin11, Isl-1 & Mec-3 (LIM) zinc finger domain of rice blast resistance protein pi21 have 12.3% similarity with the nuclear transcription factor, X-box binding-like 1 (NFX) type zinc finger domain of Pi54 protein. For the first time, we reported that Pi54 (Pi-k(h)-Tetep), a rice blast resistance (R) protein have a small zinc finger domain of NFX type located on the C-terminal in between NBS and LRR domains of the R-protein. Compositional analysis depicted by the helical wheel diagram revealed the presence of a hydrophobic region within this domain which might help in exposing the LRR region for a possible R-Avr interaction. This domain is unique among all other cloned plant disease resistance genes and might play an important role in broad-spectrum nature of rice blast resistance gene Pi54.

ABSTRACT: BACKGROUND: R genes are a key component of genetic interactions between plant and biotrophic bacteria and they regulate resistance against bacterial invasion. The most common R proteins contain a nucleotide-binding site and leucine-rich (NBS-LRR) domain. Some NBS-LRR genes in the soybean genome have also been reported to function in disease resistance. Here, we show the correlation between the number of NBS-LRR genes and the number of disease resistance quantitative trait locus (QTL) on each chromosome of soybean. We also surveyed the functional redundancy of disease resistance on recently duplicated regions known to harbor NBS-LRR genes. Moreover, we analyzed NBS-LRR gene expression in the bacterial leaf pustule (BLP)-induced soybean transcriptome. RESULTS: A total of 319 genes were determined to be putative NBS-LRR genes in the soybean genome and the number of NBS-LRR genes on each chromosome was highly correlated with the number of disease resistance QTL on each chromosome. In addition, the recently duplicated regions contained duplicated NBS-LRR genes and duplicated disease resistance QTL. These recently duplicated regions possessed either an uneven or even number of NBS-LRR genes for each pair of regions. By supporting the disease resistance functions of the NBS-LRR genes in BLP-induced transcriptome, significant differences in the expression of NBS-LRR genes between resistant near isogenic line (NIL) and susceptible NIL supports the disease resistance function of NBS-LRR genes induced by BLP. CONCLUSIONS: The correlation between the number of NBS-LRR genes and disease resistance QTL for each chromosome and for each recently duplicated region indicates that NBS-LRR genes have a disease resistance function in soybean. In addition, NBS-LRR gene expression was significantly different in the BLP-resistant NIL compared to the BLP-susceptible NIL. Moreover, the recently duplicated regions, which have each undergone a different duplication history with respect to the NBS-LRR gene content, showed putative functional diversification of the genes based on different QTL information on the duplicated regions. We concluded that NBS-LRR genes in soybean confer disease resistance in a direct and indirect way and also presented evolutional models of these genes. Therefore, NBS-LRR genes could be used to develop markers for resistance against diverse diseases in order to develop an elite cultivar with marker assisted breeding.

We used a comparative genomics approach to investigate the evolution of a complex nucleotide-binding (NB)-leucine-rich repeat (LRR) gene cluster found in soybean (Glycine max) and common bean (Phaseolus vulgaris) that is associated with several disease resistance (R) genes of known function, including Rpg1b (for Resistance to Pseudomonas glycinea1b), an R gene effective against specific races of bacterial blight. Analysis of domains revealed that the amino-terminal coiled-coil (CC) domain, central nucleotide-binding domain (NB-ARC [for APAF1, Resistance genes, and CED4]), and carboxyl-terminal LRR domain have undergone distinct evolutionary paths. Sequence exchanges within the NB-ARC domain were rare. In contrast, interparalogue exchanges involving the CC and LRR domains were common, consistent with both of these regions coevolving with pathogens. Residues under positive selection were overrepresented within the predicted solvent-exposed face of the LRR domain, although several also were detected within the CC and NB-ARC domains. Superimposition of these latter residues onto predicted tertiary structures revealed that the majority are located on the surface, suggestive of a role in interactions with other domains or proteins. Following polyploidy in the Glycine lineage, NB-LRR genes have been preferentially lost from one of the duplicated chromosomes (homeologues found in soybean), and there has been partitioning of NB-LRR clades between the two homeologues. The single orthologous region in common bean contains approximately the same number of paralogues as found in the two soybean homeologues combined. We conclude that while polyploidization in Glycine has not driven a stable increase in family size for NB-LRR genes, it has generated two recombinationally isolated clusters, one of which appears to be in the process of decay.

Many software tools for comparative analysis of genomic sequence data have been released in recent decades. Despite this, it remains challenging to determine evolutionary relationships in gene clusters due to their complex histories involving duplications, deletions, inversions, and conversions. One concept describing these relationships is orthology. Orthologs derive from a common ancestor by speciation, in contrast to paralogs, which derive from duplication. Discriminating orthologs from paralogs is a necessary step in most multispecies sequence analyses, but doing so accurately is impeded by the occurrence of gene conversion events. We propose a refined method of orthology assignment based on two paradigms for interpreting its definition: by genomic context or by sequence content. X-orthology (based on context) traces orthology resulting from speciation and duplication only, while N-orthology (based on content) includes the influence of conversion events. We developed a computational method for automatically mapping both types of orthology on a per-nucleotide basis in gene cluster regions studied by comparative sequencing, and we make this mapping accessible by visualizing the output. All of these steps are incorporated into our newly extended CHAP 2 package. We evaluate our method using both simulated data and real gene clusters (including the well-characterized α-globin and β-globin clusters). We also illustrate use of CHAP 2 by analyzing four more loci: CCL (chemokine ligand), IFN (interferon), CYP2abf (part of cytochrome P450 family 2), and KIR (killer cell immunoglobulin-like receptors). These new methods facilitate and extend our understanding of evolution at these and other loci by adding automated accurate evolutionary inference to the biologist's toolkit. The CHAP 2 package is freely available from http://www.bx.psu.edu/miller_lab.

Commercial banana varieties are highly susceptible to fungal pathogens, as well as bacterial pathogens, nematodes, viruses, and insect pests. The largest known family of plant resistance genes encodes 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 the isolation of candidate genes in banana that may be involved in plant defense. Six degenerate PCR primers were designed to target NBS and additional domains were tested on commercial banana species Musa acuminata subsp malaccensis and the Musa AAB Group propagated in vitro and plants maintained in a greenhouse. Total DNA was isolated by a modified CTAB extraction technique. Four resistance gene analogs were amplified and deposited in GenBank and assigned numbers HQ199833-HQ199836. The predicted amino acid sequences compared to the amino acid sequences of known resistance genes (MRGL1, MRGL2, MRGL3, and MRGL4) revealed significant sequence similarity. The presence of consensus domains, namely kinase-1a, kinase-2 and hydrophobic domain, provided evidence that the cloned sequences belong to the typical non-Toll/interleukin-1 receptor-like domain NBS-LRR gene family.

The leucine-rich repeat (LRR) receptor kinase (RLK) proteins constitute a large superfamily in the plant genome, and carry out key functions in a variety of biological pathways. In an effort to determine the evolutionary fate of members of a large gene family such as plant LRR RLK proteins we conducted in silico analysis using complete genome sequencing datasets, genome-wide transcriptome databases, and bioinformatics tools. A total of 292 and 165 LRR RLK genes were retrieved from the rice and Arabidopsis genomes, respectively, formed by diverse duplication events for gene expansion. The phylogenic analyses of the LRR RLK genes suggested combinations of LRR domains and RLK domains in the ancient plant genome prior to the divergence of rice and Arabidopsis, followed by massive independent expansions during speciation. The somewhat high frequencies (50-73%) of expressional divergence of members of duplicate gene pairs formed by whole/segmental genome duplication (W/SGD) and tandem duplication (TD) events of Arabidopsis and TD events of rice support the idea of their functional diversity for gene retention. By contrast, a relatively low degree (at least 20%) of members of rice LRR RLK gene pairs formed by W/SGD appear to be divergent in expression following the duplication event. At least 7 pairs of co-expressed gene clusters, including each of the tentative orthologous LRR RLK genes between rice and Arabidopsis, were enriched to an orthologous set between members of each of the pairs as compared to those of the random pairs, suggesting some degree of functional conservation of individual genes. These results may shed some light on the crucial functions of the plant LRR RLK genes with regard to a variety of biological processes.

Majority of the plant disease resistance genes expresses cytoplasmic receptor-like proteins characterized by an N-terminal nucleotide-binding site (NBS) and a leucine-rich repeat (LRR) domain. Degenerative primers based on these conserved motifs were used to isolate NBS type sequences in Kaempferia galanga. Cloning and sequencing identified 12 Kaempferia NBS-type sequences called resistance gene candidates (RGCs) classified into four classes. The amino acid sequences of the RGCs detected the presence of conserved domains, viz., kinase-1a, kinase-2, and hydrophobic GLPL, categorizing them with the NBS-LRR class gene family. Structural and phylogenetic characterization grouped the RGCs with the non-toll interleukin receptor (non-TIR) subclasses of the NBS sequences. Reverse transcription PCR with 10 Kaempferia RGC specific primers revealed 7 out of 10 Kaempferia RGCs to be expressive. The isolation and characterization of Kaempferia RGCs has been reported for the first time in this study. This will provide a starting point towards characterization of candidate resistance genes in Kaempferia and can act as a source pool for disease resistance development in other asexually reproducing plants.

Most disease-resistance (R) genes in plants encode NBS-LRR proteins and belong to one of the largest and most variable gene families among plant genomes. However, the specific evolutionary routes of NBS-LRR encoding genes remain elusive. Recently in coffee tree (Coffea arabica), a region spanning the SH3 locus that confers resistance to coffee leaf rust, one of the most serious coffee diseases, was identified and characterized. Using comparative sequence analysis, the purpose of the present study was to gain insight into the genomic organization and evolution of the SH3 locus. Sequence analysis of the SH3 region in three coffee genomes, Ea and Ca subgenomes from the allotetraploid C. arabica and Cc genome from the diploid C. canephora, revealed the presence of 5, 3 and 4 R genes in Ea, Ca, and Cc genomes, respectively. All these R-gene sequences appeared to be members of a CC-NBS-LRR (CNL) gene family that was only found at the SH3 locus in C. arabica. Furthermore, while homologs were found in several dicot species, comparative genomic analysis failed to find any CNL R-gene in the orthologous regions of other eudicot species. The orthology relationship among the SH3-CNL copies in the three analyzed genomes was determined and the duplication/deletion events that shaped the SH3 locus were traced back. Gene conversion events were detected between paralogs in all three genomes and also between the two sub-genomes of C. arabica. Significant positive selection was detected in the solvent-exposed residues of the SH3-CNL copies. The ancestral SH3-CNL copy was inserted in the SH3 locus after the divergence between Solanales and Rubiales lineages. Moreover, the origin of most of the SH3-CNL copies predates the divergence between Coffea species. The SH3-CNL family appeared to evolve following the birth-and-death model, since duplications and deletions were inferred in the evolution of the SH3 locus. Gene conversion between paralog members, inter-subgenome sequence exchanges and positive selection appear to be the major forces acting on the evolution of SH3-CNL in coffee trees.

The largest group of plant resistance (R) genes contain the regions that encode the nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains (NBS-LRR genes). To gain new resistance, amino acid substitutions and changes in number of the LRRs that recognize the presence of pathogens are considered important. In this study, we focus on the evolution of the number of LRRs and analyze the genome data of five plant species, Arabidopsis thaliana, Oryza sativa, Medicago truncatula, Lotus japonicus and Populus trichocarpa. We first categorized the NBS-LRR genes in each species into groups and subgroups based on the phylogenetic relationships of their NBS domain sequences. Then we estimated the evolutionary rate of the number of LRRs relative to the synonymous divergence in the NBS domain sequences by a maximum likelihood method assuming the single stepwise mutation model. The estimates ranged from 4.5 to 600 and differed between groups in the same species or between species. This indicated different roles played by different groups of the NBS-LRR genes within a species or the effects of various life history characteristics, such as generation time, of the species. We also tested the fit of the model to the data using the variance of number of LRRs in each subgroup. In some subgroups in some plants (16 out of 174 subgroups), the results of simulation using the estimated rates significantly deviated from the observed data. Those subgroups may have undergone different modes of selection from the other subgroups.

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