Evolution of the alcohol dehydrogenase gene family in diploid and tetraploid Gossypium L

Molecular data have had a profound effect on the field of plant evolutionary biology, yet the potential wealth of data stored in low-copy nuclear-encoded genes has been virtually ignored, relative to studies of chloroplast and ribosomal DNA. In this dissertation I present an analysis of a model nuclear-encoded gene family (alcohol dehydrogenase, Adh) in a model organismal system, the cotton genus (Gossypium L., Malvaceae);A combination of PCR- and Southern hybridization-based approaches was employed to isolate, sequence, and map multiple Adh gene family members. Diploid Gossypium contain at least seven Adh loci. Sequence analysis reveals extensive intron variation between loci, and one locus has lost two introns. Evolutionary rates differ between loci and between lineages. Finally, the Adh gene family appears dynamic in that examples of gene duplication, pseudogenization, and elimination were found;We have also employed Adh genes for phylogenetic analyses. We tested the relative utility of seven noncoding cpDNA regions and a pair of homoeologous nuclear genes for resolving recent divergences, using tetraploid cottons as a model system. We sequenced over 7 kb of CPDNA per taxon, yet obtained incomplete phylogenetic resolution. We also sequenced a 1.65-kb region of a homoeologous pair of Adh genes and obtained a robust and completely resolved topology. This enhanced resolution reflects an approximately three- to six-fold increase in evolutionary rate relative to the cpDNA sequences;Finally we have exploited Adh sequences to study intraspecific genetic diversity. We estimated nucleotide diversity for a pair of homoeologous Adh loci in allotetraploid G. hirsutum. Nucleotide diversity for AdhA in Gossypium is lower than for any plant nuclear gene yet described. This low diversity appears to reflect a history severe genetic bottlenecks supplemented by an unusually slow nucleotide substitution rate and an autogamous breeding system. While not statistically supportable, the sum of the observations also suggest differential evolutionary dynamics at each of the homoeologous loci

The complete chloroplast genome sequence of Gossypium hirsutum: organization and phylogenetic relationships to other angiosperms

BACKGROUND: Cotton (Gossypium hirsutum) is the most important fiber crop grown in 90 countries. In 2004–2005, US farmers planted 79% of the 5.7-million hectares of nuclear transgenic cotton. Unfortunately, genetically modified cotton has the potential to hybridize with other cultivated and wild relatives, resulting in geographical restrictions to cultivation. However, chloroplast genetic engineering offers the possibility of containment because of maternal inheritance of transgenes. The complete chloroplast genome of cotton provides essential information required for genetic engineering. In addition, the sequence data were used to assess phylogenetic relationships among the major clades of rosids using cotton and 25 other completely sequenced angiosperm chloroplast genomes. RESULTS: The complete cotton chloroplast genome is 160,301 bp in length, with 112 unique genes and 19 duplicated genes within the IR, containing a total of 131 genes. There are four ribosomal RNAs, 30 distinct tRNA genes and 17 intron-containing genes. The gene order in cotton is identical to that of tobacco but lacks rpl22 and infA. There are 30 direct and 24 inverted repeats 30 bp or longer with a sequence identity ≥ 90%. Most of the direct repeats are within intergenic spacer regions, introns and a 72 bp-long direct repeat is within the psaA and psaB genes. Comparison of protein coding sequences with expressed sequence tags (ESTs) revealed nucleotide substitutions resulting in amino acid changes in ndhC, rpl23, rpl20, rps3 and clpP. Phylogenetic analysis of a data set including 61 protein-coding genes using both maximum likelihood and maximum parsimony were performed for 28 taxa, including cotton and five other angiosperm chloroplast genomes that were not included in any previous phylogenies. CONCLUSION: Cotton chloroplast genome lacks rpl22 and infA and contains a number of dispersed direct and inverted repeats. RNA editing resulted in amino acid changes with significant impact on their hydropathy. Phylogenetic analysis provides strong support for the position of cotton in the Malvales in the eurosids II clade sister to Arabidopsis in the Brassicales. Furthermore, there is strong support for the placement of the Myrtales sister to the eurosid I clade, although expanded taxon sampling is needed to further test this relationship

Cytonuclear Evolution of Rubisco in Four Allopolyploid Lineages

Allopolyploidization in plants entails the merger of two divergent nuclear genomes, typically with only one set (usually maternal) of parental plastidial and mitochondrial genomes and with an altered cytonuclear stoichiometry. Thus, we might expect cytonuclear coevolution to be an important dimension of allopolyploid evolution. Here, we investigate cytonuclear coordination for the key chloroplast protein rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase), which is composed of nuclear-encoded, small subunits (SSUs) and plastid-encoded, large subunits. By studying gene composition and diversity as well as gene expression in four model allopolyploid lineages, Arabidopsis, Arachis, Brassica, and Nicotiana, we demonstrate that paralogous nuclear-encoded rbcS genes within diploids are subject to homogenization via gene conversion and that such concerted evolution via gene conversion characterizes duplicated genes (homoeologs) at the polyploid level. Many gene conversions in the polyploids are intergenomic with respect to the diploid progenitor genomes, occur in functional domains of the homoeologous SSUs, and are directionally biased, such that the maternal amino acid states are favored. This consistent preferential maternal-to-paternal gene conversion is mirrored at the transcriptional level, with a uniform transcriptional bias of the maternal-like rbcS homoeologs. These data, repeated among multiple diverse angiosperm genera for an important photosynthetic enzyme, suggest that cytonuclear coevolution may be mediated by intergenomic gene conversion and altered transcription of duplicated, now homoeologous nuclear genes

Molecular systematics in Gossypium and its relatives

Gossypium L. is an economically important genus as it harbors four species that provide the cotton of commerce. Although many aspects of the evolution and taxonomy of Gossypium are well understood, there has been little work devoted to assessing relationships among Gossypium and its allied genera. In addition, newly discovered species, of Gossypium sect. Grandicalyx from Australia, have prompted a need for more research into the phylogeny of these new species and their relationships to other Gossypium taxa. Multiple data sets were used to address questions regarding (1) phylogenetic relationships among Gossypium and its relatives, and (2) the phylogeny and evolution of Gossypium sect. Grandicalyx. A protocol for handling multiple and sometimes competing data sets was developed, and was applied in the first study. This same protocol was not applicable for the second study, where amounts of sequence divergence were too low. Phylogenetic analyses of Gossypium and its allies indicated that Gossypium is a monophyletic group and that it is closely related to a clade represented by the Hawaiian genus Kokia and the east African/Madagascan genus Gossypioides. Phylogenetic inferences also suggested that Cienfuegosia may represent the first group to diverge during tribal radiation, and that Thespesia is not monophyletic. Studies of the Australian Gossypium sect. Grandicalyx showed that sect. Grandicalyx is monophyletic, with divergences inferred to be relatively recent. Molecular data sets provide little resolution with respect to the relationship among species within the section. Species are suggested to have radiated in the late Pliocene or Pleistocene after a much earlier (Miocene) divergence from the other extant Australian cottons. The palaeoclimatic record, sequence divergence estimates and phylogenetic data are congruent in suggesting a phytogeographic scenario for the diversification of species in sect. Grandicalyx, through range fragmentation of a more widely distributed ancestor or ancestors. This evolutionary history was accompanied by the development of a prostrate to reclining herbaceous habit, adaptation to seasonal fires, and a suite of features associated with myrmecochory

Rapid evolutionary divergence of Gossypium barbadense and G. hirsutum mitochondrial genomes

Background The mitochondrial genome from upland cotton, G. hirsutum, was previously sequenced. To elucidate the evolution of mitochondrial genomic diversity within a single genus, we sequenced the mitochondrial genome from Sea Island cotton (Gossypium barbadense L.). Methods Mitochondrial DNA from week-old etiolated seedlings was extracted from isolated organelles using discontinuous sucrose density gradient method. Mitochondrial genome was sequenced with Solexa using paired-end, 90 bp read. The clean reads were assembled into contigs using ABySS and finished via additional fosmid and BAC sequencing. Finally, the genome was annotated and analyzed using different softwares. Results The G. barbadense (Sea Island cotton) mitochondrial genome was fully sequenced (677,434-bp) and compared to the mitogenome of upland cotton. The G. barbadensemitochondrial DNA contains seven more genes than that of upland cotton, with a total of 40 protein coding genes (excluding possible pseudogenes), 6 rRNA genes, and 29 tRNA genes. Of these 75 genes, atp1, mttB, nad4, nad9, rrn5, rrn18, and trnD(GTC)-cp were each represented by two identical copies. A single 64 kb repeat was largely responsible for the 9 % difference in genome size between the two mtDNAs. Comparison of genome structures between the two mitochondrial genomes revealed 8 rearranged syntenic regions and several large repeats. The largest repeat was missing from the master chromosome in G. hirsutum. Both mitochondrial genomes contain a duplicated copy of rps3 (rps3-2) in conjunction with a duplication of repeated sequences. Phylogenetic and divergence considerations suggest that a 544-bp fragment of rps3 was transferred to the nuclear genome shortly after divergence of the A- and D- genome diploid cottons. Conclusion These results highlight the insights to the evolution of structural variation between Sea Island and upland cotton mitochondrial genomes

Use of Nuclear Genes for Phylogeny Reconstruction in Plants

Molecular data have had a profound impact on the field of plant systematics, and the application of DNA-sequence data to phylogenetic problems is now routine. The majority of data used in plant molecular phylogenetic studies derives from chloroplast DNA and nuclear rDNA, while the use of low-copy nuclear genes has not been widely adopted. This is due, at least in part, to the greater difficulty of isolating and characterising low-copy nuclear genes relative to chloroplast and rDNA sequences that are readily amplified with universal primers. The higher level of sequence variation characteristic of low-copy nuclear genes, however, often compensates for the experimental effort required to obtain them. In this review, we briefly discuss the strengths and limitations of chloroplast and rDNA sequences, and then focus our attention on the use of low-copy nuclear sequences. Advantages of low-copy nuclear sequences include a higher rate of evolution than for organellar sequences, the potential to accumulate datasets from multiple unlinked loci, and bi-parental inheritance. Challenges intrinsic to the use of low-copy nuclear sequences include distinguishing orthologous loci from divergent paralogous loci in the same gene family, being mindful of the complications arising from concerted evolution or recombination among paralogous sequences, and the presence of intraspecific, intrapopulational and intraindividual polymorphism. Finally, we provide a detailed protocol for the isolation, characterisation and use of low-copy nuclear sequences for phylogenetic studies

Universal Plant DNA Barcode Loci May Not Work in Complex Groups: A Case Study with Indian Berberis Species

BACKGROUND: The concept of DNA barcoding for species identification has gained considerable momentum in animals because of fairly successful species identification using cytochrome oxidase I (COI). In plants, matK and rbcL have been proposed as standard barcodes. However, barcoding in complex genera is a challenging task. METHODOLOGY AND PRINCIPAL FINDINGS: We investigated the species discriminatory power of four reportedly most promising plant DNA barcoding loci (one from nuclear genome--ITS, and three from plastid genome--trnH-psbA, rbcL and matK) in species of Indian Berberis L. (Berberidaceae) and two other genera, Ficus L. (Moraceae) and Gossypium L. (Malvaceae). Berberis species were delineated using morphological characters. These characters resulted in a well resolved species tree. Applying both nucleotide distance and nucleotide character-based approaches, we found that none of the loci, either singly or in combinations, could discriminate the species of Berberis. ITS resolved all the tested species of Ficus and Gossypium and trnH-psbA resolved 82% of the tested species in Ficus. The highly regarded matK and rbcL could not resolve all the species. Finally, we employed amplified fragment length polymorphism test in species of Berberis to determine their relationships. Using ten primer pair combinations in AFLP, the data demonstrated incomplete species resolution. Further, AFLP analysis showed that there was a tendency of the Berberis accessions to cluster according to their geographic origin rather than species affiliation. CONCLUSIONS/SIGNIFICANCE: We reconfirm the earlier reports that the concept of universal barcode in plants may not work in a number of genera. Our results also suggest that the matK and rbcL, recommended as universal barcode loci for plants, may not work in all the genera of land plants. Morphological, geographical and molecular data analyses of Indian species of Berberis suggest probable reticulate evolution and thus barcode markers may not work in this case

Phylogenetic Analyses of Vitis (Vitaceae) Based on Complete Chloroplast Genome Sequences: Effects of Taxon Sampling and Phylogenetic Methods on Resolving Relationships Among Rosids

Background The Vitaceae (grape) is an economically important family of angiosperms whose phylogenetic placement is currently unresolved. Recent phylogenetic analyses based on one to several genes have suggested several alternative placements of this family, including sister to Caryophyllales, asterids, Saxifragales, Dilleniaceae or to rest of rosids, though support for these different results has been weak. There has been a recent interest in using complete chloroplast genome sequences for resolving phylogenetic relationships among angiosperms. These studies have clarified relationships among several major lineages but they have also emphasized the importance of taxon sampling and the effects of different phylogenetic methods for obtaining accurate phylogenies. We sequenced the complete chloroplast genome of Vitis vinifera and used these data to assess relationships among 27 angiosperms, including nine taxa of rosids. Results The Vitis vinifera chloroplast genome is 160,928 bp in length, including a pair of inverted repeats of 26,358 bp that are separated by small and large single copy regions of 19,065 bp and 89,147 bp, respectively. The gene content and order of Vitis is identical to many other unrearranged angiosperm chloroplast genomes, including tobacco. Phylogenetic analyses using maximum parsimony and maximum likelihood were performed on DNA sequences of 61 protein-coding genes for two datasets with 28 or 29 taxa, including eight or nine taxa from four of the seven currently recognized major clades of rosids. Parsimony and likelihood phylogenies of both data sets provide strong support for the placement of Vitaceae as sister to the remaining rosids. However, the position of the Myrtales and support for the monophyly of the eurosid I clade differs between the two data sets and the two methods of analysis. In parsimony analyses, the inclusion of Gossypium is necessary to obtain trees that support the monophyly of the eurosid I clade. However, maximum likelihood analyses place Cucumis as sister to the Myrtales and therefore do not support the monophyly of the eurosid I clade. Conclusion Phylogenies based on DNA sequences from complete chloroplast genome sequences provide strong support for the position of the Vitaceae as the earliest diverging lineage of rosids. Our phylogenetic analyses support recent assertions that inadequate taxon sampling and incorrect model specification for concatenated multi-gene data sets can mislead phylogenetic inferences when using whole chloroplast genomes for phylogeny reconstruction