Correction: DNA Sequence Evolution and Rare Homoeologous Conversion in Tetraploid Cotton.

[This corrects the article DOI: 10.1371/journal.pgen.1006012.].

DNA Sequence Evolution and Rare Homoeologous Conversion in Tetraploid Cotton.

Allotetraploid cotton species are a vital source of spinnable fiber for textiles. The polyploid nature of the cotton genome raises many evolutionary questions as to the relationships between duplicated genomes. We describe the evolution of the cotton genome (SNPs and structural variants) with the greatly improved resolution of 34 deeply re-sequenced genomes. We also explore the evolution of homoeologous regions in the AT- and DT-genomes and especially the phenomenon of conversion between genomes. We did not find any compelling evidence for homoeologous conversion between genomes. These findings are very different from other recent reports of frequent conversion events between genomes. We also identified several distinct regions of the genome that have been introgressed between G. hirsutum and G. barbadense, which presumably resulted from breeding efforts targeting associated beneficial alleles. Finally, the genotypic data resulting from this study provides access to a wealth of diversity sorely needed in the narrow germplasm of cotton cultivars.

Repeated polyploidization of Gossypium genomes and the evolution of spinnable cotton fibres.

Polyploidy often confers emergent properties, such as the higher fibre productivity and quality of tetraploid cottons than diploid cottons bred for the same environments. Here we show that an abrupt five- to sixfold ploidy increase approximately 60 million years (Myr) ago, and allopolyploidy reuniting divergent Gossypium genomes approximately 1-2 Myr ago, conferred about 30-36-fold duplication of ancestral angiosperm (flowering plant) genes in elite cottons (Gossypium hirsutum and Gossypium barbadense), genetic complexity equalled only by Brassica among sequenced angiosperms. Nascent fibre evolution, before allopolyploidy, is elucidated by comparison of spinnable-fibred Gossypium herbaceum A and non-spinnable Gossypium longicalyx F genomes to one another and the outgroup D genome of non-spinnable Gossypium raimondii. The sequence of a G. hirsutum A(t)D(t) (in which 't' indicates tetraploid) cultivar reveals many non-reciprocal DNA exchanges between subgenomes that may have contributed to phenotypic innovation and/or other emergent properties such as ecological adaptation by polyploids. Most DNA-level novelty in G. hirsutum recombines alleles from the D-genome progenitor native to its New World habitat and the Old World A-genome progenitor in which spinnable fibre evolved. Coordinated expression changes in proximal groups of functionally distinct genes, including a nuclear mitochondrial DNA block, may account for clusters of cotton-fibre quantitative trait loci affecting diverse traits. Opportunities abound for dissecting emergent properties of other polyploids, particularly angiosperms, by comparison to diploid progenitors and outgroups.

A novel approach for multi-SNP GWAS and its application in Alzheimer's disease.

BACKGROUND: Genome-wide association studies (GWAS) have effectively identified genetic factors for many diseases. Many diseases, including Alzheimer's disease (AD), have epistatic causes, requiring more sophisticated analyses to identify groups of variants which together affect phenotype. RESULTS: Based on the GWAS statistical model, we developed a multi-SNP GWAS analysis to identify pairs of variants whose common occurrence signaled the Alzheimer's disease phenotype. CONCLUSIONS: Despite not having sufficient data to demonstrate significance, our preliminary experimentation identified a high correlation between GRIA3 and HLA-DRB5 (an AD gene). GRIA3 has not been previously reported in association with AD, but is known to play a role in learning and memory.

Re-evaluating the phylogeny of allopolyploid Gossypium L.

The formation of allopolyploid cotton precipitated a rapid diversification and colonization of dry coastal American tropical and subtropical regions. Previous phylogenetic analyses, combined with molecular divergence analyses, have offered a temporal framework for this radiation, but provide only weak support for some of the resolved branches. Moreover, these earlier analyses did not include the recently recognized sixth polyploid species, G. ekmanianum Wittmack. Here we use targeted sequence capture of multiple loci in conjunction with both concatenated and Bayesian concordance analyses to reevaluate the phylogeny of allopolyploid cotton species. Although phylogenetic resolution afforded by individual genes is often low, sufficient signal was attained both through the concatenated and concordance analyses to provide robust support for the Gossypium polyploid clade, which is reported here.

Methods for mapping and categorization of DNA sequence reads from allopolyploid organisms.

Genome read categorization determines the genome of origin for sequence reads from an allopolyploid organism. Different techniques have been used to perform read categorization, mostly based on homoeo-SNPs identified between extant diploid relatives of allopolyploids. We present a novel technique for read categorization implemented by the software PolyDog. We demonstrate its accuracy and improved categorization relative to other methods. We discuss the situations in which one method or another might be most appropriate.

Polyploidy and the petal transcriptome of Gossypium.

BACKGROUND: Genes duplicated by polyploidy (homoeologs) may be differentially expressed in plant tissues. Recent research using DNA microarrays and RNAseq data have described a cacophony of complex expression patterns during development of cotton fibers, petals, and leaves. Because of its highly canalized development, petal tissue has been used as a model tissue for gene expression in cotton. Recent advances in cotton genome annotation and assembly now permit an enhanced analysis of duplicate gene deployment in petals from allopolyploid cotton. RESULTS: Homoeologous gene expression levels were quantified in diploid and tetraploid flower petals of Gossypium using the Gossypium raimondii genome sequence as a reference. In the polyploid, most homoeologous genes were expressed at equal levels, though a subset had an expression bias of AT and DT copies. The direction of gene expression bias was conserved in natural and recent polyploids of cotton. Conservation of direction of bias and additional comparisons between the diploids and tetraploids suggested different regulation mechanisms of gene expression. We described three phases in the evolution of cotton genomes that contribute to gene expression in the polyploid nucleus. CONCLUSIONS: Compared to previous studies, a surprising level of expression homeostasis was observed in the expression patterns of polyploid genomes. Conserved expression bias in polyploid petals may have resulted from cis-acting modifications that occurred prior to polyploidization. Some duplicated genes were intriguing exceptions to general trends. Mechanisms of gene regulation for these and other genes in the cotton genome warrants further investigation.

Genome-wide analysis of the omega-3 fatty acid desaturase gene family in Gossypium.

BACKGROUND: The majority of commercial cotton varieties planted worldwide are derived from Gossypium hirsutum, which is a naturally occurring allotetraploid produced by interspecific hybridization of A- and D-genome diploid progenitor species. While most cotton species are adapted to warm, semi-arid tropical and subtropical regions, and thus perform well in these geographical areas, cotton seedlings are sensitive to cold temperature, which can significantly reduce crop yields. One of the common biochemical responses of plants to cold temperatures is an increase in omega-3 fatty acids, which protects cellular function by maintaining membrane integrity. The purpose of our study was to identify and characterize the omega-3 fatty acid desaturase (FAD) gene family in G. hirsutum, with an emphasis on identifying omega-3 FADs involved in cold temperature adaptation. RESULTS: Eleven omega-3 FAD genes were identified in G. hirsutum, and characterization of the gene family in extant A and D diploid species (G. herbaceum and G. raimondii, respectively) allowed for unambiguous genome assignment of all homoeologs in tetraploid G. hirsutum. The omega-3 FAD family of cotton includes five distinct genes, two of which encode endoplasmic reticulum-type enzymes (FAD3-1 and FAD3-2) and three that encode chloroplast-type enzymes (FAD7/8-1, FAD7/8-2, and FAD7/8-3). The FAD3-2 gene was duplicated in the A genome progenitor species after the evolutionary split from the D progenitor, but before the interspecific hybridization event that gave rise to modern tetraploid cotton. RNA-seq analysis revealed conserved, gene-specific expression patterns in various organs and cell types and semi-quantitative RT-PCR further revealed that FAD7/8-1 was specifically induced during cold temperature treatment of G. hirsutum seedlings. CONCLUSIONS: The omega-3 FAD gene family in cotton was characterized at the genome-wide level in three species, showing relatively ancient establishment of the gene family prior to the split of A and D diploid progenitor species. The FAD genes are differentially expressed in various organs and cell types, including fiber, and expression of the FAD7/8-1 gene was induced by cold temperature. Collectively, these data define the genetic and functional genomic properties of this important gene family in cotton and provide a foundation for future efforts to improve cotton abiotic stress tolerance through molecular breeding approaches.

Deep sequencing of amplicons reveals widespread intraspecific hybridization and multiple origins of polyploidy in big sagebrush (Artemisia tridentata; Asteraceae).

PREMISE OF THE STUDY: Hybridization has played an important role in the evolution and ecological adaptation of diploid and polyploid plants. Artemisia tridentata (Asteraceae) tetraploids are extremely widespread and of great ecological importance. These tetraploids are often taxonomically identified as A. tridentata subsp. wyomingensis or as autotetraploids of diploid subspecies tridentata and vaseyana. Few details are available as to how these tetraploids are formed or how they are related to diploid subspecies. • METHODS: We used amplicon sequencing to assess phylogenetic relationships among three recognized subspecies: tridentata, vaseyana, and wyomingensis. DNA sequence data from putative genes were pyrosequenced and assembled from 329 samples. Nucleotide diversity and putative haplotypes were estimated from the high-read coverage. Phylogenies were constructed from Bayesian coalescence and neighbor-net network analyses. • KEY RESULTS: Analyses support distinct diploid subspecies of tridentata and vaseyana in spite of known hybridization in ecotones. Nucleotide diversity estimates of populations compared to the total diversity indicate the relationships are predominately driven by a small proportion of the amplicons. Tetraploids, including subspecies wyomingensis, are polyphyletic occurring within and between diploid subspecies groups. • CONCLUSIONS: Artemisia tridentata is a species comprising phylogenetically distinct diploid progenitors and a tetraploid complex with varying degrees of phylogenetic and morphological affinities to the diploid subspecies. These analyses suggest tetraploids are formed locally or regionally from diploid tridentata and vaseyana populations via autotetraploidy, followed by introgression between tetraploid groups. Understanding the phylogenetic vs. ecological relationships of A. tridentata subspecies will have bearing on how to restore these desert ecosystems.

Genome-wide divergence, haplotype distribution and population demographic histories for Gossypium hirsutum and Gossypium barbadense as revealed by genome-anchored SNPs.

Use of 10,129 singleton SNPs of known genomic location in tetraploid cotton provided unique opportunities to characterize genome-wide diversity among 440 Gossypium hirsutum and 219 G. barbadense cultivars and landrace accessions of widespread origin. Using the SNPs distributed genome-wide, we examined genetic diversity, haplotype distribution and linkage disequilibrium patterns in the G. hirsutum and G. barbadense genomes to clarify population demographic history. Diversity and identity-by-state analyses have revealed little sharing of alleles between the two cultivated allotetraploid genomes, with a few exceptions that indicated sporadic gene flow. We found a high number of new alleles, representing increased nucleotide diversity, on chromosomes 1 and 2 in cultivated G. hirsutum as compared with low nucleotide diversity on these chromosomes in landrace G. hirsutum. In contrast, G. barbadense chromosomes showed negative Tajima's D on several chromosomes for both cultivated and landrace types, which indicate that speciation of G. barbadense itself, might have occurred with relatively narrow genetic diversity. The presence of conserved linkage disequilibrium (LD) blocks and haplotypes between G. hirsutum and G. barbadense provides strong evidence for comparable patterns of evolution in their domestication processes. Our study illustrates the potential use of population genetic techniques to identify genomic regions for domestication.

Independent Domestication of Two Old World Cotton Species.

Domesticated cotton species provide raw material for the majority of the world's textile industry. Two independent domestication events have been identified in allopolyploid cotton, one in Upland cotton (Gossypium hirsutum L.) and the other to Egyptian cotton (Gossypium barbadense L.). However, two diploid cotton species, Gossypium arboreum L. and Gossypium herbaceum L., have been cultivated for several millennia, but their status as independent domesticates has long been in question. Using genome resequencing data, we estimated the global abundance of various repetitive DNAs. We demonstrate that, despite negligible divergence in genome size, the two domesticated diploid cotton species contain different, but compensatory, repeat content and have thus experienced cryptic alterations in repeat abundance despite equivalence in genome size. Evidence of independent origin is bolstered by estimates of divergence times based on molecular evolutionary analysis of f7,000 orthologous genes, for which synonymous substitution rates suggest that G. arboreum and G. herbaceum last shared a common ancestor approximately 0.4-2.5 Ma. These data are incompatible with a shared domestication history during the emergence of agriculture and lead to the conclusion that G. arboreum and G. herbaceum were each domesticated independently.

Evolutionary Conservation and Divergence of Gene Coexpression Networks in Gossypium (Cotton) Seeds.

The cotton genus (Gossypium) provides a superior system for the study of diversification, genome evolution, polyploidization, and human-mediated selection. To gain insight into phenotypic diversification in cotton seeds, we conducted coexpression network analysis of developing seeds from diploid and allopolyploid cotton species and explored network properties. Key network modules and functional associations were identified related to seed oil content and seed weight. We compared species-specific networks to reveal topological changes, including rewired edges and differentially coexpressed genes, associated with speciation, polyploidy, and cotton domestication. Network comparisons among species indicate that topologies are altered in addition to gene expression profiles, indicating that changes in transcriptomic coexpression relationships play a role in the developmental architecture of cotton seed development. The global network topology of allopolyploids, especially for domesticated G. hirsutum, resembles the network of the A-genome diploid more than that of the D-genome parent, despite its D-like phenotype in oil content. Expression modifications associated with allopolyploidy include coexpression level dominance and transgressive expression, suggesting that the transcriptomic architecture in polyploids is to some extent a modular combination of that of its progenitor genomes. Among allopolyploids, intermodular relationships are more preserved between two different wild allopolyploid species than they are between wild and domesticated forms of a cultivated cotton, and regulatory connections of oil synthesis-related pathways are denser and more closely clustered in domesticated vs. wild G. hirsutum. These results demonstrate substantial modification of genic coexpression under domestication. Our work demonstrates how network inference informs our understanding of the transcriptomic architecture of phenotypic variation associated with temporal scales ranging from thousands (domestication) to millions (speciation) of years, and by polyploidy.

Insights into transcriptomes of big and low sagebrush.

We report the sequencing and assembly of three transcriptomes from Big (Artemisia tridentata ssp. wyomingensis and A. tridentata ssp. tridentata) and Low (A. arbuscula ssp. arbuscula) sagebrush. The sequence reads are available in the Sequence Read Archive of NCBI. We demonstrate the utilities of these transcriptomes for gene discovery and phylogenomic analysis. An assembly of 61,883 transcripts followed by transcript identification by the program TRAPID revealed 16 transcripts directly related to terpene synthases, proteins critical to the production of multiple secondary metabolites in sagebrush. A putative terpene synthase was identified in two of our sagebrush samples. Using paralogs with synonymous mutations we reconstructed an evolutionary time line of ancient genome duplications. By applying a constant mutation rate to the data we estimate that these three ancient duplications occurred about 18, 34 and 60 million years ago. These transcriptomes offer a foundation for future studies of sagebrush, including inferences in chemical defense and the identification of species and subspecies of sagebrush for restoration and preservation of the threatened sage-grouse.

BamBam: genome sequence analysis tools for biologists.

BACKGROUND: Massive computational power is needed to analyze the genomic data produced by next-generation sequencing, but extensive computational experience and specific knowledge of algorithms should not be necessary to run genomic analyses or interpret their results. FINDINGS: We present BamBam, a package of tools for genome sequence analysis. BamBam contains tools that facilitate summarizing data from BAM alignment files and identifying features such as SNPs, indels, and haplotypes represented in those alignments. CONCLUSIONS: BamBam provides a powerful and convenient framework to analyze genome sequence data contained in BAM files.

Ancient gene duplicates in Gossypium (cotton) exhibit near-complete expression divergence.

Whole genome duplication (WGD) is widespread in flowering plants and is a driving force in angiosperm diversification. The redundancy introduced by WGD allows the evolution of novel gene interactions and functions, although the patterns and processes of diversification are poorly understood. We identified ∼ 2,000 pairs of paralogous genes in Gossypium raimondii (cotton) resulting from an approximately 60 My old 5- to 6-fold ploidy increase. Gene expression analyses revealed that, in G. raimondii, 99.4% of the gene pairs exhibit differential expression in at least one of the three tissues (petal, leaf, and seed), with 93% to 94% exhibiting differential expression on a per-tissue basis. For 1,666 (85%) pairs, differential expression was observed in all tissues. These observations were mirrored in a time series of G. raimondii seed, and separately in leaf, petal, and seed of G. arboreum, indicating expression level diversification before species divergence. A generalized linear model revealed 92.4% of the paralog pairs exhibited expression divergence, with most exhibiting significant gene and tissue interactions indicating complementary expression patterns in different tissues. These data indicate massive, near-complete expression level neo- and/or subfunctionalization among ancient gene duplicates, suggesting these processes are essential in their maintenance over ∼ 60 Ma.

The Li2 mutation results in reduced subgenome expression bias in elongating fibers of allotetraploid cotton (Gossypium hirsutum L.).

Next generation sequencing (RNA-seq) technology was used to evaluate the effects of the Ligon lintless-2 (Li2) short fiber mutation on transcriptomes of both subgenomes of allotetraploid cotton (Gossypium hirsutum L.) as compared to its near-isogenic wild type. Sequencing was performed on 4 libraries from developing fibers of Li2 mutant and wild type near-isogenic lines at the peak of elongation followed by mapping and PolyCat categorization of RNA-seq data to the reference D5 genome (G. raimondii) for homeologous gene expression analysis. The majority of homeologous genes, 83.6% according to the reference genome, were expressed during fiber elongation. Our results revealed: 1) approximately two times more genes were induced in the AT subgenome comparing to the DT subgenome in wild type and mutant fiber; 2) the subgenome expression bias was significantly reduced in the Li2 fiber transcriptome; 3) Li2 had a significantly greater effect on the DT than on the AT subgenome. Transcriptional regulators and cell wall homeologous genes significantly affected by the Li2 mutation were reviewed in detail. This is the first report to explore the effects of a single mutation on homeologous gene expression in allotetraploid cotton. These results provide deeper insights into the evolution of allotetraploid cotton gene expression and cotton fiber development.

PolyCat: a resource for genome categorization of sequencing reads from allopolyploid organisms.

Read mapping is a fundamental part of next-generation genomic research but is complicated by genome duplication in many plants. Categorizing DNA sequence reads into their respective genomes enables current methods to analyze polyploid genomes as if they were diploid. We present PolyCat-a pipeline for mapping and categorizing all types of next-generation sequence data produced from allopolyploid organisms. PolyCat uses GSNAP's single-nucleotide polymorphism (SNP)-tolerant mapping to minimize the mapping efficiency bias caused by SNPs between genomes. PolyCat then uses SNPs between genomes to categorize reads according to their respective genomes. Bisulfite-treated reads have a significant reduction in nucleotide complexity because nucleotide conversion events are confounded with transition substitutions. PolyCat includes special provisions to properly handle bisulfite-treated data. We demonstrate the functionality of PolyCat on allotetraploid cotton, Gossypium hirsutum, and create a functional SNP index for efficiently mapping sequence reads to the D-genome sequence of G. raimondii. PolyCat is appropriate for all allopolyploids and all types of next-generation genome analysis, including differential expression (RNA sequencing), differential methylation (bisulfite sequencing), differential DNA-protein binding (chromatin immunoprecipitation sequencing), and population diversity.

Insights into the evolution of cotton diploids and polyploids from whole-genome re-sequencing.

Understanding the composition, evolution, and function of the Gossypium hirsutum (cotton) genome is complicated by the joint presence of two genomes in its nucleus (AT and DT genomes). These two genomes were derived from progenitor A-genome and D-genome diploids involved in ancestral allopolyploidization. To better understand the allopolyploid genome, we re-sequenced the genomes of extant diploid relatives that contain the A1 (Gossypium herbaceum), A2 (Gossypium arboreum), or D5 (Gossypium raimondii) genomes. We conducted a comparative analysis using deep re-sequencing of multiple accessions of each diploid species and identified 24 million SNPs between the A-diploid and D-diploid genomes. These analyses facilitated the construction of a robust index of conserved SNPs between the A-genomes and D-genomes at all detected polymorphic loci. This index is widely applicable for read mapping efforts of other diploid and allopolyploid Gossypium accessions. Further analysis also revealed locations of putative duplications and deletions in the A-genome relative to the D-genome reference sequence. The approximately 25,400 deleted regions included more than 50% deletion of 978 genes, including many involved with starch synthesis. In the polyploid genome, we also detected 1,472 conversion events between homoeologous chromosomes, including events that overlapped 113 genes. Continued characterization of the Gossypium genomes will further enhance our ability to manipulate fiber and agronomic production of cotton.

RNA-Seq transcriptome profiling of upland cotton (Gossypium hirsutum L.) root tissue under water-deficit stress.

An RNA-Seq experiment was performed using field grown well-watered and naturally rain fed cotton plants to identify differentially expressed transcripts under water-deficit stress. Our work constitutes the first application of the newly published diploid D5 Gossypium raimondii sequence in the study of tetraploid AD1 upland cotton RNA-seq transcriptome analysis. A total of 1,530 transcripts were differentially expressed between well-watered and water-deficit stressed root tissues, in patterns that confirm the accuracy of this technique for future studies in cotton genomics. Additionally, putative sequence based genome localization of differentially expressed transcripts detected A2 genome specific gene expression under water-deficit stress. These data will facilitate efforts to understand the complex responses governing transcriptomic regulatory mechanisms and to identify candidate genes that may benefit applied plant breeding programs.