Curt L. Brubaker

A rapid method for extraction of cotton (Gossypium spp. ) genomic DNA suitable for RFLP or PCR analysis.

Extraction of high-quality genomic DNA fromGossypium (cotton) species is difficult due to high levels of polysaccharide, oxidizable quinones, and other interfering substances. We describe a procedure that consistently permits isolation of cotton genomic DNA of satisfactory size and quality for RFLP and PCR analysis, as well as for most routine cloning applications. Several antioxidants, phenol-binding reagents, and phenol oxidase inhibitors are used throughout the procedure, and most polysaccharides are eliminated early in the procedure by isolation of nuclei.

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 AtDt (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 3347-Locus Genetic Recombination Map of Sequence-Tagged Sites Reveals Features of Genome Organization, Transmission and Evolution of Cotton (Gossypium)

We report genetic maps for diploid (D) and tetraploid (AtDt) Gossypium genomes composed of sequence-tagged sites (STS) that foster structural, functional, and evolutionary genomic studies. The maps include, respectively, 2584 loci at 1.72-cM (∼600 kb) intervals based on 2007 probes (AtDt) and 763 loci at 1.96-cM (∼500 kb) intervals detected by 662 probes (D). Both diploid and tetraploid cottons exhibit negative crossover interference; i.e., double recombinants are unexpectedly abundant. We found no major structural changes between Dt and D chromosomes, but confirmed two reciprocal translocations between At chromosomes and several inversions. Concentrations of probes in corresponding regions of the various genomes may represent centromeres, while genome-specific concentrations may represent heterochromatin. Locus duplication patterns reveal all 13 expected homeologous chromosome sets and lend new support to the possibility that a more ancient polyploidization event may have predated the A-D divergence of 6–11 million years ago. Identification of SSRs within 312 RFLP sequences plus direct mapping of 124 SSRs and exploration for CAPS and SNPs illustrate the “portability” of these STS loci across populations and detection systems useful for marker-assisted improvement of the worlds leading fiber crop. These data provide new insights into polyploid evolution and represent a foundation for assembly of a finished sequence of the cotton genome.

Polyploid formation in cotton is not accompanied by rapid genomic changes

Recent work has demonstrated that allopolyploid speciation in plants may be associated with non-Mendelian genomic changes in the early generations following polyploid synthesis. To address the question of whether rapid genomic changes also occur in allopolyploid cotton (Gossypium) species, amplified fragment length polymorphism (AFLP) analysis was performed to evaluate nine sets of newly synthesized allotetraploid and allohexaploid plants, their parents, and the selfed progeny from colchicine-doubled synthetics. Using both methylation-sensitive and methylation-insensitive enzymes, the extent of fragment additivity in newly combined genomes was ascertained for a total of approximately 22,000 genomic loci. Fragment additivity was observed in nearly all cases, with the few exceptions most likely reflecting parental heterozygosity or experimental error. In addition, genomic Southern analysis on six sets of synthetic allopolyploids probed with five retrotransposons also revealed complete additivity. Because no alterations were observed using methylation-sensitive isoschizomers, epigenetic changes following polyploid synthesis were also minimal. These indications of genomic additivity and epigenetic stasis during allopolyploid formation provide a contrast to recent evidence from several model plant allopolyploids, most notably wheat and Brassica, where rapid and unexplained genomic changes have been reported. In addition, the data contrast with evidence from repetitive DNAs in Gossypium, some of which are subject to non-Mendelian molecular evolutionary phenomena in extant polyploids. These contrasts indicate polyploid speciation in plants is accompanied by a diverse array of molecular evolutionary phenomena, which will vary among both genomic constituents and taxa.

Toward Sequencing Cotton (Gossypium) Genomes

Despite rapidly decreasing costs and innovative technologies, sequencing of angiosperm genomes is not yet undertaken lightly. Generating larger amounts of sequence data more quickly does not address the difficulties of sequencing and assembling complex genomes de novo. The cotton ( Gossypium spp.)

Evolution and Natural History of the Cotton Genus

We present an overview of the evolution and diversity in Gossypium (the cotton genus). This framework facilitates insight into fundamental aspects of plant biology, provides the necessary underpinnings for effective utilization of cotton genetic resources, and guides exploration of the genomic basis of morphological diversity in the genus. More than 50 species of Gossypium are distributed in arid to semi-arid regions of the tropics and subtropics. Included are four species that independently have been domesticated for their fiber, two each in Africa-Asia and the Americas. Gossypium species exhibit extraordinary morphological variation, ranging from trailing herbaceous perennials to ∼15 m trees with a diverse array of reproductive and vegetative characteristics. A parallel level of cytogenetic and genomic diversity has arisen during the global radiation of the genus, leading to the evolution of eight groups of diploid (n = 13) species (genome groups A through G, and K). Data implicate an origin for Gossypium about 5–10 million years ago and a rapid early diversification of the major genome groups. Allopolyploid cottons appear to have arisen within the last 1–2 million years, as a consequence of trans-oceanic dispersal of an A-genome taxon to the New World followed by hybridization with an indigenous D-genome diploid. Subsequent to formation, allopolyploids radiated into three modern lineages, two of which contain the commercially important species G. hirsutum and G. barbadense.

The Origin and Evolution of Gossypium

The genus Gossypium has a long history of taxonomic and evolutionary study. Much of this attention has been stimulated by the fact that the genus includes four domesticated species, the New World allopolyploids G. hirsutum and G.barbadense (2n = 52), and the Old World diploids G. arboreum and G. herbaceum (2n = 26). These cultivated species embody considerable genetic diversity, but this diversity is dwarfed by that included in the genus as a whole, whose 50 species have an aggregate geographic range that encompasses most tropical and subtropical regions of the world.

A molecular perspective on terpene variation in Australian Myrtaceae

The terpenoid-dominated essential oils in Australian Myrtaceae mediate many ecological interactions and are important industrially. Of all the significant essential oil-producing families, Myrtaceae is the only one for which there is no molecular information on terpene biosynthesis. Here we summarise available knowledge on terpene biosynthesis and its relevance to the Myrtaceae to provide a foundation for ecological and genetic studies of chemical diversity. There are several steps in the terpene biosynthesis pathway that have potential for influencing the oil yield, profile and composition of leaf oils in Myrtaceae. The biochemical steps that influence oil yield in Myrtaceae probably occur in the steps of the pathway leading up to the synthesis of the terpene backbone. Qualitative differences in oil profiles are more likely to be due to variation in terpene synthases and terpene-modifying enzymes. Most of the information on molecular variation in terpene biosynthesis is based on the analysis of artificially derived mutants but Australian Myrtaceae can provide examples of the same mechanisms in an ecological context.

Phylogeny of Hibiscus and the Tribe Hibisceae (Malvaceae) Using Chloroplast DNA Sequences of ndhF and the rpl16 Intron

Abstract Circumscriptions of the genus Hibiscus and the tribe Hibisceae (Malvaceae) are based on morphological features that are not unique in the family. An examination of the literature regarding putatively ancestral morphological features revealed that Hibiscus and Hibisceae may be defined by shared ancestral features, and thus are unlikely to be monophyletic groups. These phylogenetic hypotheses were tested using two chloroplast DNA sequences (a coding region—ndhF, and a non-coding region—the rpl16 intron). Several genera usually placed in Hibisceae were found to occupy positions sister to the rest of the family, as was predicted from our reevaluation of their morphological features. Although the earliest divergences in the family were not resolved by chloroplast DNA topologies alone, several morphological features, when analysed in combination with ndhF, suggested a possible resolution of the basal polytomy. These early divergences are represented by extant genera with relatively restricted distributions, which all possess Australasian species that are sister to more widespread and diverse lineages. This suggests the novel hypothesis that eastern Gondwana may be the centre of origin of the family. The pollen fossil record is consistent with this possibility, but does not support it unambiguously. Unexpectedly the tribes Decaschistieae and Malvavisceae as well as other genera of Hibisceae nest within Hibiscus. Nomenclatural upheavals concerning Hibiscus, one of the worlds most popular horticultural plant genera, will be difficult to avoid. Communicating Editor: James F. Smith

Production of fertile hybrid germplasm with diploid Australian Gossypium species for cotton improvement

The 17 wild Australian Gossypium species are distant diploid relatives of the commercial tetraploid cottons, G. barbadense L. and G. hirsutum L. They interest cotton breeders as a source of terpenoid-aldehyde-free seeds, a trait only found in five Australian Gossypium species. They elicit further interest because some species grow near current and projected cotton growing areas in Australia and thus could serve as unintentional recipients of transgenes from genetically engineered cotton cultivars. The utility of the wild Australian Gossypium species in cotton breeding depends on the ability to generate fertile hybrids, and to the extent this is possible under glasshouse conditions, it allows predictions regarding the probability that fertile hybrids between the transgenic cottons and spatially associated populations of wild species will arise without human manipulation. The Australian Gossypium species fall into three morphologically and cytologically distinct groups designated the C, G, and K genomes, The G-genome species hybridize most readily with G. arboretum (a diploid A-genome cultivated cotton), while the C- and K-genome species are more compatible with G. hirsutum (a tetraploid AD-genome cultivated cotton). These intergenomic hybrids are sterile, and the chromosome complement of the hybrids must be doubled prior to backcrossing to G. hirsutum. The only exceptions were four G. hirsutum × K-genome triploids, which exhibited limited female fertility when backcrossed to G. hirsutum. Two of the three diploid species geographically associated with commercial cotton fields (G. australe F. Mueller & G. rotundifolium Fryxell, Craven & Stewart) failed to produce hybrid progeny when pollinated with G. hirsutum pollen; the third species (G. sturtianum J.H. Willis) produced only 5 sterile triploids from 25 pollinations. Thus, the probability that wild species could serve as recipients of transgenes is functionally zero, especially in conjunction with the profound prezygotic barriers that separate the cultivated tetraploid cottons from their wild Australian relatives. Eighteen new fertile synthetic polyploids and 23 self-fertile derivatives of two synthetic hexaploids were produced. Synthetic tetraploids require greater effort to backcross than do synthetic hexaploids. These fertile hybrids represent a new avenue of introgression of genes from wild Australian Gossypium species into commercial cotton cultivars, an avenue limited only by the level of recombination.