J. McD. Stewart

Molecular phylogeny of Gossypium species by DNA fingerprinting

Abstract Total genomic DNA from 31 available Gossypium species, three subspecies and one interspecific hybrid, were analysed to evaluate genetic diversity by RAPD, using 45 random decamer primers. A total of 579 amplified bands were observed, with 12.9 bands per primer, of which 99.8% were polymorphic. OPJ-17 produced the maximum number of fragments while the minimum number of fragments was produced with primer OPA-08. Cluster analysis by the unweighted paired group method of arithmetic means (UPGMA) showed six main clusters. Cluster ’A’ consisted of two species and one subspecies of the A-genome, with a 0.78–0.92 Nei’s similarity range. Cluster B, composed of all available tetraploid species and one interspecific hybrid, showed the same sister cluster. Nei’s similarity ranged from 0.69 to 0.84. The B-genome formed the UPGMA sister cluster to the E-genome species. Cluster ’C’ consisted of five Gossypium species of which three belong to the B-genome, with Nei’s similarity values of 0.81 to 0.86. Although there was considerable disagreement at lower infra-generic ranks, particularly among the D- genome (diploid New World species) and C-genome (diploid Australian species) species. The sole F-genome species Gossypium longicalyx was resolved as a sister group to the D-genome species. Gossypium herbaceum and G. herbaceum Africanum showed the maximum Nei’s similarity (0.93). Minimum similarity (0.29) was observed between Gossypium trilobum and Gossypium nelsonii. The average similarity among all studied species was 50%. The analysis revealed that the interspecific genetic relationship of several species is related to their centre of origin. As expected, most of the species have a wide genetic base range. The results also revealed the genetic relationships of the species Gossypium hirsutum to standard cultivated Gossypium barbadense, G. herbaceum and Gossypium arboreum. These results correspond well with previous reported results. The level of variation detected in closely related genotypes by RAPD analysis indicates that it may be a more efficient marker than morphological marker, isozyme and RFLP technology for the construction of genetic linkage maps.

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.

Comparative analysis of gene expression between CMS-D8 restored plants and normal non-restoring fertile plants in cotton by differential display

CMS-D8 and its restorer were developed by introducing the cytoplasm and nuclear gene Rf2from the wild diploid Gossypium trilobum (D8) into the cultivated tetraploid Upland cotton (Gossypium hirsutum). No information is available on how the Rf2 gene interacts with CMS-associated genes and how CMS-D8 cytoplasm affects nuclear gene expression. The objective of this study was to identify differentially expressed genes in anther tissues between the non-restoring fertile maintainer ARK8518 (rf2rf2) and its isogenic heterozygous D8 restorer line, ARK8518R (Rf2rf2) with D8 cytoplasm, by mRNA differential display (DD). Out of more than 3,000 DDRT-PCR bands amplified by 31 primer combinations from 12 anchor primers and 8 arbitrary decamer primers, approximately 100 bands were identified as being qualitatively differentially displayed. A total of 38 cDNA fragments including 12 preferentially expressed cDNA bands in anther were isolated, cloned and sequenced. Reverse northern blot analysis showed that only 4 genes, including genes encoding a Cys-3-His zinc finger protein and aminopeptidase, were up-regulated, while 22 genes, including genes for phosphoribosylanthranilate transferase (PAT), starch synthase (SS), 4-coumarate-CoA ligase, electron transporter, calnexin, arginine decarboxylase, and polyubiquitin, were down-regulated in the heterozygous restorer ARK8518R. The down-regulation of SS explains the lack of starch accumulation in sterile rf2 pollen grains in the heterozygous restored plants. The molecular mechanism of CMS and its restoration, specifically the possible roles of SS and PAT genes in relation to restoration of Rf2 to CMS-D8, are discussed. This investigation represents the first account of such an analysis in cotton.

MAGAININ 2 EFFECTS ON THE ULTRASTRUCTURE OF FIVE PLANT PATHOGENS

Magainin 2 from the African clawed frog is a member of a host defense peptide family with broad-spectrum antimicrobial activity related to its ef- fects on membrane integrity. This bioactive peptide was examined for its effects on the ultrastructure of the plant pathogens Thielaviopsis basicola, Rhizocton- ia solani, Fusarium oxysporum, Verticillium dahliae, and Pythium ultimum. Magainin 2 at 0.05 jig/jiL in potato dextrose broth in microtiter plate wells com- pletely inhibited all hyphal growth observable with a dissecting scope at 40 X, except for P. ultimum. These organisms were subsequently grown on potato dex- trose agar and the leading edge of each colony treat- ed with 1 pxg of magainin 2 in 10uL. Hyphal plugs from the treated area were taken at 12 and 24 h after treatment for transmission electron microscopic ex- amination. Observations on the ultrastructure of ma- gainin 2-treated hyphae revealed changes in the size and shape of the mitochondria, degradation of the mitochondrial and cytoplasmic matrices, reduction in the number of ribosomes and vacuolization of cy- toplasm after 12 h of treatment. The structural deg- radation of the cells was more severe after 24 h treat- ment. Magainin 2 at the concentration tested had no significant effect on the growth of P ultimum, per- haps due to the disparate metabolic chemistry of the Oomycetes compared to the other organisms. The low molecular weight and bioactivity of magainin 2 makes it a candidate for additional investigation into its use in bioengineered host resistance.

Ultrastructural localization of ATPase activity in cotton fiber during elongation

SummaryThe ultrastructural distribution of potassium chloride stimulated adenosine triphosphatase activity was investigated in the outer integument of a linted cultivar of cotton and a lintless (naked seed) mutant from one day preanthesis to eight days postanthesis by using a heavy metal simultaneous capture reaction technique. No enzyme activity other than in mitochondria was observed in the lintless mutant. In the linted cultivar no ATP-specific enzyme activity was seen in non-elongating epidermal cells, subepidermal cells of the outer integuments or any controls. As fiber initials started elongating, enzyme activity gradually appeared on the tonoplasts of enlarging vacuoles. Heavier lead phosphate deposits were observed on the membrane of small vacuoles compared to the tonoplast. This activity continued at least to eight days postanthesis. The enzyme inhibitor, N,N-dicyclohexylcarbodiimide inhibited, while KCl stimulated, tonoplast ATPase activity. The gradual increase of ATPase activity on the tonoplast of expanding fibers, but not on the tonoplasts of non-fiber cells, suggests the active transport of osmotically active compounds, presumably potassium and malate, into the vacuoles of expanding fibers. Fusion of smaller vacuoles with the large central vacuole indicates that these structures contribute additional membrane components along with their enzyme activity to the tonoplast of expanding fibers. The occurrence of ATPase activity, of ER-derived vesicular structures, and the organized pattern of deposition of these structures on the tonoplast indicate ER-originated ATPase activity. This study supports the theory of osmoregulation in cotton fiber where ATPase provides the energy for active accumulation of osmotically active compounds, (K+, malate) into the vacuoles, thereby generating and maintaining the turgor pressure required for fiber expansion.

Evaluation of Gossypium arboreum L. Germplasm for resistance to thrips

SummaryAs part of a project to assess the U.S. Asiatic Cotton Germplasm Collection as a source of genes for pest resistance, forty-three accessions of Gossypium arboreum L. were evaluated for resistance to thrips. Thrips, Thysanoptera spp., are a minor, but widespread pest on cotton, G. hirsutum L. and G. barbadense L. Accessions were planted in free-choice field tests in 1988, 1989, and 1990 at the Cotton Branch Experiment Station, Marianna, Arkansas. Damage ratings, based on a scale of 0 (no damage) to 7 (severe damage), were made approximately one month after planting. Relative score was computed by dividing the rating of each accession by the rating of the reference, G. hirsutum ‘Stoneville 506’. The relative score averaged over all G. arboreum accessions by year varied with the year, but was less than 100 (relative score for Stoneville 506) in each case. When data were combined for all three years, twenty-five accessions were significantly better than Stoneville 506. Introgression of the better accessions into G. hirsutum is now in progress.

Identification of differentially expressed genes associated with semigamy in Pima cotton (Gossypium barbadense L.) through comparative microarray analysis

BackgroundSemigamy in cotton is a type of facultative apomixis controlled by an incompletely dominant autosomal gene (Se). During semigamy, the sperm and egg cells undergo cellular fusion, but the sperm and egg nucleus fail to fuse in the embryo sac, giving rise to diploid, haploid, or chimeric embryos composed of sectors of paternal and maternal origin. In this study we sought to identify differentially expressed genes related to the semigamy genotype by implementing a comparative microarray analysis of anthers and ovules between a non-semigametic Pima S-1 cotton and its doubled haploid natural isogenic mutant semigametic 57-4. Selected differentially expressed genes identified by the microarray results were then confirmed using quantitative reverse transcription PCR (qRT-PCR).ResultsThe comparative analysis between isogenic 57-4 and Pima S-1 identified 284 genes in anthers and 1,864 genes in ovules as being differentially expressed in the semigametic genotype 57-4. Based on gene functions, 127 differentially expressed genes were common to both semigametic anthers and ovules, with 115 being consistently differentially expressed in both tissues. Nine of those genes were selected for qRT-PCR analysis, seven of which were confirmed. Furthermore, several well characterized metabolic pathways including glycolysis/gluconeogenesis, carbon fixation in photosynthetic organisms, sesquiterpenoid biosynthesis, and the biosynthesis of and response to plant hormones were shown to be affected by differentially expressed genes in the semigametic tissues.ConclusionAs the first report using microarray analysis, several important metabolic pathways affected by differentially expressed genes in the semigametic cotton genotype have been identified and described in detail. While these genes are unlikely to be the semigamy gene itself, the effects associated with expression changes in those genes do mimic phenotypic traits observed in semigametic plants. A more in-depth analysis of semigamy is necessary to understand its expression and regulation at the genetic and molecular level.

RFLP analysis of mitochondrial DNA in two cytoplasmic male sterility systems (CMS-D2 and CMS-D8) of cotton

Cytoplasmic male sterility (CMS) in higher plants is a maternally inherited trait and CMS-associated genes are known to be located in the mitochondrial genome. However, CMS-inducing genes in CMS-D2 and CMS-D8 of Upland cotton (Gossypium hirsutum L., AD1) are currently unknown. The objective of this study was to identify potential candidate DNA or gene sequences for CMS-D2 and CMS-D8 through restriction fragment length polymorphism (RFLP) analysis. Seven mtDNA gene probes and five restriction enzymes were first used to compare D2 (from G. harknessii Brandegee) and AD1 cytoplasms. With cox1, cox2, and atp1 as probes, RFLP polymorphisms were detected with one or more restriction enzyme digestions. The most notable difference was an additional fragment in the normal AD1 cytoplasm detected by cox2 in digests of three enzymes, and by cox1 and atp1 in digests with PstI. The RFLP analysis was then conducted among CMS-D2, CMS-D8 (from G. trilobum (DC.) Skovst.), and AD1 cytoplasms. Two probes from maize, atp1 and atp6, detected polymorphism among the different cytoplasmic lines. However, no difference in RFLP patterns was noted between male sterile (A) and restorer (R) lines with the D2 or D8 cytoplasm, indicating that the presence of the D2 or D8 restorer gene does not affect mtDNA organization in Upland cotton. The results demonstrate that RFLP using atp1 and atp6 as probes can distinguish the three cytoplasms. The atp1 and atp6 in CMS-D8 and these two genes together with cox1 and cox2 in CMS-D2 could be the candidates of CMS-associated genes in the mitochondrial genome, providing information for further molecular studies and developing PCR-based markers for the CMS cytoplasms in breeding. This research represents the first work using RFLP to analyze the genetic basis of CMS in cotton.

Response of A-genome cotton germplasm to the seedling disease pathogens, Rhizoctonia solani and Pythium ultimum

SummaryApproximately two hundred A-genome cotton (Gossypium arboreum L. and G. herbaceum L.) accessions were evaluated for resistance to the seedling disease pathogens Rhizoctonia solani Kühn [Thanatephorus cucumeris (A. B. Frank) Donk], and Pythium ultimum Trow. Susceptibility rating was based on a scale of 1 to 6 where 1 = no symptoms and 6 = dead. Variation was found among A-genome accessions in response to R. solani and P. ultimum, but none were highly resistant to these pathogens. As a group, the A-genome cottons were more susceptible to the pathogens than the G. hirsutum control.

Identification of Differentially Expressed Genes in Semigametic Pima Cotton by Differential Display

Haploid seed-producing semigamy in cotton is controlled by a single incompletely dominant gene (Se). However, its molecular mechanism is currently poorly understood. In this study, the mRNA differential display (DD) was employed to compare gene expression between non-semigametic Pima S-1 (Gossypium barbadense) and its naturally occurring semigametic mutant 57–4. Of the 70 DD cDNA fragments isolated from a total of 3,000 cDNA fragments, some exhibited semigamy-specific expression across anthers and/or ovules. Of the 61 cDNA fragments cloned and sequenced, nine showed high homology to genes encoding for α-tubulin, SPK1, Rab11, sucrose synthase, isocitrate lyase, phosphoethanolamine N-methyltransferase, phospholipase D, MADS 7, and WRKY 2. From cDNA–single-strand conformational polymorphism using primers designed from DD sequences, qualitative expression differences in six genes between the semigametic and non-semigametic genotypes were validated. However, a primer pair designed from an α-tubulin gene sequence yielded PCR products with quantitative difference in band intensity, which is linked to the semigamy gene at a recombination frequency of 0.316 in a (57–4 × Pima S-1) × Pima S-1 BC1F1 population. Its expression in the semigametic 57–4 anthers was suppressed by 2-fold as compared to that in Pima S-1 using a quantitative reverse transcription PCR analysis. This study identified several novel genes that were missed in prior microarray analysis, and their potential roles with regards to reproductive biology are discussed.