Prunus, which includes peach/nectarine, almond, apricot, cherry, and plum, is a large and economically important genus in the family Rosaceae. The size and long generation time of these tree crops have hampered improvement through classical breeding and long-term selection programs. With the advent of DNA-based molecular diagnostics, an exciting era in germplasm improvement has dawned. Efforts are underway, notably in the United States (e.g., California, Michigan, North Carolina, and South Carolina) and the European Community (e.g., England, France, Italy, and Spain), to apply the tools of molecular mapping and marker-assisted selection to this important genus. The objective of these projects is to develop molecular linkage maps of sufficient marker density to tag phenotypic trait loci of agronomic importance. These include traits controlled by single genes (e.g., flower color, compatibility, flesh color, pest resistance), as well as more-complex, quantitative traits (e.g., cold hardiness, tree architecture, sugar content). An immediate outcome of these mapping efforts has been the development of DNA “fingerprints,” allowing for the discrimination of cultivars—both scion and rootstock. The maps will be used by breeders and molecular biologists to monitor gene introgression from wild species into elite lines, for marker-assisted selection of desired trait combinations, and for map-based cloning of specific genes. The molecular markers used in these mapping projects include RFLPs, RAPDs, and microsatellites. Each has their appropriate applications and advantages depending upon the resources at hand and the project's specific goals.
Linghe Zeng and Wm. Vance Baird
Dinitroaniline herbicides exert their phytotoxic effect by interfering with tubulin dimer polymerization; thus, these and other anti-mitotic drugs destabilize cytoplasmic and spindle microtubules. The dinitroanilines are particularly effective on monocotyledonous species. A naturally occurring mutant of goosegrass [Eleusine indica (L.) Gaertn.], resistant (R) to the DNHs, and the widely distributed susceptible, wild-type (S) have been collected from a number of agricultural sites throughout the southeastern U.S. Pairs of these accessions were cross-pollinated to create F1 individuals, from which F2 and F3 generations were developed through natural self-pollination. Analysis of the dinitroaniline herbicide response phenotype (DRP) has shown the F1s to be susceptible, and the F2 and F3 to be segregating 3:1 for susceptibility and resistance, respectively (i.e., 3S:1R). This genetic data is consistent with the DRP being encoded by a single, nuclear locus. Random amplified polymorphic DNA (RAPD) analysis of a segregating F2 population (N = 60), which identified 32 linked and 33 unlinked molecular markers, supports this hypothesis of simple Mendelian inheritance. Furthermore, this RAPD analysis coupled with restriction fragment length polymorphism (RFLP) analysis, localized the DRP locus to a single chromosomal region and identified two RAPD-markers, and at least one RFLP-marker, flanking the DRP locus. This information provides a starting point for map-based (i.e., positional) cloning of the resistance (DRP r) and susceptibility (DRP s) alleles.
Xingping Zhang, Bill Rhodes, Vance Baird, and Halina Skorupska
A spontaneous watermelon mutant, previously named branch less, was re-evaluated in this study. The mutant watermelon plants from genetic stock Bl-91 and derived from F2 and BC1 populations, did not produce tendrils under field or greenhouse conditions. The mutants stopped producing branches after the fifth or sixth node. Leaf shape changed during development of the mutants. Early leaves were normal, but later leaves had fewer and fewer lobes, finally becoming triangular toward the end of the shoot. The most distinct effect of the mutant gene was to convert vegetative meristems into floral meristems; tendrils and axillary buds were replaced by flowers at the node. The mutant plants were determinate. A grafting experiment showed that the rootstock had no effect on the mutant phenotype. Genetic analysis of F1, F2, and BC1 populations suggested that the mutant is inherited as a single, recessive nuclear gene. Based on the phenotype, a new name is suggested for this mutant: tendrilless, with a new gene symbol tl.
W. Vance Baird, Agnes S. Estager, and John K. Wells
Using laser flow cytometry, nuclear DNA amounts were estimated for 12 Prunus species, representing three subgenera [Prunophora (Prunus), Amygdalus, and Cerasus (Lithocerasus)], two interspecific hybrids, four cultivars, and a synthetic polyploid series of peach consisting of haploids, diploids, triploids, and tetraploids (periclinal cytochimeras). Peach nuclear DNA content ranged from 0.30 pg for the haploid nuclei to 1.23 pg for the tetraploid nuclei. The diploid genome of peach is relatively small and was estimated to be 0.60±0.03 pg (or 5.8×108 nucleotide base pairs). The polyploid series represented the expected arithmetic progression, as genome size positively correlated with ploidy level (i.e., DNA content was proportional to chromosome number). The DNA content for the 12 diploid species and two interspecific diploid hybrids ranged from 0.57 to 0.79 pg. Genome size estimates were verified independently by Southern blot analysis, using restriction fragment length polymorphism clones as gene-copy equivalents. Thus, a relatively small and stable nuclear genome typifies the Prunus species investigated, consistent with their low, basic chromosome number (× = 8).
L. Zeng, K. S. Mysore, and Wm. Vance Baird
Goosegrass (Poaceae) is a member of a small but economically important genus, containing approximately 10 species. Goosegrass is a noxious weed in cotton, soybean and turf production, but was effectively controlled by the application of dinitroaniline herbicides (DNH). However, in the early 1970's two DNH tolerant biotypes were discovered. The highly resistant (R-) biotype is now found in many areas in the southeastern US, while the intermediately resistant (I-) biotype is known only from SC. We have investigated the inheritance and expression of resistance, using radicle/root growth bioassays, in F2 progeny derived from F1 hybrids (created by outcrossing this autogamous weed). The hybrid nature of presumptive F1 seedlings was confirmed by isoenzyme analysis. The DNH's effect their herbicidal action by disrupting the assembly of tubulin dimers into microtubules (MTs). The R-biotype has been shown to possess MTs that are hyperstable in the presence of the herbicide. Mutation(s) in a tubulin protein would be manifested in the gene and might be detectable at the nucleic acid level. We found that the alpha-, beta- and gamma-tubulins are encoded by multigene families (∼ 5.7 and 8 members, respectively). DNA polymorphisms were detected, but were not strictly correlated with biotype. Therefore, the differences in herbicide response phenotype cannot be attributed to large deletions and/or insertions in a tubulin gene(s).
John Wells, Jiyu Yan, Melissa Riley, Suresh Samala, and Vance Baird
Bermudagrass (Cynodon dactylon) cultivars may exhibit increased tolerance to cold following periods of exposure to moderately cold temperature (i.e., acclimation). We are evaluating biochemical changes and the regulation of gene expression in two cultivars—'Midiron' and U-3—during this acclimation period. Total membrane lipid fatty acids per unit of total lipids (MLFA/TL; μg·mg–1) increased in crowns over the 4-week exposure to chilling temperatures (8C day/2C night). Of the fatty acids comprising 95% of total MFLA, concentrations of short-chain and saturated FAs declined significantly while unsaturated longer-chain FA concentration increased. As a result, the double bond index (percent of each FA x number of double bonds in the FA) increased during the period of low temperature exposure, indicative of increasing membrane fluidity. Changes in MFLA were evident as early as 4 days following exposure to chilling temperatures. Identification of mRNA species expressed in response to low temperature utilized differential display-PCR. Initial screening with paired T11N1N 2 3'-anchor and 5'-random decamer primers has identified transcripts differentially expressed as early as 23 h post-exposure and was maintained for at least an additional 36 h. Isolation, reamplification, and cloning of these identified PCR products is in progress.
J. Kevin Parris, Thomas G. Ranney, Halina T. Knap, and W. Vance Baird
The genus Magnolia includes over 250 species that range in ploidy level from diploid to hexaploid. Although there is basic information on ploidy levels of various species, sampling has been limited and little information on specific cultivars and hybrids is available. The objective of this research was to determine relative genome sizes and relationships to ploidy levels among a diverse collection of species, hybrids, and cultivars using flow cytometry. Nuclei were extracted, stained with 4′, 6-diamidino-2-phenylindole (DAPI), and analyzed using a flow cytometer. Relative genome sizes were determined using Pisum sativum as the reference genome. Genome size was calibrated with ploidy level for species with documented chromosome numbers. Relative genome size for a given ploidy level varied significantly among most taxonomic sections indicating these groups have undergone considerable genomic divergence. These data also indicate it is desirable to calibrate ploidy level with relative genome size for each section separately. Within a section, relative 2C genome sizes, for a given ploidy level, had narrow ranges and could be used to clearly distinguish between euploid levels. Genome size estimates, determined with DAPI or propidium iodide fluorochromes, varied (by 0% to 14%) as a function of species and base pair (bp) composition. Both methods were suitable for determining euploid level. Base pair composition of representative Magnolia species ranged from 61.6% to 63.91% AT. Genome sizes and ploidy levels are presented for a broad range of species and hybrids within genus Magnolia. This information also provides further insight into reproductive biology, substantiation of numerous hybrids and induced polyploids, and comparison of methods for determining genome size that will help facilitate the development of improved hybrids in the future.
David J. Weston*, David J. Weston, Ginger A. Swire-Clark, and Wm. Vance Baird
Rubisco, the primary enzyme governing carbon assimilation, is dependent upon Rubisco activase. The heat sensitivity of activase, including its expression and thermal stability, varies among species and is considered a key component governing photosynthetic performance in response to moderate heat stress (32-35 °C). However, the Rubsico-Rubsico activase association has yet to be examined among woody plants or varieties within a species, the understanding of which will assist cultivar improvement strategies. Using molecular and physiological techniques to study the role of activase in thermal regulation of photosynthesis, we found that net photosynthesis decreased in Acer rubrum L. `Northwood' at 31°C, whereas the southern variety, A. rubrum `Florida Flame', maintained optimal assimilation rates up to 36 °C. Additionally, the maximal carboxylation rate of Rubisco (Vcmax) at 35 °C was 31.7% lower for Northwood in comparison to Florida Flame. The cloned activase sequences from both cultivars show 97% nucleotide homology and 98% amino acid identity, indicating the potential for similar protein product formation and function. Interestingly, sequence analysis indicates that both cultivars produce at least two isoforms of activase derived from alternative transcript splicing. We will discuss activase mRNA processing and protein isoform abundance in relation to Rubisco kinetic properties as a function of heat tolerance in these two thermally contrasting woody plant genotypes.
Vance Baird, L. Belthoff, R. Ballard, R. Scorza, A. Callahan, R. Monet, and A. Abbott
Flow cytometric analysis, of leaf nuclei from three cultivars, was used to estimate the DNA content of peach (∼0.61 pg or ∼0.59 × 109 bp/diploid nucleus; 2x=16), and ndicated that the peach genome is only slightly larger than that of Arabidopsis. This value was indirectly confirmed by measurements of nuclei from haploid, triploid and “tetraploid” (cytochimera) peach accessions. cDNA and genomic clones have been used to determine the level of polymorphism among various peach cultivars and related species. Overall, ∼33% of the clones detected polymorphic loci. As expected, the highest level of polymorphism was found in interspecific hybrids (∼50%); whereas in intraspecific populations, only 1 in 5 genomic clones, and 1 in 3 cDNA clones were able to detect polymorphisms (RFLPs). These clones, as well as RAPD primers, are being used to construct a genetic linkage map by analyzing their segregation in 3 intraspecific peach populations (an Fl from France and two F2s from the U.S.). Taken together, these populations are segregating for 12 Mendelian traits and a number of quantitative traits. Our results have enabled us to identify a number of linkage groups, some composed of both molecular and phenotypic markers. The current structure of the peach map is reported.
Elisha Otieno Gogo, Mwanarusi Saidi, Jacob Mugwa Ochieng, Thibaud Martin, Vance Baird, and Mathieu Ngouajio
French bean [Phaseolus vulgaris (L.)] is among the leading export vegetable in Africa, mostly produced by small-scale farmers. Unfavorable environmental conditions and heavy infestations by insect pests are among the major constraints limiting production of the crop. Most French bean producers grow their crop in open fields outdoors subject to harsh environmental conditions and repeatedly spray insecticides in a bid to realize high yield. This has led to rejection of some of the produce at the export market as a result of stringent limits on maximum residue levels. Two trials were conducted at the Horticulture Research and Teaching Field, Egerton University, Kenya, to evaluate the potential of using agricultural nets (herein referred to as agronets) to improve the microclimate, reduce pest infestation, and increase the yield and quality of French bean. A randomized complete block design with five replications was used. French bean seeds were direct-seeded, sprayed with an alpha-cypermethrin-based insecticide (control), covered with a treated agronet (0.9 mm × 0.7 mm average pore size made of 100 denier yarn knitted into a mesh impregnated with alpha-cypermethrin), or covered with an untreated-agronet (0.9 mm × 0.7 mm average pore size made of 100 denier yarn knitted into a mesh not impregnated with insecticide). Alpha-cypermethrin and agronets were manufactured by Tagros Chemicals (India) and A to Z Textile Mills (Tanzania), respectively. Covering French bean with the agronets modified the microclimate of the growing crop with air temperature increased by ≈10%, relative humidity by 4%, and soil moisture by 20%, whereas photosynthetic active radiation (PAR) and daily light integral (DLI) were decreased by ≈1% and 11.5%, respectively. Populations of silverleaf whitefly [Bemisia tabaci (Gennadius)] and black bean aphids [Aphis fabae (Scopoli)] were reduced under agronet covers as contrasted with control plots. Furthermore, populations of both pests were reduced on French bean grown under impregnated agronets compared with untreated agronets, but only on three of the five sampling dates [30, 44, and 72 days after planting (DAP)] for silver leaf whitefly or at only one of the five sampling dates (30 DAP) for black bean aphid. Covering French bean with agronets advanced seedling emergence by 2 days and increased seedling emergence over 90% compared with control plots. French bean plants covered with both agronet treatments had faster development, better pod yield, and quality compared with the uncovered plants. These findings demonstrate the potential of agronets in improving French bean performance while minimizing the number of insecticide sprays within the crop cycle, which could lead to less rejection of produce in the export market and improved environmental quality.