Cost-effective detection of genome-wide signatures for 2,4-D herbicide resistance adaptation in red clover.

Herbicide resistance is a recurrent evolutionary event that has been reported across many species and for all major herbicide modes of action. The synthetic auxinic herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) has been widely used since the 1940s, however the genetic variation underlying naturally evolving resistance remains largely unknown. In this study, we used populations of the forage legume crop red clover (Trifolium pratense L.) that were recurrently selected for 2,4-D resistance to detect genome-wide signatures of adaptation. Four susceptible and six derived resistant populations were sequenced using a less costly approach by combining targeted sequencing (Capture-Seq) with pooled individuals (Pool-Seq). Genomic signatures of selection were identified using: (i) pairwise allele frequency differences; (ii) genome scan for overly differentiated loci; and (iii) genome-wide association. Fifty significant SNPs were consistently detected, most located in a single chromosome, which can be useful for marker assisted selection. Additionally, we searched for candidate genes at these genomic regions to gain insights into potential molecular mechanisms underlying 2,4-D resistance. Among the predicted functions of candidate genes, we found some related to the auxin metabolism, response to oxidative stress, and detoxification, which are also promising for further functional validation studies.

Clover, red (Trifolium pratense).

Genetic modification of plants by the insertion of transgenes can be a powerful experimental approach to answer basic questions about gene product function. This technology can also be used to make improved crop varieties for use in the field. To apply this powerful tool to red clover, an important forage legume, a population of red clover with high potential for regeneration in tissue culture has been developed. Here we provide a detailed procedure for Agrobacterium-mediated transformation of genotypes derived from this regenerable population. We have successfully used this methodology to express ß-glucuronidase (GUS) reporter genes as well as for hairpin RNA-mediated silencing of endogenous genes for polyphenol oxidase and a transferase crucial in phaselic acid accumulation.

Apomixis and ploidy barrier suppress pollen-mediated gene flow in field grown transgenic turf and forage grass (Paspalum notatum Flüggé).

Bahiagrass (Paspalum notatum Flüggé) is the predominant forage grass in the southeastern US. The commercially important bahiagrass cultivar 'Argentine' is preferred for genetic transformation over sexual diploid cytotypes, since it produces uniform seed progeny through apomixis. Pseudogamous apomictic seed production in Argentine bahiagrass may contribute to transgene confinement. It is characterized by embryo development which is independent of fertilization of the egg cell, but requires fertilization with compatible pollen to produce the endosperm. Pollen-mediated gene transfer from transgenic, glufosinate-resistant apomictic bahiagrass as pollen donor at close proximity (0.5-3.5 m) with non-transgenic sexual or apomictic bahiagrass cultivars as pollen receptors was evaluated under field conditions. Hybridization frequency was evaluated by glufosinate herbicide resistance in >23,300 seedlings derived from open-pollinated (OP) pollen receptor plants. Average gene transfer between transgenic apomictic, tetraploid and sexual diploid bahiagrass was 0.03%. Herbicide-resistant hybrids confirmed by immuno-chromatographic detection of the PAT protein displayed a single copy bar gene identical to the pollen parent. Hybrids resulting from diploid pollen receptors were confirmed as triploids or aneu-triploids with significantly reduced vigor and seed set as compared to the parents. Transmission of transgenes to sexual bahiagrass is severely restricted by the ploidy difference between tetraploid apomicts and diploid sexual bahiagrass. Average gene transfer between transgenic apomictic tetraploid and non-transgenic, apomictic tetraploid bahiagrass was 0.17%, confirming a very low frequency of amphimixis in apomictic bahiagrass cultivars. While not providing complete transgene containment, gene transfer between transgenic apomictic and non-transgenic bahiagrass occurs at a much lower frequency than reported for other cross-pollinating or facultative apomictic grasses.

Risk assessment of transgenic apomictic tetraploid bahiagrass, cytogenetics, breeding behavior and performance of intra-specific hybrids.

Pollen-mediated gene transfer from stress tolerant or herbicide-resistant transgenic plants may cause environmental or agronomic problems. Apomictic seed production found in some bahiagrass cultivars may serve as a natural transgene containment system. Under greenhouse conditions, the average gene transfer frequency from an herbicide-resistant apomictic tetraploid to a population of sexual diploid bahiagrass genotypes or apomictic tetraploid bahiagrass was 0.16% when the transgenic pollen donor was placed at 0.5-1.5 m distance from the non-transgenic pollen receptors. The herbicide-resistant hybrids were characterized for transgene integration, expression and ploidy, by Southern blot analysis, immuno-chromatography and flow cytometry, respectively. Hybrids resulting from open pollination of non-transgenic diploid female plants with transgenic tetraploid male plants were triploids or near-triploids, with 2n = 26-34. These hybrids displayed a wide range of phenotypic variability, including some non-persistent or non-flowering dwarf-type hybrids with good vigor, or hybrids with vegetative growth similar to non-transgenic plants, but with significantly reduced seed set. Non-flowering aneu-triploids with good vigor/field performance will provide the highest level of transgene containment. Embryo sac analysis of pollinated spikelets confirmed a high proportion of aborted ovules. An apospory-linked RFLP marker was detected in 13 of the 15 near-triploid hybrids. All flowering aneuploid hybrids displayed significantly reduced seed set, and none of the sexual near-triploid hybrids produced any seeds. All tetraploid gene transfer events carried the apospory-linked RFLP marker, suggesting that despite the presence of the aposporus locus, a low degree of sexuality co-exists in apomictic tetraploid cultivars. Thus, tetraploid apomictic bahiagrass does not provide complete transgene containment, although intra-specific gene transfer is drastically reduced compared to sexually reproducing perennial grasses.

Genetics of growth habit and development of new coleus (Solenostemon scutellarioides (L.) Codd) varieties with trailing habit and bright color.

A high level of genetic variability for growth habit types is observed in tetraploid, cultivated coleus (Solenostemon scutellarioides (L.) Codd). Very few cultivars with trailing habit exist, and those that are available generally have dark green or purple foliage color. Coleus with trailing growth habit is desirable as it increases its utility for use in hanging baskets, mixed containers, and as ground cover. There is a lack of published information on the genetic mechanism controlling growth habit and the development of new trailing types with orange colors. Two commercial cultivars, "Red Trailing Queen" (RQ) with trailing habit and "Sedona" (S) with upright stature, were selfed and crossed (RQ x S) to produce self and F(1) populations. F(2) populations were produced by selfing plants in the F(1) population. For each population analyzed, growth habit was rated on a visual 1-5 phenotypic scale, where 1 = upright, 2 = semi-upright, 3 = prostrate, 4 = semitrailing, and 5 = trailing. Genotypes were assigned to each phenotype, assuming that upright was dominant to trailing. In this study, growth habit was observed to be controlled by a single gene (U) with additive effects, with upright growth habit designated with a UUUU genotype and trailing growth habit designated with a uuuu genotype. In addition, foliage color was rated on a visual 1-5 phenotypic scale, and purple foliage color was found to be dominant to yellow-orange color. Several new coleus selections with trailing growth habit and orange foliage color were successfully developed.

Genetic diversity and DNA content of three South American and three Eurasiatic Trifolium species

Six species of Trifolium (T. polymorphum Poir., T. riograndense Burkart, T. argentinense Speg., T. medium L., T. pratense L. and T. repens L.) were analyzed using inter-simple sequence repeats (ISSR) markers. Six selected primers generated 186 polymerase chain reaction (PCR) products exploring 112 loci in 34 genotypes analyzed with molecular sizes ranging from 200 to 1300 bp. These primers were able to discriminate among and within species, with the PCR products being on average 41.6 percent species-specific and 59.9 percent polymorphic at the within species level. Nuclear DNA content was determined by flow cytometry and revealed variation among species. The 1Cx genome size values were calculated and were found to range from 0.46 pg (T. pratense) to 0.96 pg (T. polymorphum). Genome size values of South American species were higher than those of Eurasiatic origin. The analyses of the molecular data grouped the six species in agreement with their geographical origin and clearly differentiate T. polymorphum from T. argentinense. The Eurasiatic group showed the highest average of species-specific bands (45.3 percent) and the South American group exhibited the highest amount of total bands (59.7). The highest level of intra-species polymorphisms was detected in T. argentinense (92.9 percent), followed by T. medium (89.5 percent).

Red clover (Trifolium pratense).

Genetic modification of plants by the insertion of transgenes can be a powerful experimental approach to answer basic questions about gene product function. This technology can also be used to make improved crop varieties for use in the field. To apply this powerful tool to red clover, an important forage legume, a population of red clover with a high potential for regeneration in tissue culture has been developed. Here we provide a detailed procedure for Agrobacterium-mediated transformation of genotypes derived from this regenerable population. We have successfully used this methodology to express a beta-glucuronidase (GUS) reporter gene and to silence an endogenous polyphenol oxidase gene in red clover.