Jennifer L. Hansen

Using Real-Time PCR to Determine Transgene Copy Number in Wheat

Transgene copy number is usually determined by means of Southern blot analysis which can be time consuming and laborious. In this study, quantitative real-time PCR was developed to determine transgene copy number in transgenic wheat. A conserved wheat housekeeping gene,puroindoline-b, was used as an internal control to calculate transgene copy number. Estimated copy number in transgenic lines using real-time quantitative PCR was correlated with actual copy number based on Southern blot analysis. Real-time PCR can analyze hundreds of samples in a day, making it an efficient method for estimating copy number in transgenic wheat.

The fertility of wheat × jointed goatgrass hybrid and its backcross progenies

Abstract The spontaneous flow of genes from wheat to jointed goatgrass is of great concern to breeders intending to release herbicide-resistant wheat. The objectives of this research were to study how genes could flow from wheat to jointed goatgrass through crossing and backcrossing between these two species and, based on this knowledge, to propose possible ways to minimize the chance of gene flow between them. Results showed that the wheat × jointed goatgrass hybrid can only serve as a female parent to produce the BC1 generation. The BC1 generation was found to have 1.8% male fertility and 4.4% female fertility, indicating that it could serve as either the male or female parent to produce a BC2 generation. The fertility of the resultant BC2 generation further increased. The average male, female, and self-fertility was 8.9, 18.0, and 6.9%, respectively. After the BC2 generation, the backcross progeny has three possible ways to reproduce: to pollinate jointed goatgrass, to be pollinated by jointed goatgrass, or to pollinate itself. Restoration of the chromosome number of jointed goatgrass continues as the BC2 generation is selfed, but some plants can contain an alien chromosome over generations. The possible ways to reduce the chance of gene flow between these two species are (1) prevent the production of hybrids, (2) prevent the production of the BC1 generation, and (3) put a herbicide-resistant gene on the A- or B-genome of wheat. Nomenclature:Jointed goatgrass; Aegilops cylindrica Host AEGCY; wheat; Triticum aestivum L.

Seed production on Triticum aestivum by Aegilops cylindrica hybrids in the field

Abstract Field experiments were conducted to determine if seeds would be produced on Triticum aestivum by Aegilops cylindrica hybrids in the field and, if it were, to determine the viability of the seeds produced. One, five, or 10 hybrids were planted into varying proportions of A. cylindrica and T. aestivum in a replacement series design. Percent seed set ranged from 0 to 5.5% in 1996 and from 0 to 9.2% in 1997. Seeds were set in all treatments. The average seed set was 2.3% in 1996 and 3.8% in 1997. No differences in seed set were found among treatments. The seeds produced were separated according to seed condition, either full or shriveled, and tested for germination. The germination of the seeds produced on the hybrids was not significantly different between years. The average germination for full seeds was 94% in both years and 79 and 84% for shriveled seeds in 1995 and 1996, respectively. Greenhouse studies were conducted to evaluate the rate of self-fertility of the BC1 generation and to identify morphological characteristics that could be used to identify the probable pollen donor parent and to predict the occurrence of seed set. In 1997 4.1% and in 1998 2.1% of BC1 plants set seeds. The average seed set was 0.3% in 1997 and 0.06% in 1998. It was not possible, using any morphological characteristic measured, to determine the identity of the parent serving as the pollen donor in the previous generation or to predict the occurrence of seed set in the BC1 generation. This is the first reported study to show that hybrids between T. aestivum and A. cylindrica have the ability, although limited, to backcross under field conditions and set seeds. Furthermore, the seeds produced are viable and will germinate and produce plants. With the millions of hectares of T. aestivum infested with A. cylindrica, even the limited ability to backcross is of concern for the movement of a herbicide-resistance gene. Nomenclature: Aegilops cylindrica Host AEGCY, jointed goatgrass; Triticum aestivum L., wheat.

Introgression of an imidazolinone-resistance gene from winter wheat (Triticum aestivum L.) into jointed goatgrass (Aegilops cylindrica Host).

Imidazolinone-resistant winter wheat (Triticum aestivum L.) is being commercialized in the USA. This technology allows wheat growers to selectively control jointed goatgrass (Aegilops cylindrica Host), a weed that is especially problematic because of its close genetic relationship with wheat. However, the potential movement of the imidazolinone-resistance gene from winter wheat to jointed goatgrass is a concern. Winter wheat and jointed goatgrass have the D genome in common and can hybridize and backcross under natural field conditions. Since the imidazolinone-resistance gene (Imi1) is located on the D genome, it is possible for resistance to be transferred to jointed goatgrass via hybridization and backcrossing. To study the potential for gene movement, BC2S2 plants were produced artificially using imidazolinone-resistant winter wheat (cv. FS-4) as the female parent and a native jointed goatgrass collection as the male recurrent parent. FS-4, the jointed goatgrass collection, and 18 randomly selected BC2S2 populations were treated with imazamox. The percentage of survival was 100% for the FS-4, 0% for the jointed goatgrass collection and 6 BC2S2 populations, 40% or less for 2 BC2S2 populations, and 50% or greater for the remaining 10 BC2S2 populations. Chromosome counts in BC2S3 plants showed a restoration of the chromosome number of jointed goatgrass, with four out of four plants examined having 28 chromosomes. Sequencing of AHASL1D in BC2S3 plants derived from BC2S2-6 revealed the sexual transmission of Imi1 from FS-4 to jointed goatgrass. Imi1 conferred resistance to the imidazolinone herbicide imazamox, as shown by the in vitro assay for acetohydroxyacid synthase (AHAS) activity.

Quantitative trait loci analysis for resistance to Cephalosporium stripe, a vascular wilt disease of wheat.

Cephalosporium stripe, caused by Cephalosporium gramineum, can cause severe loss of wheat (Triticum aestivum L.) yield and grain quality and can be an important factor limiting adoption of conservation tillage practices. Selecting for resistance to Cephalosporium stripe is problematic; however, as optimum conditions for disease do not occur annually under natural conditions, inoculum levels can be spatially heterogeneous, and little is known about the inheritance of resistance. A population of 268 recombinant inbred lines (RILs) derived from a cross between two wheat cultivars was characterized using field screening and molecular markers to investigate the inheritance of resistance to Cephalosporium stripe. Whiteheads (sterile heads caused by pathogen infection) were measured on each RIL in three field environments under artificially inoculated conditions. A linkage map for this population was created based on 204 SSR and DArT markers. A total of 36 linkage groups were resolved, representing portions of all chromosomes except for chromosome 1D, which lacked a sufficient number of polymorphic markers. Quantitative trait locus (QTL) analysis identified seven regions associated with resistance to Cephalosporium stripe, with approximately equal additive effects. Four QTL derived from the more susceptible parent (Brundage) and three came from the more resistant parent (Coda), but the cumulative, additive effect of QTL from Coda was greater than that of Brundage. Additivity of QTL effects was confirmed through regression analysis and demonstrates the advantage of accumulating multiple QTL alleles to achieve high levels of resistance.

Vernalization Responses of Jointed Goatgrass (Aegilops cylindrica), Wheat, and Wheat by Jointed Goatgrass Hybrid Plants

Abstract To assess the risk of gene movement between winter wheat and jointed goatgrass, information about the reproductive development of jointed goatgrass, winter wheat, and related hybrid plants is required. Seedlings from five jointed goatgrass populations, winter wheat, spring wheat, and jointed goatgrass by wheat reciprocal hybrid plants were exposed to 4, 7, or 10 C temperatures for 0, 2, 4, 5, 6, 6.5, 7, or 8 wk. Vernalized seedlings were transferred to a greenhouse set to 30/18 C day/night temperatures and 16-h photoperiod. Growth stages on all plants were recorded twice a week. All spring wheat and spring wheat related hybrid plants reproduced (as measured by the first reproductive node) in the absence of vernalization. Plants of jointed goatgrass population A-R, winter wheat, and winter wheat related hybrids were unlikely to reproduce in the absence of vernalization. Plants of jointed goatgrass populations B-W, G-S, E-S, and F-W reproduced in the absence of vernalization, and the likelihood that these plants would reproduce was different from all other plants. Plants that entered their reproductive phases together were not in synchronous development at anthesis. Plants in these studies differentially passed through the reproductive phases between the first reproductive node and anthesis. Our results demonstrate that variation in vernalization response exists among several jointed goatgrass populations, and reveal that the reproductive behavior of vernalized jointed goatgrass plants at anthesis is delayed compared to vernalized winter wheat and related hybrid plants. Hybrid plants produced between spring wheat and jointed goatgrass were vernalization insensitive. We hypothesize that hybridization between wheat and jointed goatgrass occurs as a result of cross-pollination between the younger reproductive tillers of jointed goatgrass and older reproductive tillers of wheat. The use of an early maturing wheat cultivar may exploit the difference in reproductive development and reduce the risk of hybrid production. Nomenclature: Jointed goatgrass, Aegilops cylindrica Host AEGCY; wheat, Triticum aestivum L.

Effect of wheat genotype on the phenotype of wheat × jointed goatgrass (Aegilops cylindrica) hybrids

Abstract Jointed goatgrass is a troublesome weed in winter wheat in the Pacific Northwest of the United States. Wheat and jointed goatgrass (JGG) can cross and produce hybrids in the field that can serve as a potential bridge for gene migration between the two species. To determine the potential for gene movement it is important to be able to identify hybrids in the field. To study the effect of wheat genotype on hybrid phenotype, reciprocal crosses were made between JGG and two common wheat cultivars: ‘Brundage 96’, ‘Hubbard’, a common-type advanced breeding line: ‘87–52814A’, and a club wheat cultivar: ‘Rhode’. Hybrids and parents were measured for plant height, spike length, flag leaf length, flag leaf width, and number of spikelets. Reciprocal effects were nonsignificant for all characteristics measured, indicating that hybrid morphology was not affected by the direction of the cross. Hybrids were different from their wheat parents for spike length, plant height, and flag leaf width. Hybrids produced from each of the wheat parents were uniform in phenotypic characters. Spikes were intermediate in circumference (size) from crosses between JGG and common wheat lines; however, club wheat × JGG crosses resulted in spikes that were more similar to common wheat. Spike size and flag leaf width for all hybrids also were intermediate between their parents. Hybrids differed in spike size and awn characteristics because of unique characteristics of the wheat parent. Based on these results, it should be possible to identify hybrids in the field accurately, regardless of the wheat parent or direction of the cross unless the parent is a club wheat. Nomenclature; Jointed goatgrass, Aegilops cylindrica Host., AEGCY; winter wheat, Triticum aestivum L.

The Effect of Imazamox Application Timing and Rate on Imazamox Resistant Wheat Cultivars in The Pacific Northwest

Grass weeds are a major problem in winter wheat fields in the Pacific Northwest (PNW). Control of these weeds is now enhanced with the use of imazamox resistant winter wheat cultivars, which have been rapidly adopted by wheat growers. However, the effect of spray rate and timing on crop injury and agronomic traits of wheat cultivars with different genetic backgrounds has not been adequately evaluated. Thus, experiments were conducted near Moscow and Genesee, ID in the 2003–2004 and 2004–2005 growing seasons to evaluate the effect of imazamox on four resistant cultivars and seven resistant breeding lines. Wheat plants were treated at the 3- to 5-leaf stage and the 3- to 7-tiller stage with 45 and 90 g ai/ha of imazamox. Visible crop injury was evaluated from 14 to 35 d after treatment (DAT). Heading date, plant height, grain yield and test weight, and end-use grain quality also were measured. The cultivar by treatment interaction was significant at 21 DAT, caused by a differential response of wheat lines to imazamox treatment. This interaction also was significant for plant height and grain yield. Although cultivars and breeding lines responded differently to imazamox treatment, two lines consistently showed the least levels (3 to 8%) of crop injury, with no reductions in plant height or grain yield following imazamox application. Orthogonal contrasts of visible crop injury at 21 DAT showed that the 2× imazamox rate caused more crop injury (12%) than the 1× rate (7%). The 2× rate of imazamox reduced plant height 1%, grain yield 8%, test weight 1%, and percent flour yield 1%. All other traits were not affected by application of imazamox. Application timing only minimally affected crop injury, and had no effect on agronomic or end-use quality traits. Nomenclature: Imazamox; wheat, Triticum aestivum L. ‘FS4’; ‘Brundage’; ‘Brundage 96’; ‘Lambert’; ‘87-52814A’.

Impact of transgene genome location on gene migration from herbicide resistant wheat (Triticum aestivum L.) to jointed goatgrass (Aegilops cylindrica Host)

BACKGROUND Wheat (Triticum aestivum) (ABD) and jointed goatgrass (Aegilops cylindrica) (CD) can cross and produce hybrids that can backcross to either parent. Such backcrosses can result in progeny with chromosomes and/or chromosome segments retained from wheat. Thus, a herbicide resistance gene could migrate from wheat to jointed goatgrass. In theory, the risk of gene migration from herbicide-resistant wheat to jointed goatgrass is more likely if the gene is located on the D genome and less likely if the gene is located on the A or B genome of wheat. RESULTS BC1 populations (jointed goatgrass as a recurrent parent) were analyzed for chromosome numbers and transgene transmission rates under sprayed and non-sprayed conditions. Transgene retention in the non-sprayed BC1 generation for the A, B and D genomes was 84, 60 and 64% respectively. In the sprayed populations, the retention was 81, 59 and 74% respectively. CONCLUSION The gene transmission rates were higher than the expected 50% or less under sprayed and non-sprayed conditions, possibly owing to meiotic chromosome restitution and/or chromosome non-disjunction. Such high transmission rates in the BC1 generation negates the benefits of gene placement for reducing the potential of gene migration from wheat to jointed goatgrass.