Identifying appropriate model species for transgenomics
Transgenomics depends on being able to easily move donor species’ genomic DNA into a recipient species. While reliable transformation methods exist for many species, we chose the model plant A. thaliana because of its great transformation efficiency and the immense body of available genetic and genomic data. An appropriate donor species would be phylogenetically close (i.e. within Brassicaceae), yet divergent from A. thaliana for as many distinct phenotypes as possible. Leavenworthia is a member of the Cardamineae clade, which is within ‘Lineage I’ of Brassicaceae, the same lineage that includes Arabidopsis (Beilstein et al., 2006). The radiation of Lineage I Brassicaceae has been dated at 8–14 Ma (Franzke et al., 2009), 18–36 Ma (Couvreur et al., 2010) or 35 ± 6 Ma (Beilstein et al., 2010). Leavenworthia and Arabidopsis differ in almost all visible traits that show discrete variation within Brassicaceae (Fig. 1), including leaf shape (pinnately compound vs simple), trichomes (absent vs present), shoot architecture (rosette flowering vs inflorescence flowering), flower size (> 1 cm diameter vs < 3 mm diameter), fruit shape (strongly flattened vs terete), seed number per fruit (c. 9–10 vs 50–70) and seed size (c. 3.1 vs 0.5 mm). Leavenworthia alabamica has a modest genome size of c. 500 Mbp (M. A. Lysak & P. Bures, unpublished), has 11 chromosomes (Lysak et al., 2009), and is the target of an ongoing genome-sequencing project (http://biology.mcgill.ca/vegi/index.html).
Figure 1. Comparison of Arabidopsis thaliana Columbia and Leavenworthia alabamica. Despite belonging to the same plant family (Brassicaceae), these species differ in many phenotypes. For example, A. thaliana Columbia (a) has simple leaves, an elongated primary inflorescence, short floral branches (pedicels), small flowers, narrow fruit, and an early flowering time in standard glasshouse conditions. By contrast, L. alabamica (b, c) has pinnately compound leaves, a suppressed primary inflorescence, elongated pedicels, large flowers, broader fruit, and a late flowering time in standard glasshouse conditions. Photographs of L. alabamica were kindly provided by J. Busch.
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Arabidopsis thaliana can be transformed with L. alabamica genomic clones in a high-throughput manner
As a first step in the transgenomic screen, we generated a plant transformation-competent genomic library of L. alabamica, with inserts of 20–25 kb. We conducted pilot analyses of alternative screening strategies and concluded that the optimal was a high-throughput clone-by-clone screening approach (Fig. 2). This method has the advantage that one can readily determine whether a clone generates the same abnormal phenotype in multiple independent transformants. Because each independent transformant is likely to have a unique insertion site, one can avoid wasting effort following up phenotypes that result from insertional mutagenesis or other artifacts of transformation.
Figure 2. The clone-by-clone transgenomic strategy used to move Leavenworthia alabamica genomic DNA into Arabidopsis thaliana for phenotypic screening. (a) Leavenworthia alabamica clones in E. coli are transferred to 96-well format. Cosmids harboring L. alabamica inserts are then moved into Agrobacterium tumefaciens using high-throughput freeze–thaw transformation. (b) A portion of each A. tumefaciens stock in the 96-well plate is grown, and then used to drip-transform one pot of A. thaliana T0 plants. Each T0 pot is then separately harvested for seeds to identify primary transformant (T1) plants. (c) A portion of each T1 seed stock is then grown on kanamycin-containing plates. At least one kanamycin-resistant T1 plant is then transferred to a pot and allowed to grow to maturity. If the TI plant is abnormal, the transformed seed collection is sampled again to identify four additional independent T1 plants to see if the abnormal phenotype recurs. If the phenotype recurs, then a third seed sowing may be used to identify additional T1 plants.
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We used this clone-by-clone transgenomic pipeline to conduct an initial screen of 14 96-well archives. We found that this workflow could be completed by a small team, comprising one graduate student and two to three undergraduates, in < 6 months with a peak rate of plant transformation of c. 160 clones (grown from two 96-well plates) per week. Eighty-five per cent of the E. coli clones were successfully transferred to A. tumefaciens and 99% of those were successfully introduced into plants.
Analysis of primary transformants yielded clones associated with repeatable phenoypic abnormalities
Because A. tumefaciens-mediated transformation of A. thaliana occurs in the female gametophyte, and because T-DNA integration is nonhomologous, T1 plants are hemizygous for the transgenic fragment. A transgene is only expected to manifest a visible phenotype in the T1 generation if it is trans-dominant, meaning that its effects are not masked by endogenous homologs in the genome. Our aim was to screen for clones associated with such trans-dominant phenotypic effects.
We used a small portion of each T1 seed stock to isolate one kanamycin-resistant T1 plant per L. alabamica clone (Fig. 2c). Of 1147 A. tumefaciens clones, 1134 were successfully introduced into A. thaliana plants. For each clone, we visually screened at least one T1 individual during development and at maturity (c. 6 wk of age) for morphological phenotypes that differed from those of untransformed plants. Eighty-four clones (7.4%) yielded initial T1 plants that were judged to deviate from wild type (Table 1).
Table 1. Effects of 1147 Leavenworthia alabamica clones
|Clone effect||Frequency||Per cent|
|No visible effect||1050||91.5|
An abnormal phenotype in a single T1 plant could result from a trans-dominant effect of an L. alabamica clone, insertional mutagenesis, background mutation, perturbation caused by screening on kanamycin plates and transplanting, or environmental variability. Of these, only effects that are attributable to inserted L. alabamica DNA are of interest. Because independent transformants of the same clone are likely to have integrated into the genome at unique insertion sites, phenotypes that appear repeatably are most likely to be attributable to the L. alabamica insert. Therefore, we excluded 70 clones that failed to repeat the abnormal phenotype after screening four additional, independent T1 plants for the same clone (Fig. 2c). We then screened additional T1 plants and excluded two further clones because of a failure to generate additional abnormal T1 plants.
We discarded four clones that were found, based on end-sequencing, to include Lambda viral DNA, probably introduced during library construction. These four clones were associated with low repeatability and involved phenotypes that occur not infrequently in wild-type plants.
After this winnowing process, we were able to identify eight clones containing L. alabamica genomic DNA that were associated with repeatable, trans-dominant phenotypes in A. thaliana (Table 2). The level of repeatability, defined as the number of T1 plants with the phenotype divided by the number of T1s screened, varied from 16 to 60%, with an average of c. 42% (Table 2). This collection of clones was associated with a broad range of phenotypes in A. thaliana: two were flagged primarily because of a change in fruit size/shape, three for plant architecture/stature, one for flower form, one for a leaf defect, and one for abnormal leaf and flower development (Figs 3, S1, S2).
Table 2. Clones assigned with repeatable phenotypes
|Clone||Phenotype||Repeatability (T1s with phenotype/T1s screened)|
|05_01C||Short fruit||13/27 (48%)|
|06_05C||Short plants withreduced seed set||9/15 (60%)|
|09_09A||Petals unevenly spaced||5/16 (31%)|
|11_01D||Rosette leaves twisted||12/35 (34%)|
|11_11B||Short fruit||9/16 (56%)|
|12_03E||Fruit cluster with very reduced internodes||5/14 (36%)|
|12_05A||Cauline leaves decurrent to stem||4/7 (57%)|
Figure 3. Repeatable morphological phenotypes identified in the transgenomic screen. Photographs of additional independent T1s with repeatable phenotypes are shown in Supporting Information Figs S1, S2. (a) Fruits of Leavenworthia alabamica (left) and Arabidopsis thaliana (right) differ in their length-to-width ratio. The two stunted valves in the center were taken from two independent T1s from clone 11_11B and are representative of fruit from each plant. Clone 05_01C also yielded a repeatable short fruit phenotype. (b) Petals from A. thaliana flowers (left) are more evenly spaced than those from clone 09_09A (right). Although a small proportion of flowers on even wild-type plants have unevenly spaced petals, repeatable T1 plants from this clone were identified as having an elevated frequency of such flowers. (c) Abnormal twisting and contortion of rosette leaves on a T1 plant from clone 11_01D. Clone 11_01D also yielded a T1 plant with some lobed rosette leaves and two T1 plants with abnormal flowers (see Fig. S2). (d) Inflorescence with clustered fruit on a T1 plant from clone 12_03E. (e) A T1 plant from clone 12_05A displays cauline leaves developed decurrently along the primary axis. (f) Close-up picture of a decurrent cauline leaf from the T1 of 12_05A (e). (g) A dwarf T1 plant from clone 12_06G.
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For 10 independent T1 plants from clone 05_01C and nine from 11_11B, we measured valve length for 20–40 fruit. This confirmed that they do, indeed, tend to have shorter fruit than wild type (Table 3). Because data were only collected for transgenic lines that had been visually flagged as having short fruit, meaningful statistical comparisons could not be made until later generations. However, the dramatic differences between wild type and several T1 plants for clones 05_01C and 11_11B are consistent with these clones having a transdominant effect on fruit length.
Table 3. Fruit valve lengths
|Line||Number of T1 plants||Number of valves per plant||Valve length (mm) (mean ± SE)|
|Plant with shortest fruit||Plant with longest fruit||Grand mean|
|Wild type||18||40||11.3 ± 1.0||14.2 ± 0.7||12.7 ± 0.2|
|05_01C||10||20||7.4 ± 2.5||13.5 ± 0.8||10.9 ± 4.5|
|11_11B||9||40||4.9 ± 0.6||12.5 ± 0.5||9.3 ± 0.3|
To identify lines with one transgene locus, we screened T2 seeds for four to eight lines per clone (Table S1). For seven of the eight clones, we found at least two lines whose KRS ratio was consistent with a single transgene locus. Of the 44 lines screened, 21 were inferred to have one transgene locus, and two to have two unlinked transgene loci (Table S1). Of the remaining 21 lines, 12 had a deficit of resistant plants beyond that expected even for a single transgene locus. To help assess the causes of non-Mendelian segregation patterns, we used PCR amplification of NPTII to genotype 23–40 T2 plants from those lines that showed a deficit of kanamycin-resistant seedlings (Table S2). In two of 12 lines (12_03E_1; 12_06G_1) we detected a significant deficit of NPTII-containing offspring. This could indicate an effect of the L. alabamica transgene on gamete, gametophyte or embryo development, or it could be attributable to insertional mutagenesis or chromosomal rearrangements during transformation (e.g. see Ray et al., 1997). In three lines (11_11B_2, 11_11B_15 and 12_06G_2) we found a significant difference between the KRS and NPTII ratios, where the latter was not significantly different from 3 : 1. This is most easily explained by Mendelian segregation of the transgene coupled with silencing of NPTII in many plants. In the remaining lines the ratio determined by PCR was consistent with both the KRS ratio and 3 : 1.
We selected one candidate line from each L. alabamica clone, choosing the line with the strongest T1 phenotype that was inferred to contain a single transgene locus. Because we found no lines for clone 06_05C that showed a 3 : 1 ratio, we selected a line (06_05C_03) that produced about half sensitive and half resistant T2 seedlings.
For each selected line, we grew a T2 family on soil so that we would be able to score plant phenotypes while being blind to genotype. We allowed T2 plants to self and set T3 seed. A portion of T3 seeds from each T2 plant were tested on kanamycin medium to infer its transgene genotype (see the Materials and Methods section).
ANOVA could not reject the null hypothesis that variation in the scored phenotype was independent of transgene genotype in six of eight T2 families (Fig. 4). In the T2 family for clone 06_05C, a two-tailed homoscedastic Student’s t-test did not detect a significant difference in fruit length between transgene-present and transgene-absent T2 plants. However, only 22 T2 plants were scored in this case. Combined with the distorted segregation ratios seen in this line, we judge this co-segregation test to be noninformative.
Figure 4. Co-segregation analyses of T2 families. Graphs (a–h) show phenotype distributions by genotype in T2 families. Each T2 family was derived from a T1 line that had shown a repeatable phenotype and was inferred to contain one transgene locus, except for 06_05C, which produced a smaller proportion of kanamycin-resistant offspring than expected from a single-locus insertion line (see the Results section). Clone name, sample size of T2 family (n), and P-value result of a one-way ANOVA which tested genotypes for differences in phenotype are indicated in each graph. T2 plants in the family from clone 11_01D (d) were scored qualitatively for rosette leaf twisting as follows: 0, three or fewer leaves with slight twisting; 1, four or more leaves with slight twisting; 2, four or more with strong twisting. Seven of eight co-segregation experiments (a–d, f–h) did not produce a significant result. However, for the family derived from clone 11_11B (e) the null hypothesis was rejected because hemizygous T2 plants had shorter fruit than either wild-type or transgene homozygous plants (corroborated by experiments shown in Figs 5, S3). Transgene genotypes: gray, absent; black, hemizygous; white, homozygous.
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For clone 11_11B, transgene-hemizygous T2 plants were found to have shorter fruit, while transgene-homozygous and transgene-absent T2 plants had fruit of normal length (one-way ANOVA, P = 0.00046; Fig. 4e). This result suggests that the transgene has a trans-overdominant effect: shortening fruit only when the transgene locus is hemizygous, and not when it is homozygous.
Co-segregation analysis suggests that 11_11B causes short fruit and ovule/seed abortion
The conclusion that 11_11B co-segregates with fruit length variation came from a single T1 line. To determine if this result could be replicated, we conducted a more thorough study of the same line (11_11B_1) plus three additional, independent 11_11B lines (5, 6, and 10). All lines had been scored with KRS ratios that were close to 3 : 1 (Table S1). We scanned the inflorescences and used image analysis software to measure fruit length for all mature fruits along the main axis. Genotypes were then inferred by sowing batches of T3 seeds on kanamycin plates.
Controlling for variation that was attributable to line, individual plant, individual fruit, and location of fruit in the inflorescence, the null hypothesis that genotype has no effect on fruit length was rejected. Once again, a significant transgene overdominance effect on fruit length was observed (Fig. 5). However, this effect was only detectable in lines 1, 5, and 6. Within these three lines transgene-absent and transgene-homozygous plants had indistinguishable fruit sizes, whereas transgene-hemizygous fruit were, on average, 1.6 ± 0.7, 3.1 ± 0.8, or 3.7 ± 0.7 mm shorter than the average transgene-absent fruit, respectively. The mean fruit length in line 10 hemizygotes was indistinguishable from that of wild type.
Figure 5. Co-segregation analysis of four independent T2 families verifies the overdominant short-fruit effect of clone 11_11B. The plot compares transgene-hemizygous (RS; left panel) and transgene-homozygous (RR; right panel) mean fruit lengths vs transgene-absent (SS) mean fruit length. Differences in mean fruit length are plotted with 95% confidence intervals. The four estimates per panel are from co-segregation data generated by four independent T2 families. Of the four independent families (mean n = 56 plants), three repeated the transgene overdominant effect observed for clone 11_11B in Fig. 4. For all four lines, the ratio of inferred wild-type, transgene-hemizygous, and transgene-homozygous T2 plants did not deviate significantly from 1 : 2 : 1, supporting the conclusion that each T1 line has a single transgene locus.
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As it is well established that normal fruit elongation requires the production of signals from developing seeds (Gillaspy et al., 1993; Chaudhury et al., 1997; Vivian-Smith et al., 2001), the fruit-length effect could be caused by defects during ovule/megagametophyte development, fertilization, or early embryogenesis. We observed aborted seeds in a subset of T2 plants. Aborted seeds were recognizable as short funicles with a knob-like ending, which persisted even after seed dispersal, whereas normal funicles were long and tapering after seed abscission. We scored one to four mature, dehisced fruit per plant from at least five individual plants for each genotypic class, for lines 1, 5, 6, and 10, all while blind to plant genotype.
As summarized in Table 4, plants carrying the 11_11B transgene showed significantly higher levels of seed abortion than wild-type plants in the same three lines (1, 5, and 6) that showed a fruit-length effect. Whereas < 5% of seeds typically aborted in wild-type lines, the mean abortion rate in line 1, 5, and 6 hemizygotes was 12, 34, and 31%, respectively. However, in contrast to the complete overdominance seen for fruit length, significantly elevated seed abortion was also seen in some transgene homozygotes. Examination of abortion rates for individual plants from lines 1, 5, and 6 suggested that among the transgene-containing lines there was an extensive variation in seed abortion rate (Fig. S3).
Table 4. Proportion of seed aborted (mean ± SD) in the three genotypic classes in four independent 11_11B lines
|11_11B_01||0.03 ± 0.03||0.12 ± 0.04||0.10 ± 0.07|
|11_11B_05||0.03 ± 0.02||0.34 ± 0.14||0.27 ± 0.19|
|11_11B_06||0.05 ± 0.03||0.31 ± 0.09||0.27 ± 0.15|
|11_11B_10||0.04 ± 0.02||0.03 ± 0.04||0.06 ± 0.02|
The aborted seeds seen in 11_11B-containing plants could be a result of disruption of processes such as ovule development and pollen maturation in parental tissues. Alternatively, the transgene could act directly on gametophytes or embryos. In the latter case, we might expect the seeds of hemizygotes to show a deficit of transgene homozygotes and hemizygotes because they would abort at a higher rate than wild-type gametophytes/embryos. However, for all four lines tested for co-segregation, the ratio of inferred wild-type, transgene-hemizygous, and transgene-homozygous T2 plants did not deviate significantly from 1 : 2 : 1. This supports the conclusion that these four T1 lines each has a single transgene locus and suggests that embryos carrying the transgene do not have an elevated abortion rate.