Genetic mapping of IZS 288

To isolate the IZS 288 gene, a map-based cloning strategy was implemented. Following the principle of linkage mapping (i.e. the closer a gene and a molecular marker are located to one another on a chromosome, the greater the chance that they will be inherited together as a unit) the genetic locus of IZS 288 was identified by means of scoring recombination events in the mapping population. The mapping population was derived through crossing IZS 288 with that of the Ler-0 ecotype. Since IZS 288 has Col background, the number of recombination events is equivalent to the number of times the Ler-0 ecotype is found on a chromosome. This means, as the recombination frequency scored by a molecular marker gets smaller and smaller the closer it gets towards the location of the mutated gene. Accordingly, after analyzing 907 individual F2 plants the mutation was found to map on chromosome two between the CAPS marker Thy1 and the SSLP marker NGA361. Subsequent fine mapping

Kbp, which lay between the perl339 markers (located at 8,775,325bp) and perl9577 (located at 8,820,243bp) within a contig of two bacterial artificial chromosome (BAC) clones (F11A3 and T13C7). 14 candidate genes located in this region were sequenced using genomic DNA of IZS 288 plants as a template. Subsequent sequence analysis revealed a single nucleotide substitution (G¹¹⁵⁶- to - A¹¹⁵⁶) in the coding sequence of the AT2G20330 gene. This substitution resulted in the replacement of a conserved amino acid namely threonine at position 377 (T³⁷⁷) by an isoleucine (I³⁷⁷) residue (Fig.3.36).

Figure 3.36. Positional cloning of IZS 288. A) Genetic mapping of IZS 288 using commercially available PCR-based markers. Genetic region where the mutation is located is represented by a contig of three BAC clones (T2G17, F11A3 and T20). Numbers in brackets represent location of a marker on the chromosome. B) Diagrammatic representation of the gene At2g20330 which is composed of 8 exons. Exons and introns are represented by filled boxes and lines, respectively. IZS 288 carries missense mutation in the sixth exon where it led to the substitution of the 377^th amino acid (threonine) by isoleucine. C) Multiple sequence alignment of the closest 10 homologues genes of At2g20330 (At2g20330, ARALYDRAFT_900383, Gmx_100815380, MTR_7g098900, RCOM_0541360, POPTR_573381, Vvi_9819, osa_4348832, SORBI_01g019930 and zma_100280725), red arrow indicates the amino acid that is mutated in IZS 288.

Furthermore, to ascertain the Zn hypersensitivity of IZS 288 resulted from the mutation of At2g20330, a complementation experiment was set up on IZS 288 using the WT At2g20330 gene under the control of its own promoter as well as the coding sequence of At2g20330 under the control of the cauliflower mosaic virus promoter (35S:At2g20330). Both constructs were able to restore WT phenotypes in IZS 288. This confirmed that the IZS 288 phenotypes were caused by the mutation in the At2g20330 gene (Fig.3.37).

Figure 3.37. Complementation test using genomic fragment and cDNA of At2g20330 under 35S promoter. A) Diagram of constructs used for the complementation assay. Exons and introns are represented by filled boxes and lines, respectively. B) Pictures of agar plates with and without Zn on which WT, IZS 288 and the two transgenic lines carrying the complementation constructs are growing. As it can be seen both the short root phenotype as well as the Zn hypersensitivity was restored. White bars represent 2cm.

In order to identify additional mutant alleles for the At2g20330 gene, three different T-DNA insertion lines (namely SALK_140479, SALK_038590 and SALK_065643) were obtainedfrom the SALK T-DNA insertion collection (Alonso et al., 2003). The reported T-DNA insertion site in the SALK_140479 line was in the first exon of the gene (i.e. 10 bp downstream of the start codon); whereas in lines SALK_065643 and SALK_038590 it was in the promoter (i.e. 213bp upstream of the start codon) and 3' untranslated region of the gene (i.e. 296 bp down stream

of the stop codon), respectively (Fig. 3.38). Homozygous T-DNA insertion mutants were sought-after through PCR screening using a combination of gene specific primers and a T-DNA left border–specific primers. The gene specific primers were designed in such a way that the forward and reverse primers flanked the T-DNA insertion site, thus a PCR product was formed only when there is no T-DNA insertion. In homozygous T-DNA insertion lines a PCR product is detected using the reverse gene specific primer and a left border T-DNA primer. Heterozygous lines (i.e. insertion in one of the pair chromosomes) would form both the WT PCR product and T-DNA insertion PCR product. On the first round of PCR screening carried out on 10 plants taken form each SALK lines no homozygous T-DNA insertion lines were detected (Fig. 3.39a). Assuming homozygous mutations in the At2g20330 gene might lead to embryo lethality; siliques from 3 individual plants from each SALK line were examined for aberrant embryo development. Since in Arabidopsis embryos within a single silique develop approximately at the same rate, individual embryos with aberrant development can be easily scored (Errampalli et al., 1991), however in case of all three SALK lines under investigation no such aberrant embryo development was observed (data is not included here). Subsequently, extensive screening was carried out on additional 30 individuals of the SALK_140479 line (given that SALK_140479 line has the T-DNA insertion in the first exon of At2g20330, it is more likely to show stronger effect) and also a second gene specific primer pair obtained using the SALK institutes T-DNA primer design tool (http://signal.salk.edu/tdnaprimers.2.html) was included in the analysis. However, contradicting results were attained; where the analysis using the first pair of gene specific primers did not detect any homozygous lines, the second gene specific primer pair identified 7 individual plants as homozygous lines (Fig. 3.39 b).

Figure 3.38. A) Schematic structure of At2g20330 gene and inserted T-DNA. Exons and introns are

represented by filled boxes and lines, respectively. T-DNA insert is not drawn to scale. The T-DNA insertion site in the SALK_065643 line is in the promoter region (i.e. 213bp upstream of the start codon), in SALK_140479 line it is in the first exon (i.e. 10bp down stream of the start codon) and in SALK_038590 line it is in the UTR region (i.e. 296 bp down stream of the stop codon).

A)

B)

Figure 3.39. A) Picture representation of gene specific primers for each T-DNA insertion lines and gel picture of PCR screening on 10 individuals of each lines. B) Gel pictures of PCR screening on additional 30 individuals of SALK_140479 line. White arrows indicate putative homozygous lines identified by the second primer pare in

In order to detect the effect of the T-DNA insertion on the integrity of the genomic sequence surrounding the insertion site, the transcript abundance of At2g20330 in four putative homozygous lines was quantified using quantitative real time PCR (qRT-PCR). Despite the fact that, within the genomic sequence the T-DNA insertion site and the binding site of the RT-PCR primers were further apart, no apparent difference in transcript level was observed among the WT and the four putatively homozygous lines (Fig. 3.40 a).

In a different approach, all three T-DNA insertion lines were crossed with one of the IZS 288 backcross lines (i.e. E1-1) and the first generation seeds were observed for possible phenotypes. In SALK_140479 and SALK_038590 lines, 33% (7 out of 21 individuals) and 43%

(6 out of 14 individuals) of the F1 progenies respectively failed to germinate. These observations further supported the notion that complete knockout of the WD40 gene can cause embryo lethality. However, the ratio of non-germinated to germinating F1 seeds failed to accurately demonstrate Mendel's Law of Segregation (Fig. 3.40 b).

Fig 3.40. A) Relative transcript levels (RTL) of the WD40 gene in roots of WT, IZS 288 and three putative homozygous T-DNA insertion lines (i.e. Salk14 2-3, 2-7 and 3-7) and one heterozygous line (2-9). RTL values are arithmetic means of three independent experiments and bars represent standard deviation. B) Pictures of agar plates with first generation progenies of a cross between E1-1 and three independent T-DNA insertion lines.

Red arrows indicate seeds that failed to germinate. The ratio of non-germinated seeds in Salk14 line was 7/21 and in Salk03 line it was 6/14. However in Salk06 line no such effect was observed. White bars represent 2cm.

3.2.4 Functional analysis and subcellular localization of the novel WD40 protein