GENE MAPPING OF PHOSPHOGLYCEROL PHOSPHOLIPASE C HOMEOLOGS TO SINGLE CHROMOSOMES IN WHEAT (Triticum aestivum L.)

hospholipases are sets of enzymes which have the ability to hydrolyze phospholipids. They have different forms in plants; including phosphlipases C, D and A. phospholipase C and D have important roles in signal transduction. The general structure of phospholipids consists of two fatty acyl chains esterified to a glycerol backbone at the sn-1 and sn-2 positions, and a phosphate at sn-3 position to which a variable head group (R) is attached. Phos-phatidylglycerol plays a specific role in photosynthesis (Sakurai et al., 2007) by helping in the organization and function of thylakoid membranes in plant cells.


GENE MAPPING OF PHOSPHOGLYCEROL PHOSPHOLIPASE C HOMEOLOGS TO SINGLE CHROMOSOMES IN WHEAT (Triticum aestivum L.)
NESREEN SABRY¹, F. M. ABDEL-TAWAB² AND P. J. GULICK³ 1. Benha University, Benha, Egypt 2. Ain-shams University, Cairo, Egypt 3. Concordia University, Montreal, Canada hospholipases are sets of enzymes which have the ability to hydrolyze phospholipids.They have different forms in plants; including phosphlipases C, D and A. phospholipase C and D have important roles in signal transduction.The general structure of phospholipids consists of two fatty acyl chains esterified to a glycerol backbone at the sn-1 and sn-2 positions, and a phosphate at sn-3 position to which a variable head group (R) is attached.Phos-phatidylglycerol plays a specific role in photosynthesis (Sakurai et al., 2007) by helping in the organization and function of thylakoid membranes in plant cells.Hagio et al. (2002) stated that the content of phosphatidylglycerol in both Arabidopsis thaliana mutants, that have an insertion in the PGP1 gene encoding a phosphatidylglycerol phosphate synthase, and wheat leaves of phosphate-deficient mutants were reduced than that of the wild type due to PGP1 gene disruption.Furthermore, the development of chloroplasts in the leaf cells was severely arrested in these mutants, evidence that PG is essential for the development of thylakoid membranes.
Most animals and many plant species are diploid, however many plant species are polyploid such as T. aestivum, bread wheat which is hexaploid.All members of the Triticeae tribe, including wheat, barley, and rye, have originated from a common ancestor.
Polyploid wheat species such as the tetraploid durum wheat Triticum turgidum, and the hexaploid bread wheat, Triticum aestivum, were formed by processes of interspecific hybridization events among different ancestral wheats followed by spontaneous chromosome doubling.The major genomes of wheat and its relatives, named A, B, D, G and S etc., have a basic chromosome number of seven (X=7) and within a polyploid species these genomes are referred to as being homeologous to one another.The polyploid wheat species constitute two evolutionary lineages.T. turgidum (AABB genome) and T. aestivum (AABBDD) provide one lineage, and Triticum timopheevii (AAGG) and Triticum zhukovskyi (AAAAGG) constitute the other lineage.In the T. aestivum T. turgidum lineage, the A genome was contributed by T. urartu while the other lineage received the A genome from Triticum P monococcum.The B genome is thought to have been contributed by wild diploid wheat closely related to Aegilops speltoides.Triticum aestivum (AABBDD) arose from spontaneous hybridization between T. turgidum (AABB) and the diploid goat grass Aegilops tauschii (DD) (Faris et al., 2001).Hexaploid bread wheat (Triticum aestivum L.) has the ability to tolerate the addition or loss of whole chromosomes or chromosome segments and this has contributed to the construction of several series of aneuploid lines.Nullisomic tetrasomic (NT) and ditelosomic lines have been used as tools for localization of molecular markers to chromosomes without the need of polymorphism at the gene level.In addition, 21 pairs of ditelosomic chromosome lines which have one pair of chromosomes being composed of either two identical short or long arms also were used to map genes to individual chromosome arms on the genetic maps.(Devos et al., 1999).
Since bread wheat is a selfpollinated allohexaploid, most genes are expected to be present in three homeologeous copies on three homeologous chromosomes derived from the three ancestoral genomes; the alleles on homologous pairs of chromosomes are expected to be identical.Homeologous copies of genes from the A, B and D genome are expected to have very high nucleotide sequences similarity.For example, the three homeologous copies for each of the five paralogous gene copies of the αtubulin gene family members in T. aestivum were found to be 97% identical within the coding regions of the gene and to have somewhat greater divergence in the un-translated region of the gene transcript (Farajalla and Gulick, 2007).
The main objectives of this study were to determine the chromosomal location of the PLC genes in wheat and survey the prevalence of a frame shift mutation identified in one Ta-PG-PLC gene, while sequencing, among twenty-one wheat cultivars and in progenitor species.

Triticum aestivum (L.) genetic stocks
Several accessions of diploid and tetraploid wheat species as well as several North American and Egyptian hexaploid T. aestivum cultivars were used in this study and are listed in Table (1).In addition, T. aestivum cytogenetic stocks were used for mapping PLC genes to individual chromosome arms.The nullisomictetrasomic (NT) lines have one chromosome pair substituted by a homeologous pair of chromosomes.The ditelosomic (DT) lines have single pairs of chromosomes which have two copies of one chromosome arm and are missing the other arm of the chromosome.Partial chromosome arm deletion lines are missing a part of one chromosome arm (Farajalla and Gulick, 2007); they were obtained from G. Scholes (University of Saskatchewan).The Egyptian cultivars which were used in this study were obtained from Plant Genetic Resources Dept., Desert Research Center, Egypt and the North American cultivars were ob-tained from Agriculture and Agri-Food Canada (Table 1).

cDNA Clones
A cDNA clone for phosphatidylglycerol phospholipase Cs, Ta-PG-PLC2-1 was obtained from the Genome Canada Functional Genomics of Abiotic Stress (FGAS) program (Houde et al., 2006) and Ta-PG-PLC2-2 was obtained from Arizona Genomics Institute (AGI).The clones were sequenced at the Genome Quebec Innovation Centre, McGill University.Additional partial cDNA clone of Ta-PG-PLC2-3 was assembled from EST sequences.Sequences were compared to the Rice gene sequences which obtained from GenBank using BLASTn and BLASTx.Homeologs genome assignment to ancestral genomes Gene specific primers were designed and used for PCR amplification with DNA from Triticum urartu (A genome progenitor), Aegliops speltoides (closely related to the B genome progenitor), Triticum tauschii (D genome progenitor) to determine the original genomes of the three PG-PLC genes.Specific primers are shown in (Table 2).

Nullisomic chromosomes mapping
The set of nullisomic-tetrasomic (NT) lines included 19 lines used for mapping the three ESTs homeologs to individual chromosomes (Farajalla and Gulick, 2007).Sequence differences between the homeologous genes were used to design gene specific primers.PCR reactions were conducted using gene specific left and right primers.The primer sequences are shown in (Table 2)

Screening for the prevalence of a mutation in Ta-PG-PLC2
One pair of primers was designed to detect the two base pair deletion mutation detected in Ta-PG-PLC2-1.
Another left primer was designed to detect a putative wild type version of Ta-PG-PLC2-1 (reverted) without the two bp deletion, and additional pair of primers was designed to detect the homeologous gene Ta-PG-PLC2-2.The primers sequences are shown in (Table 3).For Ta-PG-PLC2-1 primers were designed to amplify the PLC gene from twenty one wheat cultivars from different origins (Table 1) to determine the prevalence of the frame shift mutation.Amplification was carried out in a Biometra T-Gradient thermocycler programmed as follows: 94C/3 min (1 cycle), 94C/30 Sec, 65C/35 Sec, 72C/2 min (40 cycles); 72C/10 min (1 cycle) and 4C (hold).

Ta-PG-PLC2-1 Sequence
Ta-PG-PLC2-1 clone is 1734 bp in length (Fig. 1) with a 1568 bp open reading frame extending from position 61 to position 1629.The wheat sequences have 87% nucleic acid and 83% amino acid sequence similarity with the rice phosphotidylglycerol specific phospholipase C, Os03g0852800.The cDNA has a full length sequence similarity to its rice ortholog, beginning with the first ATG in the cDNA sequence.However, Ta-PG-PLC2-1 has a frame shift mutation, a deletion of two bases following the G at position +30 relative to the first ATG.This deletion is seen by sequence comparison to the rice phosphotidylglycerol specific phospholipase C gene and by comparison to an apparent homolog from wheat, as described below (Figs 2, 3 and 4).The apparent mutation was confirmed by the fact that the sequence quality is excellent in this region, a FRED score of 20 or more indicates higher than 99% sequence accuracy, and two independent sequences con-firmed the DNA sequence in this region.The apparent frame shift is followed by second ATG 28 nucleotides downstream of the frame shift that is in-frame with the major ORF that may serve as the start codon (Fig. 1).

Nullisomic-tetrasomic (NT) analysis
The three cDNA clones for Ta-PG-PLC had 93% nucleotides sequence identity within their coding regions and were hypothesized to be homologous copies of the gene, since similar degrees of identity were seen among the homologs of alphatubulins in wheat (Farajalla and Gulick, 2007).Cytogenetic stocks of hexaploid wheat cultivars Chinese Spring (T.aestivum L.) were used for chromosomal mapping of gene family members.Cytogenetic DNA stocks were screened by PCR with gene specific primers for each homolog.A primer set that was specific to Ta-PG-PLC 2-1 that could detect the 2 nt deletion amplified fragments in T. urartu (the A genome progenitor), T. trugidum and T. aestivum but not in Aegilops speltoides or in Aegilops tauschii, the B and D genome relatives.This indicated that Ta-PG-PLC2-1 is likely in the A genome (Fig. 5).Further PCR screening of the A genome nullisomic-tetrasomic (NT) chromosome lines 1-7 A, ditelosomic DT6AS and DT6aL, and selected chromosome deletion lines indicated that Ta-PG-PLC2-1 is on the short arm of chromosome number 6A.Ta-PG-PLC2-2 and Ta-PG-PLC2-3 gene specific primers amplified products from A. speltoides, the B genome progeni-tor and A. tauschii (D genome progenitor), respectively (Fig. 5).Further analysis with NT, DT and chromosome deletion lines indicated that Ta-PG-PLC2-2 homeolog is on the long arm of chromosome 6B and Ta-PG-PLC2-3 homeolog is on the short arm of chromosome number 6D (Fig. 6).
The presence of Ta-PG-PLC2-1 and Ta-PG-PLC2-3 was present on the short arms of A and D chromosome, respectively, whereas Ta-PG-PLC2-2 was present on the long arm of B chromosome may be due to duplication and then elimination or silence of the gene hence in the wheat evolution early stages genes may be duplicated and then they may be eliminated or silenced during domestication.Qi et al. (2004) found that from 5762 ESTs, 1086 hybridized to restriction fragments on chromosomes in different homologous groups.Within this group (75%) detected loci were in two homologous groups, 17% detected loci in three groups, and 10% were detected loci in more than three groups.Like BE443755 which hybridized to 17 fragments that could be located at 1A, 5A, all B-genome chromosomes, and 7D.Nine loci of those were mapped on opposite arms within homologous groups on chromosomes 1AL 1BS and 5AS-5BL and on both arms of chromosomes 4B and 7B.In several cases of intrachromosomal duplications, the duplicated fragments were detected in both arms.These events may represent peculiarities of chromosome behavior during which these duplications occurred.For example, opposite arms may be aligned together during prophase of meiosis, giving opportunities for gene-conversion to happen.When these duplicated genes may be eliminated, and/or silenced, fragment would be mutated to give new functions, or epigenetically regulated for tissue-specific expression (Adams et al., 2003).This is a reasonable explanation for the presence of two homologs on short arm chromosomes and the third one on the long arm.

Mutation detection in different wheat cultivars
Twenty one wheat cultivars were tested to detect the 2 nt deletion found in Ta-PG-PLC2-1 (Table 1).The specific primers a aforementioned were designed to characterize the region of this mutation in the three wheat ancestral genomes of hexaploid wheat, T. urartu (A genome), A. speltoides (B genome), A. tauschii (D genome), T. turgidum (AB genome) and T. aestivum L (ABD genomes) (Fig. 7).PCR primers were designed to amplify the Ta-PG-PLC2-1 sequence containing the deletion mutation amplified products from T. urartu, T. turgidum and T. aestivum L. The Ta-GP-PLC2-1 primer pair amplified PCR products in all T. aestivum cultivars that were screened.A modified Ta-GP-PLC2-1 primer pair in which the two nucleotide sequences were reinserted failed to amplify any PCR products in T. aestivum cultivars or in related wheat species.From these results it was apparent that the mutation is widespread in wheat plant and it is quite clear that it is not unique to polyploid wheat but it is also present in its ancestral species (Fig. 8).This also suggests that the gene is func-tional and that the 2 nt deletion may impart a novel function to the gene since it's persistence would appear to be under positive selection.
In Summation phospholipase C gene family plays a crucial role in signal transduction as well as in changes in membrane lipid composition.The two full length wheat PLCs cDNA clones were sequenced and related wheat PLC sequences identified in the GenBank data base and characterized.Homeologs to one of the wheat full length clones, Ta-PG-PLC2, with 93% nucleotide sequence identity were identified.The origins of the three members of the PG-PLC gene family in the ancestral genomes of T. aestivum were suggested by cytogenetic analysis, i.e. by both nullisomic-tetrasomic and ditelosomic analysis coupled with the amplification with gene-specific primers for each homolog of each bread wheat ancestors (T.urartu, A. speltoides and A. tauschii).In addition, a frame shift mutation was found in genome A progenitor and in all twenty one bread wheat cultivars.This mutation likely provided some positive selective advantage, it's identification in the diploid progenitor suggests that it is an old mutation.If the gene were not functional, we might expect the gene to accumulate other mutations, but the cDNA suggests an intact and functional gene.In relation to the diploid wheat, other members of the gene family may compensate for the change of function of this gene family member.

SUMMARY
Phospholipasase Cs (PLCs) has been recognized as important enzymes for their roles in regulation, signal transduction and membrane composition.Two full length wheat PLCs cDNAs, Ta-PG-PLC2-1 and Ta-PG-PLC2-2 from wheat, Triticum aestivum L., were sequenced and mapped to wheat chromosome arms using PCR amplification in diploid wheat ancestors, Triticum urartu, Aegilops speltoides, Aegilops tauschii and cytogenetic stocks of Triticum aestivum and found to have very high sequence similarity and one, PG-PLC2-1, was found to contain a frame shift mutation 29 nt downstream of the first ATG start codon.This sequence was used to survey the prevalence of the mutation in twenty one wheat cultivars and was found to be present in all wheat cultivars tested and in the ancestral diploid species, Triticum urartu.

Fig. ( 5
Fig. (5): Detection of the three PG-PLC2 homeologs in different wheat species, A is T. urartu, B is A. speltoides and D is A. tauschii.

Table ( 1
): Wheat species and cultivar's used to survey mutation in PG-PLC2.

Table ( 3
): Primers used for mutation survey in wheat.