- Research article
- Open Access
Delineating the structural, functional and evolutionary relationships of sucrose phosphate synthase gene family II in wheat and related grasses
© Sharma et al; licensee BioMed Central Ltd. 2010
- Received: 5 February 2010
- Accepted: 30 June 2010
- Published: 30 June 2010
Sucrose phosphate synthase (SPS) is an important component of the plant sucrose biosynthesis pathway. In the monocotyledonous Poaceae, five SPS genes have been identified. Here we present a detailed analysis of the wheat SPSII family in wheat. A set of homoeologue-specific primers was developed in order to permit both the detection of sequence variation, and the dissection of the individual contribution of each homoeologue to the global expression of SPSII.
The expression in bread wheat over the course of development of various sucrose biosynthesis genes monitored on an Affymetrix array showed that the SPS genes were regulated over time and space. SPSII homoeologue-specific assays were used to show that the three homoeologues contributed differentially to the global expression of SPSII. Genetic mapping placed the set of homoeoloci on the short arms of the homoeologous group 3 chromosomes. A resequencing of the A and B genome copies allowed the detection of four haplotypes at each locus. The 3B copy includes an unspliced intron. A comparison of the sequences of the wheat SPSII orthologues present in the diploid progenitors einkorn, goatgrass and Triticum speltoides, as well as in the more distantly related species barley, rice, sorghum and purple false brome demonstrated that intronic sequence was less well conserved than exonic. Comparative sequence and phylogenetic analysis of SPSII gene showed that false purple brome was more similar to Triticeae than to rice. Wheat - rice synteny was found to be perturbed at the SPS region.
The homoeologue-specific assays will be suitable to derive associations between SPS functionality and key phenotypic traits. The amplicon sequences derived from the homoeologue-specific primers are informative regarding the evolution of SPSII in a polyploid context.
- Bread Wheat
- Hexaploid Wheat
- Genome Copy
- Einkorn Wheat
- Genome Progenitor
Sucrose plays an important role in the plant life cycle. As the major photosynthetic product, it is essential for growth, the synthesis of biomass and as a carbon and energy source. In the cereals, it is converted into starch and storage proteins, while many dicotyledonous species use it to form lipids and/or storage proteins [1, 2]. Under stressful conditions (e.g, low temperature or drought ), the plant cell typically accumulates sucrose as a protective osomoticum [3, 4]. Sucrose phosphate synthase (SPS) is one of the main regulatory enzymes involved in sucrose biosynthesis pathway in wheat and many other crop species . It catalyzes the conversion of Fructose-6-Phosphate and UDP-glucose into Sucrose-6-phosphate, which is subsequently hydrolysed to sucrose by the action of sucrose phosphate phosphatase (SPP).
In important crop plants like maize, rice and sugarcane, plant growth and productivity have been correlated with SPS activity. SPS activity has been correlated with sucrose accumulation in sugarcane stems [6–8], while in maize, correlations have been demonstarted with vigour and biomass yield [9, 10]. Genetic studies in maize have also shown that grain yield QTL (quantitative trait loci) are linked to SPS activity QTL, as are ADP-glucose pyrophosphorylase (another starch biosynthesis enzyme) activity QTL [11–13]. In rice, the location of plant height QTL appears to coincide with that of OsSPS1, as the transgenic plants with increased SPS activity grew taller than the non-transformed control . In tobaccco lines engineered to over-express SPS, UDP- glucose pyrophosphorylase and sucrose synthase, plant height was increased and flowering time delayed , while the heterologous expression of AtSPS induced longer stems and greater biomass . Finally, the over-expression of ZmSPS in potato was shown to improve a number of yield-realted characters , while in tomato, it altered the pattern of carbohydrate partitioning in the leaf .
The regulation of SPS activity is rather complex, and involves fine tuning at both the transcriptional and the post-translational level . Among the dicotyledonous plant species, three SPS gene families have been recognized, while in the monocotyledonous Poaceae species wheat, maize and rice, there are five gene families . Here, we report the temporal patterns of expression of SPS and SPP, and the discovery of structural polymorphisms between homoeologous copies of SPSII. We have also identified the presence of unspliced introns in one of its homoeologues. Evolutionary relationships among the SPSII homoeologues have been illustrated by comparisons between the gene sequences present in hexaploid wheat and its progenitors. Finally, we extend the phylogeny to other grass species.
Gene expression analysis of sucrose biosynthesis genes during wheat plant ontogeny
As SPSI was prominently expressed in the seedling leaf and the mature stem (Figure 1) its function may be associated with the re-synthesis of sucrose from fructan. SPSII (Ta.4256.1.S1_at), SPSIII (Ta.20568.1.S1_at), TaSPSIV and TaSPSVa were prominently expressed during microsporogenesis (at premeiosis, throught meiosis and in immature pollen) and in the mature anther. All the SPP families were highly expressed during anther development (Figure 1). Thus, these genes are clearly important for normal pollen/anther development. Interestingly, note that in the Arabidopsis thaliana mutant kns2-1, an altered SPS glycosyltransferase I domain disrupts the function of sucrose synthesis, and this leads to changes in the synthesis of the pollen primexine or callose . In the wild type, SPS5.2 (KNS2) is expressed in uni- and bicellular microspores as well in more mature anthers. The present data showed that probably all the SPS and SPP genes were constitutively expressed throughout caryopsis development, indicating that more than one SPS isoforms contributes to overall SPS activity (Figure 1).
Furthermore, it is important to know how these different SPS members are spatially expressed in different seed tissues and possible co-expression of these members in the same cell for making hetero-oligomeric complexes to be functional. The developing caryopsis imports sucrose to driving its growth and allow storage of metabolites. The expression of SPS and SPP members within the developing caryopsis suggests that sucrose cleavage, transport and re-synthesis during its internal transport can be differentially regulated within various distinct tissues. Of particular interest was the observation that both variants of SPSII are differentially expressed in the developing caryopsis. Thus, Ta.4256.1.S1_at was highly expressed both throughout anther development and during late embryo development, while TaAffx.8003.1.S1_at was expressed most strongly during early caryopsis development. The contribution of the individual SPSII homeologues and the occurance of alternative splicing are expanded upon in the following section. Besides alternative splicing, a contributory reason for the present exclusive focus on the SPSII family was that there were four SPSII cDNA sequences available in the public domain, which gave the opportunity to design homoeologue-specific primers for three wheat genomes. Insufficient sequence data are presently available for the other SPS families. Therefore, in order to study polyploidization and phylogenetics relationship between three wheat genomes, SPSII was selected as best suited candidate.
Structural analysis of SPSII genes in wheat and its relatives
Genetic mapping of TaSPSII genes in wheat
Detection of SNP diversity
Haplotypes for SPSII gene obtained in wheat A and B genomes
Haplotypes -A Genome
SNP1 (Exonic, 1432 bp
SNP2* Exonic, 1554 bp)
SNP3* (Exonic, 1599 bp)
Cultivars per haplotype
SNP1 (Intronic, 451 bp)
SNP2 (Intronic, 523 bp)
SNP3, (Intronic 793 bp)
Cultivars per haplotype
Total = 27
Total = 27
Three genomes differentially contributes to the expression of TaSPSII
Genomic sequence analysis of SPSII genes
Primer sequences used for the amplification of SPSII gene in wheat and other related grasses
Forward primer (5'-3')
A. Triticum aestivum- A genome
1. w-spsII-207L* (CGTTTTATCCCCCCATAAGAGA)
2. w-spsII-5L (AGCACAAATGGAGCTGTTTTG)
3. w-spsII-9L (GCTTATGGTCTACCTATGGTTGCT)
4#. w-spsII-dL* (AGAAAAGAATAGCGAGAGTGGAA)
B. Triticum aestivum -B genome
1. w-spsII-205L (AGCTGCTACGGTCGTGAAATG)
2#. w-spsII-65L (CATTTGGTGAACACCATGAGCTA)
w-spsII-73R* ( CCGGTGTATATCGACAGGT)
3. w-spsII-2L (GCTGGTTAAGGCATTTGGT)
4. w-spsII-86L* (GAAAAATCAGGCAACATGTCG)
5. w-spsII-iL (CACTAAGGTTTGGTTATTCCTATAACTGT)
w-spsII-nR * (CCTGCACAAAGATGAACAAACG)
6#. w-spsII-mL* (AACCACAAAGTCCTGATGGTACG)
7#. w-spsII-12L (AAACTATTTGTCACGGGTTGGT)
C. Triticum aestivum -D genome
1 w-spsII-D1L* (CCAAAATGGTGGGCCTGTTG)
2a. w-spsII-D1L (CCAAAATGGTGGGCCTGTTG)
D. Triticum urartu , Triticum speltoides , Aegilops tauschii , Hordeum vulgare
1. w-spsII-205L (AGCTGCTACGGTCGTGAAATG)
2. w-spsII-2L (GCTGGTTAAGGCATTTGGT)
3. w-spsII-5L (AGCACAAATGGAGCTGTTTTG)
4. w-spsII-9L (GCTTATGGTCTACCTATGGTTGCT)
5. w-spsII-22L (TTCATACCTGTTCTAGCATCACGA)
6. w-spsII-14L (GAGCAGCATCAGAGATACATCCTT)
7. w-spsII-20L (AAAGCTGGGGCCACCAC)
8. w-spsII-22L (TTCATACCTGTTCTAGCATCACGA)
9. w-spsII-22L (TTCATACCTGTTCTAGCATCACGA)
10. w-spsII-11L (AATCAAAATGATATAGCTGAGGCACTT)
11. w-spsII-15L (GTTAACATCTGGGGGCATAGAAAT)
12. w-spsII-5L (AGCACAAATGGAGCTGTTTTG)
Frequency of SNPs
The low level of SNP within SPSII suggests that variation in its sequence is constrained by selection against changes in its physiological activity. Other starch metabolism enzyme encoding genes have similarly been associated with little sequence variation [22, 28]. The observed SNP frequency was 1 per 797 bp within the A genome copy, and 1 per 916 bp within the B genome copy, frequencies which are typical for bread wheat [29–31]. Most of the SPSII SNPs were located within either an intron or the 3' UTR, with only two exonic non-synonymous ones uncovered. These latter substitutions may be of physiological significance, given that an alanine to valine change compromised the processing of a 22 kDa protein in maize , and that an arginine to lysine substitution in the bZIP domain of the maize opaque gene abolished the specificity of its DNA binding .
Presence of an unspliced intron in TaSPSII cDNA is mapped to B genome
Contribution of A and D genome for the expression of TaSPSII
All five SPS members appeared to be both spatially and temporally regulated. Of special interest in SPSII with two different variants, Ta.4256.1.S1_at: highly expressed throughout anther development as well also during late seed development in embryo and TaAffx.8003.1.S1_at: expressed mainly during early seed development. The expression pattern of Ta.4256.1.S1_at was consistent with that previously exposed by Northern blot analysis , but the expression of the latter gene has not been documented before. Although microarray-based expression data reflect very clearly any differences between distinct genes, they are not informative at the level of homoeologues, because the array features are not homoeologue-specific. In this case, homoeologue specific primers could play a vital role in knowing the contribution of three different genomes in terms of gene expression pattern. Similar strategy was also used earlier for studying expression patterns of benzoxazinones biosynthesis in wheat contributed by different genomes . The present data have shown that the A genome copy of SPSII dominated the global expression of SPSII and that the B genome copy - despite its two alternative transcripts - the least.
Synteny to rice is perturbed at SPSII locus
At the cDNA sequence level, SPSII is highly similar to its rice orthologue, which is located on chromosome 8 . Gene order is largely conserved among the major cereal genomes [36–38], which has allowed for the identification of a syntenic relationship between rice chromosome 8 and the wheat chromosomes of homoeologous group seven. The prediction is therefore that wheat SPSII should map to wheat chromosomes 7A, 7B and 7D, and not, as is the case, to chromosomes 3A, 3B and 3D. The chromosomal location that we have deduced is secure, as it relies on a combination of anaeuploid analysis, genetic mapping, the placement of SPSII ESTs http://wheat.pw.usda.gov/cgi-bin/westsql/map_locus.cgi, and the presence of SPSII sequence on flow-sorted chromosome 3A template. Rice chromosome 1 has been reported to be highly syntenic to wheat chromosome three [38, 39]. In the present study, it appears that a segment at least of the group 3 chromosomes has lost synteny with rice chromosome 1. Failure of synteny and breakdown of colinearity is well documented [24, 38, 40, 41]. Prominent examples involve the regions around both GBSS1  and the Pbf orthologues in wheat and barley . Of 537 mapped ESTs mapping to the wheat group 3 chromosomes, ~15% shared homology with rice genes mapping outside of chromosome 1, and various complexities associated within co-linear regions suggests that the relationship between these chromosomes is not straightforward . Similarly, the analysis of 1 Gb of chromosome 3B has identified the presence of gene sequences whose rice orthologues do not map to chromosome 1 , while the annotation of 17 Mb of various regions of chromosome 3B has shown that only half of the predicted genes present had orthologues on rice chromosome 1 . The SPSII genes lie in the distal region of their respective wheat chromosome, which suggests that co-linearity with rice may be lost there. Complex duplications, rearrangements and polyploidization events have led to the erosion of synteny and colinearity between rice and wheat [45–47]. The imminent completion of the sequence of the entire chromosome 3B will allow the formulation of a better picture of orthologous relationships between wheat, rice, sorghum and false purple brome, as the genomes of later three species have already been fully sequenced.
Phylogenetic relationship of TaSPSII in the context of polyploidization among hexaploid wheat and its progeniotors
The sequence alignments and subsequent phylogenetic analyses was carried out considered either exons or introns or both (Figure 4, Additional files 2, 3, 4, 5, 6). The hexaploid wheat A genome copy consistently appeared closest to that of einkorn wheat, the A genome progenitor. At the level of exons only, the B genome copy appeared closely related to that of T. speltoides, a species considered to be taxonomically close to the B genome progenitor. Branching patterm in dendrogram was little disturbed when considered only introns depicting nonconserved nature of introns. With respect to the D genome copy, the comparison with its goatgrass orthologue was only possible on the basis of the sequence of one large exon. This showed that the D genome copy was more similar to the goatgrass orthologue than to any of the others. The level of homology in the exonic sequence between the A genome and the einkorn copy was 99.9%, decreasing to 99.0% when intronic sequences were compared (Additional file 6). The equivalent similarities between the B genome and the T. speltoides copies were 97.9% and 95.1% (Additional file 6). and that between the D genome and the goatgrass exonic sequence was 98.0%. Thus, polyploidization has left exonic sequence largely unchanged, while intronic sequence has been able to drift somewhat, as has been repeatedly observed in such comparisons [22, 34]. The origin of bread wheat has involved two separate allopolyploidization events, the first between einkorn and a species closely related to T. speltoides, and the second between the resulting wild tetraploid T. dicoccoides and goatgrass . The phylogeny predicted from the sequences of the SPSII homoeologues and wild species orthologues is fully consistent with this settled origin (Figure 4) and supports the previous published studies [49, 50].
Phylogenetic relationship of TaSPSII between different grasses
The phylogenetic analysis showed that the purple false brome SPSII sequence is more similar to those of wheat and barley than to that of rice, not only in terms of the sequence itself, but in terms of the gene's overall length. The purple false brome SPSII fragment extending from exon 8 to the 3'end of the gene (a region available for nine of the ten sequences) was 518 bp longer than the longest wheat or barley sequence, while this difference was 3,199 bp in rice and 3,663 bp in sorghum. A number of phylogenetic studies have shown that the taxonomic separation between the Triticeae and purple false brome is much less than between them and rice [51–53], which is the reason why purple false brome has been so readily adopted as a model genome for the Triticeae [53–55].
A study of the expression of sucrose biosynthesis gene family members has revealed that SPS genes are highly regulated both spatially and temporally. We have been able to locate the SPSII homoeoloci to the short arms of their respective group 3 chromosomes, and have shown that their divergence is consistent with the known evolutionary relationships between the component genomes of hexaploid wheat. A phylogenetic study confirmed that purple false brome is a more appropriate model for the Triticeae than rice. We have provided evidence that the syntenic relationship between rice chromosome 8 and wheat group 3 chromosomes is perturbed in the region where the SPSII genes map. We have shown that the transcript of the B genome copy of SPSII exists as two alternative forms, one with, and the other without an intron. The B genome copy contributed least to the global expression of SPSII expression. The development of homoeologue-specific markers for, and SNPs within the wheat SPSII genes will enable the definition of any associations between SPSII genotype and plant phenotype in terms of vigour, biomass productivity or source-sink relationships.
Plant materials and DNA isolation
Details of wheat accessions used for sequencing
Country of origin
Thatcher (TRI 1308)
Maris Huntsman (TRI 10287)
Chinese spring (TRI 23666)
W7984 (mapping parent)
- (synthetic line)
Triticum urartu (AA) (TRI 17140)
Aegilops speltoides (SS BB) (AE 413 )
Aegilops tauschii (DD) (TRI 145/96)
Triticum dicoccoides (AABB) (TRI 18495)
Triticum durum (AABB) (TRI 19197)
Expression analysis of SPS and SPP members
ESTs/cDNAs sequences for each of the five SPS gene families and three different SPP gene families in wheat were blasted against the probesets available on the wheat 61 K microarray platform on which corresponding wheat SPSI to SPSV and SPP1 to SPP3 cDNA gene sequences were identified with a significant e value of 10-10 or higher. To assess gene expression patterns of SPS and SPP gene family members during wheat plant development we downloaded CEL files of Affymetrix chip from the publicly available reference experiments (Series GSE12508; GSE9767; GSE6027) covering various tissues during germination (coleoptile, root, embryo), seedling stage (crown, leaf), stem at anthesis, reproductive tissues (immature inflorescence, floral bracts, pistil, mature anthers), various developmental stages of microsporogenesis and seed development. The transcriptome data of 61 K genes was subjected to RMA normalization with linear model using limma package. The log2 expression values were derived, differential expression is calculated with P value correction mode of Benzamini Hochberg and heat maps were generated for the differentially expressed SPS and SPP gene family members covering the complete wheat plant ontogeny using Genesis software .
The designing of SPSII family specific primers and subsequent PCR and cloning
The cDNA sequences of each SPS gene was retrieved from GenBank (accessions AF347064, AF347065, AF347066, AF347067, AF347068, AF347069, AF354298, AF534907 and AY4257109) and aligned using the Multalin  program. The SPSII gene family is represented by AF347064, AF347065, AF347066 and AF354298. Stretches of sequences limited to a particular gene family formed the basis for designing family specific primers, and in particular for SPSII (Table 2). To test the specificity of these family specific primers, the sequence of the resulting amplicons was used as a BLAST query against the GenBank sequence database http://blast.ncbi.nlm.nih.gov/Blast, applying high stringent conditions. Template DNA for the subsequent PCR was represented by cvs. 'Chinese Spring', 'M6' (a synthetic hexaploid wheat) and 'Opata'. Each 25 μl PCR comprised 50-100 ng template, 2.5 μl of buffer containing 1.5 mM MgCl2, 0.2 mM dNTP, 10 μM of each primer and 1 U Taq polymerase. The amplification conditions consisted of an initial denaturation step (94°C/4 min), followed by four cycles of 94°C/1 min, Ta/1 min, 72°C/2 min (where Ta began at 62°C and was reduced by 1°C per cycle), then 30 cycles of 94°C/1 min, 58°C/1 min, 72°C/2 min and completed by a final extension step of 72°C/5 min. This profile is referred to as "PCR profile 1". Amplicons were cloned into the pDrive cloning vector (QIAGEN, Hilden Germany). Plasmids were recovered from 20 clones from each of the three donor varieties using the QIAGEN plasmid purification kit (QIAGEN, Hilden Germany), and these were subjected to sequencing from both ends using a BigDye terminator v3.1 ready reaction cycle sequencing kit in combination with an ABI3730x1 sequencer (Applied Biosystems).
Phred-Phrap-Consed analysis of sequenced clones
Phred-Phrap-Consed analysis http://www.phrap.org/phredphrapconsed.html was applied to ensure the quality of the sequences taken forward [59, 60]. SNPs were detected by polyPhred  and visual inspection of Phrap assemblies. Two high quality contigs, assumed to represent two of the predicted three wheat homoeologues, were aligned using Sequencher software (Gene Codes Corporation, Ann Arbor, Michigan), and their consensus sequence formed a megablast [62, 63] query against the GenBank database. The outcome of this search was a confirmation that these two sequences were homologous to SPSII and not to any of the other SPS genes. Primer walking was employed to extend the sequence in both directions beyond the cloned region, and the sequence thereby acquired was subjected to megablast to ensure that it was SPSII specific.
Gene annotation, designing of homoeologue-specific primers, sequencing
A provisional gene structure was obtained by aligning the cDNA sequences with the rice genome sequence, and this was later confirmed by direct alignment with wheat genomic sequence. These alignments was performed using Genseqer http://www.plantgdb.org/PlantGDB-cgi/GeneSeqer/PlantGDBgs.cgi software, applying the the rice specific splice site model and a high stringency . Exon anchored primers were designed to span one or more intron using Primer3 software . Homoeologue-specific primers targeted indels and/or base substitutions mainly in the intronic sequence (Table 2), and their specificity was tested by the amplification of DNA from nulli-tetrasomic template. Four primer pairs specific for the 3A copy of SPSII were obtained, and six for the 3B copy (Table 2, Figure 2). However, no 3D copy primers were identified in this way. To obtain an assay for the 3D copy, primers designed to amplify an exonic fragment from bread wheat were applied to the goatgrass accession TRI 145/96, and a region of this amplicon which was specific to goatgrass (839-880 bp, see Additional file 3) was then targeted for successful primer design (Table 2, Figure 2). The A and B genome copies were amplified using PCR profile 1, while this was modified for the D genome copy to an initial denaturation step (94°C/4 min), followed by 35 cycles of 94°C/1 min, 65°C/1 min, 72°C/2 min and completed by a final extension step of 72°C/5 min (PCR profile 2). The primer sets were applied to obtain orthologous sequence from einkorn, T. speltoides, goatgrass and barley. For einkorn and T. speltoides it was possible to derive the sequence from intron 7 to the 3'end of the gene in this way, and from goatgrass and barley from exon 6 to the 3'end of the gene. For rice, sorghum and false purple brome, sequence information was retrieved from, respectively, TIGR http://rice.plantbiology.msu.edu/, Phytozome http://www.phytozome.net and BrachyBase http://www.brachypodium.org/. All sequences are shown in Additional file 1, and have been deposited in GenBank as accession numbers GU797178, GU797179, GU797180, GU797181, GU797182, GU797183 and GU929217.
Mapping of homeologue specific amplicons
Homeologue specific markers showing polymorphism between parents of International Triticeae Map ping Initiative (ITMI) population were used for genetic mapping. For genetic mapping 112 recombinant inbred (RI) lines from the ITMI population were used. This population was derived from the cross between W-7984, an amphi hexaploid wheat and Mexican wheat variety Opata 85 from CIMMYT (Centro Internacional de Me joramiento de Maizy Trigo) . The SPSII markers were integrated into the framework map composed of RFLP markers using software package MAPMAKER/Exp version 3.0b . Recombination fractions were converted to centimorgans with the Kosambi mapping function .
SNP identification, synteny study and cluster analysis
Primer pairs which produced a locus-specific amplicon were used amplify template DNA from 27 wheat cultivars, using the appropriate PCR profiles described above. The PCR products were purified with the MinElute™ UF PCR purification kit (QIAGEN, Hilden Germany) following the manufacturer's instructions, and subjected to sequencing from both ends. SNPs were identified by Sequencher v4.06 software (Gene Codes Cooperation, Ann Arbor, MI, USA). Position of the various SNPs shown in Additional file 3. Sequence alignment and phylogeny construction were performed using the MegAlign module of DNASTAR Lasergene® v8.0. software. The resulting sequence alignments and phylogenetic trees are shown in Additional files 2, 3, 4, 5 and Figure 4. Phylogenetic trees were subjected to bootstrap analysis, based on 10,000 replicates.
RNA isolation, cDNA synthesis and qRT-PCR
Total RNA was extracted from cv. 'Prinz' seedlings and developing caryopses at 4, 8 and 12 days after anthesis, and from seedlings of cv. 'Chinese Spring', einkorn, goatgrass and T.durum using the TRIzol reagent (Invitrogen GmbH, Karlsruhe, Germany) and RNAeasy columns (Qiagen, Hilden, Germany), as prescribed in the manufacturers' protocols, followed by in-column DNAse digestion. RNA concentration was measured using a NanoDrop photometer (Peqlab), following the manufacturer's instructions. A 2 μg sample of RNA was used as template for cDNA synthesis, using the SuperScript™III kit (Invitrogen GmbH), according to the manufacturer's instructions. cDNA yield was tested by quantitative PCR employing a serine/threonine protein phosphatase gene (TaPP2A) as reference. The cDNA was diluted to so that the concentration of all samples lay between ± 1 CT. A qRT-PCR assay of the A and D genome SPSII homoeologue targeted exonic regions, following recommended guidelines , while the B genome homoeologue targeted the unspliced intron. PCRs were performed in optical 384-well plates using the ABI PRISM® 7900 HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA) and SYBR® Green to monitor dsDNA synthesis. Each 10 μl reaction contained 5 μl 2 × Power SYBR® Green Master mix reagent (Applied Biosystems), 1 μl cDNA and 200 nm of each homoeologue-specific primer. The PCR profile was 50°C/2 min and 95°C/10 min, followed by 45 cycles of 95°C/15 s, 60°C/60 s. Melting curves were recorded after cycle 45 by heating from 60°C to 95°C at 1.9°C min-1. Data were analysed using SDS2.2.1 software (Applied Biosystems). To generate a baseline-subtracted plot of the logarithmic increase in fluorescence signal (ΔRn) versus cycle number, the baseline data were collected between cycles 3 and 15. To obtain CT values, the amplification plots were analysed at an Rn threshold of 0.2. To compare data from different cDNA samples, the SPSII CT values were normalized against the expression of TaPP2A. PCR efficiency (E) was estimated from the exponential phase of each amplification, following . Since these lay between 1.8 and 2.0 for all samples, the expression level was calculated as 2-ΔCT, where ΔCT represented the difference between the CT of the SPSSII homoeologue and that of TaPP2A.
The financial assistance received from the Alexander von Humboldt foundation for carrying out this study is gratefully acknowledged. The research work was carried under Humboldt Research Fellowship awarded to Shailendra Sharma. Authors also thanks to Dr. Christof Pietsch for Phred-Phrap-Consed analysis.
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