BACKGROUND
stripe rust, caused by the fungus puccinia striiformis f. sp.
tritici , is one of the most destructive diseases affecting wheat  in many regions of the world.
in china, stripe rust infects millions of hectares of wheat annually  <cit> , causing large yield losses over the past two decades due to the emergence of new virulent races in the fungus population  <cit> .
as a foliar disease, stripe rust has a considerable effect on grain yield, depending on the disease response  <cit>  and the host growth stage at which pst infection is initiated  <cit> .
stripe rust epidemics can begin at any growth stage in wheat  <cit> , and therefore, they have a marked effect on plant vigor in addition to grain yield  <cit> .
severe stripe rust infection also alters the distribution of assimilates among the various organs of the plant  <cit> .
stripe rust infection is closely linked to both leaf photosynthesis and senescence, and many of the expressed sequence tags  induced by puccinia striiformis are related to those two processes .
a recent study in wheat showed that the senescence-associated gene  tasag <dig> plays an important role in the defense against stripe rust  <cit> .
in addition, the multi-gene pathogen resistance factor ‘lr34/yr18/pm38’ involves leaf senescence  <cit> .
these findings imply that senescence-and photosynthesis-related processes might be part of the resistance response to stripe rust infection.
wheat line l <dig>  is from f6: <dig> families of a cross between my <dig> and yu <dig>  the latter being a derivative of a wide cross involving thinopyrum intermedium  <cit> .
genetic analysis demonstrated that stripe rust resistance observed in l <dig> is conferred by a single dominant gene, yrl <dig>  which was mapped to chromosome 1b by linkage analysis with simple sequence repeat  markers and chinese spring nulli-tetrasomic lines  <cit> .
although the pedigree of l <dig> includes th.
intermedium, the question of whether yrl <dig> is derived directly from th.
intermedium remains unanswered.
the objectives of the present study were to assess whether resistant plants maintained the normal timing of leaf senescence during grain filling and to examine the physiological effects of any leaf senescence traits that were affected by infection.
we also explored the molecular mechanism of resistance to stripe rust in wheat and attempted to determine the origin of yrl <dig> by high-density mapping and comparative genomic analysis.
we demonstrated that it is possible to map resistance genes using the identified ests between isogenic lines after pathogen infection, and we outline a putative novel genetic mechanism of resistance conferred by this gene.
RESULTS
identification of unique ests induced by pst infection
l <dig> was highly resistant and l <dig> highly susceptible to pst race cyr <dig> .
the response tests performed at the adult  and seedling stages  showed similar results.
random sequencing of bacterial colonies from the suppression subtractive hybridization  library identified  <dig> ests, with an average length of  <dig> bp, which were submitted to ncbi under the accession numbers  though  and  through  .
of these,  <dig> unique ests  with an average length of  <dig> bp , including  <dig> contigs with an average length of  <dig> bp and  <dig> singletons with an average length of  <dig> bp, were found.
most of the uniests were between  <dig> bp and  <dig> bp in length  and were further identified through clustering and assembly of putative full-length cdnas based on the blast analysis option in ncbi genbank .fig.
<dig> seedling  and adult  responses to a) l <dig>  b) l <dig>  c) l <dig>  d) l <dig>  c functional classes of differentially expressed sequences induced by pst inoculation
both l <dig> and l <dig> were sampled  <dig>   <dig> and  <dig> h after inoculation with pst cyr <dig>  cdna prepared from l <dig> leaves infected with pst cyr <dig> was used as the tester, and cdna prepared from l <dig> leaves infected with pst cyr <dig> was used as the driver
functional categorization of the ests, as well as gene network analysis, predicted that pst infection in l <dig> induces differential regulation of leaf senescence-associated-genes .
of the  <dig> sequences,  <dig> showed high sequence similarity to known or annotated genes from cdna libraries from wheat, rice, barley, pea and arabidopsis thaliana .
of the genes that were upregulated in l <dig>   <dig> % were involved in photosynthesis-like and leaf senescence-like pathways,  <dig> % in cellular process pathways,  <dig> % in protein biosynthesis pathways,  <dig> % in the hydrolase pathway,  <dig> % in the oxidoreduction pathway,  <dig> % in the transmembrane transport pathway,  <dig> % in the electron transport pathway, and  <dig> % in glycolysis.
the remaining  <dig>  of upregulated sequences had unspecified or unknown functions,  <dig> of which closely matched the sequences of putative senescence-associated genes .
polymorphisms between ests and linkage analysis of stripe rust resistance
out of  <dig> primers designed, one primer pair   produced polymorphic amplicons between l <dig> and l <dig> as well as between the resistant parent yu <dig> and the susceptible parent my <dig> .
the design of the ls <dig> primers was from the sequence of , which we mapped to wheat chromosome 1a by in silico mapping.
the l <dig>  l <dig>  yu <dig> and my <dig> lines produced two amplicons, one  <dig> bp amplicon, which was the same among all genotypes, and a second amplicon that was polymorphic between l <dig> and l <dig> and between yu <dig> and my <dig>  in l <dig> and susceptible parents my <dig> and cm <dig>  this amplicon was  <dig> bp, and in the resistant lines l <dig>  l <dig>  l <dig> and l <dig>  as well as yu <dig> and sister line yu <dig>  the amplicon was  <dig> bp.
amplification of genomic dna from the nullisomic-tetrasomic and ditelosomic lines  revealed that the amplicon common among all lines and cs was absent in the nullisomic 1a lines .fig.
<dig> identification of polymorphism between stripe rust-resistant and stripe rust-susceptible genotypes.
the ls <dig> primer designed from an est sequence  produced polymorphic amplicons among the various genotypes.
a a  <dig> bp amplicon was amplified in the susceptible l <dig> line and its susceptible parents my <dig> and cm <dig>  a  <dig> bp amplicon was amplified in resistant l <dig> line, resistant sister lines l <dig>  l <dig>  and l <dig>  and their resistant parent, yu <dig>  a  <dig> bp amplicon was also amplified from yu <dig>  one of the resistant sister lines of yu <dig>  a  <dig> bp fragment was amplified in all genotypes.
b chromosomal localizations of the amplicons that were polymorphic between l <dig> and l <dig>  with cs nulli-tetrasomic lines of homoeologous group  <dig> and ditelosomic lines of wheat chromosome 1b.
pcr was performed to map the gene using genomic dna from the cs and various aneuploids.
pcr products were resolved on  <dig> % polyacrylamide gels.
no pcr product was generated from nullisomic 1b  or the ditelosomic 1bs  lines.
c silver-stained polyacrylamide gels showing polymorphic markers generated using the lsc <dig> primer linked to the stripe rust resistance gene in l <dig>  l <dig>  susceptible parent; l <dig>  resistant parent; r <dig> and r <dig>  resistant f <dig> individuals; br, the resistant f <dig> dna pool; h <dig>  h <dig> and h <dig>  resistant f <dig> individuals; s <dig>  s <dig> and s <dig>  susceptible f <dig> individuals; bs, the susceptible f <dig> dna pool.
l <dig>  s <dig>  s <dig>  s <dig> and bs showed amplification of  <dig> bp and  <dig> bp fragments; l <dig>  r <dig>  r <dig> and br showed amplification of  <dig> bp and  <dig> bp fragments; h <dig>  h <dig> and h <dig> showed amplification of all three fragments, indicating heterozygosity
the polymorphic  <dig> bp amplicon was located on chromosome arm 1bl, as no pcr products were generated from the nullisomic 1b lines  or the ditelosomic 1bs line .
sequencing of the polymorphic amplicon found that there was an extra repeat of  <dig> bp sequence in the susceptible allele compared with the resistant allele.
sequence similarity searches with the polymorphic amplicon found a contig  with high sequence similarity located on wheat chromosome 1b.
we designed four pairs of primers for linkage analysis based on differences in the number of copies of the  <dig> bp sequence .
analysis using the lsc <dig> primer pair  detected a  <dig> bp product closely linked to the yrl <dig> resistance gene in l <dig>  analysis of  <dig> f2: <dig> lines with additional primer pairs  further demonstrated a co-dominant marker on chromosome 1b co-segregating with yrl <dig>
fine mapping and comparative genomic analysis of wcbp <dig> indicated that it co-segregated with yrl693
genomic in situ hybridization  failed to detect an alien chromosome segment from th.
intermedium in l <dig> .
a high-density integrated genetic map was constructed .
the linkage map consisted of nine ssr markers and eight est-sts markers .
the wcbp <dig> locus fell within a genetic interval of  <dig>  cm.
wcbp <dig> co-segregated with the yrl <dig> locus.
two flanking est-sts markers, bf <dig> and be <dig>  were linked to the wcpb <dig> locus at  <dig>  and  <dig>  cm, respectively, and mapped to the chromosome 1b bin c–1bl-6- <dig> .
therefore, wcbp <dig> is located in the same chromosomal region as yrl <dig>  the contigs of the two ssr markers xwmc269-1b and xcfd65-1b on one side of wcbp <dig> mapped to wheat chromosomal arm 1bl, whereas those of the seven other ssr markers on the other side of wcbp <dig> mapped to 1bs according to the published ssr linkage map  <cit> .fig.
<dig> genetic and comparative genomic linkage map of the candidate gene wcbp <dig> and yrl <dig>  a physical bin map of wcpb1; wcbp <dig> was mapped to the bin c-1bl6- <dig> .
b genetic map of wcbp <dig> with a total length of  <dig>  cm on wheat chromosome 1b.
the genetic distance in cm is shown on the left and genetic markers are shown on the right.
c the consensus ssr map for a total of  <dig>  cm of wheat chromosome 1b.
d, e, f, orthologous regions of wcbp <dig> in brachypodium distachyon chromosome  <dig>  and oryza sativa japonica chromosome  <dig>  and sorghum bicolor chromosome  <dig>  respectively
orthologs of wheat ests be <dig> and be <dig> were detected on b. distachyon chromosome  <dig>  rice chromosome  <dig>  and sorghum chromosome  <dig>  comparative genomic analysis established the collinearity of wcbp <dig> genomic regions with a  <dig>  mb  region on b. distachyon chromosome  <dig>  a  <dig>  mb region  on rice chromosome  <dig>  and a  <dig>  mb region  on sorghum chromosome  <dig>  regions that carry  <dig>   <dig>  and  <dig> genes, respectively.
the gene order was highly conserved among wheat, b. distachyon, and rice , but showed an intra-chromosomal inversion in the  <dig>  mb chromosome region of sorghum.
we also found an orthologous genomic region  of wheat contig 1bs- <dig> carrying the ssr marker xgwm18-1b at  <dig>  mb on b. distachyon chromosome  <dig> , but there was no corresponding ortholog in either rice or sorghum .
an ortholog of wcbp <dig> was found on bradi3g <dig>  at  <dig> mb on b. distachyon chromosome  <dig>  on os10g <dig>  at  <dig> mb on rice chromosome  <dig>  and on sb01g <dig>  at  <dig>  mb on sorghum chromosome  <dig> .fig.
<dig> comparative genomic mapping of ests in the wcbp <dig>  chromosomal region of wheat chromosome 1b  with brachypodium distachyon chromosome  <dig> , oryza sativa japonica chromosome  <dig> , and sorghum bicolor chromosome  <dig> .
the gene order in all species is clockwise
pcr-based cloning and functional analysis of the candidate stripe rust resistance gene
we obtained a candidate resistance gene by sequencing pcr products amplified by seven primers  from genomic dna and then comparing the amplified sequence with that of the contig .
this gene was  <dig>  bp in the resistant lines l <dig> and yu <dig> but  <dig>  bp in the susceptible lines l <dig> and my <dig> .
the genomic sequence of the candidate resistance gene isolated from l <dig> and yu <dig> included four exons  and three introns .
the fourth exon contained a single repeat of the  <dig> bp sequence in the resistant l <dig> line and its parent yu <dig> as well as two repeats in the susceptible l <dig> line and its parent my <dig>  we also identified and verified a single nucleotide polymorphism  in the first exon, with an adenine  in the resistant lines and a cytosine  in susceptible lines l <dig> and my <dig> .
the open reading frame   of the candidate resistance gene encodes a putative wheat copper binding protein  <dig>  predicted to contain  <dig> amino acids in resistant lines  and  <dig> amino acids in susceptible lines.
the snp in the first exon  does not change the deduced amino acid sequence.
the  <dig> bp repeated sequence in the fourth exon  caused an addition of  <dig> amino acids  in the susceptible line  at amino acids 242– <dig>  the deduced amino acid sequence forms a protein containing two predicted copper-binding domains, at amino acids 34– <dig> , and at amino acids 170– <dig>  .
the full-length, deduced wcbp <dig> protein shows sequence similarity with known heavy metal-binding proteins from diverse species.
phylogenetic analysis indicated that wcbp <dig> shared the highest similarity with the ems <dig> protein , but they did not cluster together.fig.
<dig> the wcbp <dig> gene, its putative protein structure, and its phylogenetic tree.
a the predicted mrna structure of wcbp <dig> is shown above the genomic sequence, including introns  and exons .
atg represents the methionine start codon, and tga represents the stop codon.
b coding regions of two heavy metal copper-binding domains in exons  <dig> and  <dig>  the major differences between the resistance-and susceptibility-associated wcbp <dig> alleles are a snp in exon  <dig> and a  <dig> bp deletion in exon  <dig>  c alignment of the amino acid sequences and secondary structures of the proteins putatively encoded by the resistance-and susceptibility-associated alleles of wcbp <dig>  d phylogenetic tree constructed using the neighbor-joining algorithm in mega  <dig>  following wcbp <dig> protein sequence alignment using the clustalw program.
accession numbers for the other heavy metal copper-binding proteins were as follows: ems <dig>  , emt <dig>  , xp_ <dig>  , eay <dig>  , xp_ <dig>  , afk <dig>  , xp_ <dig>  , xp_ <dig>  , bak <dig>  , xp_ <dig>  , daa <dig>  , np_ <dig>  , eoa <dig>  , xp_ <dig>  , xp_ <dig>  , afk <dig>  , eay <dig>  , xp_ <dig>   and afw <dig>
sequence analysis identified a highly similar gene, wcbp <dig>  on chromosome 1a.
this gene had the same dna sequence in l <dig> and l <dig>  wcbp <dig> and wcbp <dig> show  <dig>  % dna sequence similarity.
moreover, no polymorphism in the length of fragments was detected in genomic dna of resistant and susceptible varieties.
wcbp <dig> and wcbp <dig> are differentially regulated in association with resistance
to examine possible involvement of wcbp <dig> in defense against stripe rust, we measured its transcript levels at different time points following pathogen inoculation .
wcbp <dig>  was upregulated in leaves following pst inoculation of both the resistant and the susceptible parent.
transcript abundance of wcbp <dig> was significantly higher  in leaves from l <dig> and l <dig> at  <dig> h post inoculation than prior to inoculation .
however, at later time points, the wcbp <dig> transcript abundance decreased in l <dig> but remained higher in l <dig>  at  <dig> h post-inoculation, the wcbp <dig> transcript abundance was significantly lower in l <dig> compared to l <dig> .fig.
<dig> qrt-pcr expression analysis of wcbp <dig> and wcbp <dig>  amplification of wcbp <dig>  and wcbp <dig>  transcripts following pst infection.
c, amplification of wcbp <dig>  and wcbp <dig>  transcripts following mock inoculation with water.
the experiment was performed using three biological replicates with three technical replicates per biological sample.
relative expression was calculated using gapdh as a reference gene to infer steady-state mrna levels, and relative expression was normalized by setting the control sample from l <dig> at  <dig> h to  <dig>  bars represent the standard error, and the significance was determined using independent sample t-tests.
asterisks represent significant differences as follows: **p ≤ <dig> , *p ≤ <dig> ; ns, no significant difference.
the raised asterisks or the ns designation represent differences in gene expression between l <dig> and l <dig> at each time point.
asterisks or the ns designation in the trend line represent the differences in gene expression between two adjacent time points in the same genotype
we also measured the transcript levels of wcbp <dig>  prior to inoculation, l <dig> and l <dig> showed similar wcbp <dig> transcript levels, but after inoculation, wcbp <dig> was upregulated only in the resistant l <dig> line.
at  <dig> h post-inoculation, wcbp <dig> transcript abundance in l <dig> increased significantly compared with its level prior to inoculation , and wcbp <dig> transcript levels were significantly higher  in l <dig> than in l <dig> at both  <dig> h and  <dig> h post-inoculation .
in addition, we found that mock inoculation with water had no significant effects on the transcript level of either wcbp <dig> or wcbp <dig> in l <dig> or l <dig> at any time point .
the effects of resistance on photosynthesis, chlorophyll fluorescence dynamics, and yield
although the net photosynthetic rates  at all time points were significantly higher  in l <dig> than l <dig>  these genotypes showed similar trends .
notably, there were no significant differences  between any two consecutive measurements in l <dig>  whereas changes in l <dig> were highly significant  .
at the heading stage, the stomatal conductance  in l <dig> was significantly lower than that in l <dig> .
the gs of l <dig> increased sharply during the early stages and decreased toward the end of the experiment, while the values obtained for l <dig> declined sharply during the early stages of the experiment and then increased.
the intercellular co <dig> concentrations  observed in l <dig> were significantly lower  than those in l <dig> at all time points, except at  <dig> days after heading .
in l <dig>  there was a notable increase in ci, whereas this value decreased sharply between heading and  <dig> days after heading in l <dig> fig.
<dig> photosynthetic parameters used to assess the different responses of l <dig> and l <dig> to pst inoculation.
a, net photosynthetic rate ; b, intercellular co <dig> concentration ; c, stomatal conductance .
the parameters were measured once every  <dig> days after heading in the field, using a li-cor  <dig>  the disease had fully developed at heading, and  <dig> plants per genotype were used for these measurements.
bars represent the standard error, and significance was determined using an independent sample t-test.
asterisks represent significant differences as follows: **p ≤ <dig>  and *p ≤ <dig> , and ns indicates no significant difference.
raised asterisks or the ns label represent the differences in gene expression between l <dig> and l <dig> at each time point.
asterisks or the ns label on the trend line represent differences in gene expression between two adjacent time points in the same genotype
although the changes in the maximal photochemical efficiency of psІІ in the dark-adapted leaves  were similar between l <dig> and l <dig>  the fv/fm value obtained for l <dig> was significantly higher than that obtained for l <dig> at  <dig> days after heading.
a significant decrease in fv/fm occurred in l <dig> between  <dig> days and  <dig> days after heading, whereas only a slight decrease occurred in this timeframe in l <dig> .
the efficiencies of excitation capture by the open psii reaction centers  observed in l <dig> were significantly lower than, similar to, and higher than those recorded in l <dig> prior to  <dig> days, at  <dig> days and at  <dig> days after heading, respectively .
the photochemical quenching coefficient  was similar in l <dig> and l <dig>  although the qp values obtained in l <dig> were greater than those in l <dig>  the differences were not significant at the p =  <dig>  level .
the changes in the quantum yield of photochemical energy conversion in psii  , the quantum yield of regulated non-photochemical energy loss in psii  , and the quantum yield of non-regulated, non-photochemical energy loss in psii   were quite similar in l <dig> and l <dig>  the value of Φpsii recorded in l <dig> was significantly lower at the heading stage and significantly greater at  <dig> days after heading, compared with that in l <dig> .
in l <dig>  the value of npq obtained was significantly greater than that in l <dig> at the heading stage , and the value of no was significantly lower than that in l <dig> at  <dig> days after heading .fig.
<dig> chlorophyll fluorescence-related parameters used to determine differences in photosystem ii  activity between the resistant line l <dig> and the susceptible line l <dig> following pst inoculation and after heading.
a, values for the maximal photochemical efficiency of psii ; b, the efficiency of excitation capture by the open psii reaction centers ; c, the photochemical quenching coefficient ; d, quantum yield of photochemical energy conversion in psii ; e, quantum yield of regulated non-photochemical energy loss in psii ; and f, quantum yield of non-regulated non-photochemical energy loss in psii  in wheat flag leaves.
the parameters Φpsii, npq and no were derived from averages of related parameters according to a previously described method  <cit> .
the disease had fully developed at heading, and  <dig> plants per genotype were used for the measurements.
bars represent the standard error, and significance was determined using independent sample t-tests.
asterisks represent significant differences as follows: **p ≤ <dig> , *p ≤ <dig> , and ns for no significant difference.
raised asterisks or ns represent the differences in gene expression between l <dig> and l <dig> at each time point.
the asterisks or the ns label on the trend line represent differences in gene expression between two adjacent time points in the same genotype
yields were compared between the various wcbp <dig> genotypes and between the resistant plants and the susceptible plants in the f <dig> population .
l <dig> exhibited a significantly greater 1000-grain weight  and total grain weight per ear  than that of l <dig> .
the number of grains per ear  was also greater .
a total of  <dig> plants from the genetic mapping population was analyzed to determine yields , and resistant plants displayed significantly higher sw, ns and tw values compared to those of susceptible plants .
in addition, homozygous resistant and heterozygous wcbp <dig> genotypes showed significantly higher sw, ns and tw values than those of homozygous susceptible wcbp <dig> genotypes.table  <dig> comparison of the main yield parameters, including the number of grains per ear , total grain weight per ear  and 1000-grain weight , between the l <dig> and l <dig> parents and among the different genotypes in the segregating f <dig> population by independent sample t-tests
ns
+wcbp1/+wcbp1
+wcbp1/-wcbp1
-wcbp1/-wcbp1
tw
+wcbp1/+wcbp1
+wcbp1/-wcbp1
-wcbp1/-wcbp1
sw
+wcbp1/+wcbp1
+wcbp1/-wcbp1
-wcbp1/-wcbp1
r indicates the resistant phenotype, and s indicates the susceptible phenotype.
in the different genotypes, r indicates the dominant resistance gene, and r indicates the recessive, susceptible allele.
p, value describing the differences between l <dig> and l661; p <dig>  value describing the differences between the rr and rr genotypes compared to rr; p <dig>  value describing the differences between the rr and rr genotypes.
asterisks represent significant differences: **p ≦ <dig> , *p ≦ <dig>
discussion
a gene encoding a heavy metal copper-transport protein is involved in response to stripe rust infection
in this study, we identified wcbp <dig> and wcbp <dig>  two genes that encode predicted copper-binding proteins.
genes encoding heavy metal copper-binding proteins have proven interesting in multiple studies.
for example, similar genes exhibited significant changes in expression in all of eight lines near-isogenic for stripe rust resistance  <cit> .
in rice, the pi <dig> gene, encoding a putative heavy metal copper-binding domain protein, confers resistance to blast disease  <cit> .
furthermore, the rice gene xa <dig>  encoding a heavy metal copper-transport protein, confers resistance to the bacterial pathogen xanthomonas oryzae  <cit> .
in humans, hah <dig> encodes a metallochaperone protein belonging to a family of proteins containing metal-binding domains and is related to the menkes/wilson disease protein  <cit> .
these findings indicate that a great diversity of copper-binding protein genes can confer resistance to various fungal and bacterial diseases in different species.
qpcr analysis  indicated that the transcription of the wcbp <dig> susceptible allele was inhibited.
in contrast, wcbp <dig> is constitutively expressed in both l <dig> and l <dig>  and its sequence is identical in these lines .
however, its expression was only activated by inoculation with pst in l <dig>  wcbp <dig> co-segregated with resistance in f2: <dig> populations .
expression of both wcbp <dig> and wcbp <dig> was not upregulated  by mock inoculation with water , indicating that these genes respond specifically during the resistance response to stripe rust.
a gene encoding a blue copper-binding protein was induced by various oxidative stresses in arabidopsis and transgenic tobacco plants .
indeed, there is an oxidative burst during the primary hypersensitive response  <cit> , which implies that the oxidative burst potentially resulting from pst attack may induce the expression of wcbp <dig>
wcbp <dig> enhances photosynthetic competence without altering the timing of leaf senescence and protects wheat yields against pst infection.
another interesting category of differentially expressed genes is the leaf senescence-associated genes .
previous studies implied that leaf senescence might be involved in the defense against pathogenic fungi  <cit> .
transcriptome analysis also confirmed that tasag <dig>  is involved in the plant defense against pst infection  <cit> .
a total of  <dig>  %  of sequences were identified as belonging to photosynthesis-like and leaf senescence-like pathways .
previous studies showed that many pathogenesis-related  genes are induced during leaf senescence in arabidopsis, and the types and numbers of sags present within this family vary from species to species and from genotype to genotype in the same species infected with the same fungal pathogen  <cit> .
fourteen of the sag-related ests  have very high sequence similarity to tasag <dig> .
this finding further suggests that unique transcripts of sags are specific to certain yr genotypes.
yrl <dig> confers effective resistance at the seedling and adult stages .
the physiological effects of stripe rust on wheat yields were estimated by measuring photosynthetic parameters at the adult stage because yield losses caused by pst infection occur mostly from disease at the adult stage  <cit> .
previous studies suggested that a leaf senescence-associated pathway might be involved in the response to stripe rust infection .
our data  suggest that pn was regulated by the photosynthetic apparatus rather than by stomatal factors in l <dig>  indicating that l <dig> could actively regulate the photosynthetic pathway following infection with pst.
this hypothesis was also supported by observed differences in gene expression.
the increase in photosynthetic competence observed in l <dig> could be due either to an increase in the photochemical efficiency of psii or an increase in the efficiency of excitation capture by open psii reaction centers.
the latter phenomenon would result from other aspects of photosynthesis, such as photosynthetic electron transport and light utilization, and could therefore be affected by pathogen infection.
the onset of leaf senescence generally occurs at  <dig> days after heading  <cit> .
at this time point, leaves from the resistant wheat line l <dig> exhibited significantly higher fv/fm, fv‘/fm’ and Φpsii values as well as significantly lower no compared with that of l <dig> leaves, although the changes in fv/fm, fv’/fm’, qp, Φpsii, and npq were very similar , especially at 20– <dig> days after heading.
these findings indicated that the higher photosynthetic competence observed in l <dig> resulted from a higher photochemical efficiency of psii, which is an important, actively regulated mechanism involved in adaptation to disease stress.
this result showed that the putative candidate stripe rust resistance gene wcbp <dig> did not alter the onset or progression of leaf senescence .
the susceptible allele possibly accelerated the progress of leaf senescence within a normal onset period; therefore, the maintenance of a higher photochemical efficiency in psii is a direct and important factor in protection of yield against pst infection.
yrl <dig> may have originated from a dna sequence change in the wheat genome
we originally thought that yrl <dig> originated from th.
intermedium because the pedigree of l <dig> includes th.
intermedium  <cit> .
however, the fine genetic map of yrl <dig> shows similar marker order and total length that is consistent with the chromosome map of 1b .
this result indicates that there is likely no alien chromosome segment in yrl <dig>  comparative genomic analysis of yrl <dig>  also demonstrated that an alien chromosome segment is unlikely.
moreover, genes orthologous to wcbp <dig> were also identified in b. distachyon, rice, and sorghum .
additionally, we did not detect th.
intermedium sequences in the genomic region of yrl <dig> using closely linked markers.
furthermore, the genetic linkage map showed that yrl <dig> is located in the centromeric region, whereas it is commonly thought that insertion or translocation of an alien chromosome segment into the centromeric region very rarely occurs in wheat  <cit> .
all available evidence supports the hypothesis that yrl <dig> was not transferred from th.
intermedium, so we conclude that yrl <dig> is a wheat gene.
wcbp <dig> is a candidate for the yrl <dig> stripe rust resistance gene
the sequence of the wcbp <dig> allele associated with resistance contains a  <dig> bp deletion compared to the sequence in the susceptible line .
this finding suggests that resistance originated with the  <dig> bp deletion, similar to the event observed for the blast resistance pi <dig> gene in rice  <cit> .
these findings implied that dna sequence changes, especially deletions, might be important mechanisms for evolution of disease resistance in plants.
previous studies confirmed that the yr <dig> resistance gene encodes a kinase with a putative lipid-binding domain  <cit> , whereas yr <dig> encodes an adenosine triphosphate-binding cassette transporter belonging to a pleiotropic drug resistance subfamily  <cit> .
these findings indicate that different yr genes have diverse and specific functions.
CONCLUSIONS
in this study, we identified the gene wcbp <dig>  which may act as a candidate gene for the wheat stripe rust resistance gene yrl <dig> based on the following evidences.
first, the dna sequence of wcbp <dig> was polymorphic between l <dig> and l <dig>  as well as between the parents yu <dig> and my <dig>  and results from the polymorphic amplification demonstrated polymorphism between the wcbp <dig> alleles in all parental pairs.
second, genetic mapping of yrl <dig> showed that wcbp <dig> co-segregated with yrl <dig>  third, qpcr analysis confirmed that the expression of wcbp <dig> differed significantly between resistant and susceptible alleles at  <dig> h after pst-inoculation.
moreover, the expression of wcbp <dig> in resistant/susceptible plants was upregulated only upon pst infection, while it did not change significantly upon mock-inoculation with water.
the fact that wcbp <dig> was not annotated previously can be explained by its putative divergence in being specific to certain yr genotypes.
it is therefore reasonable to hypothesize that wcbp <dig> is a candidate gene for yrl <dig>  but positive confirmation will come with appropriate transformation experiments.
materials and methods
plant materials
the isogenic wheat lines l <dig> with yrl <dig> and l <dig> without yrl <dig> were used to examine differential gene expression after the plants were inoculated with pst.
their parents, yu <dig> and my <dig>  were employed as controls for the analysis of the candidate resistance gene.
th.
intermedium was employed for in situ hybridization to check for alien chromatin in yu <dig> and l <dig>  a set of chinese spring  nulli-tetrasomic  and relevant ditelosomic lines were used to identify the chromosomal location of the candidate resistance gene.
genotypes of  <dig> f2: <dig> families derived from the cross l661/l <dig> were used to construct the fine genetic map of the candidate resistance gene.
gene expression in plants challenged with pst
seedlings at the three-leaf stage, grown in a greenhouse, were challenged with urediniospores of pst race cyr <dig>  pst inoculation was conducted according to a previously described method  <cit> .
leaves from l <dig> and l <dig> plants were harvested at  <dig>   <dig> and  <dig> h post-inoculation.
a mixture of l <dig> rna samples containing equal amounts of rna extracted at each time point was used as the tester.
a mixture of l <dig> rna collected at the same time points was used as the driver for the construction of the cdna library.
non-inoculated leaves from l <dig> and l <dig> plants at  <dig> h, as well as leaves mock-inoculated with water, were harvested for use as controls for gene expression analyses.
a sample was collected from l <dig> at  <dig> days post-inoculation to represent full disease development .
construction of the ssh library
total rna was extracted from l <dig> and l <dig> leaves using the rna extraction reagent trizol .
the dynabeads oligo dt <dig> system  was then employed to purify mrna and to construct cdna libraries produced by rt-pcr.
an ssh library was constructed using the pcr-select cdna subtraction kit  according to a previously described protocol  <cit> .
positive colonies were sequenced using an abi prism  <dig> automated sequencer .
the resulting ests were analyzed against the genbank database using blastx and blastn.
the threshold probability for a sequence match was set at 10− <dig>
plant growth conditions
all wheat materials were planted in the field at the yaan agricultural research station of sichuan agricultural university  during the 2010– <dig> wheat-growing season .
seeds were sown in a clay soil on october  <dig>   <dig>  each experiment was executed as a randomized complete block design with three replicates.
approximately  <dig> seeds from each line were planted in  <dig>  m rows with  <dig> cm spacing.
the susceptible wheat line sy95- <dig>  serving as a vector of the pathogen, was planted in every third row of each population to ensure that all plants had an equal chance of infection.
sy95- <dig> was artificially inoculated with pst race cyr <dig> at the seedling stage according to a previously described method  <cit> .
the average air temperatures from sowing to grain maturity ranged from  <dig>  to  <dig>  °c.
the heading date of both l <dig> and l <dig> was approximately march  <dig>   <dig>  the plants received  <dig>   <dig>   <dig> and  <dig> g n m− <dig> ammonium nitrate at the following growth stages, respectively: one-node, meiosis, heading and anthesis.
after planting,  <dig> % imidacloprid wettable powder  was applied to control pests.
thirty plants from each genotype showing identical growth and developmental characteristics at heading were marked for all subsequent measurements and observations.
measurement of photosynthetic indices and psii activity
photosynthetic parameters and chlorophyll fluorescence were measured in l <dig> and l <dig> using a portable photosynthesis system  with a red-blue light source and a modulated photosynthesis system , respectively.
data collection was carried out once every  <dig> days, starting from heading on march  <dig>   <dig>  through april  <dig>   <dig>  at each sampling date, the mean of three independent measurements from each plant represented the plant phenotypic value, and the average values from  <dig> plants were employed for comparisons between genotypes.
measurements, parameter recording, and parameter calculations followed previously described methods  <cit> .
in silico mapping of candidate genes
the genomic locations of the wheat ests were determined in silico via blastn searches against the mapped expressed wheat sequences using graingenes  <dig>   <cit>  at a 10− <dig> threshold probability and via a blastx search against the wheat draft genome  <cit> .
sequences without a blastx hit or those showing an e-value of less than 10− <dig> were excluded from the in silico mapping to the wheat genome and the brachypodium genome.
pcr-based mapping of polymorphic ests and linkage analysis for resistance
primers were designed using primer <dig>  <cit>  to amplify ests from wheat genomic dna.
wheat genomic dna was extracted using a previously described method  <cit> .
primer sequences were listed in additional file 2: table s <dig>  pcr amplification and product analysis were performed according to previously described methods  <cit> .
polymorphic primers were used to genotype the segregating population of  <dig> f2: <dig> families .
linkage analysis of markers and resistance genes was performed with joinmap  <dig>  <cit> .
the linkage group was declared at a lod threshold of  <dig> .
subsequently, the chromosomal locations of the linked markers were confirmed using chinese spring nullisomic-tetrasomic and the ditelosomic lines of chromosome 1b  <cit> .
pcr-based cloning of putative stripe rust resistance genes
the primers designed with primer <dig>  according to the contig sequence containing the co-segregated markers and the polymorphic amplicon sequence  <cit>  were employed to amplify the entire length of the candidate gene.
these primers were listed in additional file 2: table s <dig>  each amplicon was purified using the genejet pcr purification kit  and cloned into the pgem t easy vector .
jm109-competent cells were transformed and cultured according to the manufacturer’s protocol.
plasmids retrieved using the genejet plasmid miniprep kit  were sequenced by shanghai majorbio bio-pharm technology co., ltd. .
qrt-pcr of the candidate gene and the key unigene conferring resistance
we used m-mlv  to synthesize complementary dna  for real-time qrt-pcr .
each  <dig> μl reaction mixture contained  <dig> μg of rna.
for qrt-pcr assays,  <dig> μl of the diluted cdna  and  <dig> μl of realmastermix   were included in  <dig> μl reaction volumes.
qrt-pcr was conducted using the miniopticon time pcr detection system with the following program: pre-incubation at  <dig> °c for  <dig> min, followed by amplification for  <dig> cycles at  <dig> °c for  <dig> s and  <dig> °c for  <dig> s. the melting curve was set at  <dig> to  <dig> °c, with a  <dig>  °c increase per step, and cooling was set at  <dig> °c for  <dig> s. each qrt-pcr run was replicated with three independent biological samples, and three technical repeats were included for each biological sample.
the primer sequences and descriptions of the genes are provided in additional file 2: table s <dig>  the crossing point value and results of the melting curve analyses were obtained using roche cfx manager software for the miniopticon system  <dig>  .
melting curve data showing only a single peak, which denotes primer specificity , were collected for all samples.
the repeatability and reliability of amplifications are demonstrated by the raw data shown in additional file 1: figure s1b.
statistical analysis
significant differences in the mean physiological parameters, gene expression, and yield between l <dig> and l <dig> and between plants carrying and lacking wcbp <dig> were observed.
differences between two adjacent time points for the same genotype were determined using independent sample t-tests in ibm spss statistics  <dig> .
additional files
additional file 1: figure s <dig>  length distributions of the wheat leaf ests  and the assembled uniests , including contigs  and singletons .
figure s <dig>  primers and the amplified region of genomic dna.
to clone the wcbp <dig> gene, primers were designed according to the chinese spring draft genome sequence.
full-length amplification was performed using the 1b- <dig> forward primer and the 1b- <dig> reverse primer.
figure s <dig>  comparison of wcbp <dig> gene sequences.
wcbp <dig> encodes a heavy metal copper-binding protein with two copper sensing domains.
the four sequences were cloned using pcr with primers designed according to the polymorphic fragment.
l <dig> is the stripe rust-resistant line; l <dig> is the stripe rust-susceptible line; yu <dig> is the resistant parent of l <dig> and l661; my <dig> is the susceptible parent of l <dig> and l <dig>  figure s <dig>  quality map of the sequences flanking the snp site.
a represents l693; b represents yu25; c represents l661; d represents my <dig>  which showed that the snp existed among the different genotypes.
figure s <dig>  gish analysis of mitotic metaphase chromosomes of tai  <dig>  l <dig>  l <dig> and yu <dig> using genomic dna of th.
intermedium as a probe .
a tai  <dig>  b l <dig>  c l <dig>  d yu <dig>  chromosomes were counterstained with dapi .
figure s <dig>  results demonstrating the high quality and good replication of q-pcr.
, the melt curve of the q-pcr product during the amplification of wcbp <dig>  , the amplification curve shows the consistent crossing points of the three technological replicates of the wcbp <dig> gene and the reference gene gapdh.
this chart shows l <dig> at  <dig> h.
additional file 2: table s <dig>  .
detailed information on  <dig> ests.
a blast search was performed using blastx in ncbi.
in silico mapping was performed using the iwgsc draft wheat sequence database.
table s <dig>  blast searches performed for the forward subtraction clones found in the stripe rust-resistant genotype l <dig>  table s <dig>  the primers used to amplify ests.
primer pairs were only designed for  <dig> out of the  <dig> ests because the remaining ests were too short to be used for primer design.
table s <dig>  primers were designed according to the sequence of the contig  carrying the polymorphic amplicon, which was located on wheat chromosome 1b .
these amplicons were used to determine the linkage relationship between the markers and the stripe rust resistance gene in l <dig>  table s <dig>  results of the amplification of  <dig> wcbp1-linked genetic markers in an f <dig> population derived from crosses between l <dig> and l <dig> and between l <dig> and l <dig>  the genotypes  of the f <dig> individuals were determined according to the phenotypes of the corresponding f2: <dig> families; "a" and "b" for the individual f <dig> populations were derived from crosses between l <dig> and l <dig> and between l <dig> and l <dig>  respectively.
r represents the same amplicon as the resistant parent l693; s represents the same amplicon as the susceptible parent l661; h represents the heterozygous condition.
the yield components of the f <dig> plants, including the number of grains per ear , the total grain weight per ear  and the 1000-grain weight , are provided.
‘\’ represents a missing yield component in the f <dig> plants.
table s <dig>  the sequences of seven primer pairs designed according to the sequence of a cloned candidate gene in silico; these primers were used for pcr-based gene cloning from genomic dna.
table s <dig>  primers for q-pcr analysis.
additional file 3:
methods s <dig>  genomic in situ hybridization .
abbreviations
bpbase pair
estsexpressed sequence tags
orfopen reading frame
prpathogenesis-related
pstpuccinia striiformis f. sp.
tritici
qrt-pcrquantitative reverse transcription pcr
sagsenescence-associated gene
snpsingle nucleotide polymorphism
sshsuppression subtractive hybridization
ssrsimple sequence repeat
wcbpwheat copper-binding protein
xin li, taiguo liu and wanquan chen contributed equally to this work.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
xl, tl, wc, and pl designed the study; xl and sz performed ssh under the instruction of ll; xl and tl carried out the comparative genomic analysis; and xl and wc performed the genetic analysis and genetic map construction.
the qrt-pcr was performed by xl, mz and hr.
xl, sz, hz, and lw performed the majority of experiments with contributions from zt, zc, rz and pl.
pl wrote the manuscript with input from xl, tl and wc.
all authors read and approved the final manuscript.
authors’ information
not applicable.
availability of data and materials
not applicable.
we are grateful for the financial support of the national natural science foundation of china , the state key laboratory for biology of plant disease and insect pests, china , the provincial science and technology foundation for young scientists of sichuan, china  and the ministry of science and technology, china .

 stripe rust, caused by the fungus puccinia striiformis f. sp. tritici, is one of the most destructive diseases affecting wheat in many regions of the world. in china, stripe rust infects millions of hectares of wheat annually, causing large yield losses over the past two decades due to the emergence of new virulent races in the fungus population. as a foliar disease, stripe rust has a considerable effect on grain yield, depending on the disease response and the host growth stage at which pst infection is initiated. stripe rust epidemics can begin at any growth stage in wheat, and therefore, they have a marked effect on plant vigor in addition to grain yield. severe stripe rust infection also alters the distribution of assimilates among the various organs of the plant. stripe rust infection is closely linked to both leaf photosynthesis and senescence, and many of the expressed sequence tags induced by puccinia striiformis are related to those two processes. a recent study in wheat showed that the senescence-associated gene tasag plays an important role in the defense against stripe rust. in addition, the multi-gene pathogen resistance factor ‘lr34/yr18/pm38’ involves leaf senescence. these findings imply that senescence-and photosynthesis-related processes might be part of the resistance response to stripe rust infection. wheat line l is from f6: families of a cross between my and yu the latter being a derivative of a wide cross involving thinopyrum intermedium. genetic analysis demonstrated that stripe rust resistance observed in l is conferred by a single dominant gene, yrl which was mapped to chromosome 1b by linkage analysis with simple sequence repeat markers and chinese spring nulli-tetrasomic lines. although the pedigree of l includes th. intermedium, the question of whether yrl is derived directly from th. intermedium remains unanswered. the objectives of the present study were to assess whether resistant plants maintained the normal timing of leaf senescence during grain filling and to examine the physiological effects of any leaf senescence traits that were affected by infection. we also explored the molecular mechanism of resistance to stripe rust in wheat and attempted to determine the origin of yrl by high-density mapping and comparative genomic analysis. we demonstrated that it is possible to map resistance genes using the identified ests between isogenic lines after pathogen infection, and we outline a putative novel genetic mechanism of resistance conferred by this gene.  identification of unique ests induced by pst infection l was highly resistant and l highly susceptible to pst race cyr. the response tests performed at the adult and seedling stages showed similar results. random sequencing of bacterial colonies from the suppression subtractive hybridization library identified ests, with an average length of bp, which were submitted to ncbi under the accession numbers though and through. of these, unique ests with an average length of bp, including contigs with an average length of bp and singletons with an average length of bp, were found. most of the uniests were between bp and bp in length and were further identified through clustering and assembly of putative full-length cdnas based on the blast analysis option in ncbi genbank.fig. seedling and adult responses to a) l b) l c) l d) l c functional classes of differentially expressed sequences induced by pst inoculation both l and l were sampled and h after inoculation with pst cyr cdna prepared from l leaves infected with pst cyr was used as the tester, and cdna prepared from l leaves infected with pst cyr was used as the driver functional categorization of the ests, as well as gene network analysis, predicted that pst infection in l induces differential regulation of leaf senescence-associated-genes. of the sequences, showed high sequence similarity to known or annotated genes from cdna libraries from wheat, rice, barley, pea and arabidopsis thaliana. of the genes that were upregulated in l % were involved in photosynthesis-like and leaf senescence-like pathways, % in cellular process pathways, % in protein biosynthesis pathways, % in the hydrolase pathway, % in the oxidoreduction pathway, % in the transmembrane transport pathway, % in the electron transport pathway, and % in glycolysis. the remaining of upregulated sequences had unspecified or unknown functions, of which closely matched the sequences of putative senescence-associated genes. polymorphisms between ests and linkage analysis of stripe rust resistance out of primers designed, one primer pair produced polymorphic amplicons between l and l as well as between the resistant parent yu and the susceptible parent my. the design of the ls primers was from the sequence of, which we mapped to wheat chromosome 1a by in silico mapping. the l l yu and my lines produced two amplicons, one bp amplicon, which was the same among all genotypes, and a second amplicon that was polymorphic between l and l and between yu and my in l and susceptible parents my and cm this amplicon was bp, and in the resistant lines l l l and l as well as yu and sister line yu the amplicon was bp. amplification of genomic dna from the nullisomic-tetrasomic and ditelosomic lines revealed that the amplicon common among all lines and cs was absent in the nullisomic 1a lines.fig. identification of polymorphism between stripe rust-resistant and stripe rust-susceptible genotypes. the ls primer designed from an est sequence produced polymorphic amplicons among the various genotypes. a a bp amplicon was amplified in the susceptible l line and its susceptible parents my and cm a bp amplicon was amplified in resistant l line, resistant sister lines l l and l and their resistant parent, yu a bp amplicon was also amplified from yu one of the resistant sister lines of yu a bp fragment was amplified in all genotypes. b chromosomal localizations of the amplicons that were polymorphic between l and l with cs nulli-tetrasomic lines of homoeologous group and ditelosomic lines of wheat chromosome 1b. pcr was performed to map the gene using genomic dna from the cs and various aneuploids. pcr products were resolved on % polyacrylamide gels. no pcr product was generated from nullisomic 1b or the ditelosomic 1bs lines. c silver-stained polyacrylamide gels showing polymorphic markers generated using the lsc primer linked to the stripe rust resistance gene in l l susceptible parent; l resistant parent; r and r resistant f individuals; br, the resistant f dna pool; h h and h resistant f individuals; s s and s susceptible f individuals; bs, the susceptible f dna pool. l s s s and bs showed amplification of bp and bp fragments; l r r and br showed amplification of bp and bp fragments; h h and h showed amplification of all three fragments, indicating heterozygosity the polymorphic bp amplicon was located on chromosome arm 1bl, as no pcr products were generated from the nullisomic 1b lines or the ditelosomic 1bs line. sequencing of the polymorphic amplicon found that there was an extra repeat of bp sequence in the susceptible allele compared with the resistant allele. sequence similarity searches with the polymorphic amplicon found a contig with high sequence similarity located on wheat chromosome 1b. we designed four pairs of primers for linkage analysis based on differences in the number of copies of the bp sequence. analysis using the lsc primer pair detected a bp product closely linked to the yrl resistance gene in l analysis of f2: lines with additional primer pairs further demonstrated a co-dominant marker on chromosome 1b co-segregating with yrl fine mapping and comparative genomic analysis of wcbp indicated that it co-segregated with yrl693 genomic in situ hybridization failed to detect an alien chromosome segment from th. intermedium in l. a high-density integrated genetic map was constructed. the linkage map consisted of nine ssr markers and eight est-sts markers. the wcbp locus fell within a genetic interval of cm. wcbp co-segregated with the yrl locus. two flanking est-sts markers, bf and be were linked to the wcpb locus at and cm, respectively, and mapped to the chromosome 1b bin c–1bl-6-. therefore, wcbp is located in the same chromosomal region as yrl the contigs of the two ssr markers xwmc269-1b and xcfd65-1b on one side of wcbp mapped to wheat chromosomal arm 1bl, whereas those of the seven other ssr markers on the other side of wcbp mapped to 1bs according to the published ssr linkage map.fig. genetic and comparative genomic linkage map of the candidate gene wcbp and yrl a physical bin map of wcpb1; wcbp was mapped to the bin c-1bl6-. b genetic map of wcbp with a total length of cm on wheat chromosome 1b. the genetic distance in cm is shown on the left and genetic markers are shown on the right. c the consensus ssr map for a total of cm of wheat chromosome 1b. d, e, f, orthologous regions of wcbp in brachypodium distachyon chromosome and oryza sativa japonica chromosome and sorghum bicolor chromosome respectively orthologs of wheat ests be and be were detected on b. distachyon chromosome rice chromosome and sorghum chromosome comparative genomic analysis established the collinearity of wcbp genomic regions with a mb region on b. distachyon chromosome a mb region on rice chromosome and a mb region on sorghum chromosome regions that carry and genes, respectively. the gene order was highly conserved among wheat, b. distachyon, and rice, but showed an intra-chromosomal inversion in the mb chromosome region of sorghum. we also found an orthologous genomic region of wheat contig 1bs- carrying the ssr marker xgwm18-1b at mb on b. distachyon chromosome, but there was no corresponding ortholog in either rice or sorghum. an ortholog of wcbp was found on bradi3g at mb on b. distachyon chromosome on os10g at mb on rice chromosome and on sb01g at mb on sorghum chromosome.fig. comparative genomic mapping of ests in the wcbp chromosomal region of wheat chromosome 1b with brachypodium distachyon chromosome, oryza sativa japonica chromosome, and sorghum bicolor chromosome. the gene order in all species is clockwise pcr-based cloning and functional analysis of the candidate stripe rust resistance gene we obtained a candidate resistance gene by sequencing pcr products amplified by seven primers from genomic dna and then comparing the amplified sequence with that of the contig. this gene was bp in the resistant lines l and yu but bp in the susceptible lines l and my. the genomic sequence of the candidate resistance gene isolated from l and yu included four exons and three introns. the fourth exon contained a single repeat of the bp sequence in the resistant l line and its parent yu as well as two repeats in the susceptible l line and its parent my we also identified and verified a single nucleotide polymorphism in the first exon, with an adenine in the resistant lines and a cytosine in susceptible lines l and my. the open reading frame of the candidate resistance gene encodes a putative wheat copper binding protein predicted to contain amino acids in resistant lines and amino acids in susceptible lines. the snp in the first exon does not change the deduced amino acid sequence. the bp repeated sequence in the fourth exon caused an addition of amino acids in the susceptible line at amino acids 242– the deduced amino acid sequence forms a protein containing two predicted copper-binding domains, at amino acids 34–, and at amino acids 170–. the full-length, deduced wcbp protein shows sequence similarity with known heavy metal-binding proteins from diverse species. phylogenetic analysis indicated that wcbp shared the highest similarity with the ems protein, but they did not cluster together.fig. the wcbp gene, its putative protein structure, and its phylogenetic tree. a the predicted mrna structure of wcbp is shown above the genomic sequence, including introns and exons. atg represents the methionine start codon, and tga represents the stop codon. b coding regions of two heavy metal copper-binding domains in exons and the major differences between the resistance-and susceptibility-associated wcbp alleles are a snp in exon and a bp deletion in exon c alignment of the amino acid sequences and secondary structures of the proteins putatively encoded by the resistance-and susceptibility-associated alleles of wcbp d phylogenetic tree constructed using the neighbor-joining algorithm in mega following wcbp protein sequence alignment using the clustalw program. accession numbers for the other heavy metal copper-binding proteins were as follows: ems, emt, xp_, eay, xp_, afk, xp_, xp_, bak, xp_, daa, np_, eoa, xp_, xp_, afk, eay, xp_ and afw sequence analysis identified a highly similar gene, wcbp on chromosome 1a. this gene had the same dna sequence in l and l wcbp and wcbp show % dna sequence similarity. moreover, no polymorphism in the length of fragments was detected in genomic dna of resistant and susceptible varieties. wcbp and wcbp are differentially regulated in association with resistance to examine possible involvement of wcbp in defense against stripe rust, we measured its transcript levels at different time points following pathogen inoculation. wcbp was upregulated in leaves following pst inoculation of both the resistant and the susceptible parent. transcript abundance of wcbp was significantly higher in leaves from l and l at h post inoculation than prior to inoculation. however, at later time points, the wcbp transcript abundance decreased in l but remained higher in l at h post-inoculation, the wcbp transcript abundance was significantly lower in l compared to l.fig. qrt-pcr expression analysis of wcbp and wcbp amplification of wcbp and wcbp transcripts following pst infection. c, amplification of wcbp and wcbp transcripts following mock inoculation with water. the experiment was performed using three biological replicates with three technical replicates per biological sample. relative expression was calculated using gapdh as a reference gene to infer steady-state mrna levels, and relative expression was normalized by setting the control sample from l at h to bars represent the standard error, and the significance was determined using independent sample t-tests. asterisks represent significant differences as follows: **p ≤, *p ≤; ns, no significant difference. the raised asterisks or the ns designation represent differences in gene expression between l and l at each time point. asterisks or the ns designation in the trend line represent the differences in gene expression between two adjacent time points in the same genotype we also measured the transcript levels of wcbp prior to inoculation, l and l showed similar wcbp transcript levels, but after inoculation, wcbp was upregulated only in the resistant l line. at h post-inoculation, wcbp transcript abundance in l increased significantly compared with its level prior to inoculation, and wcbp transcript levels were significantly higher in l than in l at both h and h post-inoculation. in addition, we found that mock inoculation with water had no significant effects on the transcript level of either wcbp or wcbp in l or l at any time point. the effects of resistance on photosynthesis, chlorophyll fluorescence dynamics, and yield although the net photosynthetic rates at all time points were significantly higher in l than l these genotypes showed similar trends. notably, there were no significant differences between any two consecutive measurements in l whereas changes in l were highly significant. at the heading stage, the stomatal conductance in l was significantly lower than that in l. the gs of l increased sharply during the early stages and decreased toward the end of the experiment, while the values obtained for l declined sharply during the early stages of the experiment and then increased. the intercellular co concentrations observed in l were significantly lower than those in l at all time points, except at days after heading. in l there was a notable increase in ci, whereas this value decreased sharply between heading and days after heading in l fig. photosynthetic parameters used to assess the different responses of l and l to pst inoculation. a, net photosynthetic rate; b, intercellular co concentration; c, stomatal conductance. the parameters were measured once every days after heading in the field, using a li-cor the disease had fully developed at heading, and plants per genotype were used for these measurements. bars represent the standard error, and significance was determined using an independent sample t-test. asterisks represent significant differences as follows: **p ≤ and *p ≤, and ns indicates no significant difference. raised asterisks or the ns label represent the differences in gene expression between l and l at each time point. asterisks or the ns label on the trend line represent differences in gene expression between two adjacent time points in the same genotype although the changes in the maximal photochemical efficiency of psІІ in the dark-adapted leaves were similar between l and l the fv/fm value obtained for l was significantly higher than that obtained for l at days after heading. a significant decrease in fv/fm occurred in l between days and days after heading, whereas only a slight decrease occurred in this timeframe in l. the efficiencies of excitation capture by the open psii reaction centers observed in l were significantly lower than, similar to, and higher than those recorded in l prior to days, at days and at days after heading, respectively. the photochemical quenching coefficient was similar in l and l although the qp values obtained in l were greater than those in l the differences were not significant at the p =  level. the changes in the quantum yield of photochemical energy conversion in psii, the quantum yield of regulated non-photochemical energy loss in psii, and the quantum yield of non-regulated, non-photochemical energy loss in psii were quite similar in l and l the value of Φpsii recorded in l was significantly lower at the heading stage and significantly greater at days after heading, compared with that in l. in l the value of npq obtained was significantly greater than that in l at the heading stage, and the value of no was significantly lower than that in l at days after heading.fig. chlorophyll fluorescence-related parameters used to determine differences in photosystem ii activity between the resistant line l and the susceptible line l following pst inoculation and after heading. a, values for the maximal photochemical efficiency of psii; b, the efficiency of excitation capture by the open psii reaction centers; c, the photochemical quenching coefficient; d, quantum yield of photochemical energy conversion in psii; e, quantum yield of regulated non-photochemical energy loss in psii; and f, quantum yield of non-regulated non-photochemical energy loss in psii in wheat flag leaves. the parameters Φpsii, npq and no were derived from averages of related parameters according to a previously described method. the disease had fully developed at heading, and plants per genotype were used for the measurements. bars represent the standard error, and significance was determined using independent sample t-tests. asterisks represent significant differences as follows: **p ≤, *p ≤, and ns for no significant difference. raised asterisks or ns represent the differences in gene expression between l and l at each time point. the asterisks or the ns label on the trend line represent differences in gene expression between two adjacent time points in the same genotype yields were compared between the various wcbp genotypes and between the resistant plants and the susceptible plants in the f population. l exhibited a significantly greater 1000-grain weight and total grain weight per ear than that of l. the number of grains per ear was also greater. a total of plants from the genetic mapping population was analyzed to determine yields, and resistant plants displayed significantly higher sw, ns and tw values compared to those of susceptible plants. in addition, homozygous resistant and heterozygous wcbp genotypes showed significantly higher sw, ns and tw values than those of homozygous susceptible wcbp genotypes.table comparison of the main yield parameters, including the number of grains per ear, total grain weight per ear and 1000-grain weight, between the l and l parents and among the different genotypes in the segregating f population by independent sample t-tests ns +wcbp1/+wcbp1 +wcbp1/-wcbp1 -wcbp1/-wcbp1 tw +wcbp1/+wcbp1 +wcbp1/-wcbp1 -wcbp1/-wcbp1 sw +wcbp1/+wcbp1 +wcbp1/-wcbp1 -wcbp1/-wcbp1 r indicates the resistant phenotype, and s indicates the susceptible phenotype. in the different genotypes, r indicates the dominant resistance gene, and r indicates the recessive, susceptible allele. p, value describing the differences between l and l661; p value describing the differences between the rr and rr genotypes compared to rr; p value describing the differences between the rr and rr genotypes. asterisks represent significant differences: **p ≦, *p ≦ discussion a gene encoding a heavy metal copper-transport protein is involved in response to stripe rust infection in this study, we identified wcbp and wcbp two genes that encode predicted copper-binding proteins. genes encoding heavy metal copper-binding proteins have proven interesting in multiple studies. for example, similar genes exhibited significant changes in expression in all of eight lines near-isogenic for stripe rust resistance. in rice, the pi gene, encoding a putative heavy metal copper-binding domain protein, confers resistance to blast disease. furthermore, the rice gene xa encoding a heavy metal copper-transport protein, confers resistance to the bacterial pathogen xanthomonas oryzae. in humans, hah encodes a metallochaperone protein belonging to a family of proteins containing metal-binding domains and is related to the menkes/wilson disease protein. these findings indicate that a great diversity of copper-binding protein genes can confer resistance to various fungal and bacterial diseases in different species. qpcr analysis indicated that the transcription of the wcbp susceptible allele was inhibited. in contrast, wcbp is constitutively expressed in both l and l and its sequence is identical in these lines. however, its expression was only activated by inoculation with pst in l wcbp co-segregated with resistance in f2: populations. expression of both wcbp and wcbp was not upregulated by mock inoculation with water, indicating that these genes respond specifically during the resistance response to stripe rust. a gene encoding a blue copper-binding protein was induced by various oxidative stresses in arabidopsis and transgenic tobacco plants. indeed, there is an oxidative burst during the primary hypersensitive response, which implies that the oxidative burst potentially resulting from pst attack may induce the expression of wcbp wcbp enhances photosynthetic competence without altering the timing of leaf senescence and protects wheat yields against pst infection. another interesting category of differentially expressed genes is the leaf senescence-associated genes. previous studies implied that leaf senescence might be involved in the defense against pathogenic fungi. transcriptome analysis also confirmed that tasag is involved in the plant defense against pst infection. a total of % of sequences were identified as belonging to photosynthesis-like and leaf senescence-like pathways. previous studies showed that many pathogenesis-related genes are induced during leaf senescence in arabidopsis, and the types and numbers of sags present within this family vary from species to species and from genotype to genotype in the same species infected with the same fungal pathogen. fourteen of the sag-related ests have very high sequence similarity to tasag. this finding further suggests that unique transcripts of sags are specific to certain yr genotypes. yrl confers effective resistance at the seedling and adult stages. the physiological effects of stripe rust on wheat yields were estimated by measuring photosynthetic parameters at the adult stage because yield losses caused by pst infection occur mostly from disease at the adult stage. previous studies suggested that a leaf senescence-associated pathway might be involved in the response to stripe rust infection. our data suggest that pn was regulated by the photosynthetic apparatus rather than by stomatal factors in l indicating that l could actively regulate the photosynthetic pathway following infection with pst. this hypothesis was also supported by observed differences in gene expression. the increase in photosynthetic competence observed in l could be due either to an increase in the photochemical efficiency of psii or an increase in the efficiency of excitation capture by open psii reaction centers. the latter phenomenon would result from other aspects of photosynthesis, such as photosynthetic electron transport and light utilization, and could therefore be affected by pathogen infection. the onset of leaf senescence generally occurs at days after heading. at this time point, leaves from the resistant wheat line l exhibited significantly higher fv/fm, fv‘/fm’ and Φpsii values as well as significantly lower no compared with that of l leaves, although the changes in fv/fm, fv’/fm’, qp, Φpsii, and npq were very similar, especially at 20– days after heading. these findings indicated that the higher photosynthetic competence observed in l resulted from a higher photochemical efficiency of psii, which is an important, actively regulated mechanism involved in adaptation to disease stress. this result showed that the putative candidate stripe rust resistance gene wcbp did not alter the onset or progression of leaf senescence. the susceptible allele possibly accelerated the progress of leaf senescence within a normal onset period; therefore, the maintenance of a higher photochemical efficiency in psii is a direct and important factor in protection of yield against pst infection. yrl may have originated from a dna sequence change in the wheat genome we originally thought that yrl originated from th. intermedium because the pedigree of l includes th. intermedium. however, the fine genetic map of yrl shows similar marker order and total length that is consistent with the chromosome map of 1b. this result indicates that there is likely no alien chromosome segment in yrl comparative genomic analysis of yrl also demonstrated that an alien chromosome segment is unlikely. moreover, genes orthologous to wcbp were also identified in b. distachyon, rice, and sorghum. additionally, we did not detect th. intermedium sequences in the genomic region of yrl using closely linked markers. furthermore, the genetic linkage map showed that yrl is located in the centromeric region, whereas it is commonly thought that insertion or translocation of an alien chromosome segment into the centromeric region very rarely occurs in wheat. all available evidence supports the hypothesis that yrl was not transferred from th. intermedium, so we conclude that yrl is a wheat gene. wcbp is a candidate for the yrl stripe rust resistance gene the sequence of the wcbp allele associated with resistance contains a bp deletion compared to the sequence in the susceptible line. this finding suggests that resistance originated with the bp deletion, similar to the event observed for the blast resistance pi gene in rice. these findings implied that dna sequence changes, especially deletions, might be important mechanisms for evolution of disease resistance in plants. previous studies confirmed that the yr resistance gene encodes a kinase with a putative lipid-binding domain, whereas yr encodes an adenosine triphosphate-binding cassette transporter belonging to a pleiotropic drug resistance subfamily. these findings indicate that different yr genes have diverse and specific functions.  in this study, we identified the gene wcbp which may act as a candidate gene for the wheat stripe rust resistance gene yrl based on the following evidences. first, the dna sequence of wcbp was polymorphic between l and l as well as between the parents yu and my and results from the polymorphic amplification demonstrated polymorphism between the wcbp alleles in all parental pairs. second, genetic mapping of yrl showed that wcbp co-segregated with yrl third, qpcr analysis confirmed that the expression of wcbp differed significantly between resistant and susceptible alleles at h after pst-inoculation. moreover, the expression of wcbp in resistant/susceptible plants was upregulated only upon pst infection, while it did not change significantly upon mock-inoculation with water. the fact that wcbp was not annotated previously can be explained by its putative divergence in being specific to certain yr genotypes. it is therefore reasonable to hypothesize that wcbp is a candidate gene for yrl but positive confirmation will come with appropriate transformation experiments. materials and methods plant materials the isogenic wheat lines l with yrl and l without yrl were used to examine differential gene expression after the plants were inoculated with pst. their parents, yu and my were employed as controls for the analysis of the candidate resistance gene. th. intermedium was employed for in situ hybridization to check for alien chromatin in yu and l a set of chinese spring nulli-tetrasomic and relevant ditelosomic lines were used to identify the chromosomal location of the candidate resistance gene. genotypes of f2: families derived from the cross l661/l were used to construct the fine genetic map of the candidate resistance gene. gene expression in plants challenged with pst seedlings at the three-leaf stage, grown in a greenhouse, were challenged with urediniospores of pst race cyr pst inoculation was conducted according to a previously described method. leaves from l and l plants were harvested at and h post-inoculation. a mixture of l rna samples containing equal amounts of rna extracted at each time point was used as the tester. a mixture of l rna collected at the same time points was used as the driver for the construction of the cdna library. non-inoculated leaves from l and l plants at h, as well as leaves mock-inoculated with water, were harvested for use as controls for gene expression analyses. a sample was collected from l at days post-inoculation to represent full disease development. construction of the ssh library total rna was extracted from l and l leaves using the rna extraction reagent trizol. the dynabeads oligo dt system was then employed to purify mrna and to construct cdna libraries produced by rt-pcr. an ssh library was constructed using the pcr-select cdna subtraction kit according to a previously described protocol. positive colonies were sequenced using an abi prism automated sequencer. the resulting ests were analyzed against the genbank database using blastx and blastn. the threshold probability for a sequence match was set at 10− plant growth conditions all wheat materials were planted in the field at the yaan agricultural research station of sichuan agricultural university during the 2010– wheat-growing season. seeds were sown in a clay soil on october each experiment was executed as a randomized complete block design with three replicates. approximately seeds from each line were planted in m rows with cm spacing. the susceptible wheat line sy95- serving as a vector of the pathogen, was planted in every third row of each population to ensure that all plants had an equal chance of infection. sy95- was artificially inoculated with pst race cyr at the seedling stage according to a previously described method. the average air temperatures from sowing to grain maturity ranged from to °c. the heading date of both l and l was approximately march the plants received and g n m− ammonium nitrate at the following growth stages, respectively: one-node, meiosis, heading and anthesis. after planting, % imidacloprid wettable powder was applied to control pests. thirty plants from each genotype showing identical growth and developmental characteristics at heading were marked for all subsequent measurements and observations. measurement of photosynthetic indices and psii activity photosynthetic parameters and chlorophyll fluorescence were measured in l and l using a portable photosynthesis system with a red-blue light source and a modulated photosynthesis system, respectively. data collection was carried out once every days, starting from heading on march through april at each sampling date, the mean of three independent measurements from each plant represented the plant phenotypic value, and the average values from plants were employed for comparisons between genotypes. measurements, parameter recording, and parameter calculations followed previously described methods. in silico mapping of candidate genes the genomic locations of the wheat ests were determined in silico via blastn searches against the mapped expressed wheat sequences using graingenes at a 10− threshold probability and via a blastx search against the wheat draft genome. sequences without a blastx hit or those showing an e-value of less than 10− were excluded from the in silico mapping to the wheat genome and the brachypodium genome. pcr-based mapping of polymorphic ests and linkage analysis for resistance primers were designed using primer to amplify ests from wheat genomic dna. wheat genomic dna was extracted using a previously described method. primer sequences were listed in additional file 2: table s pcr amplification and product analysis were performed according to previously described methods. polymorphic primers were used to genotype the segregating population of f2: families. linkage analysis of markers and resistance genes was performed with joinmap. the linkage group was declared at a lod threshold of. subsequently, the chromosomal locations of the linked markers were confirmed using chinese spring nullisomic-tetrasomic and the ditelosomic lines of chromosome 1b. pcr-based cloning of putative stripe rust resistance genes the primers designed with primer according to the contig sequence containing the co-segregated markers and the polymorphic amplicon sequence were employed to amplify the entire length of the candidate gene. these primers were listed in additional file 2: table s each amplicon was purified using the genejet pcr purification kit and cloned into the pgem t easy vector. jm109-competent cells were transformed and cultured according to the manufacturer’s protocol. plasmids retrieved using the genejet plasmid miniprep kit were sequenced by shanghai majorbio bio-pharm technology co., ltd.. qrt-pcr of the candidate gene and the key unigene conferring resistance we used m-mlv to synthesize complementary dna for real-time qrt-pcr. each μl reaction mixture contained μg of rna. for qrt-pcr assays, μl of the diluted cdna and μl of realmastermix were included in μl reaction volumes. qrt-pcr was conducted using the miniopticon time pcr detection system with the following program: pre-incubation at °c for min, followed by amplification for cycles at °c for s and °c for s. the melting curve was set at to °c, with a °c increase per step, and cooling was set at °c for s. each qrt-pcr run was replicated with three independent biological samples, and three technical repeats were included for each biological sample. the primer sequences and descriptions of the genes are provided in additional file 2: table s the crossing point value and results of the melting curve analyses were obtained using roche cfx manager software for the miniopticon system. melting curve data showing only a single peak, which denotes primer specificity, were collected for all samples. the repeatability and reliability of amplifications are demonstrated by the raw data shown in additional file 1: figure s1b. statistical analysis significant differences in the mean physiological parameters, gene expression, and yield between l and l and between plants carrying and lacking wcbp were observed. differences between two adjacent time points for the same genotype were determined using independent sample t-tests in ibm spss statistics. additional files additional file 1: figure s length distributions of the wheat leaf ests and the assembled uniests, including contigs and singletons. figure s primers and the amplified region of genomic dna. to clone the wcbp gene, primers were designed according to the chinese spring draft genome sequence. full-length amplification was performed using the 1b- forward primer and the 1b- reverse primer. figure s comparison of wcbp gene sequences. wcbp encodes a heavy metal copper-binding protein with two copper sensing domains. the four sequences were cloned using pcr with primers designed according to the polymorphic fragment. l is the stripe rust-resistant line; l is the stripe rust-susceptible line; yu is the resistant parent of l and l661; my is the susceptible parent of l and l figure s quality map of the sequences flanking the snp site. a represents l693; b represents yu25; c represents l661; d represents my which showed that the snp existed among the different genotypes. figure s gish analysis of mitotic metaphase chromosomes of tai l l and yu using genomic dna of th. intermedium as a probe. a tai b l c l d yu chromosomes were counterstained with dapi. figure s results demonstrating the high quality and good replication of q-pcr., the melt curve of the q-pcr product during the amplification of wcbp, the amplification curve shows the consistent crossing points of the three technological replicates of the wcbp gene and the reference gene gapdh. this chart shows l at h. additional file 2: table s. detailed information on ests. a blast search was performed using blastx in ncbi. in silico mapping was performed using the iwgsc draft wheat sequence database. table s blast searches performed for the forward subtraction clones found in the stripe rust-resistant genotype l table s the primers used to amplify ests. primer pairs were only designed for out of the ests because the remaining ests were too short to be used for primer design. table s primers were designed according to the sequence of the contig carrying the polymorphic amplicon, which was located on wheat chromosome 1b. these amplicons were used to determine the linkage relationship between the markers and the stripe rust resistance gene in l table s results of the amplification of wcbp1-linked genetic markers in an f population derived from crosses between l and l and between l and l the genotypes of the f individuals were determined according to the phenotypes of the corresponding f2: families; "a" and "b" for the individual f populations were derived from crosses between l and l and between l and l respectively. r represents the same amplicon as the resistant parent l693; s represents the same amplicon as the susceptible parent l661; h represents the heterozygous condition. the yield components of the f plants, including the number of grains per ear, the total grain weight per ear and the 1000-grain weight, are provided. ‘\’ represents a missing yield component in the f plants. table s the sequences of seven primer pairs designed according to the sequence of a cloned candidate gene in silico; these primers were used for pcr-based gene cloning from genomic dna. table s primers for q-pcr analysis. additional file 3: methods s genomic in situ hybridization. abbreviations bpbase pair estsexpressed sequence tags orfopen reading frame prpathogenesis-related pstpuccinia striiformis f. sp. tritici qrt-pcrquantitative reverse transcription pcr sagsenescence-associated gene snpsingle nucleotide polymorphism sshsuppression subtractive hybridization ssrsimple sequence repeat wcbpwheat copper-binding protein xin li, taiguo liu and wanquan chen contributed equally to this work. competing interests the authors declare that they have no competing interests. authors’ contributions xl, tl, wc, and pl designed the study; xl and sz performed ssh under the instruction of ll; xl and tl carried out the comparative genomic analysis; and xl and wc performed the genetic analysis and genetic map construction. the qrt-pcr was performed by xl, mz and hr. xl, sz, hz, and lw performed the majority of experiments with contributions from zt, zc, rz and pl. pl wrote the manuscript with input from xl, tl and wc. all authors read and approved the final manuscript. authors’ information not applicable. availability of data and materials not applicable. we are grateful for the financial support of the national natural science foundation of china, the state key laboratory for biology of plant disease and insect pests, china, the provincial science and technology foundation for young scientists of sichuan, china and the ministry of science and technology, china.
in silico mapping placed one of these tags on chromosome 1a. stripe rust, a highly destructive foliar disease of wheat, causes severe losses, which may be accompanied by reduced photosynthetic activity and accelerated leaf senescence. the unique chromosomal location and expression mode of wcbp suggested that wcbp is the putative candidate gene of yrl which was involved in leaf senescence and photosynthesis related to plant responses to stripe rust infection during the grain-filling stage. primers based on amplified a polymorphic band, which co-segregated with yrl we cloned a candidate gene encoding wheat copper-binding protein by amplifying the polymorphic region, and we mapped wcbp to a cm genetic interval. we used suppression subtractive hybridization to examine the mechanisms of resistance in the resistant wheat line l, which was derived from a lineage that includes a wide cross between common and thinopyrum intermedium. qrt-pcr confirmed that wcbp expression changes in response to pathogen infection. sequencing of an ssh cdna library identified expressed sequence tags.
 stripe rust, a highly destructive foliar disease of wheat, causes severe losses, which may be accompanied by reduced photosynthetic activity and accelerated leaf senescence. methods we used suppression subtractive hybridization to examine the mechanisms of resistance in the resistant wheat line l, which was derived from a lineage that includes a wide cross between common and thinopyrum intermedium. sequencing of an ssh cdna library identified expressed sequence tags.  in silico mapping placed one of these tags on chromosome 1a. primers based on amplified a polymorphic band, which co-segregated with yrl we cloned a candidate gene encoding wheat copper-binding protein by amplifying the polymorphic region, and we mapped wcbp to a cm genetic interval. brachypodium, rice, and sorghum have genes and genomic regions syntenic to this region. discussion sequence analysis suggested that the resistant wcbp allele might have resulted from a deletion of 36-bp sequence of the wheat genomic sequence, rather than direct transfer from th. intermedium. qrt-pcr confirmed that wcbp expression changes in response to pathogen infection.  the unique chromosomal location and expression mode of wcbp suggested that wcbp is the putative candidate gene of yrl which was involved in leaf senescence and photosynthesis related to plant responses to stripe rust infection during the grain-filling stage. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords quantitative reverse transcription pcrpuccinia striiformistriticum aestivumsuppression subtractive hybridizationphotosynthesisissue-copyright-statement© the author 2015 