Functional diversity at the Rc (red coleoptile)gene in bread wheat
E.K.Khlestkina ÆM.S.Ro
¨der ÆT.A.Pshenichnikova ÆA.Bo
¨rner Received:21April 2009/Accepted:8July 2009/Published online:7August 2009
冰激凌三明治
ÓSpringer Science+Business Media B.V.2009
Abstract The prence of the allele Rc-A1b on chromosome 7A specified the expression profile of the F3h-1(encoding flavanone 3-hydroxyla)genes and anthocyanin pigmentation in coleoptiles of Russian bread wheat cultivar ‘Saratovskaya 29’.A quantitative RT-PCR analysis compared the temporal expression profile of F3h-A1,F3h-B1,and F3h-D1in the coleoptiles of ‘Saratovskaya 29’and the standard cytogenetic stock ‘Chine Spring’(‘Hope’7A),both of which carry Rc-A1b .There was no within-geno-type variation for expression level of the F3h-1homoeologues at any of the sampling times,but the expression profiles varied markedly between the two genotypes.This result suggested that there may be functional allelic diversity at Rc-A1,which affects the transcription of the F3h-1genes in colored coleop-tiles.Microsatellite-bad genetic mapping was ud to locate Rc-A1along
cryonmyshoulder歌词with the new loci Pc-A1(purple culm),Plb-A1(purple leaf blade),and Pls-A1(purple leaf sheath)in a single cluster on the short arm of chromosome 7A.
Keywords Wheat ÁGene expression ÁAnthocyanin biosynthesis ÁFlavanone 3-hydroxyla ÁGenetic mapping ÁAnthocyanin pigmentation genes
Introduction
第83届奥斯卡Genetic analysis in wheat has defined at least 100major genes affecting various aspects of plant phenotype (McIntosh et al.2008),but for most of the,neither their genomic quence and gene product nor cellular function have been ascertained yet.Allelic variation at the majority of the ‘‘phe-notypic’’genes is frequently limited to two alleles.However,this paucity of variation probably reflects difficulties in detecting subtle differences in gene action,rather than a lack of genetic variation itlf.Modern methods for gene transcription profiling may facilitate investigation of functional allelic diversity.The diversified wheat anthocyanin biosynthesis genes reprent a uful model to investigate functional specialization in the bread wheat (Triticum aestivum L.)genome (Ahmed et al.2003,2006;Himi and Noda 2004;Himi et al.2005;Khlestkina et al.2008a ).Furthermore,the anthocyanin biosynthesis genes can be implicated in plant resistance to a number of abiotic and bioti
c stress factors (Bogda-nova et al.2002;Christie et al.1994;Giovanini et al.2006;Gould 2004;Izdebski 1992;Liu et al.2007;Pozolotina et al.2007).
E.K.Khlestkina (&)ÁT.A.Pshenichnikova
Institute of Cytology and Genetics,Siberian Branch
of the Russian Academy of Sciences,Lavrentjeva ave.10,630090Novosibirsk,Russia e-mail:khlest@bionet.nsc.ru
M.S.Ro
自考365网¨der ÁA.Bo ¨rner Leibniz Institute of Plant Genetics and Crop Plant Rearch (IPK),Corrensstr.3,06466Gatersleben,Germany
Mol Breeding (2010)25:125–132DOI 10.1007/s11032-009-9312-9
Broadly,the anthocyanin biosynthesis genes are divided into the regulatory and the target genes.ABP enzymes are encoded by the structural genes repre-nting the target genes in the network.Regulatory genes control tissue-specific expression of the target genes(Jaakola et al.2002).
The mode of inheritance of the anthocyanin pigmentation of various wheat tissues has been clear for many years(Capron1918; Clark1924;Goulden et al.1928).The chromosomal location of the genes responsible was defined initially thankful to the development of a range of cytogenetic stocks.In particular,the placed the coloration genes Rc(coleoptile),Pc(culm),and Pan(anther)all on the homoeologous group7chromosomes(Sears 1954;Kuspira and Unrau1958;Jha1964;Gale and Flavell1971;Rowland and Kerber1974;Sutka1977; Maystrenko1992),whereas Ra(auricles)genes were located on chromosomes1D(Gulyaeva1984),4B, and6B(Melz and Thiele1990),and Pp(pericarp) ones to2A and7B(Arbuzova et al.1998).Micro-satellite-bad mapping has defined the intrachromso-mal location of many of the genes,including all three Rc-1homoeologues,Pc-B1,Pc-D1,Pan-D1, Pls-B1(purple leaf sheath),Plb-B1(purple leaf blade)and Plb-D1on the homoeologous group7 chromosomes(Khlestkina et al.2002a,b,2008b, 2009a),complementary Pp genes on chromosomes 2A and7B,and purple glume gene on chromosome 2A(Dobrovolskaya et al.2006;Khlestkina et al. 2009b).
One of the structural anthocyanin biosynthesis genes(F3h-1)encodesflavanone3-hydroxyla,a key component of the biosynthesis,and its expression in coleoptiles is determined by the allelic status at the Rc-1genes(Khlestkina et al.2008a).F3h-1is reprented in hexaploid wheat by three homoeo-log
ues(F3h-A1,F3h-B1,and F3h-D1),which map, respectively,to chromosomes2A,2B,and2D (Khlestkina et al.2008a).The expression of all three F3h-1homoeologues in coleoptiles is governed by the dominant allele at each Rc-1gene(Khlestkina et al.2008a).
In the current study,we t out to demonstrate that the pigmentation of the coleoptile of the Russian cultivar‘Saratovskaya29’(‘S29’)is determined by the allelic status at Rc-A1,to genetically map Rc-A1with respect to the novel wheat loci Pc-A1,Plb-A1,and Pls-A1,and with respect to the F3h-1gene activity in coleoptiles,and to compare the temporal expression profiles of the F3h-1homoeologues in the coleoptiles of both‘S29’and the standard cytogenetic stock ‘Chine Spring’(‘Hope’7A)[‘CS’(‘H’7A)]. Materials and methods
Plant materials and phenotypic evaluation
An F1hybrid was made by pollinating‘S29’with ‘S29’(‘YP’4D)—a single chromosome substitu-tion line in which chromosome4D of‘S29’had been replaced by its homologue from the cultivar ‘Yanetzkis Probat’(‘YP’)(Gaidalenok et al.1995). The two parents differ from one another with respect to coleoptile pigmentation(Khlestkina et al.2008b). From this hybrid,a t of108doubled haploid(DH) progeny was obtained by pollination with maize (Laurie and Reymondie1991).In the cytogenetic stoc
k‘CS’(‘H’7A),chromosome7A from cv.‘Hope’replaces its cv.‘Chine Spring’homologue in a genetic background of cv.‘Chine Spring’(Kuspira and Unrau1958).The anthocyanin pigmen-tation of the coleoptiles was scored as described by Khlestkina et al.(2002a).The anthocyanin pigmen-tation of the culm,leaf sheaths,and leaf blades was assd in adultfield-grown plants. Microsatellite marker analysis and mapping Genomic DNA was extracted from each DH progeny, according to Plaschke et al.(1995),and ud as template for microsatellite-bad genotyping(Ganal and Ro¨der2007;Ro¨der et al.1998).The amplicons were parated on an automated larfluorescence quencer(ALF express,Amersham Biosciences), and fragment sizes were calculated with the aid of Fragment Analyr v1.02(Amersham Biosciences) software,on the basis of relative migration compared to internal size standards.Genetic mapping was achieved using MAPMAKER software(Lander et al. 1987).Recombination fractions were converted into centimorgan(cM)distances by applying the Kosambi (1944)map unit function.
RNA extraction and RT-PCR
The QIAGEN(/)Plant Rneasy kit,followed by a DNA treatment,was ud to extract
RNA from coleoptile samples taken at24h intervals from3to6-day old edlings of‘S29’and‘CS’(‘H’7A) and5-day old edlings of the DH-lines,grown at20°C under a12h day/12h night regime.Single-stranded cDNA was synthesized from1l g total RNA using a (dT)15primer and the QIAGEN Omniscript Rever Transcription kit in a20l l reaction mixture.RT-PCR was performed with F3h primers published earlier (Himi et al.2005)amplifying three F3h-1homoeo-logues(Khlestkina et al.2008a),while the standardi-zation of cDNA template was performed using primers for the constitutively expresd gene encoding glycer-aldehyde-3-phosphate dehydrogena(Gapdh).PCR products were parated by2%agaro gel electrophoresis.
A quantitative RT-PCR was carried out with PCR primers designed to specifically amplify fragments of each of F3h-A1,F3h-B1,and F3h-D1(Khlestkina et al.2008a),using the QIAGEN QuantiTect SYBR Green kit.Gapdh primers were ud to normalize the cDNA template in qRT-PCR.PCRs were performed in an Applied Biosystems7900HT fast real time PCR system.Each sample was run in three replicates. Statistical significance of differences in F3h expres-sion level either between F3h homoeologues or between‘Saratovskaya29’and‘Chine Spring’(‘Hope’7A)was assd by Student’s t-test for matched pairs.
Results
hi little babyGenetic mapping of pigmentation genes inherited from‘S29’
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Of the108DH lines,51had pigmented and57 nonpigmented coleoptiles,consistent with a mono-genic1:1gregation(v2=0.33,P[0.50).Pig-mentation of the culm,leaf sheath,and leaf blade also gregated as monogenic traits in the population (culm:v2=0.09,P[0.70;leaf sheath:v2=1.96, P[0.10;leaf blade:v2=0.79,P[0.30).The abnce of coleoptile pigmentation in the single chromosome intervarietal substitution line‘S29’(‘YP’4D)suggested that an Rc gene may be prent on chromosome4D(Khlestkina et al.2008b),even though all the Rc-1homoeoloci are located on the homoeologous group7chromosomes(Khlestkina et al.2002a).To clarify whether the group7chromosomes prent in‘S29’(‘YP’4D)had all been derived from‘S29’,the microsatellite profiles of the substitution line,and both its donor and recurrent parents were compared using loci known to map in the vicinity of Rc-1on chromosomes7A(Xgwm0870, Xgwm0060),7B(Xgwm0400,Xgwm0046)and7D (Xgwm0437).‘YP’microsatellite alleles at both Xgwm0870and Xgwm0060were prent in‘S29’(‘YP’4D).Further microsatellite genotyping revealed that the line carries a fragment of‘YP’chromosome 7A extending from Xgwm0060on the short arm to Xgwm0748on the long arm(Fig.1).The substitution line designation was changed to‘S29’(‘YP’4D*7A). Although the substitution line had received eight backcross to‘S29’in the cour of its development,
it is clear that this was not sufficient to fully reconstitute the recipient genetic background. Clearly,therefore,it is important when basing trait genetic analysis on substitution lines,that conclu-sions are verified by appropriate linkage mapping.
Mapping in the DH lines was performed using both chromosome4D and chromosome7A microsat-ellite markers.The chromosome4D linkage map comprid14microsatellite loci,and had a length of 96.1cM(data not shown).There was no linkage between any of the4D loci and any of the pigmen-tation genes gregating in the mapping population. On chromosome7A,five microsatellite loci were polymorphic between‘YP’and‘S29’,allowing a linkage map of49.5cM to be constructed(Fig.1). The four pigmentation genes clustered in a proximal region of the short arm of chromosome7A,but were parated from one another by at least three recom-bination events.The location of the gene controlling coleoptile pigmentation in‘S29’coincided with Rc-A1,whereas Pc,Pls,and Plb mapped to sites comparable with tho where similar genes have previously been located on chromosomes7B and7D (Khlestkina et al.2008b,2009a).The latter three genes were therefore designated Pc-A1,Pls-A1,and Plb-A1(Fig.1).
F3h expression:genetic mapping of‘F3h activity’,temporal pattern and the Rc-1
面部毛孔粗大allele-dependent specificity
cDNA derived from5-day old coleoptiles of the DH-lines was analyzed with RT-PCR using the primer pair amplifying all three F3h-1homoeologues.The
expression patterns of a number of the DH-lines are given at Fig.2.The RT-PCR data were converted to the row data for MAPMAKER analysis and ud together with the microsatellite and Rc-A1row data to construct genetic map.‘F3h-1activity’in coleop-tiles co-gregated with the Rc-A1locus(Fig.3), suggesting that transcription of F3h-1in‘S29’coleoptiles is regulated solely by the Rc-A1gene. Previously,the same has been shown for‘CS’(‘H’7A)(Khlestkina et al.2008a).Thefindings suggest F3h-1to be appropriate for indirect evaluation of Rc-A1activity in‘S29’and‘CS’(‘H’7A)coleoptiles.
The variation in the expression level of F3h-1,as measured by quantitative RT-PCR bad on cDNA sampled from3to6-day old edlings of‘S29’and ‘CS’(‘H’7A),is illustrated in Fig.4.All three F3h-1 homoeoloci were expresd in both lines(Fig.4),and there was no discernable within-genotype variation in the level of expression of any of the three genes at any of the sampling times(Table1).However,there was a significant genotypic difference[t=2.73, P[0.95(df=11)],with F3h-1expression being lower in‘S29’than in‘CS’(‘H’7A).In‘S29’,the peak of expression was reached5days after germi-nation,while by this time,the expression of all three ‘CS’(‘H’7A)F3h-1homoeologues had
already declined(Fig.4).Thus,in the prence of the same ‘‘phenotypic’’Rc-A1b allele,there can still be variation in the F3h-1expression profile.The delayed initiation of F3h-1expression,and its overall lower level of expression in‘S29’compared to‘CS’(‘H’7A)were consistent with the pattern of development of coleoptile pigmentation.‘Saratovskaya29’coleoptiles became pigmented later,and their peak level of pigmentation was less inten than tho of ‘CS’(‘H’7A).
Discussion
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The bread wheat genome is compod of the three homoeologous genomes A,B,and D(Kihara1944, 1954).Becau of the clo relationship between the progenitors of the genomes,their gene content is largely conrved.Thus,there are commonly three reprentatives of each single copy gene prent in bread wheat,and expression analysis has shown that for about30%of the,at least one copy is silent, while for the remaining*70%,and all three are co-expresd(Bottley et al.2006).The expression level of co-expresd homoeologues can sometimes be equal(Wknox-1,Morimoto et al.2005;Wp,Shit-sukawa et al.2007;F3h-1,Khlestkina et al.2008a)or vary(TaBx,Nomura et al.2005;TaGA20ox1,Apple-ford et al.2006;Wlhs-1,Shitsukawa et al.2007;Rc-1, Khlestkina et al.2008a).In some cas,such differ-ential expression of homoeologues is thought to be a conquence of the allopolyploidization event(Shit-sukawa et al.2007),while in other cas gen
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omic bias in transcription probably originates from the diploid progenitors themlves(Nomura et al.2005).The expression of the F3h-1homoeologues evaluated in the current(Fig.4;Table1)and our previous study (Khlestkina et al.2008a)was equal within each genotype.However,the regulatory homoeologues Rc-A1,Rc-B1,and Rc-D1differentially contribute to the F3h-1expression regulation,which may reflect
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differential expression of Rc-A1(Khlestkina et al. 2008a).
The Rc-1homoeologues have not been isolated and quenced thus far.This hinders direct analysis of their expression profiles.However,their activity may be accesd indirectly by analysis of expression patterns of their target genes.In the current study,it was shown that the target genes F3h-1are not active in nonpig-mented coleoptiles(Fig.2).In different species,F3h may be either active(Vitis vinifera,Boss et al.1996; Petunia hybrida,Quattrocchio et al.1993;Arabidopsis thaliana,Stracke et al.2007)or not(Antirrhinum majus,Martin et al.1991;Pertillaflutescens,Gong et al.1997;Triticum aestivum,Khlestkina et al.2008a; Zea mays,Deboo et al.1995)in nonpigmented tissues. Expression of some anthocyanin biosynthesis struc-tural genes in tissues not pigmented with anthocyanins can
be explained by that they are required for production of otherflavonoid compounds.For exam-ple,A.thaliana Chs(chalcone syntha),Chi(chal-cone-flavanone isomera),and F3h gene promoters are responsive to transcription factors,controlling flavonol accumulation(Stracke et al.2007).In wheat, unlike other anthocyanin biosynthesis genes who expression has been studied in coleoptiles,F3h-1 shows differential expression patterns between pig-mented and nonpigmented coleoptiles,confirmed for different genotypes(Fig.2;Khlestkina et al.2008a and unpublished data).Thus,F3h-1is the most appropriate target gene,which may be ud for indirect evaluation of Rc-1activity.Using this approach to study variation among Rc-1homoeologues,it has been shown that the highest level of expression is associated with the prence of Rc-A1,and the lowest of Rc-D1.Rc-D1, however,induces F3h-1earlier than the other Rc-1 genes do(Khlestkina et al.2008a).The extent to which a given gene’s expression profile is constant for
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