Comparative molecular marker-based genetic mapping of flavanone 3-hydroxylase genes in wheat, rye and barley
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- Khlestkina, E.K., Salina, E.A., Matthies, I.E. et al. Euphytica (2011) 179: 333. doi:10.1007/s10681-010-0337-2
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The F3h gene encoding flavanone 3-hydroxylase, one of the key enzymes of the flavonoid biosynthesis pathway, is involved in plant defense response, however, it has not yet been genetically mapped in such important crop species as wheat, barley and rye. In the current study, the F3h genes were for the first time genetically mapped in these species, using microsatellite and RFLP markers. The three wheat F3h homoeologous copies F3h-A1, F3h-B1 and F3h-D1, and rye F3h-R1 were mapped close to the microsatellite loci Xgwm0877 and Xgwm1067 on chromosomes 2AL, 2BL, 2DL, and 2RL, respectively. Wheat F3h-G1 and barley F3h-H1 were also mapped at the homoeologous F3h-1 position on chromosomes 2GL and 2HL, respectively. The non-homoeologous F3h gene (F3h-B2) was mapped on wheat chromosome 2BL about 40 cM distal to the F3h-1 map position. The results obtained in the current study are important for further studies aimed on manipulation with F3h expression (and, hence, plant defense) in wheat, barley and rye.
KeywordsFlavonoid biosynthesisFlavanone 3-hydroxylasePlant defense responseWheatRyeBarleyGenetic mapping
Cultivated plants are constantly attacked by a large number of pathogenes and are exposed to different kinds of abiotic stress factors. Plant defense involves a broad class of genes known as defense response (DR) genes which include the F3h gene encoding flavanone 3-hydroxylase (F3H; EC 188.8.131.52). F3H catalyses one of the key steps of the flavonoid biosynthesis pathway (conversion of flavanones to dihydroflavonols) yielding a large family of flavonoid compounds which are involved in many biological activities including coloration of flowers, seeds and other plant organs, seed dormancy and longevity, seed coat permeability, ultraviolet radiation protection, antimicrobial activity, antioxidant activity, plant defense response to a broad spectrum of abiotic and biotic stress factors and medicinal properties (Marbach and Meyer 1974; Freed et al. 1976; Halloin 1982; Ryan et al. 2001; Winkel-Shirley 2002; Gould 2004).
Direct relationship between F3h expression level and disease resistance in different plant species has been shown (Ardi et al. 1998; Cho et al. 2005; Giovanini et al. 2006). The F3h gene was the only one among 12,000 transcripts with differential expression pattern in leaves of chickpea susceptible and resistant to a necrotrophic fungal pathogen, Ascochyta rabiei. Constitutive expression of F3h was lower in lines susceptible to pathotypes I and II of A. rabiei, than in lines resistant to both pathotypes (Cho et al. 2005). In avocado fruits, activity of F3h was higher in cultivars naturally resistant to fungal decay than in susceptible cultivars. F3h activity was closely followed by the pattern of epicatechin (Ardi et al. 1998). In wheat seedlings, F3h mRNA level was higher in plants infested by hessian fly than in uninfested control (Giovanini et al. 2006). Recently, association between sequence polymorphism of F3h gene and resistance to soybean mosaic virus has been shown (Cheng et al. 2010).
Thus, the F3h gene is involved in plant defense response, however, it has not yet been genetically mapped in such important crop species as wheat, barley and rye. In wheat and rye, four and one copy of F3h, respectively, have been assigned to the long arms of homoeologous group 2 chromosomes (Khlestkina et al. 2008, 2009a). Chromosome allocation of barley F3h remains unknown. Molecular-marker based genetic mapping of the F3h loci in wheat, barley and rye is an important step on the way to isolation of the complete F3h gene sequences, which is necessary for further manipulation with F3h expression (and hence, plant defense) in these species.
In the current study, we performed genetic mapping of a total of seven F3h gene copies in polyploid wheats (Triticum aestivum genome AABBDD, T. timopheevii AAGG), barley (Hordeum vulgare HH) and rye (Secale cereale RR) relative to microsatellite or RFLP markers and compared their locations with each other and with those for rice and maize F3h.
Materials and methods
Populations selected for linkage mapping, segregation ratios at F3h loci, and number of microsatellite (SSR) or RFLP markers used for maps construction
Reference, resource used
Number of markers on a map
T.timopheevii k-38555/T. militinae
Salina et al. 2006, DNA, SSR data
T. aestivum ‘Saratovskaya 29’/T.spelta
Dobrovolskaya O. (unpublished data), DNA
T. aestivum ‘CS/Hope7A’/T. aestivum TRI 15010
Khlestkina et al. 2002, DNA
T. aestivum ‘Mironovskaya 808’/T. aestivum ‘Aibian’
Khlestkina et al. 2002, DNA
T. aestivum ‘Skala’/line 842*
Leonova et al. 2002, DNA, SSR data
S. cereale ‘Vyatka’/S. cereale ‘Monstrous’
Khlestkina et al. 2004, DNA, SSR/RFLP data
4 SSRs, 3 RFLPs
H. vulgare ‘Steptoe’/H. vulgare ‘Morex’
Kleinhofs et al. 1993, RFLP-data
Primer design and allele-specific PCR amplifications
F3h gene copy-specific and pyrosequencing primers designed for wheat, rye and barley used for screening parents of the mapping populations
Designation of the primer (primer pair no)
PCR fragment length (bp)
Portion of lines showed reproducible non-amplification (%)
Gene region containing primer annealing sight
Primers are described in Khlestkina et al. (2008)
Exon 1–Intron 1
Exon 1–Intron 1
0 (T. timopheevii) and 100 (T. aestivum)
Pyrosequencing and SNP-marker development in barley
In barley, primer pairs for F3h were designed using a consensus sequence of the 30 single sequences derived from the UniGene cluster hv. 143 (http://www.ncbi.nlm.nih.gov). A 650 bp fragment could be PCR-amplified which showed highest similarity (99% identity) using BLAST-search (Altschul et al. 1990, 1997) to F3h mRNA (X58138, gi: 18975; Meldgaard 1992). For SNP-detection suitable primer combinations were designed with biotinylated reverse primer and forward sequencing primer (Table 2) by using PSQ Assay Design Software version 1.0.6 (2004) provided by Biotage, Sweden (Table 2). The sequencing primers F3H-H1 (2S) and F3H-H1 (6S) (Table 2) of the Pyrosequencing-assays are optimized by the design software (Biotage, Uppsala, Sweden) and are specific for these SNPs in those generated PCR fragments. The PCR-conditions were as described in Malysheva-Otto and Röder (2006). Pyrosequencing was carried out according to manufacturer’s protocol on a PSQTMHS9A (Biotage, Sweden). Data were evaluated by using the manufacturer’s software.
Linkage maps construction
Monogenic segregation ratios in genotyped mapping population at the F3h loci were examined by the χ2 test (Table 1). The F3h loci were mapped relative to RFLP or microsatellite markers. Linkage maps were constructed with MAPMAKER 2.0 (Lander et al. 1987). For microsatellite analysis, Gatersleben wheat microsatellite (GWM) markers mapping to chromosomes 2B and 2D were chosen. The characteristics of the microsatellites, and the experimental procedures associated with their use are described by Röder et al. (1998). Unpublished primer sequences are available upon request.
Genotyping of mapping populations at the F3h loci
Two SNP sites showed polymorphism between the parents of barley mapping population ‘Steptoe’/‘Morex’ (Table 1); one represented a silent mutation and was found in the exon 2, whereas another one was located in the intron 2 (Table 2). Both polymorphic SNPs were used for genotyping 94 double haploid lines of ‘Steptoe’/‘Morex’ mapping population. Co-segregation of the two SNPs in the mapping population was observed. Therefore, only one of them (Table 2) was used for further mapping. The segregation ratio in the barley mapping population conformed to the expected 1:1 (Table 1).
Comparative mapping of the F3h genes in wheat, rye and barley
The rye F3h-R1 gene was also found at the homoeologous map position, 11.7 cM distal to the anchor marker Xgwm0877 on the long arm of chromosome 2R. The F3h gene in barley was mapped on the long arm of chromosome 2H closely linked to Xabc252 (0.0 cM), distal to Xabg072 (21.4 cM), and proximal to Xabc157 (8.8 cM) and Xcmwg720 (32.8 cM) (Fig. 2). Considering the close linkage (0.8 cM) of RFLP loci Xcmwg720 and Xcmwg660 (Börner et al. 1999), and based on the fact of that Xcmwg660 was mapped 32 cM distal to Xgwm0877 in rye (Khlestkina et al. 2004), we can infer that barley F3h is located at the homoeologous F3h-1 position (Fig. 2). This gene was designated F3h-H1.
DNA markers, in particular RFLPs and microsatellites, have facilitated the precise mapping of several sets of wheat genes, and this has provided good support for the existence of homoeologous series. Homoeologous sets of genes have been defined for red grain color (R) (Flintham and Gale 1995), glume color (Rg) (Khlestkina et al. 2006, 2009b), awn color (Bla) (Börner et al. 2002), sphaerococcoid spike morphology (S) (Salina et al. 2000), anthocyanin pigmentation of different plant organs (Rc, Pc, Pan, Plb) (Khlestkina et al. 2002, 2009c, 2010a) and for ent-kaurenoic acid oxidase (KAO) which catalyses three steps in the gibberellin biosynthesis pathway (Khlestkina et al. 2010b). Comparative genetic mapping in wheat, rye and barley has been performed for the homoeoloci affecting plant height and development (Börner et al. 1998) and the homoeoloci controlling waxless plant, waxy endosperm and absence of ligules (Korzun et al. 1997).
In the current study, the homoeologous genes (F3h-1) encoding flavanone 3-hydroxylase involved in plant defense were mapped close to the microsatellite loci Xgwm0877 and Xgwm1067 on the long arm of chromosome 2 in wheat and rye (Fig. 2). Barley F3h was mapped at the homoeologous position on chromosome 2HL (Fig. 2), and therefore was assigned to the F3h-1 homoeologous gene series. This is in agreement with the assumed microsynteny of Triticeae genomes and may suggest that the mutant locus ant17 mapped to chromosome 3HS (Franckowiak 1997) is not a structural gene for barley flavanone 3-hydroxylase as was proposed earlier (Meldgaard 1992). In rice, the gene encoding flavanone 3-hydroxylase was found in the BAC clone Os04g0662600 (GenBank accession NM_001060692) assigned to chromosome 4 (Ohyanagi et al. 2006) having synteny with chromosome 2 of barley (Stein et al. 2007) and wheat (Conley et al. 2004). In maize, in spite of widespread of gene duplications in the genome, a single copy of F3h is present which has been mapped on chromosome 2 (Deboo et al. 1995) having synteny with rice chromosome 4 (Ahn and Tanksley 1993).
Thus, a total of seven functional copies of the F3h gene were genetically mapped in wheat, barley and rye (Fig. 2). The results obtained are important for further studies aimed on manipulation with F3h expression (and, hence, plant defense) in these crop species.
The study was supported by the Russian Foundation for Basic Research (08-04-00368-a), SB RAS (Lavrentjev project 6.5), L’Oreal-UNESCO fellowship and the Federal Targeted Program of the Russian Federation (state contract P409). The part considering the mapping in barley was conducted in the frame of a GABI-project (0313125A) funded by the BMBF (Bundesministerium für Bildung und Forschung) in Germany.