The Bulgarian orange-fruited mutant cultivar Oranzheva kapiya is characterized with a high content of the antioxidant β-carotene. To identify the mutation leading to the accumulation of β-carotene and develop a suitable molecular marker for this

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    Comptes rendus de l’Acad´emie bulgare des Sciences   SCIENCES AGRAIRES  Culture de plantes  MOLECULAR CHARACTERIZATION OF ADVANCEDMUTANTS FOR EARLY DETECTION OF HIGH β -CAROTENE CONCETRATION IN PEPPERBREEDING PROGRAMMS Veselin Petrov, Iliya Denev, Marian Draganov, Oleg Timin ∗ , IvelinPanchev ∗∗ , Nasya Tomlekova ∗∗∗ ( Submitted by Academician A. Atanassov on October 30, 2012  ) Abstract The Bulgarian orange-fruited mutant cultivar Oranzheva kapiya is charac-terized with a high content of the antioxidant  β  -carotene. To identify the muta-tion leading to the accumulation of   β  -carotene and develop a suitable molecu-lar marker for this trait, the genes for geranyl-geranyl pyrophosphate synthase,capsanthin-capsorubin synthase and  β  -carotene hydroxylase were studied bothin the mutant and in the parental cultivar – Pazardzhishka kapiya. Our resultssuggest that the gene for  β  -carotene hydroxylase in the cv. Oranzheva kapiyais affected by a mutation, as well as the polymorphism between initial and mu-tant plants in this locus could be used as a molecular marker in the breedingprogrammes towards high  β  -carotene content. Key words:  molecular marker,  Capsicum annuum  , sweet pepper,  β  -carotene Introduction.  Carotenoids are among the most important metaboliteswhich contribute to the fruit quality of sweet pepper ( Capsicum annuum   Linn.)as a non-nutritional source with antioxidant properties and/or bioactivity. They This research was carried out with the support of the IAEA under CRP15406 andRER/5/017.10  303  are the main non-enzymatic antioxidants which, thanks to their multiple func-tions, can improve human health [ 1 ]. Since the human organism is not able tosynthesize them by itself, the main source of carotenoids is plant food. Dailydoses of carotenoids, varying from 180 to 300 mg, can prevent the developmentof certain types of cancer and a number of severe chronic diseases [ 2 ]. Their defi-ciency is most often associated with protein/calorie malnutrition and affects over120 million children worldwide [ 3 ].Pepper is a traditionally-important vegetable crop in Bulgaria, which is con-sumed in large quantities and is an important source of bioactive substances like β  -carotene. The expanding “healthy food” market justifies breeding new culti-vars with improved quality and high added food value [ 4, 5 ]. Breeding programmesbased on induced mutations could generate cultivars with improved phytonutri-ent level. For example, the orange-fruited pepper cultivar Oranzheva kapiya,characterized with an elevated content of   β  -carotene, was developed in a similarfashion out of the parental red-fruited cultivar Pazardzhishka kapiya 794. Re-combinant inbred lines (RILs) through three consecutive back-crosses betweeninitial (parental, wild type, wt) plants with red fruit and mutant plants with or-ange fruit were developed, followed by self-pollination and development of nearisogenic lines (NILs) till M 8  generation, and the mutation was introduced intothe pepper breeding programmes [ 6, 7 ].The objective of the present study was to assess suitable breeding markersfor early detection of high  β  -carotene concentration based on molecular charac-terization of the mutant pepper cultivar Oranzheva kapiya. Materials and methods.  Plant material  .  For this study,  Capsicum annuum   Linn. genotypes from the red-fruited local cultivar Pazardzhishka kapiya794 (PK rf  ) and the orange-fruited mutant cultivar Oranzheva kapiya (OK of  ) wereused. Seeds from the parent Pazardzhishka kapiya 794 were treated with X-rayirradiation, orange-coloured fruit plants (induced mutant – M) were identified,and a cultivar was developed through subsequent breeding [ 8 ]. DNA manipulations.  Genomic DNA was isolated from the first true leavesof the PK rf  and OK of  , using Nucleon PhytoPure Kit (Amersham) following therecommendations of the manufacturer.PCR reactions were performed using 100 ng DNA template with PuReTaqReady-To-Go PCR Beads and 5–25 pmol primers (Amersham). Primer anneal-ing temperature was estimated with the online tool: The amplified PCR products were isolated from the agarose by QIAquick Gelextraction kit (Qiagen) according to the srcinal protocol, and then used for A/Ucloning by applying Qiagen PCR Cloning Kit. The ligation reactions were mixedwith 250    L freshly-prepared chemically-competent bacterial ( E. coli  -TOP 10 –Invitrogen) cells and the transformation was induced by thermal shock (42 ◦ C for30 s). Successful transformants were determined by colony PCR and were further 304  V. Petrov, I. Denev, M. Draganov et al.  propagated for plasmid preparation by QIAprep Spin Miniprep Kit (Qiagen) sentfor sequencing by external service company.Homology identification of the obtained sequences as well as protein structureanalyses were performed using the services at NCBI web site Results and discussion.  Biochemical evaluation performed preliminarilyon PK rf  and OK of  genotypes demonstrated higher  β  -carotene levels in the ob-tained orange-fruited mutants [ 5, 12 ]. To shed light on the molecular basis of thisadvantageous mutation, we decided to isolate and characterize key genes of thecarotenoid biosynthesis. Isolation of geranyl-geranyl pyrophosphate synthase   ( GGPPS  ) frominitial and mutant pepper cultivars.  Geranyl-geranyl pyrophosphate syn-thase catalyzes an important reaction in the acetate pathway for secondary metabo-lite biosynthesis and serves as a branching point toward biosynthesis of carotenoids,chlorophylls, quinones, etc. Since GGPPS is the first enzyme in the carotenoidbiosynthetic pathway, a mutation in its gene can cause dramatic changes of thedownstream metabolic flows and can ultimately result in different pigment dis-tribution. Therefore,  Ggpps  was the first target gene to be tested for differencesbetween PK rf  and OK of  genotypes. Ggpps  was amplified and sequenced from parent and mutant peppercultivars.The analysis demonstrated amplification of a product with the expected length.The sequenced fragments were homologous to the sequence published in NCBIGene Bank except for nucleotide 558, where there is a difference (a transversionfrom G to C) (data not shown). The observed transversion is presented in boththe parental and the mutant cultivars and is silent since it is located in the 3 ′ end of the alanine codon GCG in the published sequence, which is converted intoGCC in PK rf  and OK of  . For these reasons, we assume that this mutation cannotlead to any functional differences in the GGPPS activity between the red- andthe orange-fruited Bulgarian cultivars. This result also strongly suggests thathigh  β  -carotene concentration does not result from increased biosynthesis of itsprecursor. Isolation of capsanthin-capsorubin synthase   ( CCS  )  from parental and mutant pepper cultivars.  The enzyme which catalyses the formation of the pepper-specific pigments with red-colour capsanthin and capsorubin from theyellow pigments antheraxanthin and violaxanthin respectively, is called capsan-thin-capsorubin synthase (CCS).Since CCS in pepper is strongly induced at the time of fruit ripening andis able to take on the function of weakly-expressed  β  -cyclases, it might be con-sidered as an active player in the regulation of the metabolic flow through thecarotenoid cycle. Furthermore, it is logical to assume that the colour differencesbetween Pazardzhishka kapiya 794 and Oranzheva kapiya are caused by a mu-tation leading to an impairment of CCS-activity and subsequent inability of the Compt. rend. Acad. bulg. Sci.,  66  , No 2, 2013   305  mutants to accumulate red pigments. To test this hypothesis, we isolated, clonedand sequenced the  Ccs  from PK rf  and OK of  cultivars.Comparison of the  Ccs  sequences of the initial and mutant cultivars revealedone difference between the two genotypes and one difference in the Bulgariancultivars with the published sequence (Acc.No.GU122933) (Fig. 1). Fig. 1. Alignment of CCS fragments from Oranzheva kapiya (row 1, M) and Pazardzhishkakapiya (row 2, W) compared with the published reference CCS sequence of   C. annuum  (row 3). Row 4 shows the consensus sequence In-frame translation of the sequences revealed two types of changes in thecorresponding CCS amino-acid sequences. The change of G to C at position 235leads to a replacement (denoted as G79R) of glycine G79 with arginine (R) in theprotein sequence. This difference is established in the Bulgarian cultivars whencompared to the sequence published in PubMed by  Bouvier  et al. [ 9 ].The change of A to T at position 243 leads to a conservative replacement(denoted as E81D) of the aspartic acid E81 with the highly-similar glutamic acid(D) in the protein sequence. This is the only detected difference in CCS betweenthe red-fruited parent cultivar and the orange-fruited mutant progeny.G79R causes the appearance of a strong positive charge in the protein mole-cule due to the presence of arginine, while E81D is a conservative substitution.Nevertheless, one could hypothesize that the R79/E81 combination in the mutantenzyme might lead to impaired catalytic activity compared to R79/D81 in theparental cultivar gene and G79/E81 in the published sequence. This possibilityneeds to be further investigated in the future. More detailed analysis will allowthe verification or rejection of the putative effect of these replacements on theenzyme structure and activity. Characterization of polymorphism in CrtZ gene in pepper.  Since β  -carotene hydroxylase ( CrtZ  ) catalyzes the direct conversion of   β  -carotene to β  -cryptoxanthin, a plausible explanation of the accumulation of   β  -carotene in themutant orange-fruited pepper cultivar could be the inactivation of this enzymeby a mutation. Therefore, the  β  -hydroxylase was chosen as the next target geneto compare the sequences in PK rf  and OK of  cultivars.In pepper, two  β  -hydroxylase genes have been described –  CtrZ   and  CrtZ  -2.The encoded proteins show 72% homology across their sequence [ 10 ]. PCR am-plifications of different fragments and internal regions of   β  -carotene hydroxylase( CrtZ  ) in both the red- and orange-fruited cultivars were performed with differ-ent combinations of specific primers. These primers were based on the published 306  V. Petrov, I. Denev, M. Draganov et al.
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