Genetic Analysis, Relevant Gene Cloning Of Purple Trait And Interspecific Hybrids Creating In Capsicum

Purple pepper plants have a high resistance to high temperature, drought and virus because of the purple color in the stem, branches, leaves, flowers and immature fruits rich in anthocyanins. The anthocyanins and degraded metabolic products in body of human and animal can effectively prevent the macromolecules such as DNA from damaging by oxidation, thus with functions of the preventing and curing diseases, protecting health and so on. Hybridizing purple pepper, as one of parent materials, with green pepper to achieve complementary function of different pigments, improve the nutritional quality of capsicum fruites and cultivate cultivars suitable for seasons of summer and autumn is one of the efficacious ways to solve the problem of the "short summer". In this paper, fruit setting rate of purple capsicum and the photosynthesis characteristics during summer conditions, inheritance rule of purple trait was investigated, and the accession of purple pepper was utilized as a parent to create purple interspecific hybrids successfully. The main contents and results are as follows:1. Classifying, fruit setting rate of purple capsicum and the photosynthesis characteristics during summer conditionsPhylogeny of 10 Capsicum accessions including purple varieties 7033,7036 from different habitats was researched using random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) technique, the results showed that 369 scored bands were amplified in total with 318 bands having polymorphism amplified with 34 primers screened from 45 RAPD primers in the genome DNA of 10 Capsicum accessions, and the percentage of polymorphic loci was 86.2%. Analysis with SPSS statistical software demonstrated that when the threshold of Squared Euclidean distance was at 10.5, the 10 Capsicum resources could be clustered into 4 groups, C. annuum, C. frutescens, C. chinense and C. baccatum, in which purple peppers 7033,7036 should be classified as C. annuum species of the genus Capsicum for there were similarity maximum, from 0.622 to 0.732, between them and other 3 representative C. annuum accessions.To delve into resistance to high temperature of purple pepper, an experiment was conducted in a plastic greenhouse to assess fruit setting rate and major photosynthetic and chlorophyll characteristics of purple and green pepper varieties in summer. For self fruit setting studies,18 and 21 purple and green pepper varieties, respectively were hand pollinated at blossom bud stage. Similarly,10 purple and 9 green peppers were randomly selected for reciprocal hybridization to evaluate cross fruit setting rate. Main parameters of photosynthesis and chlorophyll fluorescence were measured from 5 pepper varieties with diverse leaf colours to assess the pigment content. The following results were obtained:(1) significantly higher fruit self-setting rate of purple (72.19%) than the green peppers (37.12%), significantly higher fruit cross-setting rate was recorded. The fruit cross setting rate was 55.99 and 21.60% when purple and green pepper varieties were used as female parents respectively. This indicates stronger tolerance ability of purple pepper variety to summer conditions; (2) the amount anthocyanin (An) and ratio of chlorophyll a and b (Chl. a/b) were significantly higher in leaves of purple peppers than the green ones, amount Chl. a and carotenoid (Car) were also higher in most purple peppers, but insignificantly in amount Chl. b; (3) transpiration rate (Tr), stomatal conductance (Gs) and net photosynthetic rate (Pn) of purple and green pepper showed a similar trend of double peak curve and double stomatal and non stomatal limitation at around noon. Light compensation point (LCP), light saturation point (LSP) and actual Pn during the day time of high temperatures were significantly higher while apparent quantum efficiency (AQY) were lower in all purple varieties than the green ones. Thus the capacity of purple varieties resistance to high light and temperature is higher than the green ones; (4) purple pepper plants could prevent excessive absorption of solar energy by pigment antennas besides by partly reversible inactivation of the reaction center (RC) of photosystemⅡ(PSⅡ) to reduce the harm of excess energy absorbed, the only strategy found in green pepper. The energy of dissipation (DIo/CS) as well as absorption (ABS/CS), capture (TRo/CS) and delivery (ETo/CS) in purple varieties 7033 and 7034 was lower, while the maximum of fluorescence (Fm), maximum of photochemical efficiency (Fv/Fm), potential photochemical efficiency (Fv/Fo) and electron transport efficiency (ETo/TRo) were higher than the green pepper. It can be concluded that energy in photosynthetic membranes of purple peppers was transferred more economically. Therefore, high photosynthetic efficiency varieties that will get high resistance to high temperature, strong light and low humidity may be bred up by hybridizing, selecting the individual plants with the appropriate ratio of chlorophyll to anthocyanin content, higher values of ABS/CS, Fm, ETo/TRo, Pn and lower Fo, and pigment functional complementary in segregating generation.2. Extraction method of pigment in purple capsicum leaves and inheritance of anthocyanin contentsCompletely randomized experiment design was employed to study the effects of temperature, acid concentration and time on extraction of pigment in purple pepper leaves, results showed that optimized combination was that of leaf material soaking in 0.1 mol·L-1 hydrochloric acid for 1 h at temperature 30℃. The pigment was a kind of water soluble pigments and its absorbency peak in visible spectra was observed at wavelength 540nm. Research on pigment stability in purple pepper presented the stability was significantly affected by sunlight, pH and metallic ions of Fe3+ and Ag+, as well as oxidants of hydrogen peroxide and reducers of sodium sulfite and sodium nitrite. To some extent, the stability was also impaired by other food additives such as ascorbic acid and starch.Three hybridization combinations and their respective six-generations, F1, F2, backcrosses B1 and B2 and their parents P1 and P2, were made with purple and green leaf peppers. Inheritance of anthocyanin contents measured with spectrophotometer was studied through joint analysis of multiple generations. The results showed that relative anthocyanin contents in leaves of segregating populations of three combinations displayed one asymmetry peak or multimodal distribution, indicating that the trait was quantitative in nature and controlled by major gene plus polygene. The heritance of anthocyanin contents in leave of chili pepper fitted the pattern of additive-dominance-epitasis major gene plus additive-dominance-epitasis polygene (E-0 model). The major gene heritability is 78.89%～91.47% and polygene heritability is 5.19%～17.00% in F2 generations in three crosses, which suggested artificial selection to purple leaves trait of pepper could be carried out in the separation of the early generations.3. AFLP molecular markers related to purple trait and genes cloning of anthocyanin biosynthesisAn extreme purple pepper varieties C. annuum7033 and green C. chinense7020 were used as parents to make F1 and then selfed to build F2 segregated population of leaf colour. Extreme purple and extreme green DNA pools were developed separately using bulked segregant analysis (BSA). AFLP technique with 64 primer combinations were employed to find the polymorphisms between these DNA pools, and in extreme purple DNA pool two specific fragments,181 and 180 bp, were amplified in the primer combination of E-AC/M-CTC. Sequence analyses showed E-AC/M-CTC181 had 76% and 83% homology with the species C. frutescens sequences named FJ597540 and FJ597541, two BAC clones of L gene that confer resistance to tobacco mosaic virus, and 83% homology with the species C. annuum sequence named AY702979, a YAC clone of Bs2 gene that confers resistance to bacterial spot of pepper, respectively. E-AC/M-CTC180 had 87% homology with the species C. annuum sequence named GU048886, a BAC clone of retrotransposon of long terminal repeat (LTR) sequence.Based on RT-PCR technique and homologous cloning strategy, primers were designed to clone the fragments relevant to anthocyanin biosynthesis in the purple pepper 7033, the structure genes PAL, CHS, CHI,5GT and transcription regulator gene R2R3-MYB, and the fragments length of 664,764,579,864 and 413 bp was obtained by sequencing, respectively. The comparison of sequence homology showed that the gene PAL fragment in purple pepper 7033 had an identity 98% to that of green species C. annuum and 97% to C. chinense in GenBank; the deduced amino acid sequence was identical with that of the former, but two amino acid differences to the latter. The gene CHS sequence had 99% homology to the green pepper C. annuum, and 3,12 and 12 deduced amino acid differences to the pepper C. annuum, potato and tomato, respectively. The gene CHI sequence had a highest homology of 95% to that of tomato PSY 2, but 4 amino acid differences. The gene 5GT sequence had an identity 90% to that of both tomato and eggplant, and low deduced amino acid consistency. The gene R2R3-MYB had 99% homology nucleotide sequence and the identical protein sequence to purple pepper C. annuum reported. The present research laid the foundation of cloning and expression of full-length genes involved in the path of anthocyanin biosynthesis in the purple pepper.4. Using embryo rescue techniques to create purple Capsicum interspecific hybrids (C. annuum×C. chinense)Interspecific hybridization experiments between green of C. chinense and purple variety of C. annuum were performed to research the effects of days after pollination (DAP) and hormone concentration on percentage of plants survived by embryo rescuing, and different culture mediums on stem segment rooting. The results showed that in treatments of 32～34 DAP hybrid embryos were rescued successfully. Number/percentage, 4.5%～4.84% of plants survived was highest using MS+8% sucrose+0.1 mg-L"1 GA3+ 0.05 NAA as rescuing medium, followed by the combination of MS+8% sucrose+0.15 mg·L-1 GA3+0.1 NAA,1.43%. Comprehensively analyzing indices of number/percentage of stem segment rooting, total number of roots, average of roots each stem segment, the most suitable medium for rooting,1/2 MS+0.5 mg·L-1 IBA, was selected. Thus, a suitable method of embryo rescue to overcome seed abortion after fertilization, one type of genetic barriers, was made.Hybridity identifying to the putative F1 plant obtained through embryo rescue technique, the purple accession (C. annuum) as female and the green (C. chinense) as male parent. Morphological observations revealed that most traits of F1 hybrids were intermediate to both parents especially the organic purple colour. Cytological investigation of F1 hybrid exhibited abnormal chromosome behaviour at meiotic stage of pollen mother cells (PMCs). Pollen grains stainability percentage was significantly lower than the corresponding parents. Both peroxidase isozyme and random amplified polymorphic DNA profiles of F1 hybrid possessed specific bands of the male parent. The nuclear ribosomal DNA internal transcribed spacer (ITS) region sequences from putative F1 and the possible parents obtained by direct sequencing method showed that 186 variable loci.139 loci (74.7%) and 23 loci (12.4%) of F1 hybrid were inherited from male and female parents, respectively. The consistency in morphological characteristics, cytological features, isozyme banding patterns and ITS region sequences thus confirmed hybridity of F1 plant.