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Abstract DNA methylation is a kind of important epigenetic phenomenon， which could regulate gene expression in cells by affecting the transcription and duplication of DNA without changing cell DNA base sequence. Azalea is one of the ten famous flowers in China， as well a famous flower in the world， and the whole plant has medicinal value. This study investigated the change profile of gene methylation during the four flowering stages of Rhododendron pulchrum by methylation sensitive amplification polymorphism （MSAP）， and 412 differential bands were obtained after selective amplification with screened 18 pairs of effective primers. Among the differential bands， 23 differential bands were selected and subjected to recovery， sequencing and data alignment， and it was found that chalcone synthase， cowpox mitochondria， birch liana mitochondria， HDR reductase in camellia， Vaccinium macrocarpon mitochondria， Cyperus rotundus mitochondria protein， Shigella flexneri plasmid and Scophthalmus maximus cDNA-AFLP fragment have a regulating effect on azalea epigenetic inheritance， and next， genes related to the flowering period of R. pulchrum would be further studied， thereby improving the economic value of R. pulchrum.
Key words Rhododendron pulchrum； DNA methylation； MSAP； Epigenetics
Azalea is a famous and precious ornamental flower with large colorful flower， rich design and color and different flower types， with very high ornamental and economical value[1-2]. It is widely used in pot culture and field cultivation， and has very broad application prospect as one of the ten famous flowers. Azalea often flowers in March-May. Under natural state， it blooming period varies， and though the plant flowers constantly， the blooming is not concentrated， with not strong visual effect and reduced the sense of beauty. In order to improve its ornamental value and economic value， the blooming period could be controlled by some technical means. Rhododendron pulchrum has widely spread branches in ficelle covered with maple strigose， and its flower looks very beautiful like brocade， in a blaze of color， and could enhance the natural landscape effect. It also could be planted aside rocks， pools and lawns， and improves garden ambience. The pot plants are usually placed in hotels， habitable rooms and public areas.
Methylation as one of the epigenetic regulation methods could activate silent genes， and thus plays a role under conditions such as differentiation， growth， development and morphogenesis of animal and plant organs and stress[3]. Demethylase plays a key role in DNA demethylation， and in embryonic development period[4-5]， the demethylation of tissue specific genes are catalyzed by some overexpression， and realized by excision repair pathway. In this study， the change profile of genome DNA methylation during the development process of azalea was studied by methylation sensitive amplification polymorphism （MSAP）[6-8]， and the influence mechanism of methylation as a kind of epigenetic modification[9-10] on the development of azalea was expounded. This study lays a foundation for the study on the regulation of flowering period， and also will provide a theoretical basis for artificial regulation of flowering period[11]. Materials and Methods
Materials
Materials were collected in January-April in 2017. R. pulchrum was located in Botanic Garden of Huanggang Normal University， and samples were collected in bud stage （Fig. 1-A）， initial flowering stage （Fig. 1-B）， full-bloom stage （Fig. 1-C） and declining stage （Fig. 1-D）. The sample were frozen in liquid nitrogen， and stored in a refrigerator at -70 ℃.
DNA methylation profile of R. pulchrum during development by MSAP method
DNA was improved by improved 2×CTAB， and the DNA quality and concentration were detected by 1% agarose gel and ultraviolet spectrophotometer， respectively， diluted to 500 ng/μl and stored in a refrigerator at -20 ℃ [12]. The digestion system had a total volume of 20 μl， and two kinds of endonuclease combinations were adopted for the digestion of DNA， obtaining four groups of digestion products： I-1， I-2， II-1， II-2， III-1， III-2， IV-1 and IV-2. After digestion， electrophoresis was performed on 1% agarose gel， so as to judge the digestion effect. A 20 μl of ligation system was designed， and the digestion products were subjected to ligation with EcoR I and HpaII/ Msp I adaptor at 16 ℃ overnight.
With 10 times dilution of each ligation product as a template， a 20 μl of system was designed for pre-amplification （forward primer： 5′-GACTGCGTACCAATTCA-3′； reverse primer： 5′-GATGAGTCTAGAACGGT-3′）. The pre-amplification was started at 94 ℃ for 5 min， followed by 21 cycles of 94 ℃ for 30 s， 56 ℃ for 1 min and 72 ℃ for 1 min， and completely at 72 ℃ for 7 min， obtaining the product， which was stored at 4 ℃.
The pre-amplification product was diluted by 40 times， and a 20 μl of selective amplification system was designed. The selective amplification was performed with primer pairs in Table 1. The amplification started at 94 ℃ for 5 min， followed by 13 cycles （Touch down PCR） of 94 ℃ for 30 s， 65S ℃ for 30 s （-0.7 ℃/cycle）， and 72℃ for 1 min， and 27 cycles of 94 ℃ for 30 s， and 72 ℃ for 1 min， and completed at 72 ℃ for 10 min， obtaining the product， which was stored at 4 ℃. Then， 5 μl of the selective amplification product was subjected to electrophoresis detection on 2% agarose gel to verify the amplification effect of primers， so as to screen the primer combination with high efficiency， clear bands and no trailing. The selected primers were used for the selective amplification of pre-amplified DNA of the four stages. The amplification products were detected with 6% denatured PAGE gel， and silver nitrate staining was performed to screen the DNA bands of the methylation changes at the four stages[13]. Recovery and sequential analysis of differential band
Each differential band was recovered and added into a PCR tube. Into the tube， 30 μl of sterilized deionized water was added to soak the gel. Centrifugation was performed at 4 000 rcf for 1 min. The tube was placed on a PCR instrument at 95 ℃ for 10 min， and then cooled at room temperature. Centrifugation was then performed at 12 000 rpm for 10 min， and the supernatant was obtained as the specific fragment. Amplification was performed with corresponding MSAP amplification primers and selective amplification system. The amplification product was transformed into Escherichia coli Top10 competent cell after TA cloning. Blue-white selection was performed to select positive clones， and after PCR detection， the positive clones were sequenced.
Analysis of differential band
The detected sequence information of differential bands was subjected to removal of vector sequences using the VecScreen tool in NCBI. The vector sequences were aligned using Blast tool in NCBI to select sequences with high matching degrees， so as to analyze their biological functions and cell information.
Results and Analysis
Methylation changes during the development of R. pulchrum
Through PCR amplification， 18 pairs were obtained from 72 pairs of primers， which were E1+HM1， E3+HM1， E6+HM1， E7+HM1， E10+HM1， E11+HM1， E4+HM2， E7+HM3， E6+HM4， E11+HM4， E5+HM2， E4+HM5， E8+HM5， E11+HM5， E2+HM6， E8+HM6， E11+HM6 and E12+HM6， respectively. Then， after PAGE electrophoresis， 412 bands were obtained. The primer pair E11+HM6 gave the most bands， 50 bands， while E8+HM5 showed the least bands， two bands. For all the primers， the average number of bands was 23 （Fig. 2）. The first batch， 23 differential bands were selected and coded， respectively， as 1-2， 2-1， 3-1， 3-2， 3-3， 3-5， 3-6， 4-1， 5-1， 5-6， 6-1， 6-4， 7-1， 8-1， 9-1， 9-2， 11-1， 13-1， 14-1， 15-2， 16-2， 16-8 and 17-1， which were subjected to recovery， cloning， sequencing， and Blast alignment finally.
Analysis of differential band
The obtained differential bands were analyzed， as shown in Table 2.
The differential bands were recovered， and 23 among them were selected and sequenced. The obtained sequence information of the differentiation bands were subjected to alignment analysis， and no matched sequences were found in the NCBI nucleotide database for 10 sequences， which might be new sequences， and requires further study. Other clones were highly matched with the information in NCBI database， with identity of 80%-100%. After E1+HM1 treatment， Rp4-1 had a matching degree up to 96% with the genome in the mitochondria of birch liana. After E4+HN2 treatment， Rp1-1 was totally matched with the Gm43 mRNA sequence in Scophthalmus maximus cDNA-AFLP fragment. After E11+HM1 treatment， Rp4-1 had a matching degree up to 89% with the complete genome sequence in cowpox mitochondria. After M4+HM2 treatment， Rp1-1 showed a matching degree up to 100% with the complete genome sequence in cowpox mitochondria. After E11+HM1 treatment， Rp4-1 had a matching degree up to 89% with HDR mRNA genome sequence in camellia. HDR is a rate-limiting enzyme gene in the synthesis of terpenes， as well as an important functional gene for terpene material metabolism.
After E11+HM1 treatment， Rp2-1 had a matching degree up to 89% with the genome sequence of CHS gene chalcone synthase of Vaccinium ashei. Chalcone synthase gene is a key gene in the phenylalanine metabolic pathway. It plays an important role in the synthesis of flavonoid substances， regulates physiological and biochemical processes including pigment synthesis， defense reaction and plant fertility， and is thus crucial to the growth and development of plant. CHS mainly functions in pigment synthesis， UV irradiation prevention， defense of pathogenic fungus infection， root nodule formation， plant fertility， regulation of auxin transportation and insect resistance. After E11+HM1 treatment， Rp4-1 had a matching degree up to 99% with the whole genome sequence of Vaccinium macrocarpon mitochondria. After E1+HM1treatment， Rp4-2 exhibited a matching degree up to 80% with the complete coding region of pwr100 ipah9.8 （ipah9.8） gene of Shigella flexneri plasmid. After E1+HM1 treatment， Rp4-1 showed a matching degree up to 89% with the sequence in Cyperus rotundus mitochondria protein.
Discussion
Azalea not only has very high ornamental value， but also is edible. There are many varieties having very high medicinal value. The whole plant is edible， and could treat diseases including internal injury， kidney vacuity deafness， irregular menstruation and rheumatism. Therefore， in order to improve its ornamental value and economic value， the flowering period could be regulated by some means， and epigenetic inheritance is the most forefront research manner of flowering period regulation[14]. Epigenetics is a branch of genetics about the heritable changes of gene expression under no change of gene nucleotide sequence[15-16].
DNA methylation as one of the ways of epigenetic regulation， in methylation process， 13 gene sequences could be matched in Blast base. There were 10 gene sequences need further investigation and verification. Tang et al.[17] discussed the adaption of Festuca arundinacea to environment during the analysis of Festuca arundinacea genome methylation under drought stress. Wang[18] studied the change of expression profile of whole genome through rice microarray analysis， and understood the gene change differences of genotypes with different salt tolerance at the whole genome expression profile， which is of great significance to the exploration of salt tolerance-related gene， the understanding of interaction between genes and genetic network， and the understanding of molecular mechanism related salt tolerance[18]. Sun et al.[19] investigated the methylation dynamics of Phyllostachys bambusoides Sieb et Zucc before and after flowering， so as to reveal its epigenetic change rule during flowering and regeneration process. Huang et al.[20] studied the DNA methylation change dynamics during the germination process of cucumber seeds and the effects of exogenous NaCl on seed germination and seed DNA methylation level， so as to investigate the action of DNA methylation in the germination process of cucumber seeds and the response under NaCl stress[20]. It could thus be seen that DNA methylation is very important in epigenetic inheritance， and next， the regulation of this technique on the flowering period of azalea would be further studied. This study showed that chalcone synthase， cowpox mitochondria， birch liana mitochondria， HDR reductase in camellia， V. macrocarpon mitochondria， C. rotundus mitochondria protein， S. flexneri plasmid and S. maximus cDNA-AFLP fragment have a regulating effect on azalea epigenetic inheritance， and next， genes related to the flowering period of R. pulchrum would be further studied， thereby improving the economic value of R. pulchrum.
References
[1] ZHU CY . Azalea resources and its landscape application research[D]. Hangzhou： Zhejiang University， 2008.
[2] LAN X， ZHANG YH， ZHANG JZ， et al. Research progress of Rhododendron breeding[J]. Acta Horticulturae Sinica， 2012（9）： 1829-1838.
[3] SUN BN. Advance in the profile of DNA methylation technology[J]. Life Science Instruments， 2009 （4）： 11-14.
[4] LI N， ZHANG Z， XIE LN， et al. Research advances in DNA methylation of plant[J]. Plant Physiology Communications， 2012， 11： 1027-1036 .
[5] ZHUO WQ， LA HG. Advances in Arabidopsis thaliana DNA methylation and demethylation[J]. South China Agriculture， 2015（9）： 129-133.
[6] HAIG D. Genomic imprinting and kinship： how good is the evidence. Annual Review of Genetics， 2004， 38： 553-585.
[7] Uhlmann K， Rohde K， Zeller C， et al. Distinct methylation profiles of glioma subtypes[J]. Int Cancer， 2003， 106（1）： 52-59.
[8] JIANG M， CHEN B， LI Z， et al. DNA methylation markers ad their application on swine production[J]. Swine Industry Science， 2015（1）： 110-112.
[9] ZHANG YH. Genomic DNA methylation analysis of muscle tissue among three local chicken breeds[D]. Jilin： Jilin University， 2014.
[10] HUANG YL. The whole genome methylation pattern of CpG island and promoter for human gamete and preimplantation embryos or morulas from in vitro maturation oocytes[D]. Guangdong： Southern Medical University， 2015.
[11] WANG GL， FANG HJ. Plant genetic engineering[M]. Beijing： Science Press.2009.
[12] WANG SZ， ZHANG CQ， ZHA SX， et al. Research and application of azalea genome DNA extraction method[J]. Forest Science and Technology， 2015（2）： 5-8.
[13] BASSAM BJ， CAETANO-ANOLLES G， GRESSHOFF PM. Fast and sensitve silver staining of DNA in ployacrylamide gels[J]. Analyytical Bicochemistry， 1991， 196（1）： 80-83
[14] LI W， BIAN YH， CHU XQ. Epigenetics and its correlation with diseases[J]. Chinese Journal of Cell Biology， 2013（2）： 229-233.
[15] WANG Q， TANG WP. Relationship between DNMT1 and DNA aberrant methylation and tumor[J]. Chongqing Medicine， 2014， 17： 2228-2231.
[16] ROBERTS LR， GORES GJ. Hepato cellular carcinoma： molecular pathways and new therapeutic targets[J]. Semin Liver Dis.， 2005， 25： 212-225.
[17] TANG XM， WANG Y， MA DW et al. Methylation analysis of Festuca arundinacea genome under drought stress[N]. Acta Prataculturae Sinica， 2015，24（4）：164-173.
[18] WANG WS. Temporal and spatial variation analysis of whole genome expression profile of rice under salt stress[D]. Beijing： Chinese Academy of Agricultural Sciences， 2010.
[19] SUN HM， YUAN JL， YUE JJ， et al. MSAP analysis of DNA methylation during the flowering and regeneration process of Phyllostachys bambusoides Sieb et Zucc[J]. Bulletin of Botanical Research， 2013， 33（6）： 723-730.
[20] HUANG YY， ZHANG HJ， XING YX， et al. Effects of NaCl stress on seed germination and DNA methylation status detected by MSAP analysis in cucumber[J]. Scientia Agricultura Sinica， 2013， 46（8）： 1646-1656.
Key words Rhododendron pulchrum； DNA methylation； MSAP； Epigenetics
Azalea is a famous and precious ornamental flower with large colorful flower， rich design and color and different flower types， with very high ornamental and economical value[1-2]. It is widely used in pot culture and field cultivation， and has very broad application prospect as one of the ten famous flowers. Azalea often flowers in March-May. Under natural state， it blooming period varies， and though the plant flowers constantly， the blooming is not concentrated， with not strong visual effect and reduced the sense of beauty. In order to improve its ornamental value and economic value， the blooming period could be controlled by some technical means. Rhododendron pulchrum has widely spread branches in ficelle covered with maple strigose， and its flower looks very beautiful like brocade， in a blaze of color， and could enhance the natural landscape effect. It also could be planted aside rocks， pools and lawns， and improves garden ambience. The pot plants are usually placed in hotels， habitable rooms and public areas.
Methylation as one of the epigenetic regulation methods could activate silent genes， and thus plays a role under conditions such as differentiation， growth， development and morphogenesis of animal and plant organs and stress[3]. Demethylase plays a key role in DNA demethylation， and in embryonic development period[4-5]， the demethylation of tissue specific genes are catalyzed by some overexpression， and realized by excision repair pathway. In this study， the change profile of genome DNA methylation during the development process of azalea was studied by methylation sensitive amplification polymorphism （MSAP）[6-8]， and the influence mechanism of methylation as a kind of epigenetic modification[9-10] on the development of azalea was expounded. This study lays a foundation for the study on the regulation of flowering period， and also will provide a theoretical basis for artificial regulation of flowering period[11]. Materials and Methods
Materials
Materials were collected in January-April in 2017. R. pulchrum was located in Botanic Garden of Huanggang Normal University， and samples were collected in bud stage （Fig. 1-A）， initial flowering stage （Fig. 1-B）， full-bloom stage （Fig. 1-C） and declining stage （Fig. 1-D）. The sample were frozen in liquid nitrogen， and stored in a refrigerator at -70 ℃.
DNA methylation profile of R. pulchrum during development by MSAP method
DNA was improved by improved 2×CTAB， and the DNA quality and concentration were detected by 1% agarose gel and ultraviolet spectrophotometer， respectively， diluted to 500 ng/μl and stored in a refrigerator at -20 ℃ [12]. The digestion system had a total volume of 20 μl， and two kinds of endonuclease combinations were adopted for the digestion of DNA， obtaining four groups of digestion products： I-1， I-2， II-1， II-2， III-1， III-2， IV-1 and IV-2. After digestion， electrophoresis was performed on 1% agarose gel， so as to judge the digestion effect. A 20 μl of ligation system was designed， and the digestion products were subjected to ligation with EcoR I and HpaII/ Msp I adaptor at 16 ℃ overnight.
With 10 times dilution of each ligation product as a template， a 20 μl of system was designed for pre-amplification （forward primer： 5′-GACTGCGTACCAATTCA-3′； reverse primer： 5′-GATGAGTCTAGAACGGT-3′）. The pre-amplification was started at 94 ℃ for 5 min， followed by 21 cycles of 94 ℃ for 30 s， 56 ℃ for 1 min and 72 ℃ for 1 min， and completely at 72 ℃ for 7 min， obtaining the product， which was stored at 4 ℃.
The pre-amplification product was diluted by 40 times， and a 20 μl of selective amplification system was designed. The selective amplification was performed with primer pairs in Table 1. The amplification started at 94 ℃ for 5 min， followed by 13 cycles （Touch down PCR） of 94 ℃ for 30 s， 65S ℃ for 30 s （-0.7 ℃/cycle）， and 72℃ for 1 min， and 27 cycles of 94 ℃ for 30 s， and 72 ℃ for 1 min， and completed at 72 ℃ for 10 min， obtaining the product， which was stored at 4 ℃. Then， 5 μl of the selective amplification product was subjected to electrophoresis detection on 2% agarose gel to verify the amplification effect of primers， so as to screen the primer combination with high efficiency， clear bands and no trailing. The selected primers were used for the selective amplification of pre-amplified DNA of the four stages. The amplification products were detected with 6% denatured PAGE gel， and silver nitrate staining was performed to screen the DNA bands of the methylation changes at the four stages[13]. Recovery and sequential analysis of differential band
Each differential band was recovered and added into a PCR tube. Into the tube， 30 μl of sterilized deionized water was added to soak the gel. Centrifugation was performed at 4 000 rcf for 1 min. The tube was placed on a PCR instrument at 95 ℃ for 10 min， and then cooled at room temperature. Centrifugation was then performed at 12 000 rpm for 10 min， and the supernatant was obtained as the specific fragment. Amplification was performed with corresponding MSAP amplification primers and selective amplification system. The amplification product was transformed into Escherichia coli Top10 competent cell after TA cloning. Blue-white selection was performed to select positive clones， and after PCR detection， the positive clones were sequenced.
Analysis of differential band
The detected sequence information of differential bands was subjected to removal of vector sequences using the VecScreen tool in NCBI. The vector sequences were aligned using Blast tool in NCBI to select sequences with high matching degrees， so as to analyze their biological functions and cell information.
Results and Analysis
Methylation changes during the development of R. pulchrum
Through PCR amplification， 18 pairs were obtained from 72 pairs of primers， which were E1+HM1， E3+HM1， E6+HM1， E7+HM1， E10+HM1， E11+HM1， E4+HM2， E7+HM3， E6+HM4， E11+HM4， E5+HM2， E4+HM5， E8+HM5， E11+HM5， E2+HM6， E8+HM6， E11+HM6 and E12+HM6， respectively. Then， after PAGE electrophoresis， 412 bands were obtained. The primer pair E11+HM6 gave the most bands， 50 bands， while E8+HM5 showed the least bands， two bands. For all the primers， the average number of bands was 23 （Fig. 2）. The first batch， 23 differential bands were selected and coded， respectively， as 1-2， 2-1， 3-1， 3-2， 3-3， 3-5， 3-6， 4-1， 5-1， 5-6， 6-1， 6-4， 7-1， 8-1， 9-1， 9-2， 11-1， 13-1， 14-1， 15-2， 16-2， 16-8 and 17-1， which were subjected to recovery， cloning， sequencing， and Blast alignment finally.
Analysis of differential band
The obtained differential bands were analyzed， as shown in Table 2.
The differential bands were recovered， and 23 among them were selected and sequenced. The obtained sequence information of the differentiation bands were subjected to alignment analysis， and no matched sequences were found in the NCBI nucleotide database for 10 sequences， which might be new sequences， and requires further study. Other clones were highly matched with the information in NCBI database， with identity of 80%-100%. After E1+HM1 treatment， Rp4-1 had a matching degree up to 96% with the genome in the mitochondria of birch liana. After E4+HN2 treatment， Rp1-1 was totally matched with the Gm43 mRNA sequence in Scophthalmus maximus cDNA-AFLP fragment. After E11+HM1 treatment， Rp4-1 had a matching degree up to 89% with the complete genome sequence in cowpox mitochondria. After M4+HM2 treatment， Rp1-1 showed a matching degree up to 100% with the complete genome sequence in cowpox mitochondria. After E11+HM1 treatment， Rp4-1 had a matching degree up to 89% with HDR mRNA genome sequence in camellia. HDR is a rate-limiting enzyme gene in the synthesis of terpenes， as well as an important functional gene for terpene material metabolism.
After E11+HM1 treatment， Rp2-1 had a matching degree up to 89% with the genome sequence of CHS gene chalcone synthase of Vaccinium ashei. Chalcone synthase gene is a key gene in the phenylalanine metabolic pathway. It plays an important role in the synthesis of flavonoid substances， regulates physiological and biochemical processes including pigment synthesis， defense reaction and plant fertility， and is thus crucial to the growth and development of plant. CHS mainly functions in pigment synthesis， UV irradiation prevention， defense of pathogenic fungus infection， root nodule formation， plant fertility， regulation of auxin transportation and insect resistance. After E11+HM1 treatment， Rp4-1 had a matching degree up to 99% with the whole genome sequence of Vaccinium macrocarpon mitochondria. After E1+HM1treatment， Rp4-2 exhibited a matching degree up to 80% with the complete coding region of pwr100 ipah9.8 （ipah9.8） gene of Shigella flexneri plasmid. After E1+HM1 treatment， Rp4-1 showed a matching degree up to 89% with the sequence in Cyperus rotundus mitochondria protein.
Discussion
Azalea not only has very high ornamental value， but also is edible. There are many varieties having very high medicinal value. The whole plant is edible， and could treat diseases including internal injury， kidney vacuity deafness， irregular menstruation and rheumatism. Therefore， in order to improve its ornamental value and economic value， the flowering period could be regulated by some means， and epigenetic inheritance is the most forefront research manner of flowering period regulation[14]. Epigenetics is a branch of genetics about the heritable changes of gene expression under no change of gene nucleotide sequence[15-16].
DNA methylation as one of the ways of epigenetic regulation， in methylation process， 13 gene sequences could be matched in Blast base. There were 10 gene sequences need further investigation and verification. Tang et al.[17] discussed the adaption of Festuca arundinacea to environment during the analysis of Festuca arundinacea genome methylation under drought stress. Wang[18] studied the change of expression profile of whole genome through rice microarray analysis， and understood the gene change differences of genotypes with different salt tolerance at the whole genome expression profile， which is of great significance to the exploration of salt tolerance-related gene， the understanding of interaction between genes and genetic network， and the understanding of molecular mechanism related salt tolerance[18]. Sun et al.[19] investigated the methylation dynamics of Phyllostachys bambusoides Sieb et Zucc before and after flowering， so as to reveal its epigenetic change rule during flowering and regeneration process. Huang et al.[20] studied the DNA methylation change dynamics during the germination process of cucumber seeds and the effects of exogenous NaCl on seed germination and seed DNA methylation level， so as to investigate the action of DNA methylation in the germination process of cucumber seeds and the response under NaCl stress[20]. It could thus be seen that DNA methylation is very important in epigenetic inheritance， and next， the regulation of this technique on the flowering period of azalea would be further studied. This study showed that chalcone synthase， cowpox mitochondria， birch liana mitochondria， HDR reductase in camellia， V. macrocarpon mitochondria， C. rotundus mitochondria protein， S. flexneri plasmid and S. maximus cDNA-AFLP fragment have a regulating effect on azalea epigenetic inheritance， and next， genes related to the flowering period of R. pulchrum would be further studied， thereby improving the economic value of R. pulchrum.
References
[1] ZHU CY . Azalea resources and its landscape application research[D]. Hangzhou： Zhejiang University， 2008.
[2] LAN X， ZHANG YH， ZHANG JZ， et al. Research progress of Rhododendron breeding[J]. Acta Horticulturae Sinica， 2012（9）： 1829-1838.
[3] SUN BN. Advance in the profile of DNA methylation technology[J]. Life Science Instruments， 2009 （4）： 11-14.
[4] LI N， ZHANG Z， XIE LN， et al. Research advances in DNA methylation of plant[J]. Plant Physiology Communications， 2012， 11： 1027-1036 .
[5] ZHUO WQ， LA HG. Advances in Arabidopsis thaliana DNA methylation and demethylation[J]. South China Agriculture， 2015（9）： 129-133.
[6] HAIG D. Genomic imprinting and kinship： how good is the evidence. Annual Review of Genetics， 2004， 38： 553-585.
[7] Uhlmann K， Rohde K， Zeller C， et al. Distinct methylation profiles of glioma subtypes[J]. Int Cancer， 2003， 106（1）： 52-59.
[8] JIANG M， CHEN B， LI Z， et al. DNA methylation markers ad their application on swine production[J]. Swine Industry Science， 2015（1）： 110-112.
[9] ZHANG YH. Genomic DNA methylation analysis of muscle tissue among three local chicken breeds[D]. Jilin： Jilin University， 2014.
[10] HUANG YL. The whole genome methylation pattern of CpG island and promoter for human gamete and preimplantation embryos or morulas from in vitro maturation oocytes[D]. Guangdong： Southern Medical University， 2015.
[11] WANG GL， FANG HJ. Plant genetic engineering[M]. Beijing： Science Press.2009.
[12] WANG SZ， ZHANG CQ， ZHA SX， et al. Research and application of azalea genome DNA extraction method[J]. Forest Science and Technology， 2015（2）： 5-8.
[13] BASSAM BJ， CAETANO-ANOLLES G， GRESSHOFF PM. Fast and sensitve silver staining of DNA in ployacrylamide gels[J]. Analyytical Bicochemistry， 1991， 196（1）： 80-83
[14] LI W， BIAN YH， CHU XQ. Epigenetics and its correlation with diseases[J]. Chinese Journal of Cell Biology， 2013（2）： 229-233.
[15] WANG Q， TANG WP. Relationship between DNMT1 and DNA aberrant methylation and tumor[J]. Chongqing Medicine， 2014， 17： 2228-2231.
[16] ROBERTS LR， GORES GJ. Hepato cellular carcinoma： molecular pathways and new therapeutic targets[J]. Semin Liver Dis.， 2005， 25： 212-225.
[17] TANG XM， WANG Y， MA DW et al. Methylation analysis of Festuca arundinacea genome under drought stress[N]. Acta Prataculturae Sinica， 2015，24（4）：164-173.
[18] WANG WS. Temporal and spatial variation analysis of whole genome expression profile of rice under salt stress[D]. Beijing： Chinese Academy of Agricultural Sciences， 2010.
[19] SUN HM， YUAN JL， YUE JJ， et al. MSAP analysis of DNA methylation during the flowering and regeneration process of Phyllostachys bambusoides Sieb et Zucc[J]. Bulletin of Botanical Research， 2013， 33（6）： 723-730.
[20] HUANG YY， ZHANG HJ， XING YX， et al. Effects of NaCl stress on seed germination and DNA methylation status detected by MSAP analysis in cucumber[J]. Scientia Agricultura Sinica， 2013， 46（8）： 1646-1656.