VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -oOo - UNDERGRADUATION THESIS TITLE: STRUCTURAL ANALYSIS OF THE RC GENE IN THE LOCAL PURPLE STICKY “YEN BAI” RICE VARIETY HANOI, 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -oOo - UNDERGRADUATION THESIS TITLE: STRUCTURAL ANALYSIS OF THE RC GENE IN THE LOCAL PURPLE STICKY “YEN BAI” RICE VARIETY Student : Bui Thi Thanh Hien Class : K62CNSHE Faculty : Biotechnology Supervisor : Nguyen Quoc Trung, MSc Le Duc Thao, PhD HANOI, 2022 COMMITMENT This thesis is composed of my original works, and contains no material previously published or written by another person Hanoi, 20th March, 2022 Student Bui Thi Thanh Hien i ACKNOWLEGEMENTS During the process of studying, researching and completing the thesis, I have received the help of many individual First and foremost, I would like to express my gratitude to my supervisor MSc Nguyen Quoc Trung, Department of Molecular Biology, Vietnam National University of Agriculture for their patience, motivation, and immense knowledge Their enthusiasm guidance helped me all the time of researching and writing of this thesis My sincere thanks also go to teachers in the Faculty of Biotechnology, Vietnam National University of Agriculture, whose encouragement, guidance, and inspiration supported me during my studying time at higher education Besides that, I am thankful to my dear classmates, class K62CNSHE, for all the time we had in the last four years Last but not least, I am grateful to my family and my friends for supporting me spiritually throughout writing this thesis and my life in general I sincerely thank you! Hanoi, 20th March , 2022 Student Bui Thi Thanh Hien ii CONTENT COMMITMENT i ACKNOWLEGEMENTS ii CONTENT iii LIST OF TABLES v LIST OF FIGURES vi LIST OF ABBREVIATIONS vii ABSTRACT viii INTRODUCTION CHAPTER LITERATURE REVIEW 1.1 Evolution and domestication of ricecultivar 1.2 Purple sticky rice 1.3 Overview of anthocyanin 1.4 Roles and characteristics of the Rc gene 11 1.5 Diversity of rice genetic resources in Vietnam 15 1.6 Studies on phylogeny of Vietnamese local varieties 19 CHAPTER MATERIAL AND METHODS 22 2.1 Material 22 2.2 Methods 22 2.2.1 Total DNAextraction 22 2.2.2 DNA fragmentamplification 23 2.2.3 Sequencing and identification of gene model 26 2.2.4 Sequence polymorphism analysis 26 2.2.5 Phylogenetic analysis 27 CHAPTER RESULTS 28 3.1 Amplification and sequencing of Rc gene 28 3.2 Sequence Rc fragments 28 3.3 Structural analysis of Rc gene 32 iii 3.3.1 Intron-exon structures : 32 3.3.2 14-base deletion detection: 33 3.3.3 Sequence polymorphism of Rc gene 35 3.4 Phylogenetic analysis 36 3.5 Protein features 37 3.6 Sub cellular localization and 3Dmodel 38 CHAPTER CONCLUSION AND SUGGESTION 40 CONCLUSION 40 SUGGESTION 40 REFERENCES 41 APPENDIX 43 iv LIST OF TABLES Table 2.1 Component of the PCR reation 24 Table 2.2 List of 18 primer pairs for amplifying DNA fragments of Rc gene 25 Table 2.3 Rc gene sequences of accessions from the NCBI 27 Table 3.1 Physical and chemical parameters of protein of NepCam YB with reference varieties 38 v LIST OF FIGURES Figure1.1.The evolution of rice (Purugganan 2010) Figure1.2 Awns of wild and cultivated rice Figure 1.3 28 samples of rice varieties after husking Figure 1.4 Basic anthocyanin structure (Khoo, Azlan et al 2017) Figure1.5 Different R radicals of anthociadin (Chaves-Silva, Dos Santos et al 2018) Figure 1.6 Metabolic pathways for the synthesis of pro‐anthocyanidins and anthocyanins (Chaves-Silva, Dos Santos et al 2018) 10 Figure 1.7 Genetic diversity of rice color 11 Figure 1.8 Rc allele phenotypes (Sweeney, Thomson et al 2006) 13 Figure 1.9 Population structure and location of the Indica and Japonica subpopulations within Vietnam (Higgins, Santos et al 2021) 20 Figure 1.10 Population structure and location of the Indica and Japonica subpopulations within Vietnam (Higgins, Santos et al 2021) 21 Figure 2.1 Rice grain of NepCam YB 22 Figure 3.1 Electrophoresis photo of Rc gene fragments amplified by PCR (DNA ladder was KAPPA universal ladder 100 bp) 28 Figure 3.2 Part of the gene sequencing results expressed through Chromas software 29 Figure 3.3 Full sequence of NepCam YB 32 Figure 3.4 Exon intron structure of Nepcam YB 33 Figure 3.5 Collinear analysis of the fifth intron's partial sequence of Rc 33 Gene of NepCam YB 33 Figure 3.6 The SNPs in RC gene of NepCam YB compared with representative rice accessions 35 Figure 3.7 G-C content, A-T content chart of each variety 35 Figure 3.8 Phylogenetic tree of Nepcam YB with rice varieties 37 Figure 3.9.Model structure Rc of each variety 39 vi LIST OF ABBREVIATIONS bHLH basic helix-loop-helix BC Before Century AD Anno Domini QTL Quantitative trait locus FNP Functional nucleotide polymorphism ABP Anthocyanins biosynthesis pathway DFR PCR Dihydroflavonol-4-reductase Polymerase chain reaction vii ABSTRACT Rc is one of the most important regulatory genes for proanthocyanidin accumulation in the rice pericarp A study on mutation and sequence polymorphism of the Rc gene will provide evidence for understanding anthocyanin synthesis and evolution of rice cultivars The aim of this study was to a Rc hive a full-length genomic DNA sequence and analyze polymorphism in the Rc gene of Nepcam YB variety, a local pigmented rice in Yen Bai province By using 18 primer pairs, full- length sequence along with down-stream and upstream segment of the Rc gene was successfully assembled and aligned by BioEdit software with 7500 bp We found that the Rc gene from NepCam YB variety contains introns and exons Sequence polymorphism analysis has revealed 14 bp deletions in the fifth introns and 54 SNPs was detected by comparing with representative rice cultivars Identification of physicochemical parameters of Rc protein showed hydrophilic and unstable properties The Rc protein was determined to be located in the mitochondria Phylogeny analysis revealed close relationship of Nepcam YB with japonica Jefferson cultivar In this study, full-length sequence of the Rc gene in Nepcam YB variety was successfully sequenced and is preliminary data for further study on domestication of Vietnamese localrice Keywords: Proanthocyanidin, pigmented rice, gene model, SNPs, phylogenetic tree, Rc gene viii proteins, the instability index greater than 40 (from 58.19 to 64.29) then NepCam YB and varieties are not stable Aliphatic index ranged from 62.45 to 77.52 The grand average of hydropathicity (GRAVY) ranges from -0.590 to 0.431 Hydrophobicity score below are more likely globular (hydrophilic protein) (table 3.1) Table 3.1 Physical and chemical parameters of protein of NepCam YB with reference varieties Number No Name of amino acids Molecular Theoretical Instability Aliphatic weight pI index index GRAVY Bengkongang 634 69699.24 5.34 60.44 71.40 -0,508 Kasalath 636 69783.26 5.34 60.66 71.18 -0,512 Gangdodo 634 69796.34 5.34 59.18 72.02 -0,590 Pae Daya Indolobye 634 69796.34 5.34 59.18 72.02 -5,509 Dhala Shaitta 616 67439.21 5.37 64.29 69.71 -0,553 Dee Geo Woo Gen 616 67340.07 5.32 64.28 69.71 -0,547 Koshihikari 445 49615.95 4.91 58.19 62.45 -0,698 Jefferson 616 67340.07 5.32 64.28 69.71 -0,547 NepCam YB 549 59967.19 5.55 59.74 77.52 -0,431 3.6.Sub cellular localization and 3Dmodel Sub cellular localization of Rc gene were identified by TargetP The Rc of the reference varieties is unkown place The Rc of NepCam YB was determined to be located in the mitochondrial with mTP value (mitochondrial targeting peptide) was 0.535 compared with cTP (chloroplast transit peptide) and SP (secretor pathway signal peptide) were 0.197 and0.017 The 3D model of Rc protein of Nepcam YB Was constructed with similarity with models of other varieties (Figure 3.9) 38 Figure 3.9.Model structure Rc of each variety 39 CHAPTER CONCLUSION AND SUGGESTION CONCLUSION The PCR conditions were optimized successfully for amplification of 18 fragments using 18 primer pairs By using 18 primer pairs, full-length sequence with down-stream and upstream of the Rc gene of NepCam YB variety was successfully assembled and aligned by BioEdit software with 7500 bp We found that the Rc gene from NepCam YB variety contains introns and exons Sequence polymorphism analysis on Rc gene of NepCam YB variety has revealed 14 bp deletion was detected in fifth intron and 54 SNPs of Rc gene in comparing with representative rice cultivars Identify physicochemical parameters of protein of NepCam YB, shows that hydrophilic protein and unstable The Rc of NepCam YB was determined to be located in the mitochondrial Phylogenetic tree was constructed based on full length sequence for understanding origin and relationship of Nepcam YB with other rice group in the world This was preliminary result for further study on domestication of Vietnamese local rice SUGGESTION I am still continuing to sequence more varieties of sticky rice in other localities to somewhat understand the domestication and evolution of Vietnamese rice 40 REFERENCES References Engish Brooks, L r., et al (2008) "FHA-RING ubiquitin ligases in cell division cycle control." Cellular and molecular life sciences65(21): 3458-3466 Cao, Z., et al (2006) "Ancient paddy soils from the Neolithic age in China‟s Yangtze River Delta." Naturwissenschaften93(5): 232-236 Chaudhary, R (2003) "Speciality rices of the world: Effect of WTO and IPR on its production trend and marketing." Chen, X Q., et al (2012) "Anti-oxidative analysis, and identification and quantification of anthocyanin pigments in different coloured rice." FoodChemistry135(4): 2783-2788 Dellaporta, A (1983) "Scleral trephination for subchoroidal effusion." ARchives of Ophthalmology101(12): 1917-1919 Furukawa, T., et al (2007) "The Rc and Rd genes are involved in proanthocyanidin synthesis in rice pericarp." The Plant Journal49(1): 91-102 Gould, K., et al (2008) Anthocyanins: biosynthesis, functions, andapplications, Springer Science & Business Media Gross, B L., et al (2010) "The molecular basis of white pericarps in African domesticated rice: novel mutations at the Rc gene." 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Bioinformatics25(11): 1451-1452 Londo, J P., et al (2006) "Phylogeography of Asian wild rice, Oryza rufipogon, reveals multiple independent domestications of cultivated rice, Oryza sativa." Proceedings of the National Academy of Sciences103(25): 9578-9583 Sweeney, M T., et al (2007) "Global dissemination of a single mutation conferring white pericarp in rice." PLoS genetics3(8): e133 41 Tamura, K., et al (2011) "MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods." Molecular biology and evolution28(10): 2731-2739 Vaughan, D A., et al (2003) "Diversity in the Oryza genus." Currentopinion in plant biology6(2): 139-146 Chaves-Silva, S., et al (2018) "Understanding the genetic regulation of anthocyanin biosynthesis in plants–tools for breeding purple varieties of fruits and vegetables." Phytochemistry153: 11-27 Furukawa, T., et al (2007) "The Rc and Rd genes are involved in proanthocyanidin synthesis in rice pericarp." The Plant Journal49(1): 91-102 Grillo, M A., et al (2009) "Genetic architecture for the adaptive origin of annual wild rice, Oryza nivara." Evolution: International Journal of Organic Evolution63(4): 870-883 Higgins, J., et al (2021) "Resequencing of 672 Native Rice Accessions to Explore Genetic Diversity and Trait Associations in Vietnam." Rice14(1): 1-16 Koes, R E., et al (1994) "The flavonoid biosynthetic pathway in plants: function and evolution." BioEssays16(2): 123-132 Khoo, H E., et al (2017) "Anthocyanidins and anthocyanins: colored pigments as food, pharmaceutical ingredients, and the potential health benefits." Food & nutrition research61(1): 1361779 Nagao, S (1947) "Ein Beitrag zu einer genotypschen Analyse der Farbeigenschaften der Spelze und der anderen Pflanzenteile bei der Reispflanze Genetische Untersuchungen uber die Reispflanzen, VI." Jap J Genet Suppl.1: 1-27 Purugganan, M D (2010) "The evolution of rice: molecular vignettes on its origins and spread." Archaeological and Anthropological Sciences2(2): 61-68 Reddy, V., et al (1995) "Anthocyanin pathway in rice (Oryza sativa L): identification of a mutant showing dominant inhibition of anthocyanins in leaf and accumulation of proanthocyanidins in pericarp." Theoretical and Applied Genetics91(2): 301-312 Sang, T and S Ge (2013) "Understanding rice domestication and implications for cultivar improvement." Current opinion in plant biology16(2): 139-146 Sweeney, M T., et al (2006) "Caught red-handed: Rc encodes a basic helix-loophelix protein conditioning red pericarp in rice." The Plant Cell18(2): 283-294 Tanaka, Y., et al (2008) "Biosynthesis of plant pigments: anthocyanins, betalains and carotenoids." The Plant Journal54(4): 733-749 References VietNamese Tươi, N T H., et al (2014) "Phân tích đa dạng di truyền mẫu giống lúa cẩm thị SSR." Tạp chí Khoa học Phát triển12(4): 485- 494 Trung, N.Q.T et al (2016)." Nghiên cứu ứng dụng DNA marker khai thác chọn tạo giống lúa có hàm lượng anthocyanin cao" 42 APPENDIX APPENDIX DNA SEQUENCE POLYMORPHISM OF Rc GENE IN 'YEN BAI' LOCAL PURPLE STICKY RICE 1 Bui Thi Thanh Hien , Nguyen Thi Yen Chi , Chu Duc Ha , Bui Van Hung , Ngo Thi Hong Tuoi , Nguyen Quoc Trung , Tran Van Quang Faculty of Biotechnology, Vietnam National University of Agriculture Faculty of Agronomy, Vietnam National University of Agriculture Faculty of Agricultural Technology, University of Engineering and Technology, Vietnam National University of Hanoi Agricultural Science Institute of Northern Central Vietnam ABSTRACT Rc is one of the most important regulatory genes for proanthocyanidin accumulation in the rice pericarp A study on mutation and sequence polymorphism of the Rc gene will provide evidence for understanding the evolution of rice cultivars The aim of this study was to archive a full-length genomic DNA sequence and analyze polymorphism in the Rc gene of Nepcam YB rice, a local pigmented rice in Yen Bai province By using 18 primer pairs, full-length sequence with down-stream and up-stream of the Rc gene of NepCam YB variety was successfully assembled and aligned by BioEdit software with 7500 bp We found that the Rc gene from NepCam YB variety contains introns and exons Sequence polymorphism analysis has revealed 14 bp deletion in the fifth introns and 54 SNPs of Rc gene was detected by comparing with representative rice cultivars Next, our phylogeny analysis revealed that Nepcam YB was closely related to the Japonica Jefferson cultivar In this study, full-length sequence of the Rc gene in red pericarp Nepcam YB was successfully archived and is preliminary data for understanding domestication of Vietnamese local rice Keywords: Proanthocyanidin, pigmented rice, gene model, SNPs, phylogenetic tree, Rc gene INTRODUCTION Rice (Oryza sativa) has been considering as one of the most important cereal crops that is widely cultivated in Asia and has a long domestication history Rice is a staple food of more than 100 countries as the main carbohydrate source for more than half of the global population Up till now, a large number of rice varieties have been cultivated in the world and vary on grain properties, like shape, amylose content and pericarp color Specifically, the color of the bran shell is caused by the pigment of the accumulation of flavonoids, carotenoids, and betalains In while, flavonoid compounds such as anthocyanins, flavonols, and proanthocyanidins are major secondary metabolites in plants and are red, purple (black), and brown in color Rc has been well-characterized as one of the most important regulatory genes in the pathway of proanthocyanidin accumulation in the rice pericarp[1] Briefly, Rc locates on rice chromosome 7, contains eight exons, and encodes a basic helix-loop-helix (bHLH) regulatory protein [2, 3] The major Rc domestication allele which has been occurred in more than 97% of non-pigmented rice cultivars, is characterized by a 14-bp fragment deletion in the seventh exon [1] This mutation generates a truncated, non-functional gene product and the non-pigmented („white‟) pericarp of the domesticated rice An independently evolved domestication allele, Rc-s is found in other white pericarp rice genotypes with the frequency of less than 3% and exhibits a base transverse (i.e., C to A) in the seventh exon instead of the absence of a 14-bp fragment [3].Furthermore, [4] identified the spontaneous mutant red pericarp rice cultivar Wells in the USA, which exhibits one G-base deletion located at the 20-bp site upstream of the absent 14-bp fragment in the seventh exon This new mutation restores the reading frame and allows the re-accumulation of proanthocyanidins in the pigmented rice [5] confirmed that the red pericarp rice variety PerlaRosso in Italy is a spontaneous mutant with one G-base deletion at the 44-bp site upstream of the absent 14-bp fragment in the seventh exon This new Rc allele is designated as Rcr [1] The red pericarp rice cultivated in most Africa appears a transversion mutation (i.e., A to T) occurs in the seventh exon transforms the pericarp color of African cultivated rice from red to white[6] “The allele of Rc is known as rc-g1 So far, six alleles of the Rc gene have been reported: Rc (wild type); rc and Rc-s, which are domestication alleles found in Asian cultivated rice; Rc-g and Rcr, revert ants of rc; and rc-g1, which is found only in African cultivars”[1] 43 The aim of this study was to analyze the sequence polymorphism in Rc gene of Nepcam pigmented YB- rice a local cultivar in Yen Bai province, Vietnam MATERIAL AND METHODS Materials Purple sticky YB (pigmented) rice variety collected in Yen Bai province used in this study was provided by Department of Molecular Biology and Applied Biotechnology, Faculty of Biotechnology, Vietnam National University of Agriculture Figure Rice grain of NepCam YB Methods Total DNA extraction Sample seeds were sown in the test field and the total DNA was extracted from young leaves using the previous procedures [7] Briefly, incubate extraction buffer was prepared (1M Tris-HCl pH 8.0, 0.5M EDTA pH 8.0, 2.5N NaCl, 10% SDS) at 65°C Cut the vacuum dried leaves sample into 0.5-4 cm sections into eppendorf tube, then put in three iron marbles Crush the sample with a multi bread shocker (Yasui Kikai) at 1800 rpm for 60 seconds, rest 10 seconds, repeat twice To each ground leaf sample, add 600μL extraction buffer, mix and incubate sample for 30 minutes at 65°C Add 200μL 5M potassium acetate and mix gently Incubate sample for 30 minutes in fridge Centrifuge for 10 minutes at 4°C (9000 rpm).Transfer the supernatant (roughly 400μL) to a new eppendorf Add equal volume of Isopropanol, mix gently Centrifuge for 30 minutes at 4°C (9000 rpm).Remove the supernatant Wash the pellet with 1000μL 70% ethanol, drop gently Centrifuge for 10 minutes at 4°C (9000 rpm), remove supernatant Dry the DNA at room temperature or 37°C until there is no smell of ethanol Dissolve the pellet in 50 μL TE (10mM Tris-HCl pH 8.0, 1mM EDTA pH 8.0), store in the fridge DNA electrophoresis for 20 minute at 100V, using 1% agarose gel (with ethidium bromide) and TAE buffer 0.1X DNA fragmentsamplification o o The PCR conditions: hot start at 95 C for min; annealing followed by 35 cycles with denaturation at 95 C for 30 o o o seconds, annealing at 45 C – 62 C for 30 seconds, elongation at 72 C for min; and final elongation for at o 72 C.In order to sequence full sequence of Rc gene about 6400 bp, we separated gene into several fragments that is less than 1000 bpand sequenced by Sanger methods Total 18 primers were used according to [1] and overlapped along Rc gene PCR conditions were first optimized the annealing temperature for each primers pair and then used to amplifying 18 fragments for sequencing (Table 1) Table List of 18 primer pairs for amplifying DNA fragments of Rc gene No Primers Forward sequence Ta (oC) Reverse sequence Target fragments Expected PCR products (nucleotides) AF1 tcaattcttccatccccaac atgccatgcgatcacaacta 55 RC_1 462 AF2 atctctccgtacaacaaaa tgccaagaacacgtaacaag 53 RC_2 522 AF3 gagggagctctacgactgg ccgcacgatcttgttctaat 58 RC_3 594 AF4 caggatagctccttttcttttgc ggcatatttcaatccaacaacc 53 RC_4 582 AF5 tcgatgcttgtgtagccaat tttgatcactctagcactaccttca 52 RC_5 541 AF6 gcctgattttatgacgtcaagt gcactgtatcggagctatagaga 45 RC_6 536 AF7 ggctaagacttttcttccacctt tgcaatgcatgctattctga 55 RC_7 485 AF8 tttgcatggcaaagaatgac cctgaagacatgcaaagcac 51 RC_8 432 44 AF9 cttgcccatgcatctttctt ccactgcatccaaagatgaac 52 RC_9 485 10 AF10 ttccctgatgtggtgcataa gagggtaggcttcatgtgga 52 RC_10 513 11 AF11 atgggcctgattcagtatgc ggaacaagggtgcttttgaa 58 RC_11 553 12 AF12 gccttgtcactcttggcatt ggttggcactgaaatcacct 60 RC_12 475 13 AF13 caccacacagagaatgctcaa catgctgccattagtgagga 62 RC_13 499 14 AF14 agcctctaacgaacattggaa cagaggagcaagaatgaaagc 60 RC_14 862 15 AF15 gcacacaaagatgaaca acgggtaggattcacttctgg 51 RC_15 986 16 AF16 tgcgatctttggtacctttca gctctcgatgatggacacct 58 RC_16 812 17 AF17 agcagctaaggaaccgcata cctcatcgatatcaccctgaa 51 RC_17 606 18 AF18 ccctttttctttgctcatgg ccttctttcgatctgctcgt 45 RC_18 576 Sequencing and identification of gene model st Eighteen amplified fragments were firstly qualified by agarose electrophoresis 2% and sent to Base Co., Singapore for purifying and sequencing by Sanger method Sequences assembly and alignment were conducted using Mega X The sequence alignment result was 7500 bp in length From the obtained sequence, the exons and introns were determined and gene model was constructed by GSDS2.0 (Gene structure display server) software Sequence polymorphism analysis Analyses nucleotide polymorphisms were calculated using BioEdit, DnaSP v6.0 [8] Analyses were performed on Rc gene sequences of accessions from the NCBI (http:// www.ncbi.nlm.nih.gov/)., including accessions representative for indica, aus, tropical and temperate japonica with both red and white grain color Table Rc gene sequences of accessionsfrom the NCBI No Name Gene code in NCBI Grain color Subpopulation Origin Bengkongang DQ885810.1 red Indica Indonesia Kasalath DQ885812.1 red Aus India Gangdodo DQ885805.1 red temperate japonica Korea Pae Daya Indolobye DQ885808.1 red tropical japonica Indonesia Dhala Shaitta Q885821.1 white aus Bangladesh Dee Geo Woo Gen DQ885818.1 white indica Taiwan Koshihikari DQ885803.1 white temperate japonica Japan Jefferson DQ885802.1 white tropical japonica USA Phylogenetic analysis was constructed using the Maximum likelihood (ML) in MEGA – X[9] RESULTS Amplification and sequencing of Rc gene To archive full sequence of Rc gene with 6400 bp[2, 3], we separated gene into 18 segments and used 18 primer pairs for amplified prior purifying and sequencing In the PCR, each primer pairs were optimized annealing st temperature for amplifying unique DNA band These PCR product with good quality and quantity were sent to Base Co Singapore for purifying and sequencing Archived sequences were checked quality based on chromatogram viewed in Chromas software Good quality sequences were then used for assembly and alignment 45 Sequence of Rc gene from purple sticky YB variety was then aligned with the reference sequence Oryza sativa with the accession number KX549256 The similarity between our targeted Rc gene and the reference sequence is observed to be 97% There was 7500 bp covering completely Rc gene >NepCam YB AGGGCTTTGCTACTAGCTCCTCACTCACTCGCCTTTACTTATCGATCTCGATCATCCACGAGCTAGCTAGCAGTGCTCGCCATGGCCGGCGGCGAGGCGCATGCGGCGCTGCAGGCGGTGGCGCAGAGCCTCCGGTGGACCTACAGCCTCCTCTGGCAGCTCTGCCCCCAC CAAGGGTACCTACCCTACCTACCTACGACACGATGCACAGTGTTCATCCATGGCCGGCCATGGCGGATCGTCGTCGTTGTCGATGATCATCGAAGGAAGCTAGAGGATATGGCTCAATACTTTGATAATATATATACTGATCTCTCCGTACAACAAAAATATAAAAATTCT AGCTAGTATCGAATGAGACATATGCTATGCTAGTACTACGAATCTAAAAAGATGTACATATTTTGATTCGTATTATTAGGATATATCACGAGTTTTTATATTTTGAGACGGATGTAATAATTCTGAATTTAGTTGTGATCACATGGCATGCAGGAGCTCGCTGGTGTGGGG GGAGGGGCACTACAACGGCGCCGTCAAGACGCGGAAGTCGACGGTGATGCAGCCGCCGCCGGCGGAGGAGGAGGACGACGCCGACCACGCGGCGCGCCACCGGAGCCGGCAGCTGAGGGAGCTCTACGACTGGCTGCAGCAGGCCGGGGAGAACTCCAGCGGCGGCGTGCA GACGTCGTCGACGACGGCGAGCCGGCGGCCGGGGGCGGCTCTGTCGCCGGAGGACCTGACGGAGACGGAGTGGTTCTTCCTCATGTCGGCATCCTACTCCTTCCCTCCCGGCATCGGGTATATAATAAAAAAGAAAGATATAAATATTAAATGCATGCATGCATCACATTA AACCACACTTCTTGTTACGTGTTCTTGGCAAAATGATGAACAATTACCACTAATTAATTGGAGCCAGAAACCCTAAAGATTTACCCACCTGGTTAATTAATCGGTGTGTTGATCCACGCATGCATGCATGCAGAAAATCAAGATCAGGATAGCTCCTTTTCTTTTGCAGGT TAATTAGCTAGATCTTCACGTATAATTAGCTAGCTAGATTTTAAAATATAATTTATTCAATTTGATTTATGATTTTTATTTTTTATTTCAAATAGATACAACTGTATACAAAATTATATTTTGGTACATACCTCCGATCCAACTACATCAGAGGTAAAAAAAAAATTAAAC CGTTGGAATTGATTAGAACAAGATCGTGCGGCCAGTCTATATCATAACTAACTTTTCTGATTCTCTAAAGCATAGAGATGTATATATACATCGTATTATTAGGCTCTATATTTCCTGATTAACACTAGATGCATATATAATTTTGATAGTCAAAATATACTTTTGATAGGC TCTAAAGAAAAACTTAATAACATGTACTCCCTCCATATACTTTTGATAGTCATATTTCATCTTGACACACAGATCAAGTATAAGTAATTCTACTTATCATCCATTTAAACACGCTACTAGTTATTCCTCATAAACAAGCGATTCATTAATATTTACATTTCTCGATGCTTG TGTAGCCCAATATTGTGTGGAAGAATGTCATTAAGAGGATAGGTTGTTGGATTGAAATATGCCTATCAAAAATAAATTTTTAGATTTGAAAATATGCCTATCAAAAGTAGATGGAGGGAGTATTAATTAATGTGAATTTCCAATCCTACTGTTGTGATATTAGGCTTTGTA CCTTCTTGTCCAGGAGGTATATATATGGCTCTTTTAAGGATGGGAGAAAATATCATCTTTAATACAACTATATATGGCTTTTGTTTGATAAATACAACTTTTATTTTGTATGAATACAAATATATTGATAAATATCCACCATTATAATCCTAACCCATTAGGATCATATGG TGTATATTTTTTTAACTATTTGTTTTTTATAAATTAATATTAAGAGATCACAATAAAAATATAGTATTATGAAAGTACTCTTAACAACATATCCAATGATAAAATTATTATTATTACAAAATATAGTGGTCAAATTGTATAGAATTCAATAGCCTGATTTTATGACGTCAA GTAAATTAAATAAAGAATGAAGGTAGTGCTAAGAGTGATCAACAATATCTCTCCTAAAATATGTCCTATAAGTTTTACTCCATAAATCCAAGGGTCAAAAGTTGTTGGGTTATTTTTTTAGATAATAACATACTACCCCTTTTCAAAATGTATGATTCTATTGACTTTTTG CACAACATTTAACCATTTGTCATATTAAAAATTAGTATAAACATCTAAAAATATAAGTTACAATTATATTTTATTTGATGATAAAACAACTCACAACAAAATAAATAATATTTATATAATCTTTTTGGAATAAAACGAATGATCAAACATTATTCAAAAAGTCAATGGTAT AGTACGTTTTGAAATTGATAGACTATGAGAGCAAAATTTTGAGATAACATGGAAAATTATCCTCTTAGACATTGCACTGTGTAATAATTAATAATAATGAATGAAAGGCTAAGACTTTTCTTCCACCTTATATAAGTGGTTGAATATATAGCAATCACATCATTACATGAT TTTGTAACCAACCGTCTCTATAGCTCCGATACAGTGCTAGTTTCACATCGTAATAATTAAAGAGTATAATAATAAATCGAGGTGTACTTCTCATCGATGAAGTGATGTGCCGCTTAGCTAAATTAAACTCGTATGCGAAAAATCAGTATATGTCCGGTTAATTTCTAAGAG AGAGATTGAGAGAGAATAATTGCGCCCCTCCAAATCCCCCTCTTGGACGTTAGGGAGCTATATAGACGGTATTGCTAAGTGCGATGTGTACATAACGTACCTGTCGTAGGAACATTTCTCATCCAAATTAAGTAGTAATGCATGGCATGAAATCCATTTTTGTATTTTGCA TGGCAAAGAATGACAACAAGGAATACACTAGCTAGCCCTGCCCTTTTTCAATTTAATTTAACATCAAACTTAGTATATCTTTTGTCAGAATAGCATGCATTGCATACTCTTTAAAAATAATTAATTAGTGTATTTTACTAGTCTTACAAAAGTATCAAGAGAGACAACTAA TTATAGTTGGGAGACACCAAACTTGTTTTTAATAATGACAATTAAAACCCTACCTCTACATCCAACATAGACGTACATAGTCCGAAGGCGCCAAATATTTGTACATTTAGCTACCAGATTTCAGTACGAGTTCTCACATTATAATTTTGATTTTTTTATTTTTTTTATAAA CAATCTGGTACCCTTTTATGTCTGGAAGGAAAAAAAAAATCTAAATTGCAACATTTTAGTCGGTGAGAATGGTACTCTGTCCTAGCTACTTTCTACACATGAGAGAGAGAGAGAGAGAGAGAGAGCCTTTAATTGCCCTTGCCCATGCATCTTTCTTTGCACACATGTGGT CCCATAGTCTTATGGAGAGAACTTGTTAAGTTGCACACATGTGTGCTTTGCATGTCTTCAGTATGGAGAGAACTTGTTAAGTTGCACACATGTGTGCTTTGCATGTCTTCAGGTTACCTGGAAGGGCATTTGCAAGGAGAGGCCATGTATGGCTCACTGGAGCAAATGAAG TTGACAGCAAAGTATTCCTAAGAGCAATTCTTGCCAAGGTTCAGCCATCACCTTCTCTTACCTATTTTTCACTCTGAATGCCAACAGTGCTTTGCACATTGTAGTCTGTTTGCAGACTGCAAATGATGACCATAATCAGATCAGAAAATAAAATAATATTATATACTTTTT GAGCCAGCTAGCAAGAATATGTAACAATAATTCTCCTTTTTTTTTCTTGTTCTTTTCCCTGATGTGGTGCATAACAAATAACCAAACTGATGAATGGCAAAGTGCTGGTATCCAGGTATTTGCCTCTAAAAGTAGCTACACGTTTACTATGAAATTTTGTGGCTTTTGTTC ATCTTTGGATGCAGTGGCCATTATCTAAAAACTATGAATTTCCAGACTGCAGTTTTTATCTAATTTTGTGACTTTGTACATCAGACAGTTGTGTGCATTCCTGTTGTCGATGGCGTCCTGGAAATTGGAACTACGGAAAAGGTGATTTCGTATATTATCAGCTGACAATCT AATTATATGGGCCATATAATTAAGTATAAATCAAAATACCTCATAATATATTATAAAGTATCTAATGTGATTATGTGAATATTGGCTATTTCAATGTAATTTGATATATGAAACTGATAATCCTCTGAAACTCCGTAAGGATCAAACTAATCAAAATGTATATATTTTCAA GGTGATTTCGTATATTATCAGCTGACAATCTAATTATATGGGCCATATAATTAAGTATAAATCAAAATACCTCATAATATATTATAAAGTATCTAATGTGATTATGTGAATATTGGCTATTTCAATGTAATTTGATATATGAAACTGATAATCCTCTGAAACTCCGTAAGG ATCAAACTAATCAAAATGTATATATTTTCAAGGTGGAGGAAGATATGGGCCTGATTCAGTATGCAAGGGGCATCTTCATGGATCAACATGGCATCCACATGAAGCCTACCCTCTCACAGCACTCAACATCCAACCCAGTCACCCACTGTACTCATCAGCATCCAATCCAGG TTCAGATGCAACTAGGTATCACCAGCCAAACAAAGTTTGATTATTCAGATGAGCTCAATGCAGATGAGGAGAATGATGACACAGAAGAAGAGGGCATGTCAGGTTCAGACACTAACAACACTGACACTGAAAGGAATTCAGGCCAGCTGCAACTTCAAATGCAAGACCAAC TGAACATGGTGAGCAATGACCACCAGACAATACCAAATAATGCAGTTTCCAGTGAGCTAATGCAGTGTGAGATGTCAGAAGTGGTAAGAGATGGCTGCTCAAATAATATTTTAGAGGATGAAATCCAAATGCTGATGGATTGCCAAAACAGTAATTGTCAGTTAAATTTGC AAGGGCCAGATGAGCCTTGTCACTCTTGGCATTTTCTCTGCGAGGAGTTACAAAATGATTACCAGCCAGGTATTACATTTGAGAAGATAATCCTTCAAAAGCACCCCTTGTTCCATCCCTTCTCATTCAATGTATCCTATATATATATATATATATATATATATATATATA TATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATAT ATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATA TATATATATATATATATATATATATATATATATATATATATATATATATATATATATATAGATATATATATATAGGATATCTATAGATGTTCCAGTAGTCACTGCAGTTACAGGGGAGCAGAAACACCTGAATCAAGGGGCGGGAAAGGCGCAAGTGGATGCCATCCAAGG TGATTTCAGTGCCAACCATGTGCTGAAAGAGAGGAGAAGAAGAGAGAAGCTCAATGAGAAGTTCATAATTCTGCGATCTTTGGTACCTTTCATGACAAAGGTAATTAAGTACTCCCTCTATTTCTATAAAGCCGTATTTGACTAGTTATCTTATTTAGAAAGTATGTGCAA ATATGTAAAATATAAGTCATACTTAAAGAACTTTTAATGTTATTAAATAATAAGTCACACCAAAAATAAAACATATATATTTTTAATAAGATAAATGATTAAATGTATATATAAAAATTAATAGCGTCACATATTTTAAAATAGAGGGGTATTTAAGTACCCACAGGATCA TCAAAATTCAGTTATCTTTTCTTAAGCCTCTAACGAACATTGGAAGATCCTCACTAATGGCAGCATGAATCTAGGGTTCACTATTTCGGAATGCAAATATGTTTTACCGGGCATCCGATTTTTAAAAAATTCAGAATGAAGAAAATTGAATCTTTTTTATGGATTTGAATA AATCTTGATAAATTCGAAAAAATTTCCGAACTTTTGGCCAGAAGTGAATCCTACCCGTATCCACCGGTAATAAACCTAAATTTTTGGGAGTAATGAATTAATGTTATATATAATCCATGAATTATATAGTTCCAAACTACTCCGTAACAAATTTTCAGGAGTAGTGAAATT AATATTATTACAATCTCAGAAAAAAATGGCAGAAACAATTAATCTGTTTTCAATTATTAATTAATTTGTTTTTGTGTCCAGATGGACAAGGCGTCGATACTAGGCGACACGATCGAGTACGTGAAGCAGCTAAGGAACCGCATACAAGAGCTCGAGTCGTCGTCGTCGTCG TCACGAGCAGCCGCCCGGGCGCCATCGGCGGCGGCCGCCGGGAGGCGGAGGAAGAGATCCGCCGCCGCCGCCACTGCCACGGCGGCGGAAGGGATGAGCAGCAGCAATGGCCGCAATGGCGGCGAGGCGGCGGAGGTGGTGCAGGTGTCCATCATCGAGAGCGACGCGCTG CTGGAGCTCCGGTGCGGTTGCGGCGGCGGCGGCGGCGGTGTGGTGCTGCTCCGGGTGATGCAGGCGATGCAGGAGCTCCAGCTGGAGGTCACCGCCGTCCAGGCCTCGTGCGCCGGTGGCGAGCTGCTCGCCGAGCTGCGCGCCAAGGTCGTCGTTATGATCCTGATCTGC ATGAAAATGCAGATGCAGATGCAAATGCAGAATTAAGCTTTCATTCTTGCTCCTCTGAATTCTGAATTTATATATTCACCCTTCTTTCGATCTGCTCGTACGTTCGTTTCGCCTAAATTATGTACAAATTAACTGAATCTTTGAACTGAAAATAACTGAATCTTTTTTGTG TGTTTTTGTGTGGGTGAATTGGTTGGCGCAGGTGAAGGGGAGGAGGAGGAGCAGCATCGCTCAGGTGAAGAGGCCCATCCTTTCCCTTTTTTCTTTGCTCATGGGGATTTTTTTCCAACGGTTATATATATAGAAAGTTCACATATATAAATCATGATAATTACCTTTTAG AAAATTCAAACAAAGAATCGATATATATAGTTCTATCTATGCATCATTTTCCTAAATCACGATAATTACCTCAAGCTTGATAAAATGGCATTACATTACTATTATTATTACTAGAGTTTTTTCACTTCCCTGATCTAACCAATTTGCCACGATGGTTAGATATATAGATGG TTCTAATTAATCTAATGTTAATGTACTCCACATATATGATATATGTACATACATATCTAGTTTTAACATTTGCAAATGAATTGCAATCAGAATATATATATGCATGCAGCACAGGTGCTTGATTGCAATTATATGATTATTACTCTCTCACATATTGATATGGTAAATTTG TATATATTTGTGGCATGCATTCATGCACGAAGCTAGATTAATTATTAATTAATTCAGGGTGATATCGATGAGGAGGAGAGGACGAGATGGATGGCCCTCTTCACCTGAGCGATGCTGCTCCTCCTCCTCCCCTTCACCTGCGCCAACCAATTCACCCACACAAAAACACAC AAAAAAGATTCAGTTATTTTCAGTTCAAAGATTCAGTTAATTGTACATATTGGAACGG Figure Full sequence of NepCam YB- Blue color: upstream and downstream; Yellow color: exons; White color: introns Red color: AF2 (forward, reverse) Grey color: AF15 (reverse) Green color: AF3 (forward, reverse) Pink color: AF17 (forward, reverse) Blue color: AF3 (forward, reverse) Purple color: AF18 (reverse) Figure Electrophoresis photo of Rc gene fragments amplified by PCR (DNA ladder is KAPPA universal ladder 1000 bp) Intron-exon structures of Rc gene Rc gene model was indentified co-referred to in the Nipponbare GRASP5.0 database and drawn according to scale There were introns (black lines) and exons (yellow boxes) in the Rc gene model from purple sticky YB rice variety Two up-stream and down-stream of Rc gene were also indexed (blue boxes) In other rice varieties, Rc gene was also constructed the model of intron-exon Rc gene coding for bHLH protein was firstly sequencing by [2] and later by[1], [10] with exons and introns, while studies of [3] have identified exons and introns These findings suggested that the structure of Rc genes in rice varieties are divergent, which may play a important role in the domestication process of these varieties 14-base deletion detection According to study of [3], the Rc allele Rc-s is found in other white pericarp rice genotypes, exhibits a base transversion (i.e., C to A) in the seventh exon instead of the absence of a 14 bp fragment in red pericarp In Rc gene of Nepcam YB, there was 14-base deletion found in fifth intron (from nucleotide position 5061 to 5075) Co 46 linearity analysis of the alleles for Rc showed that NepCam YB are rc genotype which is responsible for red pericarp and was domestication alleles found in Asian cultivated rice [1] Figure Collinear analysis of the fifth intron's partial sequence of Rc Gene of NepCam YB Sequence polymorphism of Rc gene in Nepcam YB variety Figure The SNPs in RC gene ofNepCam YB compared with representative rice accessions Comparison of the sequence of Rc Gene of NepCam YB with reference sequences of the Oryza sativa indica group, japonica group and aus group [3] There were 54 SNPs obtained 20 SNPs, in which 33 SNPs in exons regions and 21 SNPs in introns regions in RC gene of Nepcam YB To understand the effect of mutation and SNPs detected and contribution to pro-anthocyanin accumulation in Nepcam YB, it is needed further analysis on amino acid sequence and protein function analysis Phylogenetic analysis In order to reveal origin and genetic relationship of Nepcam YB with other rice varieties, phylogenetic tree was constructed with representative Rc gene sequences In figure 6, we found that Nepcam YB was closely related to japonica Jefferson cultivar which is originated from USA Genetic distance displayed in tree was not grouping clearly red and white pericarp rice Figure Phylogenetic tree of Nepcam YB with rice varieties based on RC gene full-length sequence CONCLUSION Sequence polymorphism analysis on Rc gene of NepCam YB variety has revealed 14 bp deletion was detected in fifth intron and 54 SNPs of Rc gene in comparing with representative rice cultivars Phylogenetic tree was constructed based on full length sequence for understanding origin and relationship of Nepcam YB with other rice group in the world Rc gene is one of most important domestication gene for studying on rice cultivar evolution and migration Full length sequence of Rc gene in red pericarp Nepcam YB was successfully archived and was preliminary data for understanding domestication of Vietnamese local rice REFERENCES 47 [1] X.-y LI, S Qiang, X.-l SONG, C Kun, Y.-n SUN, Z.-h SHI, et al., "Allele Types of Rc Gene of Weedy Rice from Jiangsu Province, China," Rice Science, vol 21, pp 252-261, 2014 [2] T Furukawa, M Maekawa, T Oki, I Suda, S Iida, H Shimada, et al., "The Rc and Rd genes are involved in proanthocyanidin synthesis in rice pericarp," The Plant Journal, vol 49, pp 91-102, 2007 [3] M T Sweeney, M J Thomson, Y G Cho, Y J Park, S H Williamson, C D Bustamante, et al., "Global dissemination of a single mutation conferring white pericarp in rice," PLoS genetics, vol 3, p e133, 2007 [4] L r Brooks, E Heimsath, G L Loring, and C Brenner, "FHA-RING ubiquitin ligases in cell division cycle control," Cellular and molecular life sciences, vol 65, pp 3458-3466, 2008 [5] D Lee, E Lupotto, and W Powell, "G-string slippage turns white rice red," Genome, vol 52, pp 490-493, 2009 [6] B L Gross, F T Steffen, and K M Olsen, "The molecular basis of white pericarps in African domesticated rice: novel mutations at the Rc gene," Journal of evolutionary biology, vol 23, pp 2747-2753, 2010 [7] A Dellaporta, "Scleral trephination for subchoroidal effusion," Archives of Ophthalmology, vol 101, pp 1917-1919, 1983 [8] P Librado and J Rozas, "DnaSP v5: a software for comprehensive analysis of DNA polymorphism data," Bioinformatics, vol 25, pp 1451-1452, 2009 [9] K Tamura, D Peterson, N Peterson, G Stecher, M Nei, and S Kumar, "MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods," Molecular biology and evolution, vol 28, pp 2731-2739, 2011 [10] L Meng, C Qi, C Wang, S Wang, C Zhou, Y Ren, et al., "Determinant Factors and Regulatory Systems for Anthocyanin Biosynthesis in Rice Apiculi and Stigmas," Rice, vol 14, pp 1-18, 2021 PHÂN TÍCH ĐA HÌNH TRÌNH TỰ GEN Rc CỦA GIỐNG NẾP CẨM YÊN BÁI 1 Bùi Thị Thanh Hiền , Nguyễn Thị Yến Chi , Chu Đức Hà , , Bùi Văn Hùng , Ngô Thị Hồng Tươi , Nguyễn Quốc Trung , Trần Văn Quang Khoa Công nghệ sinh học, Học viện Nông nghiệp Việt Nam Khoa Nông học, Học viện Nông nghiệp Việt Nam Khoa Công nghệ Nông nghiệp, i học Công nghệ, i học u c gia H N i Viện Khoa học kỹ thuật nông nghiệp Bắc Trung B TĨM TẮT Rc gen điều hịa quan trọng quuyết định hàm lượng proanthocyanidin vỏ lụa hạt gạo Các phân tích đột biến gen tính đa hình trình tự gen Rc sở để nghiên cứu xác định trình tiến hóa lúa trồng Mục đích nghiên cứu giải trình tự tồn gen phân tích tính đa hình trình tự gen Rc Nepcam YB, giống lúa cẩm địa phương tỉnh Yên Bái Sử dụng 18 cặp mồi để giải trình tự, tồn chiều dài với vùng upstream downstream gen Rc giống NepCam YB lắp ráp hoàn chỉnh phần mềm BioEdit với chiều dài7500 bp Cấu trúc gen Rc xác định có intron exon Phân tích cho thấy có vị trí đoạn 14 bp intron thứ năm có 54 SNPs gen Rc phát so sánh với giống lúa đại diện cho nhóm lúa màu sắc vỏ lụa đỏ/trắng Cây phát sinh loài xây dựng cho thấy giống Nepcam YB có quan hệ họ hàng gần với giống japonica Jefferson Như vậy, nghiên cứu này, chúng tơi giải trình tự tồn gen Rc giống Nepcam YB có vỏ cám nâu đỏ liệu bước đầu để có nghiên cứu rõ lịch sử hóa lúa Việt Nam Từ khóa: proanthocyanidin, lúa màu, mơ hình gen, SNPs, phát sinh lồi, gen Rc 48 APPENDIX Table sequences of 18 fragments in Nepcam YB Name Rc gene fragment Sequence of Rc gene fragment AGGGCTTTGCTACTAGCTCCTCACTCACTCGCCTTTACTTATCGATCTCGATCATCCACGAGC TAGCTAGCAGTGCTCGCCATGGCCGGCGGCGAGGCGCATGCGGCGCTGCAGGCGGTGGCGCAG AGCCTCCGGTGGACCTACAGCCTCCTCTGGCAGCTCTGCCCCCACCAAGGGTACCTACCCTAC CTACCTACGACACGATGCACAGTGTTCATCCATGGCCGGCCATGGCGGATCGTCGTCGTTGTC GATGATCATCGAAGGAAGCTAGAGGATATGGCTCAATACTTTGATAATATATATACTGATCTC TCCGTACAACAAAAATATAAAAATTCTAGCTAGTATCGAATGAGACATATGCTATGCTAGTAC TACGAATCTAAAAAGATGTACATATTTTGATTCGTATTATTAGGATATATCACGAGTTTTTAT ATTTTGAGACGGATGTAATAATTCTGAATTTAGTTGTGATCGCATGGCATA AF1 RC_1 AF2 RC_2 TCCGATGAGACTATGGCTATGCTAGTACTACGAATCTAAAAAGATGTACATATTTTGATTCGT ATTATTAGGATATATCACGAGTTTTTATATTTTGAGACGGATGTAATAATTCTGAATTTAGTT GTGATCACATGGCATGCAGGAGCTCGCTGGTGTGGGGGGAGGGGCACTACAACGGCGCCGTCA AGACGCGGAAGTCGACGGTGATGCAGCCGCCGCCGGCGGAGGAGGAGGACGACGCCGACCACG CGGCGCGCCACCGGAGCCGGCAGCTGAGGGAGCTCTACGACTGGCTGCAGCAGGCCGGGGAGA ACTCCAGCGGCGGCGTGCAGACGTCGTCGACGACGGCGAGCCGGCGGCCGGGGGCGGCTCTGT CGCCGGAGGACCTGACGGAGACGGAGTGGTTCTTCCTCATGTCGGCATCCTACTCCTTCCCTC CCGGCATCGGGTATATAATAAAAAAGAAAGATATAAATATTAAATGCATGCATGCATCACATT ATACCTCTCTTTTTGTTA AF3 RC_3 GGGGGGGAATCCAAGCGGCGGCGTGCAGACGTCGTCGACGACGGCGAGCCGGCGGCCGGGGGC GGCTCTGTCGCCGGAGGACCTGACGGAGACGGAGTGGTTCTTCCTCATGTCGGCATCCTACTC CTTCCCTCCCGGCATCGGGTATATAATAAAAAATATAGATATAAATATTTAAGCATGCATGCA TAAATTAAACCACACTTCTTGTTACGTGTTCTTGGCAAAATGATGAACAATTACCACTAATTA ATTGGAGCCAGAAACCCTAAAGATTTACCCACCTGGTTAATTAATCGGTGTGTTGATCCACGC ATGCATGCATGCAGAAAATCAAGATCAGGATAGCTCCTTTTCTTTTGCAGGTTAATTAGCTAG ATCTTCACGTATAATTAGCTAGCTAGATTTTAAAATATAATTTATTCAATTTGATTTATGATT TTTATTTTTTATTTCAAATAGATACAACTGTATACAAAATTATATTTTGGTACATACCTCCGA TCCAACTACATCAGAGGTAAAAAAAAAATTAAACCGTTGGAATTGATTAGAACAAGATCGTGC GGCCAGTCGTGAGAGCTCCCGTCACAA AF4 RC_4 GAATTCCAATTCTTCCACACAATATTGGGCTACACAAGCATCGAGAAATGTAAATATTAATGA ATCGCTTGTTTATGAGGAATAACTAGTAGCGTGTTTAAATGGATGATAAGTAGAATTACTTAT ACTTGATCTGTGTGTCAAGATGAAATATGACTATCAAAAGTATATGGAGGGAGTACATGTTAT TAAGTTTTTCTTTAGAGCCTATCAAAAGTATATTTTGACTATCAAAATTATATATGCATCTAG TGTTAATCAGGAAATATAGAGCCTAATAATACGATGTATATATACATCTCTATGCTTTAGAGA ATCAGAAAAGTTAGTTATGATATAATTTGACCGCACGATCTTGTTCTAATCAATTCCAACGGT TTAATTTTTTTTTTACCTCTGATGTAGTTGGATCGGAGGTATGTACCAAAATAAAATTTTGTA TACAGTTGTATCTATTTGAAATAAAAAATAAAAATCATAAATCAAATTGAATAAATTATATTT TAAAATCTAGCTAGCTAATTATACGTGAAGATCTAGCTAATTAACCTGCAAAAGAAAGGGAGC TATCAACAA AF5 RC_5 TAGGAAGGATGTCATTAAGAGGATAGGTTGTTGGATTGAAATATGCCTATCAAAAATAAATTT TTAGATTTGAAAATATGCCTATCAAAAGTAGATGGAGGGAGTATTAATTAATGTGAATTTCCA ATCCTACTGTTGTGATATTAGGCTTTGTACCTTCTTGTCCAGGAGGTATATATATGGCTCTTT TAAGGATGGGAGAAAATATCATCTTTAATACAACTATATATGGCTTTTGTTTGATAAATACAA CTTTTATTTTGTATGAATACAAATATATTGATAAATATCCACCATTATAATCCTAACCCATTA GGATCATATGGTGTATATTTTTTTAACTATTTGTTTTTTATAAATTAATATTAAGAGATCACA ATAAAAATATAGTATTATGAAAGTACTCTTAACAACATATCCAATGATAAAATTATTATTATT ACAAAATATAGTGGTCAAATTGTATAGAATTCAATAGCCTGATTTTATGACGTCAAGTAAATT AAATAAAGAATGAAGGTAGTGCTAAGTGGGATCAAAA AF6 RC_6 TGAGGGTAGTGCTAGAGTGATCAACAATATCTCTCCTAAAATATGTCCTATAAGTTTTACTCC ATAAATCCAAGGGTCAAAAGTTGTTGGGTTATTTTTTTAGATAATAACATACTACCCCTTTTC AAAATGTATGATTCTATTGACTTTTTGCACAACATTTAACCATTTGTCATATTAAAAATTAGT ATAAACATCTAAAAATATAAGTTACAATTATATTTTATTTGATGATAAAACAACTCACAACAA AATAAATAATATTTATATAATCTTTTTGGAATAAAACGAATGATCAAACATTATTCAAAAAGT CAATGGTATAGTACGTTTTGAAATTGATAGACTATGAGAGCAAAATTTTGAGATAACATGGAA AATTATCCTCTTAGACATTGCACTGTGTAATAATTAATAATAATGAATGAAAGGCTAAGACTT TTCTTCCACCTTATATAAGTGGTTGAATATATAGCAATCACATCATTACATGATTTTGTAACC 49 AACCGTCTCTATAGCTCCGAATACCAGTGCAA AF7 RC_7 AAAAATACTAAGTTTGATGTTAAATTAAATTGAAAAAGGGCAGGGCTAGCTAGTGTATTCCTT GTTGTCATTCTTTGCCATGCAAAATACAAAAATGGATTTCATGCCATGCATTACTACTTAATT TGGATGAGAAATGTTCCTACGACAGGTACGTTATGTACACATCGCACTTAGCAATACCGTCTA TATAGCTCCCTAACGTCCAAGAGGGGGATTTGGAGGGGCGCAATTATTCTCTCTCAATCTCTC TCTTAGAAATTAACCGGACATATACTGATTTTTCGCATACGAGTTTAATTTAGCTAAGCGGCA CATCACTTCATCGATGAGAAGTACACCTCGATTTATTATTATACTCTTTAATTATTACGATGT GAAACTAGCACTGTATCGGAGCTATAGAGACGGTTGGTTACAAAATCATGTAATGATGTGATT GCTATATATTCAACCACTTATATAAGGTGGAAGAAAGTTCTTAGCCAAA AF8 RC_8 CTTTTGTCAGAATAGCATGCATTGCATACTCTTTAAAAATAATTAATTAGTGTATTTTACTAG TCTTACAAAAGTATCAAGAGAGACAACTAATTATAGTTGGGAGACACCAAACTTGTTTTTAAT AATGACAATTAAAACCCTACCTCTACATCCAACATAGACGTACATAGTCCGAAGGCGCCAAAT ATTTGTACATTTAGCTACCAGATTTCAGTACGAGTTCTCACATTATAATTTTGATTTTTTTAT TTTTTTTATAAACAATCTGGTACCCTTTTATGTCTGGAAGGAAAAAAAAAATCTAAATTGCAA CATTTTAGTCGGTGAGAATGGTACTCTGTCCTAGCTACTTTCTACACATGAGAGAGAGAGAGA GAGAGAGAGAGCCTTTAATTGCCCTTGCCCATGCATCTTTCTTTGCACACATGTA AF9 RC_9 CGTGCTTTCATTGTCATGAGGAGAGAACTTGTTAAGTTGCACACATGTGTGCTTTGCATGTCT TCAGGTTACCTGGAAGGGCATTTGCAAGGAGAGGCCATGTATGGCTCACTGGAGCAAATGAAG TTGACAGCAAAGTATTCCTAAGAGCAATTCTTGCCAAGGTTCAGCCATCACCTTCTCTTACCT ATTTTTCACTCTGAATGCCAACAGTGCTTTGCACATTGTAGTCTGTTTGCAGACTGCAAATGA TGACCATAATCAGATCAGAAAATAAAATAATATTATATACTTTTTGAGCCAGCTAGCAAGAAT ATGTAACAATAATTCTCCTTTTTTTTTCTTGTTCTTTTCCCTGATGTGGTGCATAACAAATAA CCAAACTGATGAATGGCAAAGTGCTGGTATCCAGGTATTTGCCTCTAAAAGTAGCTACACGTT TACTATGAAATTTTGTGGCTTTTGTTCATCTTTGGATGCAGTGG AF10 RC_10 AAACGGAGAAGGCAGAGTGCTGGTATCCAGGTATTTGCCTCTAAAAGTAGCTACACGTTTACT ATGAAATTTTGTGGCTTTTGTTCATCTTTGGATGCAGTGGCCATTATCTAAAAACTATGAATT TCCAGACTGCAGTTTTTATCTAATTTTGTGACTTTGTACATCAGACAGTTGTGTGCATTCCTG TTGTCGATGGCGTCCTGGAAATTGGAACTACGGAAAAGGTGATTTCGTATATTATCAGCTGAC AATCTAATTATATGGGCCATATAATTAAGTATAAATCAAAATACCTCATAATATATTATAAAG TATCTAATGTGATTATGTGAATATTGGCTATTTCAATGTAATTTGATATATGAAACTGATAAT CCTCTGAAACTCCGTAAGGATCAAACTAATCAAAATGTATATATTTTCAAGGTGGAGGAAGAT ATGGGCCTGATTCAGTATGCAAGGGGCATCTTCATGGATCAACATGGCATCCACATGAACCCT AACCCTCAA AF11 RC_11 TCATCCACATGAAGCCTACCCTCTCACAGCACTCAACATCCAACCCAGTCACCCACTGTACTC ATCAGCATCCAATCCAGGTTCAGATGCAACTAGGTATCACCAGCCAAACAAAGTTTGATTATT CAGATGAGCTCAATGCAGATGAGGAGAATGATGACACAGAAGAAGAGGGCATGTCAGGTTCAG ACACTAACAACACTGACACTGAAAGGAATTCAGGCCAGCTGCAACTTCAAATGCAAGACCAAC TGAACATGGTGAGCAATGACCACCAGACAATACCAAATAATGCAGTTTCCAGTGAGCTAATGC AGTGTGAGATGTCAGAAGTGGTAAGAGATGGCTGCTCAAATAATATTTTAGAGGATGAAATCC AAATGCTGATGGATTGCCAAAACAGTAATTGTCAGTTAAATTTGCAAGGGCCAGATGAGCCTT GTCACTCTTGGCATTTTCTCTGCGAGGAGTTACAAAATGATTACCAGCCAGGTATTACATTTG AGAAGATAATCCTTCAAAAGCACCCCTTGTTCCATCCCTTCTCATTCAA AF12 RC_12 GTAAATGTATCCTATATATATATATATATATATATATATATATATATATATATATATATATAT ATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATA TATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATAT ATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATA TATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATAT ATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATA TATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATAT ATATATAGATATATATATATAGGATATCTATAGA AF13 RC_13 AGAGATGTTCCAGTAGTCACTGCAGTTACAGGGGAGCAGAAACACCTGAATCAAGGGGCGGGA AAGGCGCAAGTGGATGCCATCCAAGGTGATTTCAGTGCCAACCATGTGCTGAAAGAGAGGAGA AGAAGAGAGAAGCTCAATGAGAAGTTCATAATTCTGCGATCTTTGGTACCTTTCATGACAAAG GTAATTAAGTACTCCCTCTATTTCTATAAAGCCGTATTTGACTAGTTATCTTATTTAGAAAGT ATGTGCAAATATGTAAAATATAAGTCATACTTAAAGAACTTTTAATGTTATTAAATAATAAGT CACACCAAAAATAAAACATATATATTTTTAATAAGATAAATGATTAAATGTATATATAAAAAT TAATAGCGTCACATATTTTAAAATAGAGGGGTATTTAAGTACCCACAGGATCATCAAAATTCA GTTATCTTTTCTTAAGCCTCTAACGAACATTGGAAGATCCTCACTAATGGCAGCATGA 50 AF14 RC_14 CCCGAGGGAGCATGAATCTAGGGTTCACTATTTCGGAATGCAAATATGTTTTACCGGGCATCC GATTTTTAAAAAATTCAGAATGAAGAAAATTGAATCTTTTTTATGGATTTGAATAAATCTTGA TAAATTCGAAAAAATTTCCGAACTTTTGGCCAGAAGTGAATCCTACCCGTATCCACCGGTAAT AAACCTAAATTTTTGGGAGTAATGAATTAATGTTATATATAATCCATGAATTATATAGTTCCA AACTACTCCGTAACAAATTTTCAGGAGTAGTGAAATTAATATTATTACAATCTCAGAAAAAAA TGGCAGAAACAATTAATCTGTTTTCAATTATTAATTAATTTGTTTTTGTGTCCAGATGGACAA GGCGTCGATACTAGGCGACACGATCGAGTACGTGAAGCAGCTAAGGAACCGCATACAAGAGCT CGAGTCGTCGTCGTCGTCGTCACGAGCAGCCGCCCGGGCGCCATCGGCGGCGGCCGCCGGGAG GCGGAGGAAGAGATCCGCCGCCGCCGCCACTGCCACGGCGGCGGAAGGGATGAGCAGCAGCAA TGGCCGCAATGGCGGCGAGGCGGCGGAGGTGGTGCAGGTGTCCATCATCGAGAGCGACGCGCT GCTGGAGCTCCGGTGCGGTTGCGGCGGCGGCGGCGGCGGTGTGGTGCTGCTCCGGGTGATGCA GGCGATGCAGGAGCTCCAGCTGGAGGTCACCGCCGTCCAGGCCTCGTGCGCCGGTGGCGAGCT GCTCGCCGAGCTGCGCGCCAAGGTCGTCGTTATGATCCTGATCTGCATGAAAATGCAGATGCA GATGCAAATGCAGAATTAAGCTTTCATTCTTGGCTCCTATGAA AF15 RC_15 CGGAATTTTCGAATTGATCAAGATTTATTCATATCCATGAGAATAAGATTCAATTCTTCGTTC ATTCTGAATTTTTTAAAAGTCGGATGCCCGGTAAAGCATATTTTGCGTACCGAAATAGTGAAC CCTAGATTCATGCTGCCATTAGTGAGGATCTTCCAATGTTCGTTAGAGGCTTAAGAAAAGATA ACTGAATTTTGATGATCCTGTGGGTACTTAAATACCCCTCTATTTTAAAATATGTGACGCTAT TAATTTTTATATATACATTTAATCATTTATCTTATTAAAAATATATATGTTTTATTTTTGGTG TGACTTATTATTTAATAACATTAAAAGTTCTTTAAGTATGACTTATATTTTACATATTTGCAC ATACTTTCTAAATAAGATAACTAGTCAAATACGGCTTTATAGAAATAGAGGGAGTACTTAATT ACCTTTGTCATGAAAGGTACCAAAGATCGCAGAATTATGAACTTCTCATTGAGCTTCTCTCTT CTTCTCCTCTCTTTCAGCACATGGTTGGCACTGAAATCACCTTGGATGGCATCCACTTGCGCC TTTCCCGCCCCTTGATTCAGGTGTTTCTGCTCCCCTGTAACTGCAGTGACTACTGGGAACAAT CATCAGGATGCTCTTGAGCATTCTCTGTGTGGTGCCTGGACTGATCATGGTCTTGTTGTCATC ACTTCCTTCAGGAGTAGTCCATCTGGAGAATGATGATTTCTCTGAAACTGGCAAGTAGTTCTT GATGTTCATCTCTTGGCTGTCGCAA AF16 RC_16 GGTCTCCCTCTATTTCTATAAAGCCGTATTTGACTAGTTATCTTATTTAGAAAGTATGTGCAA ATATGTAAAATATAAGTCATACTTAAAGAACTTTTAATGTTATTAAATAATAAGTCACACCAA AAATAAAACATATATATTTTTAATAAGATAAATGATTAAATGTATATATAAAAATTAATAGCG TCACATATTTTAAAATAGAGGGGTATTTAAGTACCCACAGGATCATCAAAATTCAGTTATCTT TTCTTAAGCCTCTAACGAACATTGGAAGATCCTCACTAATGGCAGCATGAATCTAGGGTTCAC TATTTCGGAATGCAAAATATGTTTTACCGGGCATCCGATTTTTAAAAAATTCAGAATGAAGAA AATTGAATCTTTTTTATGGATTTGAATAAATCTTGATAAATTCGAAAAAATTTCCGAACTTTT GGCCAGAAGTGAATCCTACCCGTATCCACCGGTAATAAACCTAAATTTTTGGGAGTAATGAAT TAATGTTATATATAATCCATGAATTATATAGTTCCAAACTACTCCGTAACAAATTTTCAGGAG TAGTGAAATTAATATTATTACAATCTCAGAAAAAAATGGCAGAAACAATTAATCTGTTTTCAA TTATTAATTAATTTGTTTTTGTGTCCAGATGGACAAGGCGTCGATACTAGGCGACACGATCGA GTACGTGAAGCAGCTAAGGAACCGCATACAAGAGCTCGAGTCGTCGTCCTCGTCGTCACGAGC AGCCGCCAGGGCGCCATCGGCCGAGGCCGCGAGGAAGGAGGAGGACGACATCCGCC AF17 RC_17 ATAGGGAAAGGATGGGCCTCTTCACCTGAGCGATGCTGCTCCTCCTCCTCCCCTTCACCTGCG CCAACCAATTCACCCACACAAAAACACACAAAAAAGATTCAGTTATTTTCAGTTCAAAGATTC AGTTAATTTGTACATAATTTAGGCGAAACGAACGTACGAGCAGATCGAAAGAAGGGTGAATAT ATAAATTCAGAATTCAGAGGAGCAAGAATGAAAGCTTAATTCTGCATTTGCATTTGCATTTGC ATTTTCATGCAGATCAGGATCATAACGACGACCTTGGCGCGCAGCTCGGCGAGCAGCTCGCCA CCGGCGCACGAGGCCTGGACGGCGGTGACCTCCAGCTGGAGCTCCTGCATCGCCTGCATCACC CGGAGCAGCACCACACCGCCGCCGCCGCCGCCGCAACCGCACCGGAGCTCCAGCAGCGCGTCG CTCTCGATGATGGACACCTGCACCACCTCCGCCGCCTCGCCGCCATTGCGGCCATTGCTGCTG CTCATCCCTTCCGCCGCCGTGGCAGTGGCGGCGGCGGCGGATCTCTTCCTCCGCCTCCCGGCG GCCGCCGCCGATGGCGCCCGGGCGGCTGCTCGTGACGACGACGACGACGACTCGAGCTCTTGT ATGCGGTCCTTTAAACCTGCTAA AF18 RC_18 CCGTTCCAATATGTACAATTAACTGAATCTTTGAACTGAAAATAACTGAATCTTTTTTGTGTG TTTTTGTGTGGGTGAATTGGTTGGCGCAGGTGAAGGGGAGGAGGAGGAGCAGCATCGCTCAGG TGAAGAGGGCCATCCATCTCGTCCTCTCCTCCTCATCGATATCACCCTGAATTAATTAATAAT TAATCTAGCTTCGTGCATGAATGCATGCCACAAATATATACAAATTTACCATATCAATATGTG AGAGAGTAATAATCATATAATTGCAATCAAGCACCTGTGCTGCATGCATATATATATTCTGAT TGCAATTCATTTGCAAATGTTAAAACTAGATATGTATGTACATATATCATATATGTGGAGTAC ATTAACATTAGATTAATTAGAACCATCTATATATCTAACCATCGTGGCAAATTGGTTAGATCA GGGAAGTGAAAAAACTCTAGTAATAATAATAGTAATGTAATGCCATTTTATCAAGCTTGAGGT AATTATCGTGATTTAGGAAAATGATGCATAGATAGAACTATATATATCGATTCTTTGTTTGAA TTTTCTAAAAGGTAATTATCATGATTTATATATGTGAACTTTCTATATATATAACCGTTGGAA AAAAATCCCCATGAGCAAAGAAAAAAGGGACGA 51 APPENDIX 52