云实的45S rDNA检测和核型分析
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摘要
该研究对药用植物云实的有丝分裂早中期、中期染色体进行了CPD(PI和DAPI组合)染色和相继的45S rDNA荧光原位杂交分析,并结合染色体测量和CPD带、45S rDNA杂交信号建立了其核型。结果显示:(1)云实的单倍基因组总长度为(30.38±1.58)μm;云实的核型公式为2n=24=14m+10sm (2SAT),染色体相对长度范围为11.22～7.12;核型不对称性参数CI、A1、A2、As K (%)、TF%、AI分别为41.63±6.70、0.27、0.16、58.18、41.82、2.57,核型属于2A类型。(2)云实的二倍体基因组具有9个45S rDNA位点,其中第3、8、9和12号染色体的短臂末端的位点成对存在,第10号染色体仅1个成员的短臂末端具有45S位点,呈现杂合性。该研究首次建立了云实的分子细胞遗传学核型,为该物种的基因组研究提供了基础资料。
In this study, CPD(combined PI and DAPI) staining and sequential fluorescence in situ hybridization(FISH) with 45 S rDNA probe were applied to analyze the prometaphase and metaphase chromosomes of Caesalpinia decapetala(Roth) Alston, a medicinal plant, and the karyotype of this species was established by combining the chromosome measurements and the CPD bands and rDNA FISH signals.(1) The mean haploid karyotype length was(30.38±1.58) μm. The karyotype formula was 2 n=24=14 m+10 sm(2 SAT) with the range of chromosome relative length being 11.22-7.12. The karyotype asymmetry indice, CI, A1, A2, As K(%), TF% and AI were 41.63±6.70, 0.27, 0.16, 58.18, 41.82 and 2.57, respectively, and the asymmetric karyotype belonged to the 2 A type of the Stebbins' category.(2) Four and a half pairs of 45 S rDNA sites were identified in the diploid complement. Among these rDNA sites, four pairs were located at the terminals of the short arms of chromosome pairs 3, 8, 9 and 12, respectively; and one site was located at the terminal of the short arm of one homologue of chromosome pair 10, showing heterozygosity of rDNA site. The investigation established the molecular cytogenetic karyotype of C. decapetala for the first time, providing basic data for the genome study of this species.
In this study, CPD(combined PI and DAPI) staining and sequential fluorescence in situ hybridization(FISH) with 45 S rDNA probe were applied to analyze the prometaphase and metaphase chromosomes of Caesalpinia decapetala(Roth) Alston, a medicinal plant, and the karyotype of this species was established by combining the chromosome measurements and the CPD bands and rDNA FISH signals.(1) The mean haploid karyotype length was(30.38±1.58) μm. The karyotype formula was 2 n=24=14 m+10 sm(2 SAT) with the range of chromosome relative length being 11.22-7.12. The karyotype asymmetry indice, CI, A1, A2, As K(%), TF% and AI were 41.63±6.70, 0.27, 0.16, 58.18, 41.82 and 2.57, respectively, and the asymmetric karyotype belonged to the 2 A type of the Stebbins' category.(2) Four and a half pairs of 45 S rDNA sites were identified in the diploid complement. Among these rDNA sites, four pairs were located at the terminals of the short arms of chromosome pairs 3, 8, 9 and 12, respectively; and one site was located at the terminal of the short arm of one homologue of chromosome pair 10, showing heterozygosity of rDNA site. The investigation established the molecular cytogenetic karyotype of C. decapetala for the first time, providing basic data for the genome study of this species.
引文
[1] 中国科学院中国植物志编辑委员会.中国植物志:第39卷[M].北京:科学出版社,1988:105.
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[9] 佘朝文,蒋向辉.慈姑45S rDNA和端粒序列检测及核型分析[J].植物科学学报,2015,33(4):507-512.SHE C W,JIANG X H.Detection of 45S r DNA and telomere sequences and karyotype analysis of Sagittaria trifolia L.[J].Plant Science Journal,2015,33(4):507-512.
[10] SHE C W,JIANG X H,OU L J,et al.Molecular cytogenetic characterisation and phylogenetic analysis of the seven cultivated Vigna species (Fabaceae)[J].Plant Biology,2015,17(1):268-280.
[11] SHE C W,WEI L,JIANG X H.Molecular cytogenetic characterization and comparison of the two cultivated Canavalia species (Fabaceae)[J].Comparative Cytogenetics,2017,11(4):579-600.
[12] 杨宇华,胡学明,周庆宏.用浓硫酸处理刺云实种子的发芽试验[J].云南林业科技,1996,(4):67-69.YANG Y H,HU X M,ZHOU Q H.Germination test on Caesalpinia spinosa seed treated with oil of vitriol[J].Yunnan Forestry Science and Technology,1996,(4):67-69.
[13] PERRY K L,PALUKAITIS P.Transcription of tomato ribosomal DNA and the organization of the intergenic spacer.Molecular & General Genetics,1990,221(1):102-112.
[14] 李懋学,陈瑞阳.关于植物核型分析的标准化问题[J].武汉植物学研究,1985,3(4):297-302.LI M X,CHEN R Y.A suggestion on the standardization of karyotype analysis in plants[J].Journal of Wuhan Botanical Research,1985,3(4):297-302.
[15] PASZKO B.A critical review and a new proposal of karyotype asymmetry indices[J].Plant Systematics and Evolution,2006,258(1-2):39-48.
[16] STEBBINS G L.Chromosomal evolution in higher plants[M].London:Edward Arnold,1971.
[17] RODRIGUES P S,SOUZA M M,CORRêA R X.Karyomorphology and karyotype asymmetry in the South American Caesalpinia species (Leguminosae and Caesalpinioideae)[J].Genetics & Molecular Research,2014,13(4):8 278-8 293.
[18] 包松莲,李志国,张建云,等.塔拉染色体核型分析[J].江苏农业科学,2012,40(7):176-177.BAO S L,LI Z G,ZHANG J Y,et al.Karyotype analysis of Caesalpinia spinosa[J].Jiangsu Agricultural Sciences,2012,40(7):176-177.
[19] GILL L S,HUSAINI S W H.Cytology of some arborescent Fabaceae of Nigeria[J].Silvae Genetica,1982,31(4):117-122.
[20] KUMARI S,BIR S S.Karyomorphological evolution in Caesalpiniaceae[J].Journal of Cytology and Genetics,1989,24:149-163.
[21] BELTR?O G T,GUERRA M.Citogenética de angiospermas coletadas em Pernambuco.III[J].Ciência e Cultura,1990,42:839-845.
[22] CANGIANO M A,BERNARDELLO G.Karyotype analysis in Argentinean species of Caesalpinia (Legu-minosae)[J].Caryologia,2005,58(3):262-268.
[23] SOUZA M G C,BENKO-ISEPPON A M.Cytogenetics and chromosome banding patterns in Caesalpinioideae and Papilionioideae species of Pará,Amazonas,Brazil[J].Botanical Journal of the Linnean Society,2004,144(2):181-191.
[24] JENA S,SAHOO P,MOHANTY S,et al.Identification of RAPD markers,in situ DNA content and structural chromosomal diversity in some legumes of the mangrove flora of Orissa[J].Genetica,2004,122(3):217-226.
[25] RODRIGUES P S,SOUZA M M,CORRêA R X.Karyomorphology of Caesalpinia species (Caesalpinioideae:Fabaceae) from Caatinga and Mata Atlantica Biomes of Brazil[J].Journal of Plant Studies,2012,1(2):82-91.
[26] BAIRIGANJAN G C,PATNAIK S N.Chromosomal evolution in Fabaceae[J].Cytologia,1989,54(1):51-64.
[27] PEDROSA A,SCHWEIZER D,GUERRA M.Cytological heterozygosity and the hybrid origin of sweet orange [Citrus sinensis (L.) Osbeck][J].Theoretical and Applied Genetics,2000,100(3-4):361-367.
[28] HASTEROK R,WOLNY E,HOSIAWA M,et al.Comparative analysis of rDNA distribution in chromosomes of various species of Brassicaceae[J].Annals of Botany,2006,97(2):205-216.
[2] ZHANG Q,LIU X T,LIANG J Y,et al.Chemical constituents from the stems of Caesalpinia decapetala[J].Chinese Journal of Natural Medicines,2008,6(3):168-172.
[3] GALLEGO M G,GORDON M H,SEGOVIA,F J,et al.Caesalpinia decapetala extracts as inhibitors of lipid oxidation in beef patties[J].Molecules,2015,20(8),13 913-13 926.
[4] GOLDBLATT P.Cytology and the phylogeny of Leguminosae[M]// Polhill R M,Raven P H,eds.Advances in Legume Systematics,Part 2.Royal Botanic Gardens,Kew,1981:427-465.
[5] LEWIS G P.Caesalpinia:A revision of the Poincianella-Erythrostemon Group[M].Royal Botanic Gardens,Kew,1998:233.
[6] SHE C W,LIU J Y,SONG Y C.CPD staining:an effective technique for detection of NORs and other GC-rich chromosomal regions in plants[J].Biotechnic & Histochemistry,2006,81(1):13-21.
[7] 佘朝文,宋运淳.植物荧光原位杂交技术的发展及其在植物基因组分析中的应用[J].武汉植物学研究,2006,24(4):365-376.SHE C W,SONG Y C.Progress of plant FISH technique and its applications in the analysis of plant genome[J].Journal of Wuhan Botanical Research,2006,24(4):365-367.
[8] 佘朝文,张礼华,蒋向辉.花生的荧光显带和rDNA荧光原位杂交核型分析[J].作物学报,2012,38(4):754-759.SHE C W,ZHANG L H,JIANG X H.Karyotype analysis of Arachis hypogaea L.using fluorescence banding and fluorescence in situ hybridization with rDNA probes[J].Acta Agronomica Sinica,2012,38(4):754-759.
[9] 佘朝文,蒋向辉.慈姑45S rDNA和端粒序列检测及核型分析[J].植物科学学报,2015,33(4):507-512.SHE C W,JIANG X H.Detection of 45S r DNA and telomere sequences and karyotype analysis of Sagittaria trifolia L.[J].Plant Science Journal,2015,33(4):507-512.
[10] SHE C W,JIANG X H,OU L J,et al.Molecular cytogenetic characterisation and phylogenetic analysis of the seven cultivated Vigna species (Fabaceae)[J].Plant Biology,2015,17(1):268-280.
[11] SHE C W,WEI L,JIANG X H.Molecular cytogenetic characterization and comparison of the two cultivated Canavalia species (Fabaceae)[J].Comparative Cytogenetics,2017,11(4):579-600.
[12] 杨宇华,胡学明,周庆宏.用浓硫酸处理刺云实种子的发芽试验[J].云南林业科技,1996,(4):67-69.YANG Y H,HU X M,ZHOU Q H.Germination test on Caesalpinia spinosa seed treated with oil of vitriol[J].Yunnan Forestry Science and Technology,1996,(4):67-69.
[13] PERRY K L,PALUKAITIS P.Transcription of tomato ribosomal DNA and the organization of the intergenic spacer.Molecular & General Genetics,1990,221(1):102-112.
[14] 李懋学,陈瑞阳.关于植物核型分析的标准化问题[J].武汉植物学研究,1985,3(4):297-302.LI M X,CHEN R Y.A suggestion on the standardization of karyotype analysis in plants[J].Journal of Wuhan Botanical Research,1985,3(4):297-302.
[15] PASZKO B.A critical review and a new proposal of karyotype asymmetry indices[J].Plant Systematics and Evolution,2006,258(1-2):39-48.
[16] STEBBINS G L.Chromosomal evolution in higher plants[M].London:Edward Arnold,1971.
[17] RODRIGUES P S,SOUZA M M,CORRêA R X.Karyomorphology and karyotype asymmetry in the South American Caesalpinia species (Leguminosae and Caesalpinioideae)[J].Genetics & Molecular Research,2014,13(4):8 278-8 293.
[18] 包松莲,李志国,张建云,等.塔拉染色体核型分析[J].江苏农业科学,2012,40(7):176-177.BAO S L,LI Z G,ZHANG J Y,et al.Karyotype analysis of Caesalpinia spinosa[J].Jiangsu Agricultural Sciences,2012,40(7):176-177.
[19] GILL L S,HUSAINI S W H.Cytology of some arborescent Fabaceae of Nigeria[J].Silvae Genetica,1982,31(4):117-122.
[20] KUMARI S,BIR S S.Karyomorphological evolution in Caesalpiniaceae[J].Journal of Cytology and Genetics,1989,24:149-163.
[21] BELTR?O G T,GUERRA M.Citogenética de angiospermas coletadas em Pernambuco.III[J].Ciência e Cultura,1990,42:839-845.
[22] CANGIANO M A,BERNARDELLO G.Karyotype analysis in Argentinean species of Caesalpinia (Legu-minosae)[J].Caryologia,2005,58(3):262-268.
[23] SOUZA M G C,BENKO-ISEPPON A M.Cytogenetics and chromosome banding patterns in Caesalpinioideae and Papilionioideae species of Pará,Amazonas,Brazil[J].Botanical Journal of the Linnean Society,2004,144(2):181-191.
[24] JENA S,SAHOO P,MOHANTY S,et al.Identification of RAPD markers,in situ DNA content and structural chromosomal diversity in some legumes of the mangrove flora of Orissa[J].Genetica,2004,122(3):217-226.
[25] RODRIGUES P S,SOUZA M M,CORRêA R X.Karyomorphology of Caesalpinia species (Caesalpinioideae:Fabaceae) from Caatinga and Mata Atlantica Biomes of Brazil[J].Journal of Plant Studies,2012,1(2):82-91.
[26] BAIRIGANJAN G C,PATNAIK S N.Chromosomal evolution in Fabaceae[J].Cytologia,1989,54(1):51-64.
[27] PEDROSA A,SCHWEIZER D,GUERRA M.Cytological heterozygosity and the hybrid origin of sweet orange [Citrus sinensis (L.) Osbeck][J].Theoretical and Applied Genetics,2000,100(3-4):361-367.
[28] HASTEROK R,WOLNY E,HOSIAWA M,et al.Comparative analysis of rDNA distribution in chromosomes of various species of Brassicaceae[J].Annals of Botany,2006,97(2):205-216.