IDENTIFIKASI KROMOSOM HOMOLOG MELALUI DETEKSI NUCLEOLUS ORGANIZER REGIONS DENGAN PEWARNAAN AgNO3 PADA TANAMAN BAWANG MERAH

Andin Puspita, Agus Budi Setiawan, Aziz Purwantoro, Endang Sulistyaningsih

Abstract


Generally, the standard procedure for karyotype analysis of shallot is sorted by chromosome sizes. Therefore, the identification of homologous chromosomes is difficult without using a specific probe. Nucleolus Organizing Regions (NORs) can be used as a probe for precise identification of homologous chromosomes. However, the use of NORs for plant karyotyping in Indonesia is poorly investigated. In this study, shallot chromosomes were prepared using modified Carnoy’s solution II, fixed in Carnoy’s solution, and stained by using aceto-carmine and AgNO3 for detecting NORs. Chromosome images were analyzed by CHIAS IV. One locus NOR bearing chromosome pair was detected at metaphase and interphase, and it was located at short arms of subtelomeric chromosome number 6. NORs can be used as a probe for precise identification of homologous chromosomes in shallot. Therefore, this technique has the potential to be applied on species closely related to shallot and on other plant species.

Keywords: AgNO3, chromosome condensation, NORs, shallot chromosome, shallot karyotype

 

ABSTRAK

Prosedur kariotipe untuk bawang merah umumnya masih disusun berdasarkan ukuran kromosom, sehingga diperlukan suatu penanda yang dapat mengidentifikasi kromosom homolog secara presisi. Identifikasi kromosom homolog secara presisi menggunakan suatu penanda, khususnya deteksi Nucleolus Organizing Regions (NORs), yang di Indonesia masih jarang dilakukan. Penelitian ini bertujuan untuk membuat kariotipe dan mengidentifikasi kromosom homolog bawang merah melalui deteksi NORs menggunakan metode pewarnaan AgNO3. Proses fiksasi akar dilakukan dengan menggunakan modifikasi larutan Carnoy II, lalu difiksasi dengan larutan Carnoy, dan kromosom diwarnai dengan aceto-carmine dan larutan AgNO3 untuk mendeteksi NORs. Selanjutnya, citra kromosom dianalisis menggunakan CHIAS IV. Hasil penelitian menunjukkan bahwa terdapat sepasang NORs yang terdeteksi pada fase metafase dan interfase yang  terletak pada bagian lengan pendek di kromosom subtelosentrik nomor 6. Hasil dari penelitian ini dapat dijadikan sebagai dasar di bidang sitogenetika bawang merah untuk mengidentifikasi kromosom homolog secara presisi menggunakan penanda NOR. Oleh karenanya, teknik ini dapat diaplikasikan pada spesies yang berdekatan dengan bawang merah dan komoditas tanaman lainnya.

Kata Kunci: AgNO3, kariotipe bawang, kondensasi kromosom, kromosom bawang, NORs


Keywords


chromosome condensation; ribosomal DNA; shallot chromosome; shallot karyotype; 45S rDNA

Full Text:

PDF

References


Anggarwulan E, Etikawati N, Setyawan AD (1999) Karyotipe kromosom pada tanaman bawang budidaya (Genus Allium; Familia Amaryllidaceae). BioSmart 1:13–19

APG (2016) An update of the Angiosperm phylogeny group classification for the orders and families of flowering plants: APG IV. The angiosperm phylogeny group. Bot J Linn Soc 181:1–20. doi: 10.1111/boj.12385

Arabbeigi M, Arzani A, Saeidi G (2011) Study of karyotype and nucleolar organizer regions (NORs) in wild, synthetic and cultivated wheats. Emir J Food Agric 23:196–203. doi: 10.9755/ejfa.v23i2.6457

Bersaglieri C, Santoro R (2019) Genome organization in and around the nucleolus. Cells 8:E579. doi: 10.3390/cells8060579

Budylin MV, Kan LY, Romanov VS, Khrustaleva LI (2014) GISH study of advanced generation of the interspecific hybrids between Allium cepa L. and Allium fistulosum L. with relative resistance to downy mildew. Russ J Genet 50:387–394. doi: 10.1134/S1022795414040036

Firbas P, Amon T (2014) Chromosome damage studies in the onion plant Allium cepa L. Caryologia 67:25–35. doi: 10.1080/00087114.2014.891696

Furukawa K, Sugiyama S, Ohta T, Ohmido N (2017) Chromosome analysis of tea plant (Camellia sinensis) and ornamental camellia (Camellia japonica). Chromosome Sci 20:9–15. doi: 10.11352/scr.20.9

Goodpasture C, Bloom SE (1975) Visualization of nucleolar organizer regions in mammalian chromosomes using silver staining. Chromosoma 53:37–50. doi: 10.1007/BF00329389

Greco A (2009) Involvement of the nucleolus in replication of human viruses. Rev Med Virol 19:201–214. doi: 10.1002/rmv.614

Herden T, Hanelt P, Friesen N (2016) Phylogeny of Allium L. subgenus Anguinum (G. Don. ex W.D.J. Koch) N. Friesen (Amaryllidaceae). Mol Phylogenet Evol 95:79–93. doi: 10.1016/j.ympev.2015.11.004

Herlina L, Reflinur, Sobir, Maharijaya A, Wiyono S (2019) The genetic diversity and population structure of shallots (Allium cepa var. aggregatum) in Indonesia based on R gene-derived markers. Biodiversitas 20:696–703. doi: 10.13057/biodiv/d200312

Huang M, Li H, Zhang L, Gao F, Wang P, Hu Y, Yan S, Zhao L, Zhang Q, Tan J, Liu X, He S, Li L (2012) Plant 45S rDNA clusters are fragile sites and their instability is associated with epigenetic alterations. PLoS One 7:e35139. doi: 10.1371/journal.pone.0035139

Jara-Seguel P, Palma-Rojas C, Contreras J, von Brand E (2012) Chromosome localisation of nucleolar organizer region in Rhodophiala bagnoldii (Herb.) Traub (Asparagales: Amaryllidaceae) determined by silver nitrate staining. Gayana Bot 69:201–203. doi: 10.4067/s0717-66432012000100023

Kalinina NO, Makarova S, Makhotenko A, Love AJ, Taliansky M (2018) The multiple functions of the nucleolus in plant development, disease and stress responses. Front Plant Sci 9:132. doi: 10.3389/fpls.2018.00132

Kato S, Ohmido N, Hara M, Kataoka R, Fukui K (2009) Image analysis of small plant chromosomes by using an improved system, CHIAS IV. Chromosome Sci 12:43–50

Khrustaleva L, Mardini M, Kudryavtseva N, Alizhanova R, Romanov D, Sokolov P, Monakhos G (2019) The power of genomic in situ hybridization (GISH) in interspecific breeding of bulb onion (Allium cepa L.) resistant to downy mildew (Peronospora destructor [Berk.] Casp.). Plants 8:E36. doi: 10.3390/plants8020036

Kim S, Lee DK, Rayburn AL (2015) Analysis of active nucleolus organizing regions in polyploid prairie cordgrass (Spartina pectinata Link) by silver staining. Cytologia  80:249–258. doi: 10.1508/cytologia.80.249

Kirov I, Divashuk M, Van Laere K, Soloviev A, Khrustaleva L (2014) An easy “SteamDrop” method for high quality plant chromosome preparation. Mol Cytogenet 7:21. doi: 10.1186/1755-8166-7-21

Kudryavtseva N, Havey MJ, Black L, Hanson P, Sokolov P, Odintsov S, Divashuk M, Khrustaleva L (2019) Cytological evaluations of advanced generations of interspecific hybrids between Allium cepa and Allium fistulosum showing resistance to Stemphylium vesicarium. Genes (Basel) 10:E195. doi: 10.3390/genes10030195

Lafontaine DLJ (2015) Noncoding RNAs in eukaryotic ribosome biogenesis and function. Nat Struct Mol Biol 22:11–19. doi: 10.1038/nsmb.2939

Levan A, Fredga K, Sandberg AA (1964) Nomenclature for centromeric position on chromosomes. Hereditas 52:201–220. doi: 10.1111/j.1601-5223.1964.tb01953.x

Major N, Goreta Ban S, Urlić B, Ban D, Dumičić G, Perković J (2018) Morphological and biochemical diversity of shallot landraces preserved along the Croatian coast. Front Plant Sci 9:1749. doi: 10.3389/fpls.2018.01749

Mancia FH, Sohn SH, Ahn YK, Kim DS, Kim JS, Kwon YS, Kim CW, Lee TH, Hwang YJ (2015) Distribution of various types of repetitive DNAs in Allium cepa L. based on dual color FISH. Hortic Environ Biotechnol 56:793–799. doi: 10.1007/s13580-015-1100-3

Maragheh FP, Janus D, Senderowicz M, Haliloglu K, Kolano B (2019) Karyotype analysis of eight cultivated Allium species. J Appl Genet 60:1–11. doi: 10.1007/s13353-018-0474-1

Maryanska-Nadachowska A, Kuznetsova VG, Golub NV, Anokhin BA (2018) Detection of telomeric sequences and ribosomal RNA genes in holokinetic chromosomes of five jumping plant-lice species: First data on the superfamily Psylloidea (Hemiptera: Sternorrhyncha). Eur J Entomol 115:632–640. doi: 10.14411/eje.2018.061

McStay B (2016) Nucleolar organizer regions: genomic ‘dark matter’ requiring illumination. Genes Dev 30:1598–1610. doi: 10.1101/gad.283838.116

Muakrong N, Kikuchi S, Fukuhara S, Tanya P, Srinives P (2018) Two jatropha karyotypes constructed from meiotic pachytene chromosomes: Pericentric distribution of heterochromatin and variation in repetitive DNAs. PLoS One 13:e0208549. doi: 10.1371/journal.pone.0208549

Mukherjee A, Roy S (2012) Karyotype analysis of five species of Allium. Ind J Fundam Appl Life Sci 2:374–383

Nagaki K, Yamamoto M, Yamaji N, Mukai Y, Murata M (2012) Chromosome dynamics visualized with an anti-centromeric histone H3 antibody in Allium. PLoS One 7:e51315. doi: 10.1371/journal.pone.0051315

O’Connor C (2008) Karyotyping for chromosomal abnormalities. Nat Educ 1:27

Ohmido N, Iwata A, Kato S, Wako T, Fukui K (2018) Development of a quantitative pachytene chromosome map and its unification with somatic chromosome and linkage maps of rice (Oryza sativa L.). PLoS One 13:e0195710. doi: 10.1371/journal.pone.0195710

Ohmido N, Shimoura A, Kato S, Isobe S, Tabata S (2013) Kudzu (Pueraria lobata Ohwi) karyotyping using FISH and Chromosome Image Analysis System IV. Chromosome Sci 16:17–21. doi: 10.11352/scr.16.17

Pederson T (2011) The nucleolus. Cold Spring Harb Perspect Biol 3:a000638. doi: 10.1101/cshperspect.a000638

Rosato M, Kovařík A, Garilleti R, Rosselló JA (2016) Conserved organisation of 45S rDNA sites and rDNA gene copy number among major clades of early land plants. PLoS One 11:e0162544. doi: 10.1371/journal.pone.0162544

Sánchez-G Y, Raymúndez MB, Imery J, Acosta MC, Moscone E (2018) Characterization of eight species of Aloe (Asphodelaceae) from the nucleolar organizing region. Rodriguesia 69:363–372. doi: 10.1590/2175-7860201869208

Scaldaferro MA, Moscone EA (2019) Cytology and DNA content variation of capsicum genomes. In: Ramchiary N, Kole C (eds) The capsicum genome. Compendium of plant genomes. Springer, Cham, pp 57–84. doi: 10.1007/978-3-319-97217-6_4

Schubert I (1984) Mobile nucleolus organizing regions (NORs) in Allium (Liliaceae s. lat.)? - Inferences from the specifity of silver staining. Plant Syst Evol 144:291–305. doi: 10.1007/BF00984139

Schubert I, Wobus U (1985) In situ hybridization confirms jumping nucleolus organizing regions in Allium. Chromosoma 92:143–148. doi: 10.1007/BF00328466

Setiawan AB, Teo CH, Kikuchi S, Sassa H, Kato K, Koba T (2018a) Cytogenetic variation among Cucumis accessions revealed by fluorescence in situ hybridization using ribosomal RNAs genes as the probes. Chromosom Sci 21:67–73.  doi: 10.11352/scr.21.67

Setiawan AB, Teo CH, Kikuchi S, Sassa H, Koba T (2018b) An improved method for inducing prometaphase chromosomes in plants. Mol Cytogenet 11:32. doi: 10.1186/s13039-018-0380-6

Setyowati M, Sulistyaningsih E, Purwantoro A (2013) Induksi poliploidi dengan kolkisina pada kultur meristem batang bawang wakegi (Allium x wakegi Araki). J Ilmu Pertan 16:58–76. doi: 10.22146/ipas.2526

Shaw P (2013) The plant nucleolus. In: Leitch IJ, Greilhuber J, Dolezel J, Wendel JF (eds) Plant genome diversity volume 2. Springer, Vienna, pp 65–76. doi: 10.1007/978-3-7091-1160-4_5

Shaw P, Brown J (2012) Nucleoli: Composition, function, and dynamics. Plant Physiol 158:44–51. doi: 10.1104/pp.111.188052

Shigyo M, Khar A, Abdelrahman M (2018) The Allium Genomes. Springer, Cham. doi: 10.1007/978-3-319-95825-5_3

Stack SM, Comings DE (1979) The chromosomes and DNA of Allium cepa. Chromosoma 70:161–181. doi: 10.1007/BF00288404

Stępiński D (2014) Functional ultrastructure of the plant nucleolus. Protoplasma 251:1285–1306. doi: 10.1007/s00709-014-0648-6

Sulistyaningsih E, Yamashita K, Tashiro Y (2002) Genetic characteristics of the Indonesian white shallot. J Jpn Soc Hortic Sci 71:504–508. doi: 10.2503/jjshs.71.504

Taliansky ME, Brown JW, Rajamäki ML, Valkonen JPT, Kalinina NO (2010) Involvement of the plant nucleolus in virus and viroid infections: Parallels with animal pathosystems. Adv Virus Res 77:119–158. doi: 10.1016/B978-0-12-385034-8.00005-3

Weis BL, Kovacevic J, Missbach S, Schleiff E (2015) Plant-specific features of ribosome biogenesis. Trends Plant Sci 20:729–740. doi: 10.1016/j.tplants.2015.07.003

Wibowo A, Setiawan AB, Purwantoro A, Kikuchi S, Koba T (2018) Cytological variation of rRNA genes and subtelomeric repeat sequences in Indonesian and Japanese cucumber accessions. Chromosome Sci 21:81–87. doi: 10.11352/scr.21.81

Xie DF, Yu HX, Price M, Xie C, Deng YQ, Chen JP, Yu Y, Zhou SD, He XJ (2019) Phylogeny of Chinese Allium species in section Daghestanica and adaptive evolution of Allium (Amaryllidaceae, Allioideae) species revealed by the chloroplast complete genome. Front Plant Sci 10:460. doi: 10.3389/fpls.2019.00460





DOI: https://doi.org/10.29122/jbbi.v7i1.3693

Refbacks

  • There are currently no refbacks.


Copyright (c) 2020 Jurnal Bioteknologi & Biosains Indonesia (JBBI)

Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.

© Copyright: BY-NC-SA