野生水稻物種B型基因組特有的RAPD之研究

A RAPD Specific to Rice Wild Species with B Genome

方紹安(Shao-An Fang)

指導教授 ： 鍾美珠

共同指導教授 ： 謝兆樞

國立臺灣大學/生物資源暨農學院/農藝學研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.02876

摘要

利用分子標記可以分辨物種基因組之組成及檢測種間雜交後代基因組中異源物種基因組滲入程度等。本論文以12種水稻為參試材料，包括栽培種和野生種，分別屬於九個物種共涵蓋六個基因組型：AA，BB，BBCC，CC，CCDD，EE。栽培種水稻參試品種中，日本晴（O. sativa, Japonica, cv. Nipponbare）為稻屬研究中經常使用的稉稻品種之一，而台農67號（O. sativa, Japonica, cv. TNG 67）為目前在台灣地區普遍栽種，為常用之育種親本和研究材料稉稻品種。由O. punctata Kotschy ex steud（W1593）分離得到兩個逢機引子擴增DNA片段（randomly amplified polymorphic DNAs，RAPD），分別命名為Opun225和Opun246。Opun225片段長度為874 bp，在基因組內為低重複數的片段，對於稻屬BB基因組型二元體物種有高度的專一性，可作為RAPD分子標誌，有效的分辨不同基因組型物種之不同族群。Opun246片段長度約為2084 bp，在O. punctata (W1593)單套基因組內約有6,320個重複，屬於一種高重複數的片段。對於稻屬B基因組型物種有高度的基因組型專一性，能有效的分辨不同基因組型物種。將Opun246序列與生物核苷序列收集資料庫中的已知序列比對，可找到少數的同為稻屬的同源序列，且大多均在其5’端約有1200 bp的同源性較高，而在3’端核苷序列的同源性較低。此外，Opun246片段75~1266 bp之間的序列與一段被註解為類似retroelements的序列之間有75％一致性。南方墨漬雜交的結果顯示，Opun246為高度重複的序列且散布在BB基因型物種之全基因組內。以螢光原位雜交（FISH）的染色體定位結果則指出，Opun246散布在BB基因組物種的染色體之異染色質區域。此外，Opun246的訊號有集中在O. punctata（BB）染色體靠近末端或中節區域的現象。然而，在四元體O. punctata（BBCC）和O. minuta（BBCC）中，Opun246只會出現在BB基因組的染色體上，且有集中在染色體末端位置的趨勢。因此認為Opun246多數分佈在靠近中節或末端附近的異染色質區域，且為一個BB基因組專一性的分子標記。

關鍵字

螢光原位雜交技術 ； 野生稻Oryza punctata ； 重複序列 ； 物種專一性逢機引子多型性標誌

並列摘要

Molecular markers are useful in discriminating specific constituents of genome and monitoring genomic introgression of interspecific hybrids. In this study, we use 12 rice materials, including nine Oryza species, both cultivated and wild species of rice, which belong to six genome types. We isolate and characterize two genome specific RAPDs, named Opun225 and Opun246, from Oryza punctata Kotschy ex Steud (W1593), an African native wild rice. The Opun225 sequence is 874 base pair in length and a low-copy sequence present in genome and is demonstrated as a repetitive sequence specific to a diploid Oryza species with B genome. On the other hand, the Opun246 is 2084 bp in length and estimated at 6320 copies in O. punctata (W1593) haploid genome. Sequence comparison analysis by BLAST found a few homologues of Opun246 in EMBL-GenBank database. Among those homologues, a relatively high similarity was found in the position ~1200 nucleotides regions at the 5’ ends, but a low similarity in the rest of the nucleotides at the 3’ends. In addition, the Opun246 sequence at position 75~1266 nucleotides with 75% identity to a putative retroelements. The results of Southern hybridization indicated that the repetitive Opun246 sequences dispersed throughout the entire genome of specues with B genome. The results of fluorescent in situ hybridization (FISH) indicated that the repetitive Opun246 sequence dispersed to the heterochromatin throughout chromosomes in the whole genome. Furthermore, obvious Opun246 sequences were detected near the centrimeric and terminal regions at chromosomes of O. punctata (W15777). The Opun246 sequence were detected near the terminal regions on part of chromosomes, which might be restricted to B genome originated chromosomes in O. punctata (W1564) and O. minuta (W0045) with BBCC.

並列關鍵字

Oryza punctata ； repetitive sequence ； species-specific RAPD ； fluorescent in situ hybridization (FISH)

參考文獻

Wu J-Y, Wu H-K, Chung M-C. (2002) Co-dominant RAPD markers closely linked with two morphological genes in rice (Oryza sativa L.). Bot Bull Acad Sin 43:171-180.

Adam-Blondon A-F, Sevignac M, Bannerot H, Dron M. (1994) SCAR, RAPD and RFLP markers linked to a dominant gene (Are) conferring resistance to anthracnose in common bean. Theor Appl Genet 88: 865-870.

Aggarwal R-K, Brar D-S, Khush G-S. (1997) Two new genomes in the Oryza complex identified on the basis of molecular divergence analysis using total genomic DNA hybridization. Mol Gen Genet 254:1-12.

Alfenito M-R and Birchler J-A. (1993) Molecular characterization of a maize B chromosome centric sequence. Genetics 135:589-597.

Appels R, Reddy P, MacIntyre C-L, Moran L-B. (1989) The molecular-cytogenetic analysis of grasses and its application to studying relationships among species of the Triticeae. Genome 31: 122-133.