石蒜及雙核型雜種45S rDNA基因與近同源重組變異研究

Abstract

根據染色體組成，石蒜 (Lycoris spp.)可區分為MT、A與MT-A三核型群種。本研究利用螢光原位雜交法標記45S 核醣體RNA基因(rDNA)在原生種、雜交種以及試交代之染色體上的分佈，以瞭解45S rDNA基因座在種間之分歧及種內之變異，並評估其是否可作為辨識染色體之依據。 MT核型群種，如L. traubii 2n=12 (10M+2T)、金花石蒜 2n=14 (8M+6T)和中國石蒜2n=16 (6M+10T)等，45S rDNA基因均位在其T型染色體短臂末端 (Tp)。 因此，MT核型種之rDNA基因座的數目會隨著該基因組內所包含的T型染色體之數目而定。 金花石蒜有三對T型同源染色體，Tp臂均具有rDNA基因座與DAPI特殊染色區域，其中第三對T染色體呈現較大的DAPI特殊染色區域，可與另兩對區別。 A核型群種2n = 22 ( 22A)，例如換錦石蒜、東引紅花與馬祖捲葉等，各有兩個45S rDNA基因座位在染色體短臂末端；紅花石蒜、玫瑰石蒜與紅藍石蒜等，則各有三個45S rDNA基因座位於染色體短臂末端；矮小石蒜為唯一具四個45S rDNA基因座，也位在短臂上。 紅花石蒜出現種內變異，其中有三選系分別觀察到二、三、四個45S rDNA基因座。 MT-A雙核型種與試交代之45S rDNA基因座位點與數目則皆符合其兩親本之累加，即所有T型染色體短臂末端均有45S rDNA標記。 較特殊者如L. albiflora 與L. houdyshelii在M型染色體之著絲點附近有較微弱的45S rDNA-FISH訊號， 顯示A核型群內45S rDNA基因座位置的歧異度高，且其中T型染色體末端為新生之45S核醣體rRNA。 金花石蒜與紅花石蒜之雜交種的核型為2n = 18 ( 4M+3T+11T)，可產生23.95 % 有機能雄配子，推測由於四對M-2A與三對T-A染色體異型配對且平衡分離，能產生雄配子染色體數為n = 7-11且總臂數維持11，這些遺傳平衡且多樣的雄配子稱為回收雄配子。 對於這些回收雄配子，可藉由核型分析，以MT與A基因組DNA為探針進行基因組原位雜交(genomic in situ hybridization, GISH)分析或流式細胞儀分析基因組大小，以探討MT與A基因組間近同源重組之變異特性。 由核型分析得知其中四條M型染色體短臂(p)與長臂(q)的平均長度分別為16.46 ± 1.36 µm與18.14 ± 1.78 µm，三條T型染色體p與q臂平均長度分別為1.03±0.11 µm與16.06±1.26 µm，而11條A型染色體p臂與q臂平均長度分別為1.80±1.39 µm與11.63±1.30 µm。 4M+3T與11A兩染色體組基因組DNA含量分別為33.70 pg與26.18 pg，前者染色體之總長度比後者多出12.44 %。 以基因組原位雜交法可以檢視雄配子內兩親基因組之組成，根據由19個遺傳平衡的雄配子所呈現的解析度良好之GISH訊號得知，染色體數為n = 7-11，平均有7.16臂發生近同源基因重組，其中約4.47臂發生單點置換，3.37臂發生雙點置換。 發生置換的區域約佔回收雄配子基因組之23.52 %，顯示MT與A兩基因組之間可以經由近同源重組而發生基因交流。 M、T與A型染色體重組置換位點多集中於中段至末端間，而近著絲點基端為非置換區域，其中T型染色體組中非置換區域達37 %，研判MT染色體長度之增加，主要發生於染色體近中節區域，因T染色體增長區域與A為非同源性或欠缺同源性以致於無法配對或發生置換。 M型染色體發生單點與雙點置換分別造成其臂長縮短16.26 %與15.05 %。 T型染色體長臂發生單點置換，使染色體長度短縮16.07 %，而雙點置換之影響較小僅短縮4.18 %。 A型發生單點與雙點置換反而會造成長度增加19.30 %。 以流式細胞儀分析基因組DNA含量，得知試交代之基因組DNA含量多於兩親本所供獻基因組量之總合，超出含量約在2.1-3.9 %之間，顯示金花與紅花石蒜之MT-A雙核型雜交種的染色體中，M-2A以及T-A染色體彼此可能呈現鬆弛配對，並可能發生不對稱置換，導致基因組含量與染色體形態多型性。 本研究共檢視30個體，獲得四個未減數雄配子，其中三個2n配子為第一次減數分裂復合所得，以GISH檢視皆未發現基因重組片段；另一2n配子由第二次減數分裂復合產生，在姐妹染色體上發現成對的基因置換位點，為四股置換之結果。雜交種石蒜能逢機產生未減數配子，經由不同的發生途徑，得以開創出多樣異質或同質結合之新核型。

Lycoris spp. were classified into MT, A and MT-A karyotype groups based on their chromosome complements. 45S ribosomal RNA genes (rDNAs) were detected at telocentrics, acrocentrics and metacentrics by using rDNA as probe in fluorescene in situ hybridization (rDNA-FISH). In the MT karyotype group, such as L. traubii (2n = 12 = 10M + 2T), L. aurea (2n = 14 = 8M + 6T) and L. chinensis (2n=16=10M+6T), 45S rDNA loci were mapped in terminal region of all telocentrics by rDNA-FISH. In the A karyotype groups, the localizations of rDNA loci were polymorphic. In L. sprengeri, L. radiata (MD) and LSM accession, the rDNA loci were mapped on terminal end of the short arm of two acrocentrics. In L. radiata, L. rosea and L. haywardii, three rDNA loci were mapped on the terminal end of p-arm of telocentrics. In L. radiata var. pumila, four rDNA loci were mapped on terminal end of acrocentrics. Two to four rDNA loci were detected in three derivational taxa of L. radiata. The number and positions of rDNA loci in interspecific hybrid taxa and testprogenies were consistent with of the combination of both parents. In natural MT-A karyotype group, rDNA loci were detected on all telocentrics and on several metacentrics near their centromeres as weak signals. These results indicated that the terminal region of telocentrics might be neo–generated and different from any segment of metacentric and acrocentric chromosomes. The interspecific hybrid between two divergent species L. aurea and L. radiata a dikaryotype hybrid, has a chromosome complement as 2n = 18 = (4M+3T)+(11A) and can generate functional male gametes in about 23.95 %. Such functional male gametes were generated due to the formation of heteromorphic bivalents by four M-2A chromosomes and by three T-A chromosomes. The chromosome numbers of these gametes are various from n = 7 - 11, but totally were 11 chromosomal arms. This study applied chromosomal karyotyping, GISH analysis, and flow cytometry to study homoeologous recombination in a dikaryotype hybrid. The karyotyping analysis described the morphologies of chromosome complements. The average lengths of the short arm (p-arm) of four metacentrics and the long arm (q-arm) of metacentrics were 16.46 ± 1.36 µm and 18.14 ± 1.78 µm, respectively. The average lengths of p-arm or q-arm of three telocentrics were about 1.03 ± 0.11 µm or 16.06 ± 1.26 µm. The average lengths of p-arms or q-arms of eleven acrocentrics were 1.80 ± 1.39 µm or 11.63 ± 1.30 µm. The total length of chromosome complement of 4M + 3T were 12.44 % longer than that of 11A, and their DNA content were estimated as 33.70 pg/C and 26.18 pg/C, respectively, by flow cytometry. By using genomic in situ hybridization (GISH), the chromosomal organization of functional gametes could be identifed. Nineteen male gametes were observed with distinct GISH signals and have 7.16 arms in average with recombinant segments. Those recombinant segments were resulted from two types of crossover, single crossover (SCO) and double crossover (DCO). Besides, the total length of those recombination segments was about 23.52 % of chromosome complement in male gamete which were resulted from SCO occurred at 4.47 arms in average and DCO occurred at 3.37 arms in average. These results suggested that gene flow between MT and A genomes were consequent on homoeologous recombinations. Most of interchanged segments were detected between interstitial and distal regions and less were detected in the proximal regions. In telocentrics, the non-crossover region, proximal region, was as much as 37 % in length. Our observations suggested that chromosome increasing in length in MT genome was a consequent of extension in the proximal region, which might result in the proximal region becoming more divergent and less, or non-homeologous pair. Owing to SCO and DCO, the length of metacentrics would be shortened about 16.26 % and 15.05 %, q-arm length of telocentrics would be shortened about 16.07 % and 4.18 %. However, the q-arm length of acrocentrics would extend about 19.30 % due to recombination. The DNA contents of testprogenies were slight increased around 2.1-3.9 % in comparing with the sum of genomes gained from both parents. Our results revealed that loose pairing and unequal crossover between M-2A and T-A in dikaryotype hybrid resulted in polymorphism of DNA contents and karyotypes. Four 2n-gametes were generated by dikaryotype hybrids, three of them were formed by first division restitution (FDR) during meiosis in pollen mother cell (PMC), and one was formed by secondary division restitution (SDR). The GISH results indicated that no recombinants were formed in FDR, while the similar recombinant segments on sister-chromatids suggested that crossover have occurred between sister-chromatins in SDR. Therefore, dikaryotype hybrids could incidentally generate 2n-gametes and accelerate the formation of variously homogenous or heterogenous neokaryotypes in Lycoris spp.