基于45S rDNA-FISH与GISH分析的草莓属（Fragaria）野生种亲缘关系与系统分类研究
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摘要
草莓属植物在世界自然分布有20个种,其中19个种均为野生种,存在倍性主要有2x、4x、6x和8x以及少量的3x、5x、9x、10x甚至12x,因此关于草莓属植物的系统分类、亲缘关系与起源尤其是多倍体种的起源研究一直是草莓研究的重要方面,对其野生资源的开发利用具有重要的意义。目前,国内外虽有利用形态学、染色体核型、孢粉学、同工酶、DNA分子标记技术等进行相关研究,但仍未有准确和完整的结论,而且利用FISH与GISH技术开展相关研究属国内外首次。
本研究收集了原产我国的6个二倍体种(2n=2x=14)、3个四倍体种(2n=4x=28)和1个五倍体种(2n=5x=35),来自日本的2个二倍体种(2n=2x=14)、1个四倍体种(2n=4x=28)和1个八倍体种(2n=8x=56)以及来自欧洲的1个六倍体种(2n=6x=42),包括来自不同地区的品种或类型共48份材料,利用45S rDNA-FISH和GISH技术,对其系统分类、亲缘关系和多倍体形成等方面进行了分析和探讨,取得了一定的进展。
1、利用45S rDNA-FISH技术对包括2x、4x、5x、6x和8x的48份材料共15个种进行了研究,观察分析了45S rDNA在所有供试材料中期染色体与间期细胞核中分布位点的数目、区域与拷贝数,并探讨了种间亲缘关系和多倍体起源,结果如下：
(1)中期染色体与间期细胞核中45S rDNA杂交位点数目与分布相对一致；种内不同品种或类型45S rDNA-FISH结果一致,而种间存在一定的差异；
(2)二倍体种(2n=2x=14)中期染色体与间期细胞核存在2个45S rDNA杂交位点,信号强弱和分布区域在同一个细胞中有三种情况,一是2个杂交信号强弱相同且分布区域相同,二是2个信号强弱不同且分布区域相同,三是2个信号强弱不同且分布区域不相同；45S rDNA杂交信号位置存在两类情况,一类是位于染色体端部,另一类是位于染色体中部区域；森林草莓的2个杂交信号均较强,且位于染色体端部,东北草莓2个杂交信号为1强1弱,分别位于染色体中部和端部,绿色草莓、五叶草莓和西藏草莓相同,2个杂交信号为1强1弱,均位于染色体端部,纤细草莓2个杂交信号为1强1弱,分别位于染色体端部和中部区域,饭沼草莓与蝦夷草莓相似,有2个位于染色体端部且亮度较弱的杂交信号；
(3)四倍体种(2n=4x=28)中期染色体与间期细胞核存在4个45S rDNA杂交位点；蝦夷草莓和东方草莓4个信号分布区域相同,均是3个位于染色体端部,另1个位于染色体中部区域,但蝦夷草莓信号均较弱,而东方草莓则均强；伞房草莓与西南草莓4个信号均分布在染色体端部,然而伞房草莓为3强1弱,而西南草莓为2强2弱；
(4)五倍体东方草莓(2n=5x=35)中期染色体与间期细胞核存在5个45S rDNA杂交位点,4个较强,另1个较弱,其中3个位于染色体端部,另1个较强和1个较弱的位于染色体中部；
(5)六倍体麝香草莓(2n=6x=42)中期染色体与间期细胞核存在6个45S rDNA杂交位点,5个亮度较强,1个亮度较弱,均位于染色体端部区域；
(6)3个八倍体种(2n=8x=56)中期染色体与间期细胞核均存在8个45S rDNA杂交位点,且均为4个亮度较强,4个亮度较弱,但弗吉尼亚草莓和择捉草莓杂交信号均位于染色体端部,而智利草莓8个杂交信号中除了2个较弱的位于染色体中部外,其余均位于染色体端部。
(7)认为森林草莓较为原始,与饭沼草莓和蝦夷草莓亲缘关系较远,与其它二倍体种亲缘关系较近；四倍体东方草莓可能是森林草莓与东北草莓杂交后再加倍形成的异源四倍体,五倍体东方草莓可能起源于四倍体东方草莓形成未减数配子与东北草莓杂交形成；伞房草莓可能是森林草莓与绿色草莓形成未减数配子杂交形成；西南草莓可能起源于西藏草莓,而不是黄毛草莓；证明弗吉尼亚草莓可能是森林草莓与饭沼草莓杂交后加倍形成。
2、以森林草莓、西藏草莓、东北草莓、绿色草莓和饭沼草莓等野生种基因组DNA为探针,对包括自身在内的48份供试材料中期染色体进行GISH分析,以四倍体东方草莓基因组DNA为探针与五倍体东方草莓中期染色体进行GISH分析,结果如下：
(1)探针与自身进行GISH分析时,所有中期染色体的全部区域均呈现出较强的杂交信号,而种间的原位杂交信号却呈现出一定的差异,表明各个种的基因组同源性不同。另外,种内不同品种或类型之间GISH结果没有明显差异,表明种内基因组同源性较高；
(2)以西藏草莓、东北草莓、绿色草莓和自身作为探针与二倍体种(饭沼草莓和蝦夷草莓除外)进行GISH分析,其14条中期染色体上均分布有较强杂交信号,而且几乎覆盖全部染色体区域,表明这些种之间基因组同源性较高；而以饭沼草莓为探针时,饭沼草莓与蝦夷草莓结果与前者类似,但其它二倍体种只有3—7条不等数目染色体存在较弱的杂交信号,而且染色体上只有部分区域有信号分布,表明饭沼草莓与这些二倍体种亲缘关系较远,而与蝦夷草莓较近。
(3)蝦夷草莓,四倍体种(2n=4x=28),饭沼草莓作探针时,有20条染色体的全部区域出现了较强的杂交信号,而其余8条染色体没有出现杂交信号；而其它二倍体种作探针时,仅有10—15条染色体出现了杂交信号,且信号较弱,其余染色体上没有信号出现。
(4)伞房草莓,四倍体种(2n=4x=28),饭沼草莓作探针时,信号较弱,且仅分布在8-10条染色体上,其余染色体上没有杂交信号出现；森林草莓作探针时,杂交信号最多,有20—24条染色体全部区域均出现了较强的杂交信号；其次为绿色草莓、西藏草莓、东北草莓,分别有18—22条、15—18条、14—17条染色体上分布有杂交信号。
(5)西南草莓,四倍体种(2n=4x=28),饭沼草莓作探针时,信号较弱,且仅分布在8—10条染色体上,其余染色体上没有杂交信号出现；西藏草莓和东北草莓作探针时,其所有染色体全部区域均覆盖有较强的杂交信号,其次为绿色草莓和森林草莓,分别有26—28条和25—28条染色体全部区域出现杂交信号。
(6)东方草莓,四倍体种(2n=4x=28),以饭沼草莓作探针时,只有12—15条染色体的部分区域出现了较弱的杂交信号,其余染色体均没有杂交信号出现；而森林草莓和东北草莓作探针时,其28条染色体全部覆盖有较强的杂交信号,西藏草莓和绿色草莓作探针,也分别有24—26条和26—28条染色体全部区域出现杂交信号。
(7)东方草莓,五倍体种(2n=5x=35),它是本文中唯一的奇数倍性野生种,与东方草莓四倍体种类似,饭沼草莓基因组DNA仅在其10—12条中期染色体的部分区域出现较弱的杂交信号；与四倍体东方草莓一样,森林草莓和东北草莓基因组DNA在五倍体所有染色体全部区域上均出现了较强的杂交信号,西藏草莓和绿色草莓作探针时,分别有32—35条和30—33条染色体出现了较强杂交信号。
(8)麝香草莓,六倍体种(2n=6x=42),以饭沼草莓作为探针时,仅有12—15条染色体部分区域出现较弱杂交信号,其余染色体没有信号出现；森林草莓和绿色草莓作探针时,均可能出现所有染色体全部区域均出现杂交信号的情况,分别为40—42条和38—42条染色体出现；而西藏草莓作探针时也有35—38条染色体出现杂交信号,东北草莓作探针时,有30—32条染色体出现杂交信号。
(9)弗吉尼亚草莓,八倍体种(2n=8x=56),与供试其它草莓野生种不同,它是较特殊的一个种,因为以饭沼草莓作探针时,在弗州草莓中期染色体出现了较强的杂交信号,位于16—18条染色体部分区域上,少数覆盖全部染色体区域,推测它的起源可能与饭沼草莓有关；而森林草莓作探针时,有40—45条染色体呈现较强杂交信号,分布于染色体整个区域,其次分别为绿色草莓(33—35条)、东北草莓(32—35条)和西藏草莓(30—32条)。
(10)择捉草莓,八倍体种(2n=8x=56),起源和分布于日本,以饭沼草莓为探针时,有22—25条染色体出现了杂交信号,较弱,分布于部分染色体区域；森林草莓作探针时杂交信号最多,有40—42条染色体呈现杂交信号,较强,分布于染色体整个区域,绿色草莓、东北草莓和西藏草莓作探针时结果相似,有30—35条染色体出现了较强的分布于染色体整个区域的杂交信号。
(11)智利草莓,八倍体种(2n=8x=56),饭沼草莓作探针时,有12—15条染色体部分区域出现了较弱的杂交信号；森林草莓、东北草莓、西藏草莓和绿色草莓作探针时,出现杂交信号的染色体数目为42—44条、32—35条、32—34条和30—32条,且信号较强,分布于染色体整个区域。
(12)综合分析可以认为：
①森林草莓、西藏草莓、东北草莓、绿色草莓、五叶草莓、纤细草莓等二倍体种亲缘关系较近；
②饭沼草莓与蝦夷草莓亲缘关系较近,它们与其它草莓种亲缘关系较远；
③伞房草莓与森林草莓亲缘关系较其它二倍体草莓更近,其次为绿色草莓,结合前人研究和本文45S rDNA-FISH结果,推测伞房草莓可能是森林草莓和绿色草莓的杂交后代；
④西南草莓与西藏草莓和东北草莓亲缘关系最近,其次为绿色草莓和森林草莓,推测其可能起源于西藏草莓或东北草莓,而前面的45S rDNA-FISH结果表明它与西藏草莓有关,二者有较一致的结果；
⑤东方草莓四倍体和五倍体种的所有染色体整个区域均能检测出森林草莓和东北草莓的基因组DNA,而其它二倍体种作探针时仅有部分染色体上有信号分布,推测东方草莓可能起源于森林草莓和东北草莓,这与45S rDNA-FISH结果一致；东方草莓五倍体体细胞中全部35条染色体均呈现出东方草莓四倍体基因组DNA杂交信号,推测五倍体可能来源于四倍体未减数配子参与杂交形成,这与45SrDNA-FISH分析结果一致；
⑥麝香草莓是供试材料中唯一的六倍体材料,它的起源也相对较为复杂,GISH结果表明,森林草莓、绿色草莓均与麝香草莓基因组同源性最高,推测其亲缘关系最近；
⑦弗吉尼亚草莓最可能包含森林草莓和饭沼草莓基因组组成；
⑧择捉草莓和智利草莓均与森林草莓、西藏草莓、东北草莓和绿色草莓亲缘关系较饭沼草莓更近；
综上所述,本文首次对收集到的涉及国内外15个草莓属野生种资源48份进行了45S rDNA-FISH和GISH研究,取得了一定的进展,对于草莓属植物系统分类与进化以及多倍体来源提供了有力的依据和参考,具有重要意义。
There are about 20 species in Fragagia and 19 of them are wild species, including 2x、4x、6x and 8x and a few 3x、5x、9x、10x, even 12x. Therefore, genetic relationship, systematic taxonomy and origination of Fragaria are always very important, which is significant for improve their characteristic. However, many techniques, such as morphology, chromosome karyotype, palynology, isoenzyme, DNA molecular etc. were used to study on these ways, but there is no clear conclusion. And FISH and GISH have not used in this research field yet.
In this paper,48 materials of 15 species from different regions, including 6 diploids (2n=2x=14)、3 tetraploids (2n=4x=28) and 1 pentaploid (2n=5x=35) originated in China, 2 diploids (2n=2x=14)、1 tetraploids (2n=4x=28) and 1 Octoploid (2n=8x=56) from Japan, and 1 hexaploid (2n=6x=42) from Europe are the experiment accesses. The 45S rDNA-FISH and GISH are carried out to investigate and analysis their systematic classification, genetic relationship and formation of polyploidy.
1.48 materials belong to 15 species including 2x、4x、5x、6x and 8x were analysed by 45S rDAN-FISH technique. The number region and intensity of 45S rDNA loci on the metaphase chromosome and interphase nucleus of all materials were observed. Genetic relationship and origination of polyploidy were discussed. The main results are as follows:
(1) In metaphase chromosome and interphase nucleus, the number and region of 45S rDNA sites were relatively consistent. The results of 45S rDNA-FISH were consistent in intraspecies, but different in interspecies.
(2) There were 2 45S rDNA hybridization sites located in diploid metaphase chromosome and interphase nucleus. According to the intensity and region, there were three situations:first, the intensity and region of 2 hybridization signal were the same; second, the intensity were different but region were the same; last, the intensity and region were different. There were 2 regions of 45S rDNA hybridization signal. One was located in the end of the chromosome, another is located in the middle of the chromosome. It was noticeable that F. vesca L. had 2 strong hybridization signals located in the end of the chromosome. F.mandschurica Staudt had 1 strong and 1 weak hybridization signals located in the middle and the end of the chromosome respectively. F. viridis Duch、F. pentaphylla Losinsk. and F. nubicola (Hook. f.) Lindl. ex Lacaita had 1 strong and 1 weak hybridization signal both located in the end of the chromosome. F. gracilis Losinsk. had 1 strong and 1 weak hybridization signals located in the end and the middle of the chromosome respectively. F. iinumae and F. yezoensis had 2 weak hybridization signals located in the end of the chromosome.
(3) There were 4 45S rDNA hybridization sites located in tetraploid metaphase chromosome and interphase nucleus. The located sites were complex. The signal region in F. yezoensis and F. orientalis Lozinsk. were the same,3 located in the end of the chromosome and 1 in the middle of the chromosome. But the hybridization signals were weak in F. yezoensis and strong in F. orientalis.4 signals of F. corym bosa Lozinsk and F. moupinensis (Franch.) Card. were located in the end of the chromosome, but 3 strong and 1 weak in F. corym bosa Lozinsk,2 strong and 2 weak in F. moupinensis (Franch.) Card.
(4) There were 5 45S rDNA hybridization sites located in pentaploid metaphase chromosome and interphase nucleus.4 signals were strong and 1 was weak.3 located in the end of the chromosome and 1 strong and 1 weak in the middle of the chromosome.
(5) There were 6 45S rDNA hybridization sites located in hexaploid metaphase chromosome and interphase nucleus.5 signals were strong and 1 was weak, all located in the end of the chromosome.
(6) There were 8 45S rDNA hybridization sites located in 3 Octoploids metaphase chromosome and interphase nucleus.4 signals were strong and the other 4 were weak. The hybridization signals of F. virginiana and F. iturupensis Staudt were located in the end of the chromosome. While,2 hybridization signals of F. chiloensis were weak and located in the middle of the chromosome, and 6 were in the end of the chromosome.
(7) The results showed that F. vesca Linn. was original and heterologous to some extent between it and F. iinumae and F. yezoensis, and homology to other diploids. F. orientalis Lozinsk (4x) was allotetraploid and originated from chromosome doubling in F. vesca Linn. X F. mandschurica Staudt.. F. orientalis Lozinsk.(5x) was obtained by hybridization of 2n gametes of F. orientalis Lozinsk. X F. mandschurica Staudt.. F. corym bosa Lozinsk was possibly formed by the 2n gametes of F. vesca Linn. X F. viridis Duch. F. moupinensis (Franch.) Card. May originated from F. nubicola (Hook. f.) Lindl. ex Lacaita not F. mandschurica. F. virginiana was obtained by chromosome doubling in F. vesca Linn. X F. iinumae.
2. Genomic in situ hybridization (GISH) using genomic DNA probes from F. vesca L., F. nubicola (Hook. f.) Lindl. ex Lacaita, Northeast strawberries, green strawberries and wild strawberries, was used to examine the genomic constitution of 48 tested materials, including their own. Genomic in situ hybridization (GISH) using genomic DNA probes from tetraploid Eastern Strawberry, was used to examine the metaphase chromosome of pentaploid East Strawberry. The results were as follows:
(1)GISH analysis of the probe with itself, bright hybridization signals were observed on all the chromosomal regions, while hybridization signals of between species preached some differences. This suggests that the genome of each species homology different. In addition,no significant difference between different varieties within species or types by GISH, indicating that intra-genomic homologous.
(2)Using genomic DNA of F. nubicola (Hook. f.) Lindl. ex Lacaita, F. mandschurica Staudt, F. viridis Duch and F. vesca L. as probe, diploid species was analyzed with genome in situ hybridization (GISH).The results showed that bright and even hybridization signals were observed on 14 metaphase chromosomes which covering almost all the chromosomal regions in these diploid species but F. iinumae and F. yezoensis were exepted. This implies these kinds are high homology between the genome. But using genomic DNA of F. iinumae as probe, only 3-7 chromosomes with weak hybridization signals existed, and only in part of the region on chromosome signal distributed, indicating that F. iinumae distantly related.
(3) Tetraploid F. yezoensis (2n=4x=28). Using F. iinumae as a probe, strong hybridization signals appeared in all the regions of 20 chromosomes, while no hybridization signal in the remaining eight chromosomes; and using other diploid species as probes, only 10-15 chromosomes appeared in weak hybridization signal, no signal appears on the other chromosome.
(4) Tetraploid F. corym bosa Lozinsk (2n=4x=28).Using F. iinumae as a probe, the signal is weak, and only distributed in the 8-10 chromosomes, no other chromosome hybridization signals; When F. vesca L. gDNA were used as probes, hybridization signals to the highest of all regions were 20-24 chromosomes appeared strong hybridization signal; and hybridization signals distributed in 18-22,15-18,14-17 chromosomes of the F. viridis Duch, F. nubicola (Hook. f.) Lindl. ex Lacaita, F. mandschurica Staudt respectively.
(5)Tetraploid F. moupinensis (Franch.) Card.(2n=4x=28). Using F. iinumae as a probe, the signal is weak, and only distributed in the 8-10 chromosomes, no other chromosome hybridization signals; Using F. nubicola (Hook. f.) Lindl. ex Lacaita and F. mandschurica Staudt as probes, blight hybridization signals occurred in all chromosomes. Followed by F. vesca L. and F. viridis Duch,26-28 and 25-28 chromosomes respectively, all regions of chromosome showed hybridization signals.
(6) Tetraploid F. orientalis Lozinsk. (2n=4x=28). Using F. iinumae as a probe, only 12-15 chromosomes appeared in some areas of weak hybridization signals, and no hybridization signal appeared in the remaining chromosomes; and when probed with gDNA from F. vesca L. and F. mandschurica Staudt, blight hybridization signals for F. nubicola (Hook. f.) Lindl. ex Lacaita and F. viridis Duch appeared in 28 chromosomes, but also 24-26 and 26-28 respectively, all regions of chromosome showed hybridization signals.
(7) Pentaploid F. orientalis Lozinsk (2n=5x=35). It is the only odd multiple of the wild species in this study and it is similar to the tetraploid F. orientalis Lozinsk. F. iinumae genomic DNA only in the 10-12 region appeared weak hybridization signals; and tetraploid F. orientalis Lozinsk as F. vesca L. and F. mandschurica Staudt, their genomic DNA of all chromosomes in pentaploid all regions have appeared on the blight hybridization signals. When probed with sequences from F. nubicola (Hook. f.) Lindl. ex Lacaita and F. viridis Duch, respectively, in 32-35 and 30-33 chromosomes appeared stronger hybridization signal.
(8) F. moschata is a hexaploid species (2n=6x=42).When F. iinumae gDNA were used as a probe, only weak hybridization signals occurred in some regions of 12-15 chromosomes. When probed with gDNA from F. viridis Duch and F. vesca L. hybridization signals could occur in all regions of all chromosomes.When hybridized with F. nubicola (Hook. f.) Lindl. ex Lacaita,35-38 chromosomes may release positive signals, while 30-32 when hybridized with F. mandschurica Staudt.
(9) F. virginiana is an octoploid species (2n=8 x=56). Be different with other tested wild speices, strong hybridization signals occurred in some regions of 16-18 metaphase chromosomes in F. virginiana as F. iinumae gDNA a probe, and even a few cover all regions of metaphase chromosomes which suggesting that its origin may be related with the F. iinumae. However, when using F. vesca L. gDNA as probe,40-45 chromosomes showing stronger hybridization signal which located in all chromosome region, followed by F. viridis Duch (33-35), F. mandschurica Staudt (32-35) and F. nubicola(Hook. f.) Lindl. ex Lacaita (Article 30-32).
(10) F. iturupensis Staudt is octoploid species (2n=8 x=56) that origin and distribution in Japan.When F. iinumae gDNA was used as probe, only weak hybridization signals occurred some regions of 12-15 chromosomes. When hybridized with F. vesca L., strong hybridization signal is the highest of 40-42 chromosomes showed hybridization signals, located in whole chromosome region. When F. nubicola (Hook. f.) Lindl. ex Lacaita, F. mandschurica Staudt, F. viridis Duch were used as probes, they all have 30-35 chromosomes showed hybridization signals which appeared all region.
(11) F. chiloensis is octoploid species (2n=8 x=56). When hybridized with F. iinumae, only weak hybridization signals occurred in some regions of 12-15 chromosomes. When probed with from F. vesca L., F. nubicola (Hook. f.) Lindl. ex Lacaita, F. mandschurica Staudt, F. viridis Duch, the stronger hybridization signals of chromosome number is 42-44,32-35,32-34 and 30-32 of all regions.
(12) Comprehensive analysis, we can get the following conclusions:
①Diploid species of F. vesca L., F. nubicola (Hook. f.) Lindl. ex Lacaita, F. mandschurica Staudt, F. viridis Duch, F. pentaphylla Losinsk., F. gracilis Losinsk. have close genetic relationship.
②F. iinumae and F. yezoensis have close genetic relationship, but they are distantly related with the other species.
③F. corym bosa Lozinsk and F. vesca L.'s genetic relationship are closer than the other diploid strawberry, followed by F. viridis Duch. Combining with previous studies and the results of this 45S rDNA-FISH have suggested that F. corym bosa Lozinsk may be the offspring of F. vesca L. and F. viridis Duch.
④F. moupinensis (Franch.) Card., F. mandschurica Staudt and F. nubicola (Hook. f.) Lindl. ex Lacaita have closest relationship, followed by F. vesca L. and F. viridis Duch, suggesting the origin of F. moupinensis (Franch.) Card. may be from F. nubicola (Hook. f.) Lindl. ex Lacaita and F. mandschurica Staudt. And the result is same as 45S rDNA-FISH.
⑤Both all regions of all chromosomes in F. orientalis Lozinsk. of tetraploid and pentaploid can be detected the signals of F. mandschurica Staudt and F. vesca L. gDNA. While only some regions of chromosomes have hybridization signals when the other diploid species were used as probes. The results showed that the origin of F. orientalis Lozinsk possible from F. vesca L. and F. mandschurica Staudt. This is consistent with the results of 45S rDNA-FISH; In all 35 chromosomes in F. orientalis Lozinsk pentaploid the signals were shown by F. orientalis Lozinsk tetraploid gDNA and we considered that pentaploid may come from non-reduced gamete of tetraploid which was same as the result of 45S rDNA-FISH analysis.
⑥F. moschata is the only hexaploid species in the tested materials, its origin is relatively more complex. GISH results showed that the F. vesca L., F. viridis Duch and F. moschata were highest homology, so that they have closest relationship;
⑦F. virginiana is most likely to contain F. vesca L. and F. iinumae genome organization;
⑧The genetic relationship between F. iturupensis Staudt and F. vesca L., F. nubicola (Hook. f.) Lindl. ex Lacaita, F. viridis Duch, F. mandschurica Staudt are more closer than the F. iinumae. And this situation is same with F. chiloensis.
48 wild materials of 15 species in Fragaria involving domestic and foreign resources were studied firstly by 45S rDNA-FISH and GISH in the world. Some important progress has been obtained which provide a strong basis and reference for the strawberry plants, the taxonomy and evolution of polyploid sources. The paper is great significance as well.
本研究收集了原产我国的6个二倍体种(2n=2x=14)、3个四倍体种(2n=4x=28)和1个五倍体种(2n=5x=35),来自日本的2个二倍体种(2n=2x=14)、1个四倍体种(2n=4x=28)和1个八倍体种(2n=8x=56)以及来自欧洲的1个六倍体种(2n=6x=42),包括来自不同地区的品种或类型共48份材料,利用45S rDNA-FISH和GISH技术,对其系统分类、亲缘关系和多倍体形成等方面进行了分析和探讨,取得了一定的进展。
1、利用45S rDNA-FISH技术对包括2x、4x、5x、6x和8x的48份材料共15个种进行了研究,观察分析了45S rDNA在所有供试材料中期染色体与间期细胞核中分布位点的数目、区域与拷贝数,并探讨了种间亲缘关系和多倍体起源,结果如下：
(1)中期染色体与间期细胞核中45S rDNA杂交位点数目与分布相对一致；种内不同品种或类型45S rDNA-FISH结果一致,而种间存在一定的差异；
(2)二倍体种(2n=2x=14)中期染色体与间期细胞核存在2个45S rDNA杂交位点,信号强弱和分布区域在同一个细胞中有三种情况,一是2个杂交信号强弱相同且分布区域相同,二是2个信号强弱不同且分布区域相同,三是2个信号强弱不同且分布区域不相同；45S rDNA杂交信号位置存在两类情况,一类是位于染色体端部,另一类是位于染色体中部区域；森林草莓的2个杂交信号均较强,且位于染色体端部,东北草莓2个杂交信号为1强1弱,分别位于染色体中部和端部,绿色草莓、五叶草莓和西藏草莓相同,2个杂交信号为1强1弱,均位于染色体端部,纤细草莓2个杂交信号为1强1弱,分别位于染色体端部和中部区域,饭沼草莓与蝦夷草莓相似,有2个位于染色体端部且亮度较弱的杂交信号；
(3)四倍体种(2n=4x=28)中期染色体与间期细胞核存在4个45S rDNA杂交位点；蝦夷草莓和东方草莓4个信号分布区域相同,均是3个位于染色体端部,另1个位于染色体中部区域,但蝦夷草莓信号均较弱,而东方草莓则均强；伞房草莓与西南草莓4个信号均分布在染色体端部,然而伞房草莓为3强1弱,而西南草莓为2强2弱；
(4)五倍体东方草莓(2n=5x=35)中期染色体与间期细胞核存在5个45S rDNA杂交位点,4个较强,另1个较弱,其中3个位于染色体端部,另1个较强和1个较弱的位于染色体中部；
(5)六倍体麝香草莓(2n=6x=42)中期染色体与间期细胞核存在6个45S rDNA杂交位点,5个亮度较强,1个亮度较弱,均位于染色体端部区域；
(6)3个八倍体种(2n=8x=56)中期染色体与间期细胞核均存在8个45S rDNA杂交位点,且均为4个亮度较强,4个亮度较弱,但弗吉尼亚草莓和择捉草莓杂交信号均位于染色体端部,而智利草莓8个杂交信号中除了2个较弱的位于染色体中部外,其余均位于染色体端部。
(7)认为森林草莓较为原始,与饭沼草莓和蝦夷草莓亲缘关系较远,与其它二倍体种亲缘关系较近；四倍体东方草莓可能是森林草莓与东北草莓杂交后再加倍形成的异源四倍体,五倍体东方草莓可能起源于四倍体东方草莓形成未减数配子与东北草莓杂交形成；伞房草莓可能是森林草莓与绿色草莓形成未减数配子杂交形成；西南草莓可能起源于西藏草莓,而不是黄毛草莓；证明弗吉尼亚草莓可能是森林草莓与饭沼草莓杂交后加倍形成。
2、以森林草莓、西藏草莓、东北草莓、绿色草莓和饭沼草莓等野生种基因组DNA为探针,对包括自身在内的48份供试材料中期染色体进行GISH分析,以四倍体东方草莓基因组DNA为探针与五倍体东方草莓中期染色体进行GISH分析,结果如下：
(1)探针与自身进行GISH分析时,所有中期染色体的全部区域均呈现出较强的杂交信号,而种间的原位杂交信号却呈现出一定的差异,表明各个种的基因组同源性不同。另外,种内不同品种或类型之间GISH结果没有明显差异,表明种内基因组同源性较高；
(2)以西藏草莓、东北草莓、绿色草莓和自身作为探针与二倍体种(饭沼草莓和蝦夷草莓除外)进行GISH分析,其14条中期染色体上均分布有较强杂交信号,而且几乎覆盖全部染色体区域,表明这些种之间基因组同源性较高；而以饭沼草莓为探针时,饭沼草莓与蝦夷草莓结果与前者类似,但其它二倍体种只有3—7条不等数目染色体存在较弱的杂交信号,而且染色体上只有部分区域有信号分布,表明饭沼草莓与这些二倍体种亲缘关系较远,而与蝦夷草莓较近。
(3)蝦夷草莓,四倍体种(2n=4x=28),饭沼草莓作探针时,有20条染色体的全部区域出现了较强的杂交信号,而其余8条染色体没有出现杂交信号；而其它二倍体种作探针时,仅有10—15条染色体出现了杂交信号,且信号较弱,其余染色体上没有信号出现。
(4)伞房草莓,四倍体种(2n=4x=28),饭沼草莓作探针时,信号较弱,且仅分布在8-10条染色体上,其余染色体上没有杂交信号出现；森林草莓作探针时,杂交信号最多,有20—24条染色体全部区域均出现了较强的杂交信号；其次为绿色草莓、西藏草莓、东北草莓,分别有18—22条、15—18条、14—17条染色体上分布有杂交信号。
(5)西南草莓,四倍体种(2n=4x=28),饭沼草莓作探针时,信号较弱,且仅分布在8—10条染色体上,其余染色体上没有杂交信号出现；西藏草莓和东北草莓作探针时,其所有染色体全部区域均覆盖有较强的杂交信号,其次为绿色草莓和森林草莓,分别有26—28条和25—28条染色体全部区域出现杂交信号。
(6)东方草莓,四倍体种(2n=4x=28),以饭沼草莓作探针时,只有12—15条染色体的部分区域出现了较弱的杂交信号,其余染色体均没有杂交信号出现；而森林草莓和东北草莓作探针时,其28条染色体全部覆盖有较强的杂交信号,西藏草莓和绿色草莓作探针,也分别有24—26条和26—28条染色体全部区域出现杂交信号。
(7)东方草莓,五倍体种(2n=5x=35),它是本文中唯一的奇数倍性野生种,与东方草莓四倍体种类似,饭沼草莓基因组DNA仅在其10—12条中期染色体的部分区域出现较弱的杂交信号；与四倍体东方草莓一样,森林草莓和东北草莓基因组DNA在五倍体所有染色体全部区域上均出现了较强的杂交信号,西藏草莓和绿色草莓作探针时,分别有32—35条和30—33条染色体出现了较强杂交信号。
(8)麝香草莓,六倍体种(2n=6x=42),以饭沼草莓作为探针时,仅有12—15条染色体部分区域出现较弱杂交信号,其余染色体没有信号出现；森林草莓和绿色草莓作探针时,均可能出现所有染色体全部区域均出现杂交信号的情况,分别为40—42条和38—42条染色体出现；而西藏草莓作探针时也有35—38条染色体出现杂交信号,东北草莓作探针时,有30—32条染色体出现杂交信号。
(9)弗吉尼亚草莓,八倍体种(2n=8x=56),与供试其它草莓野生种不同,它是较特殊的一个种,因为以饭沼草莓作探针时,在弗州草莓中期染色体出现了较强的杂交信号,位于16—18条染色体部分区域上,少数覆盖全部染色体区域,推测它的起源可能与饭沼草莓有关；而森林草莓作探针时,有40—45条染色体呈现较强杂交信号,分布于染色体整个区域,其次分别为绿色草莓(33—35条)、东北草莓(32—35条)和西藏草莓(30—32条)。
(10)择捉草莓,八倍体种(2n=8x=56),起源和分布于日本,以饭沼草莓为探针时,有22—25条染色体出现了杂交信号,较弱,分布于部分染色体区域；森林草莓作探针时杂交信号最多,有40—42条染色体呈现杂交信号,较强,分布于染色体整个区域,绿色草莓、东北草莓和西藏草莓作探针时结果相似,有30—35条染色体出现了较强的分布于染色体整个区域的杂交信号。
(11)智利草莓,八倍体种(2n=8x=56),饭沼草莓作探针时,有12—15条染色体部分区域出现了较弱的杂交信号；森林草莓、东北草莓、西藏草莓和绿色草莓作探针时,出现杂交信号的染色体数目为42—44条、32—35条、32—34条和30—32条,且信号较强,分布于染色体整个区域。
(12)综合分析可以认为：
①森林草莓、西藏草莓、东北草莓、绿色草莓、五叶草莓、纤细草莓等二倍体种亲缘关系较近；
②饭沼草莓与蝦夷草莓亲缘关系较近,它们与其它草莓种亲缘关系较远；
③伞房草莓与森林草莓亲缘关系较其它二倍体草莓更近,其次为绿色草莓,结合前人研究和本文45S rDNA-FISH结果,推测伞房草莓可能是森林草莓和绿色草莓的杂交后代；
④西南草莓与西藏草莓和东北草莓亲缘关系最近,其次为绿色草莓和森林草莓,推测其可能起源于西藏草莓或东北草莓,而前面的45S rDNA-FISH结果表明它与西藏草莓有关,二者有较一致的结果；
⑤东方草莓四倍体和五倍体种的所有染色体整个区域均能检测出森林草莓和东北草莓的基因组DNA,而其它二倍体种作探针时仅有部分染色体上有信号分布,推测东方草莓可能起源于森林草莓和东北草莓,这与45S rDNA-FISH结果一致；东方草莓五倍体体细胞中全部35条染色体均呈现出东方草莓四倍体基因组DNA杂交信号,推测五倍体可能来源于四倍体未减数配子参与杂交形成,这与45SrDNA-FISH分析结果一致；
⑥麝香草莓是供试材料中唯一的六倍体材料,它的起源也相对较为复杂,GISH结果表明,森林草莓、绿色草莓均与麝香草莓基因组同源性最高,推测其亲缘关系最近；
⑦弗吉尼亚草莓最可能包含森林草莓和饭沼草莓基因组组成；
⑧择捉草莓和智利草莓均与森林草莓、西藏草莓、东北草莓和绿色草莓亲缘关系较饭沼草莓更近；
综上所述,本文首次对收集到的涉及国内外15个草莓属野生种资源48份进行了45S rDNA-FISH和GISH研究,取得了一定的进展,对于草莓属植物系统分类与进化以及多倍体来源提供了有力的依据和参考,具有重要意义。
There are about 20 species in Fragagia and 19 of them are wild species, including 2x、4x、6x and 8x and a few 3x、5x、9x、10x, even 12x. Therefore, genetic relationship, systematic taxonomy and origination of Fragaria are always very important, which is significant for improve their characteristic. However, many techniques, such as morphology, chromosome karyotype, palynology, isoenzyme, DNA molecular etc. were used to study on these ways, but there is no clear conclusion. And FISH and GISH have not used in this research field yet.
In this paper,48 materials of 15 species from different regions, including 6 diploids (2n=2x=14)、3 tetraploids (2n=4x=28) and 1 pentaploid (2n=5x=35) originated in China, 2 diploids (2n=2x=14)、1 tetraploids (2n=4x=28) and 1 Octoploid (2n=8x=56) from Japan, and 1 hexaploid (2n=6x=42) from Europe are the experiment accesses. The 45S rDNA-FISH and GISH are carried out to investigate and analysis their systematic classification, genetic relationship and formation of polyploidy.
1.48 materials belong to 15 species including 2x、4x、5x、6x and 8x were analysed by 45S rDAN-FISH technique. The number region and intensity of 45S rDNA loci on the metaphase chromosome and interphase nucleus of all materials were observed. Genetic relationship and origination of polyploidy were discussed. The main results are as follows:
(1) In metaphase chromosome and interphase nucleus, the number and region of 45S rDNA sites were relatively consistent. The results of 45S rDNA-FISH were consistent in intraspecies, but different in interspecies.
(2) There were 2 45S rDNA hybridization sites located in diploid metaphase chromosome and interphase nucleus. According to the intensity and region, there were three situations:first, the intensity and region of 2 hybridization signal were the same; second, the intensity were different but region were the same; last, the intensity and region were different. There were 2 regions of 45S rDNA hybridization signal. One was located in the end of the chromosome, another is located in the middle of the chromosome. It was noticeable that F. vesca L. had 2 strong hybridization signals located in the end of the chromosome. F.mandschurica Staudt had 1 strong and 1 weak hybridization signals located in the middle and the end of the chromosome respectively. F. viridis Duch、F. pentaphylla Losinsk. and F. nubicola (Hook. f.) Lindl. ex Lacaita had 1 strong and 1 weak hybridization signal both located in the end of the chromosome. F. gracilis Losinsk. had 1 strong and 1 weak hybridization signals located in the end and the middle of the chromosome respectively. F. iinumae and F. yezoensis had 2 weak hybridization signals located in the end of the chromosome.
(3) There were 4 45S rDNA hybridization sites located in tetraploid metaphase chromosome and interphase nucleus. The located sites were complex. The signal region in F. yezoensis and F. orientalis Lozinsk. were the same,3 located in the end of the chromosome and 1 in the middle of the chromosome. But the hybridization signals were weak in F. yezoensis and strong in F. orientalis.4 signals of F. corym bosa Lozinsk and F. moupinensis (Franch.) Card. were located in the end of the chromosome, but 3 strong and 1 weak in F. corym bosa Lozinsk,2 strong and 2 weak in F. moupinensis (Franch.) Card.
(4) There were 5 45S rDNA hybridization sites located in pentaploid metaphase chromosome and interphase nucleus.4 signals were strong and 1 was weak.3 located in the end of the chromosome and 1 strong and 1 weak in the middle of the chromosome.
(5) There were 6 45S rDNA hybridization sites located in hexaploid metaphase chromosome and interphase nucleus.5 signals were strong and 1 was weak, all located in the end of the chromosome.
(6) There were 8 45S rDNA hybridization sites located in 3 Octoploids metaphase chromosome and interphase nucleus.4 signals were strong and the other 4 were weak. The hybridization signals of F. virginiana and F. iturupensis Staudt were located in the end of the chromosome. While,2 hybridization signals of F. chiloensis were weak and located in the middle of the chromosome, and 6 were in the end of the chromosome.
(7) The results showed that F. vesca Linn. was original and heterologous to some extent between it and F. iinumae and F. yezoensis, and homology to other diploids. F. orientalis Lozinsk (4x) was allotetraploid and originated from chromosome doubling in F. vesca Linn. X F. mandschurica Staudt.. F. orientalis Lozinsk.(5x) was obtained by hybridization of 2n gametes of F. orientalis Lozinsk. X F. mandschurica Staudt.. F. corym bosa Lozinsk was possibly formed by the 2n gametes of F. vesca Linn. X F. viridis Duch. F. moupinensis (Franch.) Card. May originated from F. nubicola (Hook. f.) Lindl. ex Lacaita not F. mandschurica. F. virginiana was obtained by chromosome doubling in F. vesca Linn. X F. iinumae.
2. Genomic in situ hybridization (GISH) using genomic DNA probes from F. vesca L., F. nubicola (Hook. f.) Lindl. ex Lacaita, Northeast strawberries, green strawberries and wild strawberries, was used to examine the genomic constitution of 48 tested materials, including their own. Genomic in situ hybridization (GISH) using genomic DNA probes from tetraploid Eastern Strawberry, was used to examine the metaphase chromosome of pentaploid East Strawberry. The results were as follows:
(1)GISH analysis of the probe with itself, bright hybridization signals were observed on all the chromosomal regions, while hybridization signals of between species preached some differences. This suggests that the genome of each species homology different. In addition,no significant difference between different varieties within species or types by GISH, indicating that intra-genomic homologous.
(2)Using genomic DNA of F. nubicola (Hook. f.) Lindl. ex Lacaita, F. mandschurica Staudt, F. viridis Duch and F. vesca L. as probe, diploid species was analyzed with genome in situ hybridization (GISH).The results showed that bright and even hybridization signals were observed on 14 metaphase chromosomes which covering almost all the chromosomal regions in these diploid species but F. iinumae and F. yezoensis were exepted. This implies these kinds are high homology between the genome. But using genomic DNA of F. iinumae as probe, only 3-7 chromosomes with weak hybridization signals existed, and only in part of the region on chromosome signal distributed, indicating that F. iinumae distantly related.
(3) Tetraploid F. yezoensis (2n=4x=28). Using F. iinumae as a probe, strong hybridization signals appeared in all the regions of 20 chromosomes, while no hybridization signal in the remaining eight chromosomes; and using other diploid species as probes, only 10-15 chromosomes appeared in weak hybridization signal, no signal appears on the other chromosome.
(4) Tetraploid F. corym bosa Lozinsk (2n=4x=28).Using F. iinumae as a probe, the signal is weak, and only distributed in the 8-10 chromosomes, no other chromosome hybridization signals; When F. vesca L. gDNA were used as probes, hybridization signals to the highest of all regions were 20-24 chromosomes appeared strong hybridization signal; and hybridization signals distributed in 18-22,15-18,14-17 chromosomes of the F. viridis Duch, F. nubicola (Hook. f.) Lindl. ex Lacaita, F. mandschurica Staudt respectively.
(5)Tetraploid F. moupinensis (Franch.) Card.(2n=4x=28). Using F. iinumae as a probe, the signal is weak, and only distributed in the 8-10 chromosomes, no other chromosome hybridization signals; Using F. nubicola (Hook. f.) Lindl. ex Lacaita and F. mandschurica Staudt as probes, blight hybridization signals occurred in all chromosomes. Followed by F. vesca L. and F. viridis Duch,26-28 and 25-28 chromosomes respectively, all regions of chromosome showed hybridization signals.
(6) Tetraploid F. orientalis Lozinsk. (2n=4x=28). Using F. iinumae as a probe, only 12-15 chromosomes appeared in some areas of weak hybridization signals, and no hybridization signal appeared in the remaining chromosomes; and when probed with gDNA from F. vesca L. and F. mandschurica Staudt, blight hybridization signals for F. nubicola (Hook. f.) Lindl. ex Lacaita and F. viridis Duch appeared in 28 chromosomes, but also 24-26 and 26-28 respectively, all regions of chromosome showed hybridization signals.
(7) Pentaploid F. orientalis Lozinsk (2n=5x=35). It is the only odd multiple of the wild species in this study and it is similar to the tetraploid F. orientalis Lozinsk. F. iinumae genomic DNA only in the 10-12 region appeared weak hybridization signals; and tetraploid F. orientalis Lozinsk as F. vesca L. and F. mandschurica Staudt, their genomic DNA of all chromosomes in pentaploid all regions have appeared on the blight hybridization signals. When probed with sequences from F. nubicola (Hook. f.) Lindl. ex Lacaita and F. viridis Duch, respectively, in 32-35 and 30-33 chromosomes appeared stronger hybridization signal.
(8) F. moschata is a hexaploid species (2n=6x=42).When F. iinumae gDNA were used as a probe, only weak hybridization signals occurred in some regions of 12-15 chromosomes. When probed with gDNA from F. viridis Duch and F. vesca L. hybridization signals could occur in all regions of all chromosomes.When hybridized with F. nubicola (Hook. f.) Lindl. ex Lacaita,35-38 chromosomes may release positive signals, while 30-32 when hybridized with F. mandschurica Staudt.
(9) F. virginiana is an octoploid species (2n=8 x=56). Be different with other tested wild speices, strong hybridization signals occurred in some regions of 16-18 metaphase chromosomes in F. virginiana as F. iinumae gDNA a probe, and even a few cover all regions of metaphase chromosomes which suggesting that its origin may be related with the F. iinumae. However, when using F. vesca L. gDNA as probe,40-45 chromosomes showing stronger hybridization signal which located in all chromosome region, followed by F. viridis Duch (33-35), F. mandschurica Staudt (32-35) and F. nubicola(Hook. f.) Lindl. ex Lacaita (Article 30-32).
(10) F. iturupensis Staudt is octoploid species (2n=8 x=56) that origin and distribution in Japan.When F. iinumae gDNA was used as probe, only weak hybridization signals occurred some regions of 12-15 chromosomes. When hybridized with F. vesca L., strong hybridization signal is the highest of 40-42 chromosomes showed hybridization signals, located in whole chromosome region. When F. nubicola (Hook. f.) Lindl. ex Lacaita, F. mandschurica Staudt, F. viridis Duch were used as probes, they all have 30-35 chromosomes showed hybridization signals which appeared all region.
(11) F. chiloensis is octoploid species (2n=8 x=56). When hybridized with F. iinumae, only weak hybridization signals occurred in some regions of 12-15 chromosomes. When probed with from F. vesca L., F. nubicola (Hook. f.) Lindl. ex Lacaita, F. mandschurica Staudt, F. viridis Duch, the stronger hybridization signals of chromosome number is 42-44,32-35,32-34 and 30-32 of all regions.
(12) Comprehensive analysis, we can get the following conclusions:
①Diploid species of F. vesca L., F. nubicola (Hook. f.) Lindl. ex Lacaita, F. mandschurica Staudt, F. viridis Duch, F. pentaphylla Losinsk., F. gracilis Losinsk. have close genetic relationship.
②F. iinumae and F. yezoensis have close genetic relationship, but they are distantly related with the other species.
③F. corym bosa Lozinsk and F. vesca L.'s genetic relationship are closer than the other diploid strawberry, followed by F. viridis Duch. Combining with previous studies and the results of this 45S rDNA-FISH have suggested that F. corym bosa Lozinsk may be the offspring of F. vesca L. and F. viridis Duch.
④F. moupinensis (Franch.) Card., F. mandschurica Staudt and F. nubicola (Hook. f.) Lindl. ex Lacaita have closest relationship, followed by F. vesca L. and F. viridis Duch, suggesting the origin of F. moupinensis (Franch.) Card. may be from F. nubicola (Hook. f.) Lindl. ex Lacaita and F. mandschurica Staudt. And the result is same as 45S rDNA-FISH.
⑤Both all regions of all chromosomes in F. orientalis Lozinsk. of tetraploid and pentaploid can be detected the signals of F. mandschurica Staudt and F. vesca L. gDNA. While only some regions of chromosomes have hybridization signals when the other diploid species were used as probes. The results showed that the origin of F. orientalis Lozinsk possible from F. vesca L. and F. mandschurica Staudt. This is consistent with the results of 45S rDNA-FISH; In all 35 chromosomes in F. orientalis Lozinsk pentaploid the signals were shown by F. orientalis Lozinsk tetraploid gDNA and we considered that pentaploid may come from non-reduced gamete of tetraploid which was same as the result of 45S rDNA-FISH analysis.
⑥F. moschata is the only hexaploid species in the tested materials, its origin is relatively more complex. GISH results showed that the F. vesca L., F. viridis Duch and F. moschata were highest homology, so that they have closest relationship;
⑦F. virginiana is most likely to contain F. vesca L. and F. iinumae genome organization;
⑧The genetic relationship between F. iturupensis Staudt and F. vesca L., F. nubicola (Hook. f.) Lindl. ex Lacaita, F. viridis Duch, F. mandschurica Staudt are more closer than the F. iinumae. And this situation is same with F. chiloensis.
48 wild materials of 15 species in Fragaria involving domestic and foreign resources were studied firstly by 45S rDNA-FISH and GISH in the world. Some important progress has been obtained which provide a strong basis and reference for the strawberry plants, the taxonomy and evolution of polyploid sources. The paper is great significance as well.
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