Study On Relationship And Systematic Taxonomy Of Wild Species In Fragaria By 45S RDNA-FISH And GISH Analysis

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.