莪术CPD染色和45S rDNA荧光原位杂交核型分析

为了对莪术[Curcuma zedoaria (Christm.) Roscoe]的染色体进行识别并对该物种基因组的结构及进化进行初步研究,利用改进的火焰干燥法及荧光原位杂交技术,对莪术中期染色体的长度,着丝粒的位置及随体的数目进行分析。PI和DAPI组合（CPD）染色后和相继的45S rDNA探针荧光原位杂交结果显示,莪术具有五对45S rDNA位点,三对位于8,22,31号染色体末端的CPD带区,二对位于4,30号染色体的短臂上。第五号短臂为富含GC对的非45S rDNA位点。该实验建立了莪术的经典核型,为非整倍体,核型公式为2n＝62+1=40m+12sm+1m,其核型不对称性为2A型。     

葱的CPD染色和45S rDNA FISH核型分析

为给葱的染色体的识别提供新标记,建立葱的分子细胞遗传学核型,本研究采用去壁火焰干燥法制备了分散且形态良好的葱中期染色体,并进行了CPD（PI和DAPI组合）染色和45S rDNA荧光原位杂交（FISH）,根据葱染色体的形态特征,结合CPD染色和FISH结果,对葱进行了核型分析。CPD染色结果:葱所有染色体臂末端都显示CPD带。FISH结果：有一对45S rDNA位点（在第5对染色体上）。葱的核型公式：2n=2x=16=2sm+12m+2st(SAT)。研究表明：利用CPD染色和45S rDNA FISH,不仅能为染色体识别提供新标记,还能了解染色体GC丰富区的分布,为葱属植物的物种鉴定、系统分类与进化等研究提供DNA分子方面的证据。     

基于荧光原位杂交技术分析沙地云杉及近缘种核型

利用小麦45S r DNA为探针,对沙地云杉及其近缘种红皮云杉与白杄进行荧光原位杂交(fluorescence in situ hybridization,FISH)鉴定,分析三种云杉染色体上45S r DNA的分布,并进行核型分析。结果表明:白杄染色体上有5对信号位点,其核型公式为2n=24=22m(6sc)+2sm;红皮云杉有6对信号位点,核型公式为2n=24=20m(4sc)+4sm;沙地云杉信号位点为7对,核型公式为2n=24=22m(6sc)+2sm。三种云杉染色体类型大多为亚中部着丝粒染色体,核型均为1A型,且白杄与红皮云杉的核型更为原始;较强的信号位点主要位于次缢痕处,沙地云杉与白杄的FISH核型模式更为接近,说明白杄与沙地云杉其亲缘关系较红皮云杉更近。     

偏穗鹅观草(Roegneria komarovii)的分子核型

以偏穗鹅观草(Roegneria komarovii)为试验材料,利用荧光原位杂交技术,进行5 S rDNA、45 S rDNA以及重复序列pAs 1 DNA的定位,观察荧光原位杂交信号的分布特征以及信号的强弱,对偏穗鹅观草进行染色体配对以及核型分析。结果表明:1)偏穗鹅观草的染色体组总长度为104.785 μm,平均绝对长度为7.485 μm,最长染色体与最短染色体长度比为2.030,臂比大于2∶1的染色体有0组,核型不对称系数为56.01%,具有2对随体,分别位于第6号染色体和第14号染色体的短臂上,故偏穗鹅观草的核型公式是2 n=4 x=28=26 m(4 SAT)+2 sm,核型类型属于1 B型。2) 5 S rDNA位于第6对染色体的短臂上和第11对染色体的长臂上;45 S rDNA位于第6对染色体的短臂上和第14对染色体的短臂上;有8对染色体检测到明显的pAs 1 DNA的荧光信号,大多分布于染色体的端部,少数定位于着丝粒区域。     

基于荧光原位杂交的‘燕山红栗’核型分析

板栗染色体核型分析是开展育种工作的基础。为明确板栗染色体形态结构特征,本研究以‘燕山红栗’为试验材料,采用体细胞染色体常规制片法及荧光原位杂交法,对其染色体进行核型检测及分析。结果表明:板栗染色体数为2n=24,核型公式为2n=2x=24=2M(sat)+18m+4sm,染色体组总长度为13.04滋m,其中长臂总长为7.31滋m,核型不对称系数为52.06%,核型相对对称。精准的染色体核型分析为板栗种质资源利用和新品种选育提供了细胞学基础。     

基于45S rDNA和雷蒙德氏棉gDNA为探针的草棉FISH核型研究

 草棉基于荧光原位杂交（FISH）的核型公式为2n = 2x = 26 = 16m + 10sm (6 sat),短臂和长臂的相对长度分别为1.43～4.14和3.34～5.18,染色体长度比(最长与最短染色体的比值)是1.63。染色体组有6个随体,都定位在最后3条染色体的短臂上,其中位于第12和第13号染色体的随体在DAPI和罗丹明镜像中明显可见,但位于第11号染色体的随体在DAPI镜像中观察不到。检测到6个(3对)NOR信号,与随体同位,1对位于染色体端粒,2对紧接着丝粒。雷蒙德氏棉基因组DNA（gDNA）作探针时,在体细胞染色体上检测到GISH-NOR,其数量、位置和大小与45S探针的NOR相同,说明FISH核型比以前常规核型（非FISH核型）更精确。结合本试验室其它FISH资料,推断A基因组棉种在作为供体形成异源四倍体棉种以来,一些串连重复序列如rDNA可能发生了很大变化,包括扩增、易位或缺失等。对于D基因组特有的GISH-NOR的一个可能解释,就是D基因组棉种的rDNA拷贝数远远多于A基因组棉种。NOR或者GISH-NOR位点等方面的进一步研究,有助于探讨rDNA基因进化和功能,并作为一种标记应用于棉属构建染色体序号定位的物理图谱。     

二倍体栽培棉45 S rDNA-FISH作图及核型比较

以45SrDNA为探针,获得了草棉体、亚洲棉体细胞染色体的荧光原位杂交(FISH)资料。从rDNA FISH实验结果看,草棉有6个杂交信号,也显示了3对核仁组织区(NOR),分别位于第3、9、13对同源染色体上;亚洲棉有4个杂交信号,显示了2对NOR,分别位于第6、13对同源染色体上。草棉、亚洲棉基于45SrDAN FISH的核型分别为:2n=2x=26=20m(4sat) 6sm(2sat)和2n=2x=26=26m(4sat)。     

45S rDNA基因在新麦草染色体上的分布

分析rDNA基因位点在染色体上的分布可以对新麦草染色体进行识别和分析其基因组特征。利用FISH和顺序C-分带-FISH技术将45S rDNA定位于新麦草细胞分裂中期染色体上,结果表明,45S rDNA在二倍体新麦草染色体上有6个主要分布位点,另外几条染色体在两臂中部或长臂末端还显示出较弱的杂交信号,信号强度显示蒙农4号新麦草基因组具有一定杂合性。分析确定新麦草的45S rDNA基因主位点分别位于N1染色体短臂末端、N3染色体短臂末端以及N5染色体短臂末端,推测这3对染色体是NOR染色体。     

杉木根尖细胞染色体C带及荧光带型的研究

对杉木的根尖有丝分裂中期染色体进行研究,结果发现,杉木的染色体核型为2n=22=20m(2SAT) 2sm,10对染色体均为中间着丝粒染色体,只有1对(最小一对)为近中着丝粒染色体,第3对为具随体的染色体,核型不对称性属于1B型.对杉木的Giemsa C-带进行研究发现,有8对染色体有C带出现,只有3对染色体无C带,C带纹均出现在染色体的两臂.且利用C带在杉木11对染色体上的分布情况,能够较容易地辨认出11对中的5对染色体.而荧光分带研究的结果则为在杉木根尖细胞的中期分裂相中,只有CMA(色霉素A3)在带有随体的染色体的次缢痕和随体处有专一的荧光带纹,而DAPI无带.CMA带比DAPI带更适宜杉木的分带研究.最后讨论了C带与荧光带的在杉木染色体研究中的应用.     

高粱属4种植物的核型及其与入侵性关系探讨

对高粱属(Sorghum)4种植物假高粱(S.halepense)、黑高粱(S.almlzm)、高粱(S.bicolor)和苏丹草(S.sudanense)的染色体数目和核型进行了研究.结果表明,假高粱的核型公式为2n=4x=40=28m+12sm,属2B型,核型不对称系数为59.92%;黑高粱的核型公式为2n=4x=40=32m+8 8m,属1B型,核型不对称系数为58.68%.苏丹草的核型公式为2n=2x=20=18m+2sm,属1A型,核型不对称系数为55.56%;高粱的核型公式为2n=2x=20=16m+4sm,属2A型,核型不对称系数为57.95%.与高粱和苏丹草两种作物相比,具有入侵性的假高粱和黑高粱为多倍体、核型不对称性程度更高.     

岷江百合根尖染色体的C-分带和FISH分析

利用Giemsa C-分带和荧光原位杂交(FISH)的方法对岷江百合(L.regale)根尖染色体进行了研究.结果表明岷江百合的带型公式为:2n=24=4I 8CL 2I 6I 2I T 2I T ,每条染色体都显示出了可以明显区别的特征带,带纹强弱差异明显.以45S rDNA为探针对岷江百合根尖染色体进行了荧光原位杂交,杂交点数为5对,分别位于A、B、H和K 4对同源染色体的着丝点区域和一对G染色体的长臂上.通过Giemsa C-带和HSH的方法可以将岷江百合的每条染色体区分开来.     

Molecular cytogenetics in hop (Humulus lupulus L.) and identification of sex chromosomes by DAPI-banding

Hop (Humulus lupulus L.) waskaryotyped by Fluorescence In SituHybridization (FISH) with probes 18S-25Sand 5S rDNA as well as DAPI banding andmeasurements of lengths of chromosome.Irrespective of the sex of the plants theseprobes revealed one signal near thetelomere of chromosome 6 and two signalseach on chromosomes 2 and 5.The shortest chromosome in a metaphaseplate of a male plant was designated to bechromosome Y. It displayed neither signalswith FISH nor DAPI bands. A middle sizedmetacentric chromosome with no telomericbut with an interstitial DAPI band on theshort arm was designated as X. Allautosomes displayed telomeric DAPI-positivebands. For the first time all mitotic hopchromosomes including the sex chromosomeswere identified by methods of molecularcytogenetics.     

Genomic changes in generations of synthetic rapeseed-like allopolyploid grown under selection

Resynthesized Brassica napus L. is an important source for broadening genetic diversity and producing lines with desired characteristics. It is also a fine model to study the processes of genomic reorganizations in recently formed polyploids. We firstly performed molecular cytogenetic characterization of newly resynthesized rapeseed (B. rapa ssp. narinosa × B. oleracea ssp. capitata) and its parental species, and also examined genomic changes in hybrids of the succeeding generations grown under pressure of selection of yellow-seeded progeny. For karyotype studies, FISH/GISH with 45S, 5S rDNA, C genome specific BoB014O06 BAC clone and genomic DNA of parental B. rapa was performed. Synthetic S0–S2 hybrids had common rapeseed karyotypes (2n = 38) including 14 loci of 45S rDNA sites and 10 loci of 5S rDNA. Progeny selection led to gradual deletion of C genome chromosomes in hybrid karyotypes. So, in karyotypes of S6 and S7 hybrids, the chromosome number was reduced to 2n = 20–22, and only chromosomes of A genome bearing 10–13 loci of 45S rDNA and 8–10 loci of 5S rDNA, variations in chromosome number, chromosome rearrangements as well as examples of trisomy and monosomy were revealed. Our findings indicate an enhanced genome instability in resynthesized rapeseed lines developed under the pressure of selection which might lead to chromosome rearrangements or/and deletions and even elimination of the whole parental genome in hybrids in succeeding generations. The approach can be useful for the development of rapeseed lines with trisomy, chromosome addition/substitution lines important for genetics and plant breeding.     

甘蓝自交不亲和基因MLPK与SSP的FISH定位

利用FISH技术, 对自交不亲和基因MLPK与SSP在甘蓝有丝分裂前中期染色体、减数分裂早粗线期染色体以及伸长DNA纤维等3种分辨率水平的靶DNA载体上进行物理定位。结果表明, 在有丝分裂前中期, MLPK探针信号位于一对近中着丝粒同源染色体的短臂中部, 距着丝粒的百分距离约为53.41±3.16;SSP探针信号位于一对具有随体的近端着丝粒同源染色体的长臂端部, 距着丝粒的百分距离约为78.36±4.26。综合3种载体上的FISH结果表明, MLPK与SSP在甘蓝染色体组中可能都只有一个同源序列座位, 具有在单倍体基因组中的单拷贝性。重复FISH杂交表明, MLPK与5S rDNA位于同一对染色体。依据Armstrong的核型分析标准, 初步判断MLPK与SSP分别位于甘蓝的2号和7号染色体, 与S位点不存在连锁关系。另从比较基因组学角度对定位结果进行了讨论。     

Karyotype analysis and physical mapping of 45S rRNA genes in Hydrangea species by fluorescence in situ hybridization

Detailed karyotypes of Hydrangea macrophylla, Hydrangea paniculata and Hydrangea quercifolia were constructed on the basis of arm lengths and centromeric index, together with 45S rDNA fluorescence in situ hybridization. Although the chromosomes were small, they were well distinguishable for all species. Chromosome morphology and karyotypes were different for the three species. H. macrophylla had six metacentric (M), eight submetacentric (SM) and four subtelocentric (ST) chromosomes. The karyotype of H. paniculata contained seven M, 10 SM and one ST chromosomes and H. quercifolia had six M, 10 SM and two ST chromosomes. The variability among three species also was expressed by 45S rDNA signals. H. macrophylla had a nucleolar organizing region on chromosome 2, H. paniculata had 45S rDNA signals on chromosomes 2, 5 and 11 and H. quercifolia on chromosomes 3 and 8. Hybridization signal always was distally on the short arm but the strength of the signals was different for the three species. The chromosome portraits made in this study will be used to trace chromosome behaviour in interspecific hybrids resulting from breeding work between the three species.     

Molecular cloning of Tulipa fosteriana rDNA and subsequent FISH analysis yields cytogenetic organization of 5S rDNA and 45S rDNA in T. gesneriana and T. fosteriana

In this study, Tulipa fosteriana was found to contain 45S rDNA repeat units of 9.7 and 9.5 kb, in which at least 7 types of 45S rDNAs were identified by restriction site analysis. For 5S rDNA, repeat units ranging from 364 bp to 396 bp were identified. The diploid cultivars (2n = 2x = 24) ‘Christmas Dream’ and ‘Queen of Night,’ representing the horticultural group T. gesneriana, and ‘Red Emperor’, belonging to T. fosteriana, were compared cytogenetically using cloned 5S and 45S rDNAs. Fluorescence in situ hybridization (FISH) analysis identified many rDNA sites located on each chromosome in the diploid genomes. For example, we identified 71 sites of 5S rDNA and 10 sites of 45S rDNA in ‘Red Emperor’. Additionally, FISH analyses enabled construction of karyotypes for these cultivars. Karyotype comparison of T. gesneriana cultivars showed conservation of repetitive rDNA unit positioning. A clear difference in chromosome size and signal pattern was observed between T. gesneriana and T. fosteriana cultivars. Here we demonstrate the unique nature of the highly repeated 5S rDNA units in these Tulipa species and the usefulness of FISH karyotyping with cloned 5S and 45S rDNAs to clearly distinguish between chromosomes from T. gesneriana and T. fosteriana. Hitoshi Mizuochi and Agnieszka Marasek contributed equally to this paper     

燕麦属植物核糖体DNA染色体定位及45S rDNA的系统进化分析

由于缺乏明确的二倍体供体信息,燕麦属植物的起源和系统进化关系一直存在争议。利用荧光原位杂交（fluorescence in situ hybridization,FISH）方法,检测45S rDNA和5S rDNA在燕麦属不同倍性植物染色体上的位点信息;并依据已公开的45S rDNA ITS区全长DNA序列构建分子进化树。探讨燕麦属植物在不同基因组中45S rDNA的位点变化、进化规律以及分化机制,为探究燕麦属物种的起源与演化提供参考。