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。     

萝卜-芥蓝异源四倍体的合成及GISH分析

通过萝卜(Raphanus sativus L.,2n=18,RR)与白花芥蓝(Brassica alboglabra Bailey,2n=18, CC)杂交,F1经秋水仙碱加倍合成萝卜-芥蓝异源四倍体（Raphanobrassica, 2n=36, RRCC）。经F₄~F₁₀代连续育性选择,F₁₀单株种子产量达32.3 g,每角粒数达14.9。基因组原位杂交显示F₁₀减数分裂行为类似于二倍体物种,表明该异源四倍体的细胞学行为已经稳定。育性观察表明,可育花粉足够各代生产种子,但低世代杂种出现高频瘪粒种子,胚珠败孕可能是其主要原因。该萝卜-芥蓝异源四倍体可以用作向油菜（B. napus L.,2n=38,AACC）转移萝卜基因的遗传桥梁。     

Verification of the introgression of Erianthus arundinaceus germplasm into sugarcane using molecular markers

Erianthus arundinaceus has a number of important agronomic traits including good ratooning ability, tolerance to both drought and water logging, disease resistance and vigour and is of interest as a potential source of parental germplasm to sugarcane breeders. However, to date, attempts to produce fertile hybrids between sugarcane (Saccharum spp.) and E. arundinaceus have been unsuccessful. Microsatellite markers that generated genus‐specific markers and were highly polymorphic within sugarcane were identified. The microsatellite markers and 5S rDNA PCR were used to screen intergeneric (F1) clones from Saccharum officinarum×E. arundinaceus crosses, and two Saccharum backcross progeny (BC1) populations derived from crosses between selected F1 clones and sugarcane (Saccharum spp.), to identify genuine Saccharum spp. BC1 progeny. The 5S rDNA PCR marker and highly polymorphic microsatellites with Erianthus‐specific bands confirmed the F1 parent of the two putative BC1 populations was a S. officinarum×E. arundinaceus hybrid and allowed the identification of the genuine BC1 progeny from selfs of the F1 parent. This is the first verification of BC1 progeny from an F1 intergeneric hybrid x sugarcane (Saccharum spp.) clone with molecular markers and confirms the introgression of E. arundinaceus germplasm into sugarcane. It should now be possible to exploit genes of value from E. arundinaceus in sugarcane breeding programmes.     

Uniqueness of the Gossypium mustelinum Genome Revealed by GISH and 45S rDNA FISH

Gossypium mustelinum [-(AD)4"] is one of five tetraploid species in Gossypium.Three pairs of nucleolar organizer regions (NOR) in (AD)4 were detected by FISH with 45S rDNA as a probe,they also were observed with genomic DNA (gDNA) from Gossypium D genome species as probes.Of the three NORs or GISH-NORs,one was super-major and other two were minor,which was distinctly different from other tetraploid cottons.     

花生的荧光显带和rDNA荧光原位杂交核型分析

建立花生准确而详细的核型对于阐明其起源和开展其基因组研究十分重要。本研究采用DAPI显带和5S、45S rDNA探针双色荧光原位杂交对花生有丝分裂中期染色体进行了分析。结果表明,花生的单倍基因组总长度为(81.06±3.74) μm,最长染色体为(4.72±0.15) μm,最短染色体为(2.62±0.14)μm;有15对染色体显示了着丝粒区DAPI⁺带,其中10对为强带,5对为弱带;有2对5S rDNA位点和5对45S rDNA位点,其中1对5S与1对45S位点同线。综合染色体测量数据、DAPI⁺带和rDNA杂交信号,对花生染色体进行了准确配对和排列,建立了详细的分子细胞遗传学核型。花生的核型公式为2n=4x=40=38m+2sm(SAT),核型不对称类型属于2A型。     

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。     

甘薯近缘野生种Ipomoea trifida(4x) GISH分析

以甘薯近缘野生种I. trifida (2x)为探针, 与I. trifida (4x) 2个株系“695104”和“697288”的体细胞染色体进行基因组荧光原位杂交, 结果显示, 2株系都与I. trifida (2x)有很近的亲缘关系, 但2株系的信号存在差异。“695104”几乎所有染色体整条都有均匀明亮的信号, 应为I. trifida (2x)基因组直接加倍而来;而 “697288”与“695104”不同, 虽然各条染色体也均有杂交信号, 但信号的区域与亮度有差异, 较为复杂, 可分为三种情况。第1种是整条染色体有均匀明亮的信号, 亮度与分布区域同“695104” , 有41条;第2种是几乎整条染色体有信号, 但亮度较第一种暗, 有14条;第3种为染色体部分区域有信号, 亮度较前二者更暗, 有5条。推测 “697288”是在加倍同时或之后又发生了基因组重组与部分变异。     

应用GISH与STS标记鉴定小麦-中间偃麦草抗黄矮病端体系

由大麦黄矮病毒引起的小麦黄矮病毒病是一个严重病害,至今在小麦属内还没有发现抗源。中间偃麦草2Ai-2染色体携带一个高抗黄矮病基因,对该基因的染色体臂定位将为制定抗病基因向小麦转移策略,筛选、开发特定的、与抗性连锁的分子标记的研究提供重要信息。本文对由小麦-中间偃麦草二体附加系Z6衍生的3个抗黄矮病端体系进行鉴定,通过分析端体的遗传构成、筛选与端体共分离的STS标记以确定端体在遗传上的染色体臂归属,从而明确BYDV抗病基因的染色体位置。以拟鹅冠草基因组[Pseudoroegneria strigosa (M. Bieb.) Löve,St]DNA为探针,中国春基因组(Triticum aestivum L., ABD) DNA作封阻分别对抗病亲本Z6及抗病端体系N530的根尖体细胞染色体进行原位杂交,结果表明,N530体细胞中有2个端体显示出与Z6中外源染色体2Ai-2短臂相似, 而与长臂不同的杂交信号。以小麦第2同源群的5个RFLP探针的DNA序列为基础,设计了6对PCR引物,对小麦-中间偃麦草二体异附加系、二体代换系和端体系进行扩增,结果表明,基于短臂探针psr126,psr131序列设计的2对引物,可在含有2Ai-2染色体及端体的抗黄矮病材料中特异扩增,而基于长臂探针psr112序列设计的1对引物,可在含有2Ai-2染色体的抗黄矮病材料中特异扩增,但不能在端体系进行特异扩增,证明外源端体为2Ai-2染色体的短臂。本研究不仅将黄矮病抗性基因定位于2Ai-2染色体的短臂上, 而且由RFLP探针psr126、psr131和psr112序列转化的标记STS₁₂₆ (sequence tagged site) STS₁₃₁和STS₁₁₂还可分别作为追踪2Ai-2染色体短臂和长臂的分子标记,用于抗病易位系辅助选择。     

利用原位杂交及CAPS标记分析芸薹属A、B和C基因组间的关系

以来源于Brassica rapa基因组(AA)的重复序列(151 bp)为探针, 分别同二倍体白菜型油菜(AA, 2n=20)、甘蓝(CC, 2n=18)和异源四倍体芥菜型油菜(AABB, 2n=36)的中期染色体杂交, 白菜型油菜和甘蓝的所有染色体上都有杂交信号, 芥菜型油菜的染色体上显示出20个明显的信号, 其余染色体上信号很弱或无, 可以区分出A和B基因组。对来源于油菜3个基本种与3个复合种FAE1基因进行CAPS分析表明, 3个基本种表现出不同的酶切式样, 用Mbo I和Msp I酶切表现出多态性, 基因组A和C非常相似, 而基因组B与A、C关系较远, 同时3个复合种也并不是2个基本种的简单相加, 表明异源四倍体在长期进化过程中可能发生了重排和重组。     

Embryo-rescue in the genus Tulipa L.; successful direct transfer of T. kaufmanniana Regel germplasm into T. gesneriana L.

Summary Embryo-rescue was studied as a means to overcome post-fertilization barriers in interspecific crosses in the genus Tulipa. With compatible T. gesneriana L. cultivar crosses, ovule culture was found to be superior to isolated embryo culture. Complete plantlet formation was possible from an embryo size of about 0.5 mm onwards.In the interspecific cross T. gesneriana x T. kaufmanniana, which is hampered by embryo breakdown, successful rescue of abortive embryos was demonstrated. Optimal embryo-rescue was achieved in cultures started seven to nine weeks after pollination. With cultures initiated at a later time, the rate of success decreased. A low number of germinative seeds were obtained after normal ripening of the seed pods, but by using ovule culture the efficiency of seedling formation could be increased dramatically.The ovule culture procedure will allow novel crosses and will offer new possibilities for the introduction of desirable genes into tulip cultivars.     

Cytological analyses of hybrids and derivatives of hybrids between durum wheat and Thinopyrum bessarabicum,using multicolour fluorescent GISH*

The wheat progenitors and other wild relatives continue to be important sources of genes for agronomically desirable traits, which can be transferred into durum wheat (Triticum turgidum; 2n = 4x = 28; AABB genomes) cultivars via hybridization. Chromosome pairing in durum × alien species hybrids provides an understanding of genomic relationships, which is useful in planning alien gene introgression strategies. Two durum cultivars, ‘Lloyd’ and ‘Langdon’, were crossed with diploid wheatgrass, Thinopyrum bessarabicum (2n = 2x = 14; JJ), to synthesize F₁ hybrids (2n = 3x = 21; ABJ) with Ph1. ‘Langdon’ disomic substitution 5D(5B) was used as a female parent to produce F₁ hybrids without Ph1, which resulted in elevation of pairing between durum and grass chromosomes – an important feature from the breeding standpoint. The F₁ hybrids were backcrossed to respective parental cultivars and BC₁ progenies were raised. ‘Langdon’ 5D(5B) substitution × Th. bessarabicum F₁ hybrids were crossed with normal ‘Langdon’ to obtain BC₁ progeny. Chromosome pairing relationships were studied in F₁ hybrids and BC₁ progenies using both conventional staining and fluorescent genomic in situ hybridization (fl‐GISH) techniques. Multicolour fl‐GISH was standardized for characterizing the nature and specificity of chromosome pairing: A–B, A–J and B–J pairing. The A–J and B–J pairing will facilitate gene introgression in durum wheat. Multicolour fl‐GISH will help in characterizing alien chromosome segments captured in the durum complement and in their location in the A and/or B genome, thereby accelerating chromosome engineering research.     

利用1RS特异标记和染色体原位杂交技术鉴定小麦1BL·1RS易位系

以小麦-黑麦1BL·1RS易位系(Kavkaz、山农030-1)、1AL·1RS易位系(Amigo)、荆州黑麦、八倍体小黑麦劲松49、1R-7R二体异附加系以及普通小麦中国春、辉县红、铭贤169、Chancellor等为材料,对65个黑麦1RS特异标记进行鉴定,从中筛选出8个稳定的标记,即NOR-1、SECA2/SECA3、SCSS30.2、Sec1Gene、Sec1Pro、ω-Sec-P1/P2、ω-Sec-P3/P4和IB-267,可用于检测1AL·1RS易位系或1BL·1RS易位系;另外3个特异标记O-SEC5′-A/O-SEC3′-R、IAG95-1和SCM-9可用于区别1RS来源不同的1AL·1RS和1BL·1RS易位系。利用这11个标记和染色体原位杂交技术对40份山东省近年育成小麦品种(系)进行检测,发现潍麦8号、鲁麦14、济宁13、山农664、山农优麦3号和烟农25为1BL·1RS易位系,而且是1RS的整臂易位系,未检测到1AL·1RS易位系和其他易位类型。     

Changes in the meiotic pairing behaviour of a winter wheat-winter barley hybrid maintained for a long term in tissue culture, and tracing the barley chromatin in the progeny using GISH and SSR markers

The aim of the present study was to produce backcross progenies in a new winter wheat (‘Asakaze komugi’) × winter barley (‘Manas’) hybrid produced in Martonvasar. As no backcross seeds were obtained from the initial hybrids, young inflorescences of the hybrids were used for in vitro multiplication in three consecutive cycles until a backcross progeny was developed. The chromosome constitution of the regenerated hybrids was analysed using genomic in situ hybridization (GISH) after each in vitro multiplication cycle. The seven barley chromosomes were present even after the third in vitro multiplication cycle but abnormalities were observed. Sixteen BC; plants containing, according to GfSH analysis, one to three complete barley chromosomes, two deletion barley chromosomes and a dicentric wheat‐barley translocation were grown to maturity from the single backcross progeny. The barley chromatin was identified using 20 chromosome‐specific barley SSR markers. All seven barley chromosomes were represented in the BC: plants. A deletion breakpoint at FL ±0,3 on the 5HL chromosome arm facilitated the physical localization of microsatellite markers.     

Alteration of the genomic composition of Solanum nigrum (+) potato backcross derivatives by somatic hybridization: selection of fusion hybrids by DNA measurements and GISH

Fusion experiments were performed with a first (BC₁‐6738) and a second (BC₂‐9017) generation backcross hybrid of 6x Solarium nigrum (+) 2x potato somatic hybrids with potato cultivars. Because no progeny was obtained from the BC₂ genotypes, alternative approaches were sought to overcome the sexual crossing barrier. Five potato genotypes, one of which contains the hygromycin resistance gene, were used in the fusion experiments. All vigorous regenerants were used for the estimation of nuclear DNA content using flow cytometry. Plants with a DNA content higher than that of the BC₁‐6738 or BC₂ genotypes were considered potential somatic hybrids. Forty‐nine potential somatic hybrids resulted from fusion experiments with BC₁‐6738, from which 20 grew vigorously in the greenhouse and flowered. After pollination with several 4x potato cultivars, eight genotypes produced seeded berries and five genotypes gave seedless berries. In addition, 11 of these 13 somatic hybrids were selected for genomic in situ hybridization (GISH) analysis to determine their genomic composition. Nine had exactly or approximately the expected number of 36 S. nigrum and 60 potato chromosomes. In one genotype, only 22 instead of 36 S. nigrum chromosomes were found and one potato chromosome was possibly missing. Only five potential somatic hybrids were detected among the 79 regenerants from BC₂‐9017 (+) 2x potato fusion experiments that were analysed by flow cytometry. Two of these hybrids were rather vigorous and did flower, but pollinations with potato have not yet set any berries.     

Application of C-banding and fluorescence in situ hybridization for the identification of the trisomics of Hordeum chilense

Hordeum chilense is a wild barley species that has a high degree of genetic variability and significant potential for use in plant breeding. To establish a series of trisomics in H. chilense (2n = 14), plants with 2n + 1 chromosome numbers were isolated from the progenies of selfed triploid plants. Based on both fluorescent in situ hybridization with pAs1 and pTa71 repetitive DNA probes and C-banding patterns, seven different trisomics were tentatively identified. Primary trisomic plants were for chromosomes 1H^ch, 4H^ch, 5H^ch, 6H^ch and 7H^ch. A secondary trisomic carrying a 5H^chS-5H^chS isochromosome as the extra chromosome and a trisomic for chromosome 3H^ch heterozygous for the 3H^chS-4H^chL and 4H^chS–3H^chL interchange were identified. The trisomic for chromosome 1H^ch cannot be phenotypically distinguished from the diploid. The rest of the trisomic types were distinguishable from the diploid by their morphological characteristics (relatively poor vigour, decreased size and shorter spikes) but they were morphologically indistinguishable from each other. The frequencies of trisomics among the progenies derived from self-fertilization of these aneuploids ranged from 10.7% to 37.5%, with an average frequency of 26.1%. This revised version was published online in July 2006 with corrections to the Cover Date.     

叶绿体5S rDNA ITS和matK基因序列应用于刚竹属植物系统分类的可行性分析

本文应用CTAB法从37种竹类植物中提取基因组DNA,根据在GenBank中发表的5S rDNAITS和matK基因设计引物,通过PCR进行扩增.结果表明:37种竹子5S rDNA ITS序列PCR产物大小约为450 bp,在刚竹属内部无长度上的差异,但是舒竹(Phyllostachys shuchengensis)与其他刚竹属植物相比,有一个位点的差异(由A变为C).因此,5S rDNA ITS在属下水平上无法提供较大的信息量,变异性较低,不适于刚竹属属下水平的系统分类研究.同样,选取37种竹子中3个竹种的,matK基因的PCR扩增产物直接进行测序,结果显示marK基因在竹亚科的属间长度上无差异,产物的长度约为1 500 bp,将测序结果进行同源性比对,在变异位点附近寻找多态性酶切位点,碱基序列上有一个位点的差异(BstN Ⅰ).PCR-RFLP结果显示,共有3种竹子在此位点发生变异分别为:浙江淡竹(Phyllostachys meyeri)、安吉金竹(Phyllostachysparvifolia)和黄古竹(Phyllostachys angusta).刚竹属植物的mark基因序列相当保守,片段中刚竹属间的绝对核苷酸差异不到1个,所提供的信息量不够充分.因此,叶绿体5S rDNA ITS和marK基因序列不适用于刚竹属植物系统分类研究,但其可能适合在属或属以上分类等级竹类植物的系统分类中应用.     

Screening and Positioning of Three Chromosome-specific BAC Clones in Gossypium raimondii

We screened a bacterial artificial chromosome(BAC) library of Gossypium barbadense acc. Pima 90-53 to identify chromosome-specific BAC clones. Using BAC-fluorescence in situ hybridization technology, we obtained three BAC clones specific to chromosome D₅01, D₅02, and D₅10 of Gossypium raimondii, which could be used as cytological markers for those three chromosomes. Comparative mapping of these three BACs between G. barbadense and in G. raimondii showed that these three BAC clones could also be used to identify chromosomes D_b01, D_b02, and D_b10 in G. barbadense. The position of the BAC clone 280G06 on chromosome 10 did not show colinearity between G. barbadense and G. raimondii, possibly because of chromosomal rearrangement.