High-resolution flow karyotyping and chromosome sorting in Vicia faba lines with standard and reconstructed karyotypes

Flow cytometric analysis has been performed on chromosomes isolated from formaldehyde-fixed root tips in a Vicia faba (2n = 12) line with a standard (wild-type) karyotype and in six V. faba translocation lines with reconstructed karyotypes. The resolution of individual chromosome types on histograms of chromosome fluorescence intensity (flow karyotypes) depended on the type of fluorochrome used for chromosome staining. The highest degree of resolution was achieved with 4,6-diamidino-2-phenylindole (DAPI). The lower resolution obtained after staining with mithramycin A (MIT) and propidium iodide (PI) was probably due to the sensitivity of these stains to changes in chromatin structure induced by formaldehyde fixation. After the staining with DAPI, only 1 chromosome type could be discriminated in the line with a standard karyotype. In the translocation lines, the number of chromosome types resolved on flow karyotypes ranged from 2 in the G and the ACB lines to all (6) chromosome types in the EFK and EF lines. Refined flow karyotyping permitted the sorting of a total of 15 different chromosome types from five of the translocation lines. It is expected that flow sorting of chromosomes from reconstructed karyotypes will become a powerful tool in the study of nuclear genome organisation in V. faba.     

Common Mechanisms of Y Chromosome Evolution

Y chromosome evolution is characterized by the expansion of genetic inertness along the Y chromosome and changes in the chromosome structure, especially the tendency of becoming heterochromatic. It is generally assumed that the sex chromosome pair has developed from a pair of homologues. In an evolutionary process the proto-Y-chromosome, with a very short differential segment, develops in its final stage into a completely heterochromatic and to a great extends genetically eroded Y chromosome. The constraints evolving the Y chromosome have been the objects of speculation since the discovery of sex chromosomes. Several models have been suggested. We use the exceptional situation of the in Drosophila mirandato analyze the molecular process in progress involved in Y chromosome evolution. We suggest that the first steps in the switch from a euchromatic proto-Y-chromosome into a completely heterochromatic Y chromosome are driven by the accumulation of transposable elements, especially retrotransposons inserted along the evolving nonrecombining part of the Y chromosome. In this evolutionary process trapping and accumulation of retrotransposons on the proto-Y-chromosome should lead to conformational changes that are responsible for successive silencing of euchromatic genes, both intact or already mutated ones and eventually transform functionally euchromatic domains into genetically inert heterochromatin. Accumulation of further mutations, deletions, and duplications followed by the evolution and expansion of tandem repetitive sequence motifs of high copy number (satellite sequences) together with a few vital genes for male fertility will then represent the final state of the degenerated Y chromosome. This revised version was published online in July 2006 with corrections to the Cover Date.     

Chromosome banding patterns of the aye-aye,Daubentonia madagascariensis (primates,Daubentoniidae)

The aye-aye (Daubentonia madagascariensis) is an endangered prosimian primate found only on the island of Madagascar. It is the only extant representative of its family Daubentoniidae. The phylogenetic relationship of this species with other prosimians is unclear. Because a G-banded karyotype forDaubentonia has not previously been reported, blood for preparation of lymphocyte cultures was obtained from one of the four aye-ayes in captivity in the United States. The diploid chromosome number was 30. The karyotype consisted of 14 autosomal metacentrics, 10 autosomal submetacentrics, and 4 autosomal acrocentrics. The similarities between the G-banded chromosomes ofDaubentonia and those ofPropithecus, a member of the Indriidae, support the notion thatDaubentonia has a closer relationship with the Indriidae than with other Lemuriformes or the lorisoids.     

Flow karyotyping and sorting of Vicia faba chromosomes

Summary Chromosome suspensions were prepared from formaldehyde-fixed, synchronized Vicia faba root tips. After staining with the DNA intercalating fluorochrome propidium iodide, the suspensions were analysed with a flow cytometer. The resulting histograms of integral fluorescence intensity contained peaks similar to those of theoretical V.faba flow karyotypes. From V. Faba cv Inovec (2n = 12) only one peak, corresponding to a single chromosome type (metacentric chromosome), could be discriminated. However, it was found that the peak also contained doublets of acrocentric chromosomes. Bivariate analysis of fluorescence pulse area (chromosome DNA content) and fluorescence pulse width (chromosome length) was necessary to distinguish the metacentric chromosome. To achieve a high degree of purity, a two-step sorting protocol was adopted. During a working day, more than 25 000 metacentric chromosomes (corresponding to 0.2 g DNA) were sorted with a purity of more than 90%. Such chromosomes are suitable for physical gene mapping by in situ hybridization or via the polymerase chain reaction (PCR) and allow the construction of chromosome-specific DNA libraries. While it was only possible to distinguish and sort one chromosome type from V. Faba cv Inovec with the standard karyotype, it was possible to sort with a high degree of purity five out of six chromosome types of the line EFK of V. Faba, which has six pairs of morphologically distinct chromosomes. This result confirmed the possibility of using reconstructed karyotypes to overcome existing problems with the discrimination and flow sorting of individual chromosome types in plants.     

Genomic instability of the DYZ1 repeat in patients with Y chromosome anomalies and males exposed to natural background radiation.

We assessed genomic instability of 3.4 kb DYZ1 repeat arrays in patients encompassing prostate cancer (PC), cases of repeated abortion (RA) and males exposed to natural background radiation (NBR) using real-time PCR and fluorescence in situ hybridization (FISH). Normal males showed DYZ1 copies ranging from 3000 to 4300, RA, 0-2237; PC, 550; and males exposed to NBR, 1577-5700. FISH showed organizational variation of DYZ1 in these samples substantiating the data obtained from real-time PCR. Of the 10 RA samples, 7 were found to be affected of which, 5 showed deletion of 265 bp from nt 25 to 290 and 773 bp from 1347 to 2119 and 2 showed deletion of 275 bp from nt 3128 to 3402. Copy number variation of DYZ1 in these males correlated with genetic constrains/anomalies. Although precise mechanisms of genomic instability of DYZ1 remains unclear, we construe that this repeat plays a critical role in maintaining the structural integrity of the Y chromosome, possibly by absorbing the load of mutations. This may be used as a marker system to analyze genetic integrity of the DYZ1 repeat array(s) across the spectrum of patients.     

Flow-cytometric characterization and sorting of plant chromosomes

Summary Flow cytometric measurements of DNA frequency distribution were used to follow the synchronization process in suspension cells from Haplopappus gracilis (2n=4). Metaphase chromosomes were isolated from these synchronized cells and both the acro- and metacentric chromosomes were sorted by flow cytometry based on the different DNA contents. Possible applications of this procedure in fundamental genetics as well as practical plant breeding are discussed.     

Accumulation of chloroplast DNA sequences on the Y chromosome of Silene latifolia

Silene latifolia is a model dioecious plant with heteromorphic sex chromosomes. The Y chromosome is the largest in this species. Theoretical models propose an accumulation of repetitive DNA sequences in non-recombining parts of the Y chromosome. In this study, we isolated a BAC7H5 clone preferentially hybridizing to the Y chromosome of S. latifolia. Sequence analysis revealed that this BAC7H5 contains part of the chloroplast genome, indicating that these chloroplast sequences have accumulated on the Y chromosome and also may contribute to its large size. We constructed Y chromosome- and X chromosome-specific libraries and screened them to find Y- and/or X-linked copies of chloroplast sequences. Sequence analysis revealed higher divergence of a non-genic region of the chloroplast sequences located on the Y chromosome while genic regions tested showed only very low (max 0.9%) divergence from their chloroplast homologues.     

A strategy for generation and balancing of autosome: Y chromosome translocations

We describe a method for generation and maintenance of translocations that move large autosomal segments onto the Y chromosome. Using this strategy we produced (2;Y) translocations that relocate between 1.5 and 4.8 Mb of the 2^nd chromosome.. All translocations were easily balanced over a male-specific lethal 1 (msl-1) mutant chromosome. Both halves of the translocation carry visible markers, as well as P-element ends that enable molecular confirmation. Halves of these translocations can be separated to produce offspring with duplications and with lethal second chromosome deficiencies . Such large deficiencies are otherwise tedious to generate and maintain.     

国产黄连属植物的染色体核型分析

中国黄连属6种1变种分属叶掌状三全裂和叶掌状五全裂2个类群,前者包括三角叶黄连、峨眉黄连、云南黄连、黄连和其变种短萼黄连;而分布于滇东南的五裂黄连和产于台湾的五叶黄连则归于后者。采用根尖压片法和卡宝品红染色法,对除五叶黄连外的中国黄连属5种1变种,以及日本黄连进行染色体核型比较分析,从细胞学角度为探讨中国黄连属植物的系统分类提供新的线索和证据。结果表明:(1)五裂黄连(2n=2x=18=2M+16m)、短萼黄连(2n=2x=18=8m+10sm)和日本黄连(2n=2x=18=12m+6sm)的染色体数目和核型均为首次报道。(2)7个材料的染色体基数均为9,除三角叶黄连为三倍体外,其余均为二倍体。(3)叶为掌状三全裂的二倍体种核型一致,为2A型,染色体类型以及不对称系数均很相似;叶为掌状五全裂的五裂黄连与五叶黄连的核型更接近,为1A型,核型特征的共性表明了这2个类群的自然属性。(4)三倍体三角叶黄连的不对称程度较高,核型为3A型,其染色体大小与峨眉黄连最接近。(5)根据核型不对称程度和染色体大小,结合地理分布,推测叶掌状五全裂种为本属的原始类群。     

Y Chromosome Diversity in Brazilians: Switching Perspectives from Slow to Fast Evolving Markers

We have previously shown that the Y chromosomes of ‘white’ Brazilians have their immediate geographical origin in Europe, with low frequency of sub-Saharan African chromosomes and virtual absence of Amerindian contribution. The typing of slow evolving polymorphisms on the Y chromosome also revealed no differences between Brazilians and Portuguese, the bulk of European immigrants to Brazil, and even among Brazilians from distinct regions of Brazil, the latter being in sharp contrast with mtDNA data. In order to test if the lack of differentiation is a sex-biased and not a marker-biased phenomenon, we decided to study faster evolving Y chromosome markers in samples from Brazil and Portugal previously studied. The population structure revealed by this work confirmed that there were indeed no significant differences between Brazil and Portugal and no population differentiation within the four geographical regions of Brazil, suggesting that this phenomenon is unrelated to the nature of the markers typed. Nevertheless the fast evolving markers did uncover a higher within population diversity in Brazil than Portugal, which could be explained by the input of diverse European Y chromosomes carried by several migration waves to Brazil. Our present data highlight the significance of typing and combining Y markers that evolve according to distinct mutational paces to usefully assess the levels of diversity in a given population, and can be applied in the study of populations derived from distinct geographical origins such as the Brazilians.     

Modeling monkeys: A comparison of computer-generated and naturally occurring foraging patterns in two species of neotropical primates

We present a series of computer-generated foraging models (random movement, olfactory navigation, and spatial memory) designed to examine the manner in which sensory cues and cognitive skills might be used by rainforest monkeys to locate patchily distributed feeding sites. These simulations are compared with data collected in the Amazon Basin of northeastern Peru on the foraging patterns of two species of neotropical primates, the moustached tamarin monkey (Saguinus mystax) and the saddle-back tamarin monkey (Saguinus fuscicollis). The results indicate that, although tamarins may rely on olfactory cues to locate nearby feeding sites, their foraging patterns are better explained by an ability to maintain a detailed spatial map of the location and distribution of hundreds of feeding trees in their home range. There is evidence that such informationis retained for a period of at least several weeks and is used to minimize the distance traveled between widely scattered feeding sites. The use of computer simulations provides a powerful research tool for generating predictive models regarding the role of memory and sensory cues in animal foraging patterns.     

3种香薷属植物染色体数目与核型分析

参照植物根尖细胞学研究的方法标准,对香薷属3种(5个居群)植物进行核形态学分析。结果表明:(1)从染色体数目看,密花香薷2居群染色体数目2n=16;野苏子2居群染色体数目2n=20,染色体数目和倍性与前人报道的一致;毛穗香薷染色体数目2n=10为首次报道。(2)聚类分析结果显示,3种(5居群)植物中野苏子和密花香薷亲缘关系较近;结合现有报道数据分析表明,该属植物仅有2种倍性(二倍体和四倍体),且二倍体占主导地位。(3)核型参数分析表明:密花香薷的稻城无名山居群1核型公式为2n=2x=16=14m+2sm,居群2为2n=2x=16=16m,着丝粒指数(CI)分别为39.57和42.32,不对称系数AI值分别为2.75和2.87,核型不对称性都为1A型;毛穗香薷的核型公式为2n=2x=10=10m,着丝粒指数(CI)为41.76,不对称系数AI值为5.25,核型不对称性为1B型;野苏子的昆明西山居群核型公式为2n=2x=20=14m+6sm,聂拉木樟木沟居群为2n=2x=20=16m+4sm,着丝粒指数(CI)分别为38.49和40.97,不对称系数AI值为4.20和4.30,核型不对称性为1B型和2B型。     

Gene Finding on the Y: Fruitful Strategy in Drosophila does not Deliver in Anopheles

The Anopheles gambiae genome project yielded almost complete sequences for the autosomes and for a large part of the X chromosome, however, no information for the Y chromosome was obtained. Yet, by design, fragmented Y chromosome sequences should be present in the resulting assembly. Here we report the search for Anopheles Y chromosome genes using a strategy successfully applied for identification of Y genes in Drosophila. A complete set of the unmapped scaffolds was targeted in a broad TBLASTN search using both A. gambiae predicted genes and all proteins from nr database as query sequences. After filtering of the BLAST report, we selected 181 scaffolds possibly containing fragments of Y chromosome genes to experimentally test their Y-linkage. Surprisingly, none of the tested sequences appeared to originate from the Y chromosome. Several factors could account for the failure to detect Y genes, including their different organization in A. gambiae compared to Drosophila and the suboptimal quality of the assembly and annotation of the Anopheles genome. Regardless of the cause, our results illuminate problems associated with the genome analysis of outbred organisms.     

黄果龙葵和龙葵染色体制片优化及核型分析

该实验以黄果龙葵和龙葵的根尖为实验材料,进行不同的预处理、固定和解离,确定出各种材料适合于核型分析的制片方法。结果表明:龙葵于15℃条件下经0.05%秋水仙素预处理2.5h,固定后用1mol/L HCl酸解后,染色观察,得到的染色体分散,易于染色体计数和形态观察。用此方法对黄果龙葵和龙葵进行核型分析,结果发现:黄果龙葵和龙葵都属于小型染色体,黄果龙葵为四倍体,核型公式为K(4n)=48=4sm+44m,核型不对称系数为56.22%,属于2B核型。龙葵为六倍体,核型公式为K(6n)=72=72m,核型不对称系数为55.89%,属于1B核型。     

Chromosome profile ofLeishmania donovani: Interstrain and interspecific variations

The genome ofLeishmania donovani AG83, a virulent strain causing kala-azar, was resolved into 29 chromosomal bands by pulsed field gel electrophoresis (pFGE) under standardized conditions. Comparison of the karyotype with those of other strains and species revealed variations. By Southern hybridization, specific genes were localized to individual chromosomes. Twenty-two copies ofβ-tubulin genes are located on band 27 (1.63 Mb); minor copies are present in band 16 (850 kb) and band 9 (650 kb). Aβ-tubulin related nontranscribed locus was isolated from a genomic library and shown to contain repetitive sequences hybridizing throughout the genome. Single chromosomes contain multicopy clusters of gp63 and rnini-exon-derived RNA genes, but interspecific variations were observed in each case. The results emphasize the importance of using a standard reference strain ofLeishmania donovani for coordinated genome mapping of this clinically important organism.     

Isolation and identification of Triticum aestivum L. em. Thell. cv Chinese Spring-T. peregrinum Hackel disomic chromosome addition lines

Analyses of RFLPs, isozymes, morphological markers and chromosome pairing were used to isolate 12 Triticum aestivum cv Chinese Spring (genomes A, B, and D)-T. peregrinum (genomes S^v and U^v) disomic chromosome addition lines. The evidence obtained indicates that each of the 12 lines contains an intact pair of T. peregrinum chromosomes. One monosomic addition line, believed to contain an intact 6S^v chromosome, was also isolated. A CS-7U^v chromosome addition line was not obtained. Syntenic relationships in common with the standard Triticeae arrangement were found for five of the seven S^v genome chromosomes. The exceptions were 4S^v and 7S^v. A reciprocal translocation exists between 4S¹ and 7S¹ in T. longissimum and evidence was obtained that the same translocation exists in T. peregrinum. In contrast, evidence for syntenic relationships in common with the standard Triticeae arrangements were found for only one U^v chromosome of T. peregrinum.; namely, chromosome 2U^v. All other U^v genome chromosomes are involved in at least one translocation, and the same translocations were found in the U genome of T. umbellulatum. Evidence was also obtained indicating that the centromeric regions of 4U and 4U^v are homoeologous to the centromeric regions of Triticeae homoeologous group-6 chromosomes, that the centromeric regions of 6U and 6U^v are homoeologous to the centromeric regions of group-4 chromosomes, and that 4U and 4U^v are more closely related overall to Triticeae homoeologous group-6 chromosomes than they are to group-4 chromosomes.     

新疆特色药食两用植物恰玛古内生放线菌的分离与鉴定

恰玛古（Qamgur, Brassica rapa L.）内生菌的研究主要集中在内生真菌,内生放线菌的研究报道较少。通过研究新疆药食两用植物恰玛古内生放线菌多样性,以期发现产新活性物质的放线菌或新种放线菌,为研究微生物药物奠定基础。从恰玛古根、茎和叶三个部位分离培养获得内生放线菌,对其菌落与个体形态进行观察,并利用序列测定方法进行鉴定,以获取其分类地位。从恰玛古三个部位共分离得到17株内生放线菌,其中12株为革兰氏阳性杆菌,3株为革兰氏阳性球菌,2株为革兰氏阳性丝状菌;17株内生放线菌分属于红球菌属（Rhodococcus）、拟诺卡氏菌属（Nocardiopsis）、链霉菌属（Streptomyces）、短杆菌属（Brevibacterium）、小短杆菌属（Brachybacterium）、两面神菌属（Janibacter）和微杆菌属（Microbacterium）。从新疆药食两用植物恰玛古中分离获得17株内生放线菌以稀有放线菌为主。     

Isolation and identification of threeCellulomonas spp. from forest soils

Three stains of cellulose-degrading, aerobic, mesophilic bacteria were isolated from forest soils and, from their cultural, biochemical, and physiological characteristics, they were identified as members of the genusCellulomonas. Unusual biochemical characteristics, e.g. urea hydrolysis, were observed in two isolates. These characteristics have not previously been reported for cellulomonads and may prove to be significant for characterization ofCellulomonas spp. The isolates were able to use urea as a N source in cellulose fermentation. All three strains were motile, with one to four peritrichous flagella observed. Amino acid and polysaccharide composition of the cell walls of the three isolates were identical.     

Inheritance of chromosome length polymorphisms inCoprinus cinereus

The sexually compatible strains ofCoprinus cinereus 5302 and Dd 13 revealed chromosome length polymorphisms in their electrophoretic karyotypes. The dikaryon derived from two monokaryons contained a mixture of the two electrophoretic patterns. F₁ progenies were isolated by crossingC. cinereus 5302 and Dd 13 strains and it showed unique karyotypes. Chromosome length polymorphisms of both parental strains were inherited at random in the F₁ progenies. As a result, several novel electrophoretic karyotypes which had not been observed in either parental strains were found in the F₁ progeny. The rDNA probe hybridized with one chromosome in both parental strains, with two chromosomes in the hybridization pattern of both parental strains in the dikaryon, and with one to two chromosomes in the F₁ progenies. The relation between mating type and hybridization pattern has thus not been made clear in the case of F₁ progeny.     

Bt新菌株资源的采集与鉴定

【目的】寻找对致倦库蚊高效的苏云金芽胞杆菌(Bacillus thuringiensis,Bt)杀蚊菌株新资源。【方法】从福建省的武夷山自然保护区、建阳、建瓯、浦城等多个地区采集土壤样品,采用热处理法从土壤中分离Bt菌株,并测定其对致倦库蚊活性的效果。【结果】从125份土壤样品中分离出71株Bt菌株,经生物测定得到4株对致倦库蚊有效菌株(QQ13、QQ42、QQ66和QQ92)。其中,QQ66和QQ92有较高的毒性,均有几丁质酶基因,没有检测到cry1、cry1Ⅰ、cry2、cry4、cry5、cry6、cry7、cry8、cry9、cry10和cry11基因,在75～100 ku处各有一条杀虫晶体蛋白条带。【结论】采集和鉴定到的Bt新菌株资源将对致倦库蚊的生物防治起到促进作用。