Extensive ribosomal DNA amplification during Andean common bean (Phaseolus vulgaris L.) evolution

The extent of 5S and 45S ribosomal DNA (rDNA) variation was investigated in wild and domesticated common beans (Phaseolus vulgaris) chosen to represent the known genetic diversity of the species. 5S and 45S rDNA probes were localized on mitotic chromosomes of 37 accessions by fluorescent in situ hybridization (FISH). The two 5S rDNA loci were largely conserved within the species, whereas a high variation in the number of 45S rDNA loci and changes in position of loci and number of repeats per locus were observed. Domesticated accessions from the Mesoamerican gene pool frequently had three 45S rDNA loci per haploid genome, and rarely four. Domesticated accessions from Andean gene pool, particularly from the race Peru, showed six, seven, eight or nine loci, but seven loci were found in all three races of this gene pool. Between three and eight loci were observed in accessions resulting from crosses between Andean and Mesoamerican genotypes. The presence of two to eight 45S rDNA loci in wild common beans from different geographic locations indicates that the 45S rDNA amplification observed in the Andean lineage took place before domestication. Our data suggest that ectopic recombination between terminal chromosomal regions might be the mechanism responsible for this variation.     

Contrasting rDNA evolution in lima bean (Phaseolus lunatus L.) and common bean (P. vulgaris L., Fabaceae)

Phaseolus vulgaris has two 5S rDNA sites in chromosomes 6 and 10 and from two up to nine 45S rDNA sites depending on the accession. The presence of three 45S rDNA sites, in chromosomes 6, 9 and 10, is considered the ancestral state for the species. For P. lunatus, only one 5S and one 45S rDNA sites in distinct chromosomes were known. In order to investigate the homeologies among these rDNA-bearing chromosomes and the stability of the rDNA sites in P. lunatus, rDNA and P. vulgaris chromosome-specific probes were hybridized in situ to P. lunatus. The chromosomes bearing the 5S and the 45S rDNA of P. lunatus are homeologous to chromosomes 10 and 6 of P. vulgaris, respectively. In contrast to the common bean, no variation in the number of rDNA loci was detected, except for a duplication of the 5S rDNA in the same chromosome in a small group of cultivars. These results suggest that the 5S rDNA site in chromosome 10 and the 45S rDNA site in chromosome 6 represent the ancestral loci in the genus. The 5S rDNA site in chromosome 10 of P. vulgaris is located in the long arm, while in P. lunatus it is present in the short arm, suggesting the occurrence of a transposition or a pericentric inversion after separation of both lineages.     

中国普通菜豆形态性状分析及分类

对129份中国普通菜豆地方品种的形态性状进行分析,结果表明,8个性状共检测到35个变异类型,平均变异类型为4.375个,平均多态信息含量为0.5638。中国普通菜豆包括安第斯和中美两个基因库种质,中美洲基因库资源在参试资源中比重较大,但安第斯基因库资源遗传多样性水平高于中美基因库材料。由中美基因库向安第斯基因库渗透的天然杂交种质可为普通菜豆高产、优质、抗逆育种提供有价值的桥梁品种。     

Phylogenetic study on wild allies of Lima bean,Phaseolus lunatus (Fabaceae), and implications on its origin

An investigation was made of the phylogenetic relationships among wild accessions of Lima bean (Phaseolus lunatus) and wild allies of Mesoamerican and Andean origins, using electrophoresis of seed storage proteins and isozymes. Mesoamerican wild species are phylogenetically more distant fromP. lunatus than Andean species, and apparently belong to the tertiary gene pool of Lima bean. The Andean wild species, which are investigated for the first time, reveal a high similarity to the Lima bean, and particularly with its Mesoamerican gene pool. These Andean species probably constitute a secondary gene pool of Lima bean, and are thus of considerable interest in the context of genetic improvement of the crop. Based on these observations, an Andean origin is suggested for the Andean wild species and forP. lunatus. These results point out the importance of collecting and conserving AndeanPhaseolus germplasm.     

Chromosomal localization of 5S and 45S ribosomal DNA in species of Linum L. section Linum (syn=Protolinum and Adenolinum)

Fluorescence in situ hybridization (FISH) was for the first time used to study the chromosomal location of the 45S (18-2.5S-26S) and 5S ribosomal genes in the genomes of five flax species of the section Linum (syn. Protolinum and Adenolinum). In L. usitatissimum L. (2n = 30), L. angustifolium Huds. (2n = 30), and L. bienne Mill. (2n = 30), a major hybridization site of 45S rDNA was observed in the pericentric region of a large metacentric chromosome. A polymorphic minor locus of 45S rDNA was found on one of the small chromosomes. Sites of 5S rDNA colocalized with those of 45S rDNA, but direct correlation between signal intensities from the 45S and 5S rDNA sites was observed only in some cases. Other 5S rDNA sites mapped to two chromosomes in these flax species. In L. grandiflorum Desf. (2n = 16) and L. austriacum L. (2n = 18), large regions of 45S and 5S rDNA were similarly located on a pair of homologous satellite-bearing chromosomes. An additional large polymorphic site of 45S and 5S rDNA was found in the proximal region of one arm of a small chromosome in the L. usitatissimum. L. angustifolium, and L. bienne karyotypes. The other arm of this chromosome contained a large 5S rDNA cluster. A similar location of the ribosomal genes in the pericentric region of the pair of satellite-bearing metacentrics confirmed the close relationships of the species examined. The difference in chromosomal location of the ribosomal genes between flax species with 2n = 30 and those with 2n = 16 or 18 testified to their assignment to different sections. The use of ribosomal genes as chromosome markers was assumed to be of importance for comparative genomic studies in cultivated flax, a valuable crop species of Russia, and in its wild relatives.     

FISH分析栽培高粱×拟高粱、甜高粱×拟高粱的染色体行为

采用荧光原位杂交技术对45S rDNA在栽培高粱×拟高粱、甜高粱×拟高粱F1的有丝分裂和减数分裂染色体进行定位研究。在有丝分裂中期染色体上2个杂种分别检测到2个杂交信号, 在减数分裂粗线期、终变期、中期Ⅰ染色体上45S rDNA位于一个二价体上, 说明这两个杂种携带45S rDNA的染色体为同源染色体。根据45S rDNA位点随细胞减数分裂过程的位置变化, 表明这两个杂种染色体配对行为正常, 平均构型为2n=2x=20(10Ⅱ), 证明45S rDNA可作为染色体的一个识别指标间接地观察细胞减数分裂过程染色体的变化行为。     

Isozyme diversity, RFLP of the rDNA and phylogenetic affinities among cultivated Lima beans,Phaseolus lunatus (Fabaceae)

Genetic variation inPhaseolus lunatus (Lima bean) was investigated at isozyme and DNA levels. Sixty cultivated accessions, including representatives of the Mesoamerican and Andean gene pools and intermediate types, were analyzed for variability at 17 isozyme loci. Some accessions were also examined for restriction fragment length polymorphism (RFLP) at the rDNA level. These data were used to construct two dendrograms showing clear separation in two distinct groups corresponding to each of the gene pools and an intermediate one probably representing a transitional group.     

水稻45S rDNA和5S rDNA的染色体定位研究

45SrDNA和5SrDNA是水稻中与核糖体RNA合成有关的2个功能片段,有关这2个序列在水稻染色体上的位置,不同研究者的研究结果不尽相同,在获得水稻染色体清晰制片的基础上,通过FISH确定了45SrDNA序列位于水稻的第9号和第10号染色体的短臂末端,并且第9号染色体上的拷贝数多于第10号染色体,5SrDNA序列位于第11号染色体短臂靠近着丝点处。     

Visualization by atomic force microscopy and FISH of the 45S rDNA gaps in mitotic chromosomes of Lolium perenne

The mitotic chromosome structure of 45S rDNA site gaps in Lolium perenne was studied by atomic force microscope (AFM) combining with fluorescence in situ hybridization (FISH) analysis in the present study. FISH on the mitotic chromosomes showed that 45S rDNA gaps were completely broken or local despiralizations of the chromatid which had the appearance of one or a few thin DNA fiber threads. Topography imaging using AFM confirmed these observations. In addition, AFM imaging showed that the broken end of the chromosome fragment lacking the 45S rDNA was sharper, suggesting high condensation. In contrast, the broken ends containing the 45S rDNA or thin 45S rDNA fibers exhibited lower density and were uncompacted. Higher magnification visualization by AFM of the terminals of decondensed 45S rDNA chromatin indicated that both ends containing the 45S rDNA also exhibited lower density zones. The measured height of a decondensed 45S rDNA chromatin as obtained from the AFM image was about 55–65 nm, composed of just two 30-nm single fibers of chromatin. FISH in flow-sorted G2 interphase nuclei showed that 45S rDNA was highly decondensed in more than 90% of the G2/M nuclei. Our results suggested that a failure of the complex folding of the chromatin fibers occurred at 45S rDNA sites, resulting in gap formation or break.     

Microsatellite marker diversity in common bean (Phaseolus vulgaris L.)

A diversity survey was used to estimate allelic diversity and heterozygosity of 129 microsatellite markers in a panel of 44 common bean (Phaseolus vulgaris L.) genotypes that have been used as parents of mapping populations. Two types of microsatellites were evaluated, based respectively on gene coding and genomic sequences. Genetic diversity was evaluated by estimating the polymorphism information content (PIC), as well as the distribution and range of alleles sizes. Gene-based microsatellites proved to be less polymorphic than genomic microsatellites in terms of both number of alleles (6.0 vs. 9.2) and PIC values (0.446 vs. 0.594) while greater size differences between the largest and the smallest allele were observed for the genomic microsatellites than for the gene-based microsatellites (31.4 vs. 19.1 bp). Markers that showed a high number of alleles were identified with a maximum of 28 alleles for the marker BMd1. The microsatellites were useful for distinguishing Andean and Mesoamerican genotypes, for uncovering the races within each genepool and for separating wild accessions from cultivars. Greater polymorphism and race structure was found within the Andean gene pool than within the Mesoamerican gene pool and polymorphism rate between genotypes was consistent with genepool and race identity. Comparisons between Andean genotypes had higher polymorphism (53.0%) on average than comparisons among Mesoamerican genotypes (33.4%). Within the Mesoamerican parental combinations, the intra-racial combinations between Mesoamerica and Durango or Jalisco race genotypes showed higher average rates of polymorphism (37.5%) than the within-race combinations between Mesoamerica race genotypes (31.7%). In multiple correspondance analysis we found two principal clusters of genotypes corresponding to the Mesoamerican and Andean gene pools and subgroups representing specific races especially for the Nueva Granada and Peru races of the Andean gene pool. Intra population diversity was higher within the Andean genepool than within the Mesoamerican genepool and this pattern was observed for both gene-based and genomic microsatellites. Furthermore, intra-population diversity within the Andean races (0.356 on average) was higher than within the Mesoamerican races (0.302). Within the Andean gene pool, race Peru had higher diversity compared to race Nueva Granada, while within the Mesoamerican gene pool, the races Durango, Guatemala and Jalisco had comparable levels of diversity which were below that of race Mesoamerica.     

45S rDNA regions are chromosome fragile sites expressed as gaps in vitro on metaphase chromosomes of root-tip meristematic cells in Lolium spp

Background

In humans, chromosome fragile sites are regions that are especially prone to forming non-staining gaps, constrictions or breaks in one or both of the chromatids on metaphase chromosomes either spontaneously or following partial inhibition of DNA synthesis and have been well identified. So far, no plant chromosome fragile sites similar to those in human chromosomes have been reported.

Methods and Results

During the course of cytological mapping of rDNA on ryegrass chromosomes, we found that the number of chromosomes plus chromosome fragments was often more than the expected 14 in most cells for Lolium perenne L. cv. Player by close cytological examination using a routine chromosome preparation procedure. Further fluorescent in situ hybridization (FISH) using 45S rDNA as a probe indicated that the root-tip cells having more than a 14-chromosome plus chromosome fragment count were a result of chromosome breakage or gap formation in vitro (referred to as chromosome lesions) at 45S rDNA sites, and 86% of the cells exhibited chromosome breaks or gaps and all occurred at the sites of 45S rDNA in Lolium perenne L. cv. Player, as well as in L. multiflorum Lam. cv. Top One. Chromatin depletion or decondensation occurred at various locations within the 45S rDNA regions, suggesting heterogeneity of lesions of 45S rDNA sites with respect to their position within the rDNA region.

Conclusions

The chromosome lesions observed in this study are very similar cytologically to that of fragile sites observed in human chromosomes, and thus we conclude that the high frequency of chromosome lesions in vitro in Lolium species is the result of the expression of 45S rDNA fragile sites. Possible causes for the spontaneous expression of fragile sites and their potential biological significance are discussed.     

不同染色技术和双色荧光原位杂交技术对宽翅菘蓝（Isatis violascens Bunge）的细胞遗传学分析

为了解十字花科菘蓝属植物宽翅菘蓝（宽翅菘蓝,Isatis violascens Bunge）的染色体结构,本文利用45SrDNA和5SrDNA双色荧光原位杂交、银染技术和CMA3对宽翅菘蓝进行分子细胞遗传学研究。45SrDNA和5SrDNA荧光原位杂交结果显示宽翅菘蓝染色体上有1对45SrDNA信号和1对5SrDNA信号;银染结果显示其中期染色体有1对银染点;CMA3染色结果发现宽翅菘蓝中期染色体存在1对CMA3信号。     

45S rDNA和5S rDNA在南瓜、丝瓜和冬瓜染色体上的比较定位

首次利用荧光原位杂交和双色荧光原位杂交技术对45S和5S rDNA在南瓜(Cucurbita moschata Duch)、丝瓜(Luffa cylindrical Roem)、冬瓜(Benincasa hispida Cogn)的有丝分裂中期染色体上进行了物理定位分析。南瓜有5对45S rDNA位点, 2对5S rDNA位点; 丝瓜具有5对45S rDNA位点, 1对5S rDNA位点; 冬瓜具有2对45S rDNA位点, 1对5S rDNA位点, 5S rDNA位点与其中一对45S rDNA位点都位于7号染色体短臂上, 并在物理位置上紧密相邻。45S rDNA在这3种作物染色体上数目变化较大, 但在染色体上都倾向分布在短臂末端, 其分布模式较为一致。5S rDNA在这3种作物染色体上数目相对保守, 但在染色体上分布的位置变化较大。文中讨论了45S rDNA和5S rDNA在植物基因组中不同的进化趋势。     

Hybrid Weakness in Phaseolus vulgaris L. II. Disruption of Root-Shoot Integration

We have been examining the importance of the root system on shoot growth and development using a developmentally disabled hybrid of the common bean Phaseolus vulgaris L. Parental cultivars (P. Vulgaris cv. Redkloud of Mesoamerican origin, and P. vulgaris cv. Batt of Andean origin) grow normally, but crosses produce F1 hybrids exhibiting hybrid weakness associated with reduced root and shoot growth. In this study, applications of benzylaminopurine (BAP) to roots of F1 hybrids increased the number of root tips and leaves. Reciprocal grafting was used to study the effects of the root system on shoots. Grafting of roots of the Mesoamerican cultivar onto shoots of F1 hybrids increased the cytokinin concentrations in leaves of F1 hybrids and removed the characteristics associated with hybrid weakness. To determine whether factors in the xylem sap enhanced leaf growth, leaf discs were incubated on sap collected from Mesoamerican and Andean cultivars. Sap from Mesoamerican plants enhanced the growth of leaf discs excised from F1 hybrids more than sap collected from Andean cultivars. Estimates of the transport of zeatin riboside (ZR)–type cytokinins from roots of F1 hybrids indicated that transport out of hybrid roots was reduced compared with those transported out of Mesoamerican or Andean roots. Results suggest that ZR-type cytokinins are involved in hormonal integration between roots and shoots of P. vulgaris and that one of the barriers to hybridization between Andean and Mesoamerican landraces is related to hormone transport. Received October 15, 1998; accepted May 12, 1999     

不同染色技术和双色荧光原位杂交技术对宽翅菘蓝 （Isatis violascens Bunge）的细胞遗传学分析

为了解十字花科菘蓝属植物宽翅菘蓝(宽翅菘蓝,Isatis violascens Bunge)的染色体结构,本文利用45SrDNA和5SrDNA双色荧光原位杂交、银染技术和CMA3对宽翅菘蓝进行分子细胞遗传学研究。45SrDNA和5SrDNA荧光原位杂交结果显示宽翅菘蓝染色体上有1对45SrDNA信号和1对5SrDNA信号;银染结果显示其中期染色体有1对银染点;CMA3染色结果发现宽翅菘蓝中期染色体存在1对CMA3信号。     

慈姑45S rDNA和端粒序列检测及核型分析

以45S r DNA和拟南芥型端粒序列为探针对慈姑(Sagittaria trifolia L.)有丝分裂中期染色体进行单色和双色荧光原位杂交分析,并用银染方法检测慈姑45S r DNA位点的表达,最后结合染色体测量数据和45S r DNA杂交信号建立慈姑的核型。结果显示,慈姑的单倍基因组总长度为76.9±1.38μm,最长染色体为11.55±0.10μm,最短染色体为4.54±0.27μm;慈姑的核型公式为:2n=22=2m+2sm+14st+4t,核型不对称性参数CI、A1、A2、As K(%)、AI分别为19.86±11.06、0.72、0.27、78.82、15.29,核型属于Stebbins类型中的3B型。慈姑具有3对45S r DNA位点,分别位于第8、9、10号染色体的短臂末端。拟南芥型端粒序列的杂交信号出现在慈姑每一条染色体的长、短臂末端。银染检测到6个Ag-NOR和6个核仁,表明3对45S r DNA位点在间期核都有表达。本研究结果为药食兼用植物慈姑提供了分子细胞遗传学基础资料。     

Genome and Chromosome Dimensions of Lotus japonicus

Lotus Japonicus , Miyakojima MG-20 and Gifu B-129. The genome sizes of Miyakojima and Gifu were determined as 472.1 and 442.8 Mbp, respectively. Both the accessions were diploid (2n=12) and six chromosomes were identified and characterized based on the condensation patterns and the locations of rDNA loci. The obvious polymorphism observed in the genome size and the chromosome morphology between the two accessions, revealed specific accumulation of heterochromatin in Miyakojima or elimination in Gifu. The chromosomes L. japonicus were numbered according to their length. A quantitative chromosome map was also developed by the imaging methods using the digital data of the condensation pattern. 45S rDNA loci were localized on chromosomes A and F, and 5S rDNA locus was localized on chromosome A by fluorescence in situ hybridization (FISH). Identification of the chromosome and genome sizes and development of the quantitative chromosome map represent significant contribution to the L. japonicus genome project as the basic information. Received 29 August 2000/ Accepted in revised form 17 October 2000     

Individual chromosome assignment and chromosomal collinearity in Gossypium thurberi, G. trilobum and D subgenome of G. barbadense revealed by BAC-FISH

The experiment on individual chromosome assignments and chromosomal diversity was conducted using a multi-probe fluorescence in situ hybridization (FISH) system in D subgenome of tetraploid Gossypium barbadense (D(b)), G. thurberi (D(1)) and G. trilobum (D(8)), which the later two were the possible subgenome donors of tetraploid cottons. The FISH probes contained a set of bacterial artificial chromosome (BAC) clones specific to 13 individual chromosomes from D subgenome of G. hirsutum (D(h)), a D genome centromere-specific BAC clone 150D24, 45S and 5S ribosomal DNA (rDNA) clones, respectively. All tested chromosome orientations were confirmed by the centromere-specific BAC probe. In D(1) and D(8), four 45S rDNA loci were found assigning at the end of the short arm of chromosomes 03, 07, 09 and 11, while one 5S rDNA locus was successfully marked at pericentromeric region of the short arm of chromosome 09. In D(b), three 45S rDNA loci and two 5S rDNA loci were found out. Among them, two 45S rDNA loci were located at the terminal of the short arm of chromosomes D(b)07 and D(b)09, whilst one 5S rDNA locus was found situating near centromeric region of the short arm of chromosome D(b)09. The positions of the BAC clones specific to the 13 individual chromosomes from D(h) were compared between D(1), D(8) and D(b). The result showed the existence of chromosomal collinearity within D(1) and D(8), and as well between them and D(b). The results will serve as a base for understanding chromosome structure of cotton and polyploidy evolution of cotton genome and will provide bio-information for assembling the sequences of finished and the on-going cotton whole genome sequencing projects.     

45S rDNA在小麦及其近缘物种染色体上的分布

将染色体C-分带和原位杂交技术相结合,系统研究了45S rDNA在栽培一粒小麦、野生二粒小麦、普通小麦、大麦、簇毛麦、硬簇麦、六倍体燕麦及鹅观草等物种染色体上的分布情况。这些物种染色体的次缢痕区都有45S rDNA位点, 某些非随体染色体上也有45S rDNA位点分布。以小麦—鹅观草1Rk^#1二体附加系为材料,通过顺序C分带-FISH技术首次将一个45S rDNA定位到1Rk^#1染色体短臂末端。     

Isolation and characterization of a new repetitive DNA family recently amplified in the Mesoamerican gene pool of the common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L., Fabaceae)

The common bean (Phaseolus vulgaris) is one of the most important crop plants. About 50% of its genome is composed of repetitive sequences, but only a little fraction was isolated and characterized so far. In this paper, a new repetitive DNA family from the species, named PvMeso, was isolated and characterized in both gene pools of P. vulgaris (Andean and Mesoamerican) and related species. Two fragments, 1.7 and 2.3 kb long, were cloned from BAC 255F18, which has previously shown a repetitive pattern. The subclone PvMeso-31 showed a terminal block in chromosome 7. This subclone contains a 1,705 bp long, AT-rich repeat with small internal repeats and shares a 1.2 kb region with PvMeso-47, derived from the 2.3 kb fragment. The presence of this repetitive block was restricted to Mesoamerican accessions of the common bean. In P. acutifolius, P. leptostachyus and Andean P. vulgaris, only a faint, 2.3 kb fragment was visualized in Southern experiments. Moreover, in Mesoamerican accessions, two other fragments (1.7 kb and 3.4 kb) were strongly labelled as well. Taken together, our results indicate that PvMeso is a recently emerged, repeat family initially duplicated in chromosome 11, on ancestral Mesoamerican accession, and later amplified in chromosome 7, after the split of the two major gene pools of the common bean.