Comparative physical mapping of the 18S-5.8S-26S rDNA in three sorghum species.

The physical locations of the 18S-5.8S-26S rDNA sequences were examined in three sorghum species by fluorescence in situ hybridization (FISH) using biotin-labeled heterologous 18S-5.8S-26S rDNA probe (pTa71). Each 18S-5.8S-26S rDNA locus occurred at two sites on the chromosomes in Sorghum bicolor (2n = 20) and S. versicolor (2n = 10), but at four sites on the chromosomes of S. halepense (2n = 40) and the tetraploid S. versicolor (2n = 20). Positions of the rDNA loci varied from the interstitial to terminal position among the four accessions of the three sorghum species. The rDNA data are useful for investigation of chromosome evolution and phylogeny. This study excluded S. versicolor as the possible progenitor of S. bicolor.     

基于Adh1基因分析高粱属的系统进化关系

高梁属中有重要的粮食作物和优良牧草, 也有农业生产上的重要杂草。文章旨在进一步从分子水平阐明高梁属种间的系统进化关系, 为有效利用种质资源进行分子育种改良作物品质提供理论依据, 并明确检疫性杂草的分类地位。根据二色高粱(Sorghum bicolor)的Adh1全基因序列(GenBank登录号: AF050456)设计引物, 扩增并测定黑高粱(S. almum)、假高粱(S. halepense)、丝克高粱(S. silk)和苏丹草(S. sudanense)共计8个植物材料约2 000 bp的Adh1基因部分序列, 结合GenBank中其他24个Sorghum属的同源序列, 以Cleistachne sorghoides的对应序列为外群, 进行了高梁属的亲缘关系分析, 用MP、ML和NJ法分别构建了分子进化树, 得到了基本相同的拓扑结构。结果显示: (1) 高梁属可明显分为三大支, 一支是蒴柄高梁(Chaetosorghum)和异高梁(Heterosorghum)二个亚属, 一支是优高梁亚属(Eusorghum), 这两个分支包含2n=20、40, 染色体较小的种类, 另一分支包括拟高梁 (Parasorghum)和有柄高梁(Stiposorghum)两个亚属, 包含2n=10的种类和它们的多倍体近缘种, 染色体相对较大; (2) S. almum的Adh1基因表现出明显的地理分化; (3) Parasorghum亚属的S. pur-pureosericeum和多色高粱(S. versicolor)、光高粱(S. nitidum)和S. leiocladum聚在一起, 而该亚属中的S. mata-rankense、S. grande、S. timorense却与亚属Stiposorghum的种聚在一起, 表现出更近的亲缘关系; (4) S. mac-rospermum和S. laxiflorum之间具有比其他高梁属种更近的亲缘关系。     

Targeted analysis of orthologous phytochrome A regions of the sorghum,maize, and rice genomes using comparative gene-island sequencing

A "gene-island" sequencing strategy has been developed that expedites the targeted acquisition of orthologous gene sequences from related species for comparative genome analysis. A 152-kb bacterial artificial chromosome (BAC) clone from sorghum (Sorghum bicolor) encoding phytochrome A (PHYA) was fully sequenced, revealing 16 open reading frames with a gene density similar to many regions of the rice (Oryza sativa) genome. The sequences of genes in the orthologous region of the maize (Zea mays) and rice genomes were obtained using the gene-island sequencing method. BAC clones containing the orthologous maize and rice PHYA genes were identified, sheared, subcloned, and probed with the sorghum PHYA-containing BAC DNA. Sequence analysis revealed that approximately 75% of the cross-hybridizing subclones contained sequences orthologous to those within the sorghum PHYA BAC and less than 25% contained repetitive and/or BAC vector DNA sequences. The complete sequence of four genes, including up to 1 kb of their promoter regions, was identified in the maize PHYA BAC. Nine orthologous gene sequences were identified in the rice PHYA BAC. Sequence comparison of the orthologous sorghum and maize genes aided in the identification of exons and conserved regulatory sequences flanking each open reading frame. Within genomic regions where micro-colinearity of genes is absolutely conserved, gene-island sequencing is a particularly useful tool for comparative analysis of genomes between related species.     

Host-plant preference and oviposition responses of the sorghum midge, Stenodiplosis sorghicola (Coquillett) (Dipt., Cecidomyiidae) towards wild relatives of sorghum

Sorghum midge, Stenodiplosis ( Contarinia ) sorghicola (Coquillett) is an important pest of grain sorghum world-wide. Considerable progress has been made in screening and breeding for resistance to sorghum midge. However, some of the sources of resistance have become susceptible to sorghum midge in Kenya, in eastern Africa. Therefore, the wild relatives of Sorghum bicolor were studied as a possible source of new genes conferring resistance to sorghum midge. Midge females did not lay eggs in the spikelets of Sorghum amplum , Sorghum bulbosum , and Sorghum angustum compared to 30% spikelets with eggs in Sorghum halepense when infested with five midge females per panicle under no-choice conditions. However, one egg was laid in S. amplum when infested with 50 midges per panicle. A larger number of midges were attracted to the odours from the panicles of S. halepense than to the panicles of Sorghum stipoideum , Sorghum brachypodum , S. angustum , Sorghum macrospermum , Sorghum nitidium , Sorghum laxiflorum , and S. amplum in dual-choice olfactometer tests. The differences in midge response to the odours from S. halepense and Sorghum intrans were not significant. Under multi-choice conditions, when the females were also allowed a contact with the host, more sorghum midge females were attracted to the panicles of S. bicolor compared with S. amplum , S. angustum , and S. halepense . In another test, numerically more midges responded to the panicles of IS 10712 compared with S. halepense , whereas the differences in midge response to the panicles of ICSV 197 ( S. bicolor ) and S. halepense were not apparent, indicating that S. halepense is as attractive to sorghum midge females as S. bicolor . The wild relatives of sorghum (except S. halepense ) were not preferred for oviposition, and they were also less attractive to the sorghum midge females. Thus, wild relatives of sorghum can prove to be an alternative source of genes for resistance to sorghum midge.     

Phylogenetic relationships of sorghum taxa inferred from mitochondrial DNA restriction fragment analysis.

To elucidate the evolutionary history and affinity of sorghum species, 41 sorghum taxa were analyzed using variability in mitochondrial DNA. Analysis of species relationships at the molecular level can provide additional data to supplement the existing classification based on morphological characters and may also furnish unexpected but useful information. Total DNA extracted from each of the sorghum accessions was digested with each of five restriction enzymes, BamHI, HindIII, EcoRI, EcoRV, and XbaI, and probed with five mitochondrial DNAs cloned from Sorghumbicolor. A total of 180 restriction fragments was detected by the 25 probe-enzyme combinations. Forty-three fragment bands were phylogenetically informative. Multiple correspondence analysis was performed to visualize associations among the accessions and suggested that section Eusorghum species may be divided into four groups, with Sorghumlaxiflorum (section Heterosorghum) and Sorghumnitidum (section Parasorghum) appearing as outliers. A phylogenetic tree was assembled from mitochondrial restriction fragment data. The taxa analyzed formed three major groups comprising section Heterosorghum (group I), section Parasorghum (group II), and all accessions in section Eusorghum (group III). Group III is further divided into four groups: (i) two sweet sorghums and shattercane; (ii) Sorghumhalepense, Sorghummiliaceum, Sorghumhewisonii, Sorghumaethiopicum, Sorghumverticilliflorum, and S. bicolor, including Sorghumsudanense (sudangrass), the Chinese Kaoliangs, and a number of commercial sorghum inbreds from the U.S.A.; (iii) Sorghumpropinquum; and (iv) Sorghumarundinaceum, Sorghumniloticum, Sorghumalmum, Sorghumcontroversum, and the Chinese material C-401 and 5-27. Results indicate that the analysis of fragmented mitochondrial DNA was diagnostic and useful in sorghum phylogenetic and taxonomic research at the species, subspecies, and race levels, and can complement results from those analyses using nuclear ribosomal DNA and chloroplast DNA that effectively distinguish taxa at species and genus levels. Key words : Sorghum, mitochondrial DNA, phylogeny, restriction fragment.     

Cloning and characterization of repetitive DNA sequences from genomes of Oryza minuta and Oryza australiensis

The value of genome-specific repetitive DNA sequences for use as molecular markers in studying genome differentiation was investigated. Five repetitive DNA sequences from wild species of rice were cloned. Four of the clones, pOm1, pOm4, pOmA536, and pOmPB10, were isolated from Oryza minuta accession 101141 (BBCC genomes), and one clone, pOa237, was isolated from Oryza australiensis accession 100882 (EE genome). Southern blot hybridization to different rice genomes showed strong hybridization of all five clones to O. minuta genomic DNA and no cross hybridization to genomic DNA from Oryza sativa (AA genome). The pOm1 and pOmA536 sequences showed cross hybridization only to all of the wild rice species containing the C genome. However, the pOm4, pOmPB10, and pOa237 sequences showed cross hybridization to O. australiensis genomic DNA in addition to showing hybridization to the O. minuta genomic DNA.     

Repetitive DNA variation and pivotal-differential evolution of wild wheats.

Several polyploid species in the genus Triticum contain a U genome derived from the diploid T. umbellulatum. In these species, the U genome is considered to be unmodified from the diploid based on chromosome pairing analysis, and it is referred to as pivotal. The additional genome(s) are considered to be modified, and they are thus referred to as differential genomes. The M genome derived from the diploid T. comosum is found in many U genome polyploids. In this study, we cloned three repetitive DNA sequences found primarily in the U genome and two repetitive DNA sequences found primarily in the M genome. We used these to monitor variation for these sequences in a large set of species containing U and M genomes. Investigation of sympatric and allopatric accessions of polyploid species did not show repetitive DNA similarities among sympatric species. This result does not support the idea that the polyploid species are continually exchanging genetic information through introgression. However, it is also possible that repetitive DNA is not a suitable means of addressing the question of introgression. The U genomes of both diploid and polyploid U genome species were similar regarding hybridization patterns observed with U genome probes. Much more variation was found both among diploid T. comosum accessions and polyploids containing M genomes. The observed variation supports the cytogenetic evidence that the M genome is more variable than the U genome. It also raises the possibility that the differential nature of the M genome may be due to variation within the diploid T. comosum, as well as among polyploid M genome species and accessions.     

Isolation and characterization of repetitive DNA sequences from Panax ginseng

Repetitive sequences constitute a significant component of most eukaryotic genomes, and the isolation and characterization of repetitive DNA sequences provide an insight into the organization and evolution of the genome of interest. We report the isolation and characterization of the major classes of repetitive sequences from the genome of Panax ginseng. The isolation of repetitive DNA from P. ginseng was achieved by the reannealing of chemically hydrolyzed (200 bp-1 kb fragments) and heat-denatured genomic DNA to low C(o)t value. The low C(o)t fraction was cloned, and fifty-five P. ginseng clones were identified that contained repetitive sequences. Sequence analysis revealed that the fraction includes repetitive telomeric sequences, species-specific satellite sequences, chloroplast DNA fragments and sequences that are homologous to retrotransposons. Two of the retrotransposon-like sequences are homologous to Ty1/ copia-type retroelements of Zea mays, and six cloned sequences are homologous to various regions of the del retrotransposon of Lilium henryi. The del retrotransposon-like sequences and several novel repetitive DNA sequences from P. ginseng were used to differentiate P. ginseng from P. quinquefolius, and should be useful for evolutionary studies of these disjunct species.     

Isolation of A/D and C genome specific dispersed and clustered repetitive DNA sequences from Avena sativa.

DNA gel-blot and in situ hybridization with genome-specific repeated sequences have proven to be valuable tools in analyzing genome structure and relationships in species with complex allopolyploid genomes such as hexaploid oat (Avena sativa L., 2n = 6x = 42; AACCDD genome). In this report, we describe a systematic approach for isolating genome-, chromosome-, and region-specific repeated and low-copy DNA sequences from oat that can presumably be applied to any complex genome species. Genome-specific DNA sequences were first identified in a random set of A. sativa genomic DNA cosmid clones by gel-blot hybridization using labeled genomic DNA from different Avena species. Because no repetitive sequences were identified that could distinguish between the A and D gneomes, sequences specific to these two genomes are refereed to as A/D genome specific. A/D or C genome specific DNA subfragments were used as screening probes to identify additional genome-specific cosmid clones in the A. sativa genomic library. We identified clustered and dispersed repetitive DNA elements for the A/D and C genomes that could be used as cytogenetic markers for discrimination of the various oat chromosomes. Some analyzed cosmids appeared to be composed entirely of genome-specific elements, whereas others represented regions with genome- and non-specific repeated sequences with interspersed low-copy DNA sequences. Thus, genome-specific hybridization analysis of restriction digests of random and selected A. sativa cosmids also provides insight into the sequence organization of the oat genome.     

Representational difference analysis reveals genomic differences between Q. robur and Q. suber: implications for the study of genome evolution in the genus Quercus

Very similar genome sizes, similar karyotypes and heterochromatin organisation, and identical number/position of ribosomal loci characterise the common oak (Q. robur) and the cork oak (Q. suber), two distantly related oak species. Representational Difference Analysis (RDA) was used to subtract the genome of Q. suber from the genome of Q. robur in order to search for genome differentiation. A library of 400 clones (bearing RDA fragments) representing genome differences between the two species was obtained. Seven Q. robur-specific DNA sequences were analysed with respect to their molecular and chromosome organisation. All belong to the dispersed repetitive component of the genome, as revealed by Southern hybridisation and in situ hybridisation. They are present in the Q. robur genome in between 100 and 700 copies, and are distributed along the length of almost all chromosomes. A search for homologies between RDA fragments and sequences in Genbank revealed similarities of all RDA fragments with known retrotransposons. The RDA fragments were also tested for their presence/absence in the genomes of six additional oak species belonging to different phylogenetic groups, in order to examine the evolutionary dynamics of these DNA sequences.     

Construction and characterization of a bacterial artificial chromosome library of Sorghum bicolor.

The construction of representative large insert DNA libraries is critical for the analysis of complex genomes. The predominant vector system for such work is the yeast artificial chromosome (YAC) system. Despite the success of YACs, many problems have been described including: chimerism, tedious steps in library construction and low yields of YAC insert DNA. Recently a new E.coli based system has been developed, the bacterial artificial chromosome (BAC) system, which offers many potential advantages over YACs. We tested the BAC system in plants by constructing an ordered 13,440 clone sorghum BAC library. The library has a combined average insert size, from single and double size selections, of 157 kb. Sorghum inserts of up to 315 kb were isolated and shown to be stable when grown for over 100 generations in liquid media. No chimeric clones were detected as determined by fluorescence in situ hybridization of ten BAC clones to metaphase and interphase S.bicolor nuclei. The library was screened with six sorghum probes and three maize probes and all but one sorghum probe hybridized to at least one BAC clone in the library. To facilitate chromosome walking with the BAC system, methods were developed to isolate the proximal ends of restriction fragments inserted into the BAC vector and used to isolate both the left and right ends of six randomly selected BAC clones. These results demonstrate that the S. bicolor BAC library will be useful for several physical mapping and map-based cloning applications not only in sorghum but other related cereal genomes, such as maize. Furthermore, we conclude that the BAC system is suitable for most large genome applications, is more 'user friendly' than the YAC system, and will likely lead to rapid progress in cloning biologically significant genes from plants.     

Organization of the Euplotes crassus micronuclear genome

Euplotes crassus, like other hypotrichous ciliated protozoa, eliminates most of its micronuclear chromosomal DNA in the process of forming the small linear DNA molecules that comprise the macronuclear genome. By characterizing randomly selected lambda phage clones of E. crassus micronuclear DNA, we have determined the distribution of repetitive and unique sequences and the arrangement of macronuclear genes relative to eliminated DNA. This allows us to compare the E. crassus micronuclear genome organization to that of another distantly related hypotrichous ciliate, Oxytricha nova. The clones from E. crassus segregate into three prevalent classes: those containing primarily eliminated repetitive DNA (Class I); those containing macronuclear genes in addition to repetitive sequences (Class II); and those containing only eliminated unique sequence DNA (Class III). All of the repetitive sequences in these clones belong to the same highly abundant repetitive element family. Our results demonstrate that the sequence organization of the E. crassus and O. nova micronuclear genomes is related in that the macronuclear genes are clustered together in the micronuclear genome and the eliminated unique sequences occur in long stretches that are uninterrupted by repetitive sequences. In both organisms a single repetitive element family comprises the majority of the eliminated interspersed middle repetitive DNA and appears to be preferentially associated with the macronuclear sequence clusters. The similarities in the sequence organization in these two organisms suggest that clustering of macronuclear genes plays a role in the chromosome fragmentation process.     

Isolation and characterization of the highly repeated fraction of the banana genome

Although the nuclear genome of banana (Musa spp.) is relatively small (1C approximately 610 Mbp for M. acuminata), the results obtained from other sequenced genomes suggest that more than half of the banana genome may be composed of repetitive and non-coding DNA sequences. Knowledge of repetitive DNA can facilitate mapping of important traits, phylogenetic studies, BAC-based physical mapping, and genome sequencing/annotation. However, only a few repetitive DNA sequences have been characterized in banana. In this work, we used DNA reassociation kinetics to isolate the highly repeated fraction of the banana genome (M. acuminata 'Calcutta 4'). Two libraries, one prepared from Cot 相似文献   

The occurrence of families of repetitive sequences in a library of cloned cDNA from human lymphocytes

A library of cloned cDNAs representative of lymphocyte total poly(A)+ RNA was screened with total DNA probes at high clone density. 10% of the recombinants showed the presence of sequences which are repeated in the genome. Further analysis of six such isolated cDNA clones indicated that they contain different families of repetitive sequences with reiteration frequencies of between 150 and 45,000 copies per haploid genomes. Five of the six clones were found to contain single copy sequences as well as a repetitive sequence. cDNA clones containing repetitive sequences have been found to be derived from high, intermediate and low abundance classes of lymphocyte poly(A)+ RNA.     

四倍体高粱与约翰逊草间的亲缘关系分析

本研究以四倍体高粱与约翰逊草为材料,利用SSR分子标记和细胞遗传学方法分析了高粱与约翰逊草间的亲缘关系,SSR分析结果表明,高粱与约翰逊草的遗传背景差异较大,SSR差异位点和相似位点在连锁群上的分布具不平衡性;按照差异引物出现频率高低,将连锁群分为两类：高度差异区和低度差异区。细胞学分析结果表明：（1）双亲及杂交种都是不规则的四倍体遗传群体。（2）花粉母细胞减数分裂中期I,双亲及杂交种染色体配对以二价体和四价体为主,杂交种平均每个细胞二价体数为17．00,四倍体高粱为15．23、约翰逊草为15．83,四价体数分别为0．95,2．15和1．60个。但杂交种减数分裂过程中也出现一定数量的单价体,减数分裂会形成一定比例的非整倍配子。SSR检测结果与细胞学分析结果具有一致性,约翰逊草与高粱的染色体组间存在一定程度的同源性。二者杂交不能形成稳定遗传的双二倍体。     

Organization of the Euplotes crassus Micronuclear Genome1

Euplotes crassus, like other hypotrichous ciliated protozoa, eliminates most of its micronuclear chromosomal DNA in the process of forming the small linear DNA molecules that comprise the macronuclear genome. By characterizing randomly selected lambda phage clones of E. crassus micronuclear DNA, we have determined the distribution of repetitive and unique sequences and the arrangement of macronuclear genes relative to eliminated DNA. This allows us to compare the E. crassus micronuclear genome organization to that of another distantly related hypotrichous ciliate, Oxytricha nova. The clones from E. crassus segregate into three prevalent classes: those containing primarily eliminated repetitive DNA (Class I); those containing macronuclear genes in addition to repetitive sequences (Class II); and those containing only eliminated unique sequence DNA (Class III). All of the repetitive sequences in these clones belong to the same highly abundant repetitive element family. Our results demonstrate that the sequence organization of the E. crassus and O. nova micronuclear genomes is related in that the macronuclear genes are clustered together in the micronuclear genome and the eliminated unique sequences occur in long stretches that are uninterrupted by repetitive sequences. In both organisms a single repetitive element family comprises the majority of the eliminated interspersed middle repetitive DNA and appears to be preferentially associated with the macronuclear sequence clusters. The similarities in the sequence organization in these two organisms suggest that clustering of macronuclear genes plays a role in the chromosome fragmentation process.     

Characterization and comparative analysis of DNA from the pericentric heterochromatin of chromosome 2 of Anopheles atroparvus V. Tiel (Culicidae, Diptera)

The minilibrary containing DNA sequences from the diffuse pericentric heterochromatin from the right arm of Anopheles atroparvus V. Tiel (Culicidae, Diptera) chromosome 2 (2R) was generated by use of chromosome microdissection technique. Southern-blot hybridization of the minilibrary fragments with the labeled genomic DNA of A. atroparvus and analysis of their primary structure showed that this heterochromatin region contained repeated DNA sequences differed by their primary structure and the number of copies. These were mostly AT-rich sequences harboring the features characteristic of the S/MAR regions. Based on the clones homology to the sequences from the An. gambiae and Drosophila melanogaster genomes, it was demonstrated that the pericentric heterochromatin from the right arm of An. atroparvus chromosome 2 contained gypsy-like transposable elements, as well as the sequences homologous to the structural genes. In situ hybridization with the chromosomes of A. atroparvus and of the two representatives of the Anopheles maculipennis species complex, A. messeae and A. beklemishevi, showed that pericentric regions of all these chromosomes contained DNA sequences homologous to the sequences from the region-specific minilibrary. Cloned fragments of conserved repetitive DNA revealed upon interspecific Southern-blot hybridization of the clones with the labeled genomic DNA of A. messeae can be utilized in further investigations of evolutionary rearrangements of the pericentric heterochromatin within the Anopheles maculipennis species complex.     

Differential detection of transposable elements between Saccharum species

Cultivars of sugarcane (Saccharum) are hybrids between species S. officinarum (x = 10, 2n = 8x = 80) and S. spontaneum (x = 8, 2n = 5 – 16x = 40 – 128). These accessions have 100 to 130 chromosomes, 80–85% of which are derived from S. officinarum, 10–15% from S. spontaneum, and 5–10% are possible recombinants between the two genomes. The aim of this study was to analyze the repetition of DNA sequences in S. officinarum and S. spontaneum. For this purpose, genomic DNA from S. officinarum was digested with restriction enzymes and the fragments cloned. Sixty-eight fragments, approximately 500 bp, were cloned, sequenced and had their identity analyzed in NCBI, and in the rice, maize, and sorghum genome databases using BLAST. Twelve clones containing partial transposable elements, one single-copy control, one DNA repetitive clone control and two genome controls were analyzed by DNA hybridization on membrane, using genomic probes from S. officinarum and S. spontaneum. The hybridization experiment revealed that six TEs had a similar repetitive DNA pattern in the genomes of S. officinarum and S. spontaneum, while six TEs were more abundant in the genome of S. officinarum. We concluded that the species S. officinarum and S. spontaneum have differential accumulation LTR retrotransposon families, suggesting distinct insertion or modification patterns.     

The use of dna sequencing (ITS and trnL-F), AFLP, and fluorescent in situ hybridization to study allopolyploid Miscanthus (Poaceae)

Two clones of Miscanthus, grown under the names M. ×giganteus and M. sacchariflorus, have been used in biomass trials in Europe, but neither the identity of these clones nor their origin has been established. DNA sequencing, amplified fragment length polymorphism (AFLP), and chromosome studies confirm that M. ×giganteus is an allotriploid (2n = 3x = 57) combining genomes from M. sinensis (2n = 2x = 38) and M. sacchariflorus (2n = 38 or 76). Two alleles of the internal transcribed spacer of 18S-25S nuclear ribosomal DNA (ITS) were discovered in polymerase chain reaction products of M. ×giganteus. Cloning of these revealed that one matched M. sinensis and the other M. sacchariflorus. Plastid trnL intron and trnL-F spacer sequences showed that the maternal lineage of M. ×giganteus was M. sacchariflorus. Fluorescent in situ hybridization, FISH, was used to investigate genome organization in Miscanthus but was unable to differentiate between the different parental genomes present in M. ×giganteus, indicating that two parental genomes are still extremely similar at the repetitive DNA level. This study is an example in which rDNA sequences and AFLP fingerprints permit identification of the parental genomes in a hybrid, but FISH methods, at the repetitive DNA level (including genomic in situ hybridization, GISH), were unable to do so because their sequences remain too similar.