Repeat turnover meets stable chromosomes: repetitive DNA sequences mark speciation and gene pool boundaries in sugar beet and wild beets.

Sugar beet and its wild relatives share a base chromosome number of nine and similar chromosome morphologies. Yet, interspecific breeding is impeded by chromosome and sequence divergence that is still not fully understood. Since repetitive DNAs are among the fastest evolving parts of the genome, we investigated, if repeatome innovations and losses are linked to chromosomal differentiation and speciation. We traced genome and chromosome-wide evolution across 13 beet species comprising all sections of the genera Beta and Patellifolia. For this, we combined short and long read sequencing, flow cytometry, and cytogenetics to build a comprehensive framework that spans the complete scale from DNA to chromosome to genome. Genome sizes and repeat profiles reflect the separation into three gene pools with contrasting evolutionary patterns. Among all repeats, satellite DNAs harbor most genomic variability, leading to fundamentally different centromere architectures, ranging from chromosomal uniformity in Beta and Patellifolia to the formation of patchwork chromosomes in Corollinae/Nanae. We show that repetitive DNAs are causal for the genome expansions and contractions across the beet genera, providing insights into the genomic underpinnings of beet speciation. Satellite DNAs in particular vary considerably between beet genomes, leading to the evolution of distinct chromosomal setups in the three gene pools, likely contributing to the barriers in beet breeding. Thus, with their isokaryotypic chromosome sets, beet genomes present an ideal system for studying the link between repeats, genomic variability, and chromosomal differentiation and provide a theoretical fundament for understanding barriers in any crop breeding effort.

Assembly of the Complete Mitochondrial Genome of Chinese Plum (Prunus salicina): Characterization of Genome Recombination and RNA Editing Sites.

Despite the significant progress that has been made in the genome sequencing of Prunus, this area of research has been lacking a systematic description of the mitochondrial genome of this genus for a long time. In this study, we assembled the mitochondrial genome of the Chinese plum (Prunus salicina) using Illumina and Oxford Nanopore sequencing data. The mitochondrial genome size of P. salicina was found to be 508,035 base pair (bp), which is the largest reported in the Rosaceae family to date, and P. salicina was shown to be 63,453 bp longer than sweet cherry (P. avium). The P. salicina mitochondrial genome contained 37 protein-coding genes (PCGs), 3 ribosomal RNA (rRNA) genes, and 16 transfer RNA (tRNA) genes. Two plastid-derived tRNA were identified. We also found two short repeats that captured the nad3 and nad6 genes and resulted in two copies. In addition, nine pairs of repeat sequences were identified as being involved in the mediation of genome recombination. This is crucial for the formation of subgenomic configurations. To characterize RNA editing sites, transcriptome data were used, and we identified 480 RNA editing sites in protein-coding sequences. Among them, the initiation codon of the nad1 gene confirmed that an RNA editing event occurred, and the genomic encoded ACG was edited as AUG in the transcript. Combined with previous reports on the chloroplast genome, our data complemented our understanding of the last part of the organelle genome of plum, which will facilitate our understanding of the evolution of organelle genomes.

Customized optical mapping by CRISPR-Cas9 mediated DNA labeling with multiple sgRNAs.

Whole-genome mapping technologies have been developed as a complementary tool to provide scaffolds for genome assembly and structural variation analysis (1,2). We recently introduced a novel DNA labeling strategy based on a CRISPR-Cas9 genome editing system, which can target any 20bp sequences. The labeling strategy is specifically useful in targeting repetitive sequences, and sequences not accessible to other labeling methods. In this report, we present customized mapping strategies that extend the applications of CRISPR-Cas9 DNA labeling. We first design a CRISPR-Cas9 labeling strategy to interrogate and differentiate the single allele differences in NGG protospacer adjacent motifs (PAM sequence). Combined with sequence motif labeling, we can pinpoint the single-base differences in highly conserved sequences. In the second strategy, we design mapping patterns across a genome by selecting sets of specific single-guide RNAs (sgRNAs) for labeling multiple loci of a genomic region or a whole genome. By developing and optimizing a single tube synthesis of multiple sgRNAs, we demonstrate the utility of CRISPR-Cas9 mapping with 162 sgRNAs targeting the 2Mb Haemophilus influenzae chromosome. These CRISPR-Cas9 mapping approaches could be particularly useful for applications in defining long-distance haplotypes and pinpointing the breakpoints in large structural variants in complex genomes and microbial mixtures.

The ribosomal intergenic spacer (IGS) in the potato and tobacco cyst nematodes, Globodera pallida, G. rostochiensis and G. tabacum.

The potato cyst nematodes Globodera pallida and G. rostochiensis (PCN), and tobacco cyst nematode (TCN), G. tabacum, are the most important parasitic nematodes of potato and tobacco worldwide. Ribosomal DNA provides useful molecular data for diagnostics, the study of polymorphisms and for evolutionary research in eukaryotic organisms including nematodes. Here we present data on the structure and organization of a rarely studied part of the intergenic spacer (IGS) region of the PCN and TCN genome of cyst nematodes. This region has shown potential for diagnostic purposes and population studies in other organisms including nematodes. In nematodes, the ribosomal RNA gene cluster comprises three genes: 5.8S, 18S and 28S rRNA, which are separated by spacer regions: the intergenic spacer (IGS), non-transcribed spacer (NTS), externally transcribed spacer (EST) and the internally transcribed spacer (ITS). The intergenic spacer (IGS) region consists of an external transcribed spacer (ETS) and a non-transcribed spacer (NTS) which is located between the 28S of one repeat and the 18S gene of the next repeat within the rRNA genes cluster. In this study, the first flanking portion of the IGS was amplified, cloned and sequenced from PCN and TCN. Primers were then designed to amplify the whole IGS sequence. PCR amplification of IGS from G. tabacum, G. pallida, and G. rostochiensis yielded respectively: a single amplicon of 3 kb, three amplicons sized 2.5, 2.6 and 2.9 kb, and two amplicons sized 2.8 and 2.9 kb. Results showed that Globodera spp. has more than one variant copy of the IGS, with both long and short repetitive DNA elements. An approximately 400 bp long region without any internal repetitive elements, were identified in a position between the two repetitive regions suggesting that there is a 5S gene in the IGS of these species.

Nondisjunction and unequal spindle organization accompany the drive of Aegilops speltoides B chromosomes.

B chromosomes (Bs) are supernumerary chromosomes, which are often preferentially inherited. When transmission rates of chromosomes are higher than 0.5, not obeying the Mendelian law of equal segregation, the resulting transmission advantage is collectively referred to as 'chromosome drive'. Here we analysed the drive mechanism of Aegilops speltoides Bs. The repeat AesTR-183 of A. speltoides Bs, which also can be detected on the Bs of Aegilops mutica and rye, was used to track Bs during pollen development. Nondisjunction of CENH3-positive, tubulin interacting B sister chromatids and an asymmetric spindle during first pollen grain mitosis are key for the accumulation process. A quantitative flow cytometric approach revealed that, independent of the number of Bs present in the mother plant, Bs accumulate in the generative nuclei to > 93%. Nine out of 11 tested (peri)centromeric repeats were shared by A and B chromosomes. Our findings provide new insights into the process of chromosome drive. Quantitative flow cytometry is a useful and reliable method to study the drive frequency of Bs. Nondisjunction and unequal spindle organization accompany during first pollen mitosis the drive of A. speltoides Bs. The prerequisites for the drive process seems to be common in Poaceae.

Identification and Phylogenetic Analysis of the Complete Chloroplast Genomes of Three Ephedra Herbs Containing Ephedrine.

Ephedrae Herba and Ephedrae Radix et Rhizoma (Mahuang) have been used as Chinese herbal medicines. Ephedra plants mainly live in deserts and have good governance of desertification. Despite their important medicinal and environmental protection value, dietary supplements containing ephedrine from Ephedra species may threaten the health of people. Morphological resemblance amongst species causes difficulty in identifying the original species of Ephedra herbs. Chloroplast (CP) genome shows good prospects in identification and phylogenetic analysis. This study introduced the structures of the CP genomes of three Ephedra species and analysed their phylogenetic relationships. Three complete CP genomes of Ephedra showed four-part annular structures, namely, two single-copy regions and two inverted repeat regions. The entire CP genomes of three Ephedra species in terms of size were 109,550 bp (E. sinica), 109,667 bp (E. intermedia), and 109,558 bp (E. equisetina). Each CP genome of the three Ephedra species encoded 118 genes, including 73 protein-coding genes, 37 tRNA genes and 8 ribosomal RNA genes. Eleven high-variation regions were screened through mVISTA to be potential specific DNA barcodes for identifying Ephedra species. Maximum likelihood and maximum parsimony trees showed that CP genomes could be used to identify Ephedra species. The Ephedra species had a close phylogenetic relationship with Gnetum species and Welwitschia mirabilis. This research provided valuable information for the identification and phylogenetic analysis of gymnosperms and drug safety of Ephedra.

Population authentication of the traditional medicinal plant Cassia tora L. based on ISSR markers and FTIR analysis.

Cassia tora is a plant of medicinal importance. Medicinal plants from different localities are believed to differ in their therapeutic potency. In this study, six populations of C. tora with different eco-geographical origins were investigated genotypically (ISSR) and phytochemically (FTIR) to establish an integrated approach for population discrimination and authentication of the origin of this medicinal herb. CHS gene expression analysis and determination of flavonoid content were carried out to substantiate the study. A total of 19 population-specific authentication bands were observed in 11 ISSR fingerprints. Authentication codes were generated using six highly polymorphic bands, including three authentication bands. FTIR spectra revealed that the peaks at wavenumber 1623 cm-1 (carbonyl group) and 1034 cm-1 (>CO- group) were powerful in separating the populations. These peaks are assigned to flavonoids and carbohydrates, respectively, were more intense for Ranchi (highland) population. Variation in the transcript level of CHS gene was observed. The findings of FTIR and RT-PCR analyses were in agreement with the TFC analysis, where, the lowest amount of flavonoids observed for Lucknow (lowland) population. All the populations of C. tora have been authenticated accurately by ISSR analyses and FTIR fingerprinting, and the Ranchi site was observed to be more suitable for the potential harvesting of therapeutic bioactive compounds.

Comparative chloroplast genomes of Paris Sect. Marmorata: insights into repeat regions and evolutionary implications.

BACKGROUND: Species of Paris Sect. Marmorata are valuable medicinal plants to synthesize steroidal saponins with effective pharmacological therapy. However, the wild resources of the species are threatened by plundering exploitation before the molecular genetics studies uncover the genomes and evolutionary significance. Thus, the availability of complete chloroplast genome sequences of Sect. Marmorata is necessary and crucial to the understanding the plastome evolution of this section and facilitating future population genetics studies. Here, we determined chloroplast genomes of Sect. Marmorata, and conducted the whole chloroplast genome comparison. RESULTS: This study presented detailed sequences and structural variations of chloroplast genomes of Sect. Marmorata. Over 40 large repeats and approximately 130 simple sequence repeats as well as a group of genomic hotspots were detected. Inverted repeat contraction of this section was inferred via comparing the chloroplast genomes with the one of P. verticillata. Additionally, almost all the plastid protein coding genes were found to prefer ending with A/U. Mutation bias and selection pressure predominately shaped the codon bias of most genes. And most of the genes underwent purifying selection, whereas photosynthetic genes experienced a relatively relaxed purifying selection. CONCLUSIONS: Repeat sequences and hotspot regions can be scanned to detect the intraspecific and interspecific variability, and selected to infer the phylogenetic relationships of Sect. Marmorata and other species in subgenus Daiswa. Mutation and natural selection were the main forces to drive the codon bias pattern of most plastid protein coding genes. Therefore, this study enhances the understanding about evolution of Sect. Marmorata from the chloroplast genome, and provide genomic insights into genetic analyses of Sect. Marmorata.

Lake sedimentary DNA accurately records 20th Century introductions of exotic conifers in Scotland.

Sedimentary DNA (sedDNA) has recently emerged as a new proxy for reconstructing past vegetation, but its taphonomy, source area and representation biases need better assessment. We investigated how sedDNA in recent sediments of two small Scottish lakes reflects a major vegetation change, using well-documented 20th Century plantations of exotic conifers as an experimental system. We used next-generation sequencing to barcode sedDNA retrieved from subrecent lake sediments. For comparison, pollen was analysed from the same samples. The sedDNA record contains 73 taxa (mainly genus or species), all but one of which are present in the study area. Pollen and sedDNA shared 35% of taxa, which partly reflects a difference in source area. More aquatic taxa were recorded in sedDNA, whereas taxa assumed to be of regional rather than local origin were recorded only as pollen. The chronology of the sediments and planting records are well aligned, and sedDNA of exotic conifers appears in high quantities with the establishment of plantations around the lakes. SedDNA recorded other changes in local vegetation that accompanied afforestation. There were no signs of DNA leaching in the sediments or DNA originating from pollen.

Small Open Reading Frames, Non-Coding RNAs and Repetitive Elements in Bradyrhizobium japonicum USDA 110.

Small open reading frames (sORFs) and genes for non-coding RNAs are poorly investigated components of most genomes. Our analysis of 1391 ORFs recently annotated in the soybean symbiont Bradyrhizobium japonicum USDA 110 revealed that 78% of them contain less than 80 codons. Twenty-one of these sORFs are conserved in or outside Alphaproteobacteria and most of them are similar to genes found in transposable elements, in line with their broad distribution. Stabilizing selection was demonstrated for sORFs with proteomic evidence and bll1319_ISGA which is conserved at the nucleotide level in 16 alphaproteobacterial species, 79 species from other taxa and 49 other Proteobacteria. Further we used Northern blot hybridization to validate ten small RNAs (BjsR1 to BjsR10) belonging to new RNA families. We found that BjsR1 and BjsR3 have homologs outside the genus Bradyrhizobium, and BjsR5, BjsR6, BjsR7, and BjsR10 have up to four imperfect copies in Bradyrhizobium genomes. BjsR8, BjsR9, and BjsR10 are present exclusively in nodules, while the other sRNAs are also expressed in liquid cultures. We also found that the level of BjsR4 decreases after exposure to tellurite and iron, and this down-regulation contributes to survival under high iron conditions. Analysis of additional small RNAs overlapping with 3'-UTRs revealed two new repetitive elements named Br-REP1 and Br-REP2. These REP elements may play roles in the genomic plasticity and gene regulation and could be useful for strain identification by PCR-fingerprinting. Furthermore, we studied two potential toxin genes in the symbiotic island and confirmed toxicity of the yhaV homolog bll1687 but not of the newly annotated higB homolog blr0229_ISGA in E. coli. Finally, we revealed transcription interference resulting in an antisense RNA complementary to blr1853, a gene induced in symbiosis. The presented results expand our knowledge on sORFs, non-coding RNAs and repetitive elements in B. japonicum and related bacteria.

The complete chloroplast genome sequence of Dendrobium nobile.

The complete chloroplast (cp) genome sequence of Dendrobium nobile, an endangered and traditional Chinese medicine with important economic value, is presented in this article. The total genome size is 150,793 bp, containing a large single copy (LSC) region (84,939 bp) and a small single copy region (SSC) (13,310 bp) which were separated by two inverted repeat (IRs) regions (26,272 bp). The overall GC contents of the plastid genome were 38.8%. In total, 130 unique genes were annotated and they were consisted of 76 protein-coding genes, 30 tRNA genes and 4 rRNA genes. Fourteen genes contained one or two introns.

Isolation and characterization of microsatellite markers and analysis of genetic diversity in Chinese jujube (Ziziphus jujuba Mill.).

Chinese jujube (Ziziphus jujuba Mill, 2n = 2× = 24, Rhamnaceae) is an economically important Chinese native species. It has high nutritional value, and its medicinal properties have led to extensive use in traditional oriental medicine. The characterization of genotypes using molecular markers is important for genetic studies and plant breeding. However, few simple sequence repeat (SSR) markers are available for this species. In this study, 1,488 unique SSR clones were isolated from Z. jujuba 'Dongzao' using enriched genomic libraries coupled with a three-primer colony PCR screening strategy, yielding a high enrichment rate of 73.3%. Finally, 1,188 (80.87%) primer pairs were amplified successfully in the size expected for 'Dongzao'. A total of 350 primer pairs were further selected and evaluated for their ability to detect polymorphisms across a panel of six diverse cultivars; among these, 301 primer pairs detected polymorphisms, and the polymorphism information content (PIC) value across all loci ranged from 0.15 to 0.82, with an average of 0.52. An analysis of 76 major cultivars employed in Chinese jujube production using 31 primer pairs revealed comparatively high genetic diversity among these cultivars. Within-population differences among individuals accounted for 98.2% of the observed genetic variation. Neighbor-joining clustering divided the cultivars into three main groups, none of which correspond to major geographic regions, suggesting that the genetics and geographical origin of modern Chinese jujube cultivars might not be linked. The current work firstly reports the large-scale development of Chinese jujube SSR markers. The development of these markers and their polymorphic information represent a significant improvement in the available Chinese jujube genomic resources and will facilitate both genetic and breeding applications, further accelerating the development of new cultivars.

Low temperature-responsive changes in the anther transcriptome's repeat sequences are indicative of stress sensitivity and pollen sterility in rice strains.

Genome-wide transcriptome analyses using microarray probes containing genes and repeat sequences have been performed to examine responses to low temperatures in rice (Oryza sativa). We focused particularly on the rice anther at the booting stage, because a low temperature at this stage can result in pollen abortion. The five rice strains examined in this study showed different pollen fertilities due to a low-temperature treatment during the booting stage. The microarray analyses demonstrated that the low-temperature stress caused genome-wide changes in the transcriptional activities not only of genes but also of repeat sequences in the rice anther. The degree of the temperature-responsive changes varied among the five rice strains. Interestingly, the low-temperature-sensitive strains revealed more changes in the transcriptome when compared with the tolerant strains. The expression patterns of the repeat sequences, including miniature inverted-repeat transposable elements, transposons, and retrotransposons, were correlated with the pollen fertilities of the five strains, with the highest correlation coefficient being 0.979. Even in the low-temperature-sensitive strains, the transcriptomes displayed distinct expression patterns. The elements responding to the low temperatures were evenly distributed throughout the genome, and the major cis-motifs involved in temperature-responsive changes were undetectable from the upstream sequences in the corresponding repeats. The genome-wide responses of transcription to the temperature shift may be associated with chromatin dynamics, which facilitates environmental plasticity. A genome-wide analysis using repeat sequences suggested that stress tolerance could be conferred by insensitivity to the stimuli.

Complete chloroplast genome sequences of Mongolia medicine Artemisia frigida and phylogenetic relationships with other plants.

BACKGROUND: Artemisia frigida Willd. is an important Mongolian traditional medicinal plant with pharmacological functions of stanch and detumescence. However, there is little sequence and genomic information available for Artemisia frigida, which makes phylogenetic identification, evolutionary studies, and genetic improvement of its value very difficult. We report the complete chloroplast genome sequence of Artemisia frigida based on 454 pyrosequencing. METHODOLOGY/PRINCIPAL FINDINGS: The complete chloroplast genome of Artemisia frigida is 151,076 bp including a large single copy (LSC) region of 82,740 bp, a small single copy (SSC) region of 18,394 bp and a pair of inverted repeats (IRs) of 24,971 bp. The genome contains 114 unique genes and 18 duplicated genes. The chloroplast genome of Artemisia frigida contains a small 3.4 kb inversion within a large 23 kb inversion in the LSC region, a unique feature in Asteraceae. The gene order in the SSC region of Artemisia frigida is inverted compared with the other 6 Asteraceae species with the chloroplast genomes sequenced. This inversion is likely caused by an intramolecular recombination event only occurred in Artemisia frigida. The existence of rich SSR loci in the Artemisia frigida chloroplast genome provides a rare opportunity to study population genetics of this Mongolian medicinal plant. Phylogenetic analysis demonstrates a sister relationship between Artemisia frigida and four other species in Asteraceae, including Ageratina adenophora, Helianthus annuus, Guizotia abyssinica and Lactuca sativa, based on 61 protein-coding sequences. Furthermore, Artemisia frigida was placed in the tribe Anthemideae in the subfamily Asteroideae (Asteraceae) based on ndhF and trnL-F sequence comparisons. CONCLUSION: The chloroplast genome sequence of Artemisia frigida was assembled and analyzed in this study, representing the first plastid genome sequenced in the Anthemideae tribe. This complete chloroplast genome sequence will be useful for molecular ecology and molecular phylogeny studies within Artemisia species and also within the Asteraceae family.

Interstitial telomeric repeats are enriched in the centromeres of chromosomes in Solanum species.

Interstitial telomeric repeats (ITRs) were reported in a number of animal and plant species. Most ITRs are organized as short tandem arrays and are likely evolutionary relics derived from chromosomal rearrangements and DNA repairs. However, megabase-sized ITR arrays were reported in Solanum species. Here, we report a fluorescence in situ hybridization (FISH) survey of ITRs in all representative diploid Solanum species, including potato, tomato, and eggplant. FISH revealed massive amplification of ITRs in the centromeric regions of chromosomes from the Solanum species containing the B and P genomes. A significant proportion of the ITR FISH signals was mapped within the primary constrictions of the pachytene chromosomes of Solanum pinnatisectum (B genome). In addition, some ITR sites overlapped with St49, a satellite repeat enriched in centromeric DNA sequences associated with CENH3 nucleosomes, in both A and B genome Solanum species. These results show that some ITR subfamilies have been amplified and invaded in the functional centromeres of chromosomes in Solanum species.

Coxiella burnetii, the agent of Q fever in Brazil: its hidden role in seronegative arthritis and the importance of molecular diagnosis based on the repetitive element IS1111 associated with the transposase gene.

Coxiella burnetii is the agent of Q fever , an emergent worldwide zoonosis of wide clinical spectrum. Although C. burnetii infection is typically associated with acute infection, atypical pneumonia and flu-like symptoms, endocarditis, osteoarticular manifestations and severe disease are possible, especially when the patient has a suppressed immune system; however, these severe complications are typically neglected. This study reports the sequencing of the repetitive element IS1111 of the transposase gene of C. burnetii from blood and bronchoalveolar lavage (BAL) samples from a patient with severe pneumonia following methotrexate therapy, resulting in the molecular diagnosis of Q fever in a patient who had been diagnosed with active seronegative polyarthritis two years earlier. To the best of our knowledge, this represents the first documented case of the isolation of C. burnetii DNA from a BAL sample.

Coxiella burnetii, the agent of Q fever in Brazil: its hidden role in seronegative arthritis and the importance of molecular diagnosis based on the repetitive element IS1111 associated with the transposase gene

Coxiella burnetii is the agent of Q fever , an emergent worldwide zoonosis of wide clinical spectrum. Although C. burnetii infection is typically associated with acute infection, atypical pneumonia and flu-like symptoms, endocarditis, osteoarticular manifestations and severe disease are possible, especially when the patient has a suppressed immune system; however, these severe complications are typically neglected. This study reports the sequencing of the repetitive element IS1111 of the transposase gene of C. burnetii from blood and bronchoalveolar lavage (BAL) samples from a patient with severe pneumonia following methotrexate therapy, resulting in the molecular diagnosis of Q fever in a patient who had been diagnosed with active seronegative polyarthritis two years earlier. To the best of our knowledge, this represents the first documented case of the isolation of C. burnetii DNA from a BAL sample.

Hereditary dentine diseases resulting from mutations in DSPP gene.

OBJECTIVES: This review groups the newest results of molecular analyses of DSPP gene for patients diagnosed either with dentinogenesis imperfecta type II/III or dentine dysplasia and tries to link the phenotypes with specific mutations in the DSPP gene. DATA: The review includes biochemical data introducing a specificity of DSPP protein which justifies it as a critical factor for dentine mineralization and maturation. The majority of the review analyzes mutations in the DSPP gene which result in phenotypes of dentinogenesis imperfecta types II or/and III or dentine dysplasia. SOURCES: An electronic search was conducted in the databases of Pub Med and supplemented by manual study of relevant references. STUDY SELECTION: 52 out of 108 references were finally selected for the review based on the novelty and/or originality of data. CONCLUSION: Hereditary dentine disorders dentinogenesis imperfecta type II/III and dentine dysplasia are currently proposed to be one disease with distinct clinical manifestations reflecting various mutations in the same DSPP gene. For years both disorders were linked exclusively to mutations in the DSP code but a growing number of papers describe mutations which manifest a similar phenotype but are localized in the strongly repetitive sequence of the 3' terminus of the DSPP which codes DPP protein. Our search suggests that the localization of mutation in the sequence of the DSPP gene might result in a different phenotype due to the diverse cellular fate of the mutated protein. Thus comprehensive research on the cellular fate and processing of both normal and mutated DSPP is still required.

Random BAC FISH of monocot plants reveals differential distribution of repetitive DNA elements in small and large chromosome species.

BAC FISH (fluorescence in situ hybridization using bacterial artificial chromosome probes) is a useful cytogenetic technique for physical mapping, chromosome marker screening, and comparative genomics. As a large genomic fragment with repetitive sequences is inserted in each BAC clone, random BAC FISH without adding competitive DNA can unveil complex chromosome organization of the repetitive elements in plants. Here we performed the comparative analysis of the random BAC FISH in monocot plants including species having small chromosomes (rice and asparagus) and those having large chromosomes (hexaploid wheat, onion, and spider lily) in order to understand a whole view of the repetitive element organization in Poales and Asparagales monocots. More unique and less dense dispersed signals of BAC FISH were observed in species with smaller chromosomes in both the Poales and Asparagales species. In the case of large-chromosome species, 75-85% of the BAC clones were detected as dispersed repetitive FISH signals along entire chromosomes. The BAC FISH of Lycoris did not even show localized repetitive patterns (e.g., centromeric localization) of signals.

[Study of EST-SSR marker system of Cordyceps].

OBJECTIVE: To establish the EST-SSR marker system for Cordyceps by using ESTs of C. bassiana and C. militaris. METHOD: The ESTs of Cordyceps were downloaded from the public database of NCBI, and the redundant ESTs with low quality were removed. The EST-SSR primers were designed by Sequece Seiner 1. 2. And the primers were screened through PAGE-Electrophoresis. RESULT: The 4 556 non-redundant ESTs which from C. bassiana with total length of 2 953 173 bp were selected. 718 EST-SSRs distributed in 616 ESTs were totally screened out, accounting for 15.8% of the non-redundant ESTs. It was discovered that the average distance of EST-SSSR was 1/4 096 bp in EST-SSRs distribution of C. bassiana. Trinucleotide repeats were the most abundant types with 419 repeated sequences. Regarding to C. militaris, totally 1 363 non-redundant ESTs were acquired, from which 1 117 EST-SSRs were screened, and rate of SSR sites in ESTs was 81.95%. The leading motif of SSR was nucleotide A. The 50 pairs of EST-SSR primers were designed according to the ESTs of C. bassiana, and preliminary test showed the 34 pairs of primers amplified clear fragments,accounting for 68% of all primers. Furthermore, the 39 of the 40 pairs of primers from the ESTs of C. militaris were found to be amplified as the clear fragments, accounting for 97.5%. The phylogenetic analysis revealed that different anamorph of Cordyceps spieces were divided into four branches. CONCLUSION: The EST-SSR of Cordyceps had comparably higher utility value. The EST-SSR markers developed from ESTs of C. bassiana and C. militaris had well transferability in Cordyceps. And it was suggested that the EST-SSR markers should be an easy and effective way to assay molecular genetic structure of Cordyceps.