Origin of the main class of repetitive DNA within selected Pennisetum species

In an attempt to identify relationships among genomes of the allotetraploid Pennisetum purpureum Schumach and closely related Pennisetum species with which it can be successfully hybridized, repetitive DNA sequences were examined. Digestion with KpnI revealed two highly repetitive fragments of 140 by and 160 bp. The possibility that these sequences could be used as genome markers was investigated. Average sequences were determined for the 140 by and 160 by KpnI families from P. purpureum and P. squamulatum Fresen. Average sequences (based upon four or five repeats) were determined for the P. glaucum (L.) R. Br. 140 by KpnI family and the diploid P. hohenackeri Hochst. ex Steud. 160 bp KpnI family. The average sequences of the 160 by KpnI families in P. purpureum and P. squamulatum differ by only nine bases. The 140 by KpnI families of the three related species, P. purpureum, P. squamulantum, and P. glaucum are nearly identical, and thus likely represent a recent divergence from a common progenitor or a common genome. Each repetitive sequence may contain internal duplications, which probably diverged following amplification of the original sequence. The 140 by KpnI repeat probably evolved from the 160 by KpnI repeat since the missing 18 by segment is part of the internal duplication that is otherwise conserved in the subrepeats. Tandemly arrayed repetitive sequences in plants are likely to be composed of subrepeats which have been duplicated and amplified.Florida Aqricultural Experiment Station series #R-02758     

Molecular and physical organization of highly repetitive,undermethylated DNA from Pennisetum glaucum

A HaeIIl monomer of a repetitive DNA family from Pennisetum glaucum (L.) R. Br. cv. Massue has been cloned and characterized. The repeat is 137 bp long and is organized in head-to-tail orientation in tandem arrays. The HaeIII monomer contains 55% A+T residues. The distribution of this highly repetitive sequence in different Pennisetum species and in other cereals was investigated. The HaeIII satellite is present in all Pennisetum species investigated but absent from other genera examined. In situ hybridization revealed a centromeric localization of this sequence on all seven chromosome pairs and indicated chromosome-specific differences in copy number. Methylation was investigated by comparative restriction enzyme analysis (Msp/HpaII) which showed a greater extent of methylation of the internal C of the enzyme recognition site 5′-CCGG. A South-Western analysis, using an anti-methylcytosine antibody to examine the methylation status in P. glaucum confirmed that the sequence is not highly methylated.     

Organization of the<Emphasis Type="Italic"> Kpn</Emphasis>I family of chromosomal distal-end satellite DNAs in<Emphasis Type="Italic"> Silene latifolia</Emphasis>

The major satellite DNAs of the dioecious plant Silene latifolia are represented by the repetitive sequences X43.1, RMY1 and members of the SacI family, which are located at the distal ends of chromosomes. To characterize the satellite DNAs at the distal ends of the chromosomes in S. latifolia (Sl-distal-satDNA), we isolated a bacterial artificial chromosome clone (number 15B12) that contained multiple repeat sequences with KpnI restriction sites, and subcloned a portion of this sequence into a plasmid vector. Sequencing analysis confirmed that recognition or degenerate sites for KpnI were repeated 26 times at intervals of 310–324 bp in the inserted DNA. The phylogenetic tree that was constructed with the 26 KpnI repeat units contained clustered branches that were independent of the SacI family. It is clear that the KpnI repeat belongs to an Sl-distal-satDNA family that is distinct from the SacI family. We designated this family as "KpnI" after the restriction enzyme that does not have a site in the SacI family. Multi-colored fluorescent in situ hybridization was performed with the KpnI family and RMY1 probes under high stringency conditions. The results suggest that chromosome 7 is unique and that it carries the KpnI family at only one end.     

Genome survey sequencing of purple elephant grass (<Emphasis Type="Italic">Pennisetum purpureum</Emphasis> Schum ‘Zise’) and identification of its SSR markers

Elephant grass (Pennisetum purpureum) is a perennial grass in the Poaceae family with high tolerance and one of the best forage plants. Despite its economic importance, the inheritance information of P. purpureum has remained largely unknown. To obtain the whole reference genome, we first conducted a genome survey of P. purpureum. Next-generation sequencing (NGS) was used to perform the de novo whole genome sequencing. As a result, the estimated genome size of elephant grass was 2.01 Gb, with 71.36% repetitive elements. The heterozygosity was 1.02%, which indicates a highly heterozygous genome. The retroelements (9.36%) were the most repetitive elements, followed by DNA transposons (3.66%). In the meantime, 83,706 high-quality genomic simple sequence repeat (SSR) markers, in which the greatest SSR unit length was 3, were developed. Thirty pairs of SSR markers were randomly selected to verify the efficiency and all of them yielded clear amplification products, among which 28 pairs (93.3%) of the primers showed polymorphism. The genome data obtained in this research provided a large amount of gene resources for further investigating Pennisetum species.     

Ribosomal DNA Intergenic Spacer of the Swimming Crab, Charybdis japonica

We have determined the full sequence of the ribosomal DNA intergenic spacer (IGS) of the swimming crab, Charybdis japonica, by long PCR for the first time in crustacean decapods. The IGS is 5376 bp long and contains two nonrepetitive regions separated by one long repetitive region, which is composed mainly of four subrepeats (subrepeats I, II, III, and IV). Subrepeat I contains nine copies of a 60-bp repeat unit, in which two similar repeat types (60 bp-a and 60 bp-b) occur alternatively. Subrepeat II consists of nine successive repeat units with a consensus sequence length of 142 bp. Subrepeat III consists of seven copies of another 60-bp repeat unit (60 bp-c) whose sequence is complementary to that of subrepeat I. Immediately downstream of subrepeat III is subrepeat IV, consisting of three copies of a 391-bp repeat unit. Based on comparative analysis among the subrepeats and repeat units, a possible evolutionary process responsible for the formation of the repetitive region is inferred, which involves the duplication of a 60-bp subrepeat unit (60 bp-c) as a prototype. Received: 13 April 1999 / Accepted: 2 August 1999     

Targeted sequencing of a complex locus within a polyploid genome using reduced representation libraries

Apospory is a form of gametophytic apomixis in which embryos develop from unreduced embryo sacs derived from aposporous initials formed from nucellar cells of ovules to produce offspring genetically identical to the female plant. Apospory in Pennisetum squamulatum (8X) and Cenchrus ciliaris (4X) is a dominant trait controlled by a physically large, hemizygous, heterochromatic chromosomal block called the apospory-specific genomic region (ASGR). Both apomictic species are polyploid, with genome sizes estimated at 2600 to 3000 Mbp for C. ciliaris and 9400 to 10,300 Mbp for P. squamulatum. A study was conducted to determine whether duplex-specific nuclease (DSN) normalization of DNA from apomictic and sexual genotypes would reduce repetitive sequences and allow bioinformatic analysis to predict sequence contigs derived from the ASGR. DSN libraries from four genotypes were sequenced using Illumina^® HiSeq 2000 technology. 39 out of 44 tested sequence characterized amplified region (SCAR) markers from in silico predicted ASGR-specific contigs were mapped to the ASGR in a Pennisetum F₁ mapping population. Eighteen SCARs showed apomict-specific amplification in C. ciliaris. The successful mapping of ~90 % of the SCAR markers to the ASGR in the Pennisetum F₁ mapping population shows that DSN normalization and Illumina sequencing can be used as an effective strategy for targeted mapping of a physically large locus rich in repetitive sequences, like that of the ASGR.     

Non-Mendelian transmission of an apospory-specific genomic region in a reciprocal cross between sexual pearl millet (Pennisetum glaucum) and an apomictic F1 (P. glaucum×P. squamulatum)

We recently showed that aposporous apomixis, a form of gametophytic apomixis, is controlled by a single apospory-specific genomic region (ASGR) in both Pennisetum squamulatum and Cenchrus ciliaris. We present evidence that in a reciprocal cross between sexual pearl millet (P. glaucum) and an apomictic F1 (P. glaucum× P. squamulatum) the ASGR is not transmitted at the same rate. When pearl millet was used as the female parent and the apomictic genotype as the pollen donor, the ASGR was transmitted at a rate of 0.41 in a progeny of 57 plants, indicating a slight transmission ratio distortion. However, in a population of 52 rare sexual progenies characterized among a large progeny of a quasi-obligate apomict (an F1 hybrid of P. glaucum×P. squamulatum), the transmission rate of ASGR was only 0.12. This strong segregation distortion may have occurred at four different levels: (1) female meiosis, (2) during female gametophyte maturation, (3) upon fertilization with differential survival of embryos being a consequence of differential gene expression controlled by parent-of-origin specific effects (imprinting) and (4) at a later developmental stage of the embryo through an embryo/endosperm genetic incompatibility system. Recevied: 13 June 2000 / Revision accepted: 23 October, 2000     

A new repetitive DNA sequence family in the olive (Olea europaea L.)

Two families of repeated DNA sequences were cloned from Olea europaea ssp sativa cv. "Picual". The first repetitive DNA is organized in a tandem repeat of monomers of 178 bp. Sequencing of several clones showed that it is relatively A-T rich (54.49%) and possesses short direct and inverted subrepeats as well as some palindromic sequences. Comparison between the monomers revealed heterogeneity of the sequence primary structure. This repetitive DNA is present in several cultivars of olive cultivates. Comparison of sequences with other repetitive DNAs described in Olea europaea has been carried out. No significant similarity was found. All the obtained results suggest that this repetitive DNA described here is a new family of repetitive DNA. The second repetitive DNA is organized in a tandem repeat of monomers of 78 bp. This second family of repetitive DNA showed significant similarity with other repetitive DNAs previously described in Olea europaea. Their existence in new cultivars of olive is shown.     

Sequence of the cryptic satellite DNA from the plant Sinapis alba

A satellite DNA with a buoyant density equal to that of main band DNA in neutral cesium chloride (‘cryptic satellite’) can be isolated from the DNA of mustard (Sinapis alba) nuclei by Ag⁺/Cs₂SO₄ density gradient centrifugation. This satellite is cleaved into 172 bp repeat units by HinfI, AluI or HaeIII. The HinfI fragments have been further cleaved by AluI, and seven AluI subfragments have been sequenced. As a result two versions of a basic 172 HinfI repeat have been found, one (A + B) with an additional HinfI site. These two sequences (A + B and C) are the most frequent versions of the basic repeat of mustard satellite DNA. The basic 172 bp unit does not contain subrepeats or palindromic sequences. It is not similar (at a criterion of 15 common bases) with any known satellite sequence. It is not unusually highly methylated in the native state.     

Evaluation of selectable markers for obtaining stable transformants in the gramineae

Cell suspension cultures of Triticum monococcum, Panicum maximum, Saccharum officinarum, Pennisetum americanum, and a double cross trispecific hybrid between Pennisetum americanum, P. purpureum, and P. squamulatum were tested for resistance to kanamycin, hygromycin, and methotrexate for use in transformation studies. All cultures showed high natural levels of resistance to kanamycin, in excess of 800 milligrams per liter, and variable levels of resistance to hygromycin. Methotrexate was a potent growth inhibitor at low concentrations with all species. Kanamycin and hygromycin were growth inhibitory only if added early (within 5 days after protoplast isolation and culture). Protoplasts of T. monococcum, P. maximum, S. officinarum, and the tri-specific hybrid were electroporated with plasmid DNA containing hygromycin (pMON410), kanamycin (pMON273), or methotrexate (pMON806) resistance genes. Resistant colonies were obtained at low frequencies (1 × 10⁻⁵ to 2 × 10⁻⁶) when selected under conditions which were growth inhibitory to protoplasts electroporated without DNA. Southern blot hybridization confirmed stable integration of plasmid DNA into T. monococcum using hygromycin vectors and P. maximum using the methotrexate vector with 1 to 10 copies integrated per haploid genome.     

Molecular and physical organization of highly repetitive, undermethylated DNA from Pennisetum glaucum

A HaeIIl monomer of a repetitive DNA family from Pennisetum glaucum (L.) R. Br. cv. Massue has been cloned and characterized. The repeat is 137 bp long and is organized in head-to-tail orientation in tandem arrays. The HaeIII monomer contains 55% A+T residues. The distribution of this highly repetitive sequence in different Pennisetum species and in other cereals was investigated. The HaeIII satellite is present in all Pennisetum species investigated but absent from other genera examined. In situ hybridization revealed a centromeric localization of this sequence on all seven chromosome pairs and indicated chromosome-specific differences in copy number. Methylation was investigated by comparative restriction enzyme analysis (Msp/HpaII) which showed a greater extent of methylation of the internal C of the enzyme recognition site 5-CCGG. A South-Western analysis, using an anti-methylcytosine antibody to examine the methylation status in P. glaucum confirmed that the sequence is not highly methylated.     

Mitochondrial DNA variation in pearl millet and related species

Summary Mitochondrial DNA (mtDNA) restriction endonuclease fragment patterns and patterns of mtDNA hybridized by mitochondrial gene probes were used to study phylogenetic relationships of seven Pennisetum species, including five P. americanum (pearl millet) ecotypes and a reference species from the distantly related genus, Panicum. The restriction patterns of the pearl millet ecotypes were uniform with the exception of the ecotype collected in Ethiopia. The probe hybridization method revealed more variability, with both the Rhodesian and Ethiopian ecotypes differing from the others and from each other. Considerable restriction pattern polymorphism was noted among different species of Pennisetum, and Panicum. Significant relationships were noted of Pennisetum polystachyon to P. pedicellatum and of P. purpureum to P. squamulatum using the restriction pattern method. In addition to those relationships, the hybridization method showed relationships of pearl millet to P. purpureum and to P. squamulatum. The relationships noted between species by the hybridization method agreed more closely to the cytological data than those indicated by the restriction pattern method. Therefore, the hybridization method appeared to be the preferred method for studying species relationships. The mitochondrial genome size of pearl millet was calculated to be 407 kb and the mitochondrial genome sizes of other Pennisetum species ranged from 341 to 486 kb.Florida Agricultural Experiment Station Journal Series No. 8485.     

Cytogenetics of double cross hybrids between Pennisetum americanum — P. purpureum amphiploids and P. americanum x Pennisetum squamulatum interspecific hybrids

Summary Pearl millet, Pennisetum americanum L. Leeke-napiergrass, Pennisetum purpureum Schum. amphiploids (2n=42) were crossed with pearl millet X Pennisetum squamulatum Fresen. interspecific hybrids (2n=41) to study the potential of germplasm transfer from wild Pennisetum species to pearl millet. These two interspecific hybrids were highly cross-compatible and more than two thousand trispecific progenies were produced from 17 double crosses. All doublecross hybrids were perennial and showed a wide range of morphological variations intermediate to both parents in vegetative and inflorescence characteristics. Some crosses resulted in sublethal progenies. Chromosomes paired mainly as bivalents (¯x15.88) or remained as univalents. At metaphase I, trivalents, quadrivalents, an occasional hexavalent and a high frequency of bivalents indicated some homeology among the genomes of the three species. Delayed separation of bivalents, unequal segregation of multivalents, lagging chromosomes, and chromatin bridges were observed at anaphase I. Although approximately 93% of the double-cross hybrids were male-sterile, pollen stainability in male-fertile plants ranged up to 94%. Seed set ranged from 0 to 37 seed per inflorescence in 71 plants under open-pollinated conditions. Apomictic embryo sac development was observed in double-cross progenies when crosses involved a pearl millet x P. squamulatum apomictic hybrid as pollen parent. These new double-cross hybrids may serve as bridging hybrids to transfer genes controlling apomixis and other plant characteristics from the wild Pennisetum species to pearl millet.     

KpnBI is the prototype of a new family (IE) of bacterial type I restriction-modification system

KpnBI is a restriction-modification (R-M) system recognized in the GM236 strain of Klebsiella pneumoniae. Here, the KpnBI modification genes were cloned into a plasmid using a modification expression screening method. The modification genes that consist of both hsdM (2631 bp) and hsdS (1344 bp) genes were identified on an 8.2 kb EcoRI chromosomal fragment. These two genes overlap by one base and share the same promoter located upstream of the hsdM gene. Using recently developed plasmid R-M tests and a computer program RM Search, the DNA recognition sequence for the KpnBI enzymes was identified as a new 8 nt sequence containing one degenerate base with a 6 nt spacer, CAAANNNNNNRTCA. From Dam methylation and HindIII sensitivity tests, the methylation loci were predicted to be the italicized third adenine in the 5′ specific region and the adenine opposite the italicized thymine in the 3′ specific region. Combined with previous sequence data for hsdR, we concluded that the KpnBI system is a typical type I R-M system. The deduced amino acid sequences of the three subunits of the KpnBI system show only limited homologies (25 to 33% identity) at best, to the four previously categorized type I families (IA, IB, IC, and ID). Furthermore, their identity scores to other uncharacterized putative genome type I sequences were 53% at maximum. Therefore, we propose that KpnBI is the prototype of a new ‘type IE’ family.     

Microsporogenesis,reproductive behavior,and fertility in five Pennisetum species

Summary Microsporogenesis, reproductive behavior, pollen fertility and seed set were studied in Pennisetum basedowii Summerhayes and C. E. Hubbard, 2n = 54; P. macrostachyum (Brough.) Trin., 2n = 54; P. macrourum Trin., 2n = 36; P. polystachion (L.) Schult, 2n = 54; and P. squamulatum Fresen 2n = 54. Meiosis was regular in P. basedowii with primarily bivalent pairing. As many as 54 univalents were observed at metaphase I in P. macrostachyum. A high frequency of univalents at metaphase I in P. macrourum resulted in lagging chromosomes and micronuclei at anaphase I and telophase I, respectively. Pennisetum polystachion and P. squamulatum showed frequent multivalent chromosome associations. Studies of megasporogenesis and embryo sac development in P. basedowii showed sexual reproduction. Pennisetum macrostachyum was highly male sterile with predominantly aposporous apomictic embryo sac development. Pennisetum macrourum, P. polystachion, and P. squamulatum had only aposporous embryo sac development. Seed propagated progenies of these latter three species were uniform and matromorphic, confirming the obligate apomixis nature.     

Analysis of the complete plastid genome of the unicellular red alga Porphyridium purpureum

We determined the complete nucleotide sequence of the plastid genome of the unicellular marine red alga Porphyridium purpureum strain NIES 2140, belonging to the unsequenced class Porphyridiophyceae. The genome is a circular DNA composed of 217,694 bp with the GC content of 30.3 %. Twenty-nine of the 224 protein-coding genes contain one or multiple intron(s). A group I intron was found in the rpl28 gene, whereas the other introns were group II introns. The P. purpureum plastid genome has one non-coding RNA (ncRNA) gene, 29 tRNA genes and two nonidentical ribosomal RNA operons. One rRNA operon has a tRNA^Ala(UGC) gene between the rrs and the rrl genes, whereas another has a tRNA^Ile(GAU) gene. Phylogenetic analyses suggest that the plastids of Heterokontophyta, Cryptophyta and Haptophyta originated from the subphylum Rhodophytina. The order of the genes in the ribosomal protein cluster of the P. purpureum plastid genome differs from that of other Rhodophyta and Chromalveolata. These results suggest that a large-scale rearrangement occurred in the plastid genome of P. purpureum after its separation from other Rhodophyta.     

A series of repetitive DNA sequences are associated with human core and H1 histone genes

Summary Repetitive DNA sequences, derived from the human β-globin gene cluster, were mapped within a series of human genomic DNA segments containing core (H2A, H2B, H3 and H4) and H1 histone genes. Cloned recombinant λCH4A phage with human histone gene inserts were analyzed by Southern blot analysis using the following³²P-labeled (nick translated) repetitive sequences as probes:Alu I,Kpn I and LTR-like. A cloned DNA designated RS002-5′C6 containing (i)a (TG)₁₆ simple repeat, (ii) an (ATTTT)n repeat and (iii)a 52 base pair alternating purine and pyrimidine sequence was also used as a radiolabelled hybridization probe. Analysis of 12 recombinant phage, containing 6 arrangements of core histone genes, indicated the presence ofAlu I,Kpn and RS002-5′C6 repetitive sequences. In contrast, analysis of 4 human genomic DNA segments, containing both core and H1 histone genes, indicated the presence of onlyAlu I family sequences. LTR-like sequences were not detected in association with any of the core or H1 histone genes examined. These results suggest that human histone and β-globin genes share certain aspects of sequence organization in flanking regions despite marked differences in their overall structure and pattern of expression.     

Cloning and characterization of chromosomal markers in alfalfa (Medicago sativa L.)

Eleven tandemly repetitive sequences were identified from a Cot-1 library by FISH and sequence analysis of alfalfa (Medicago sativa). Five repetitive sequences (MsCR-1, MsCR-2, MsCR-3, MsCR-4, and MsCR-5) were centromeric or pericentromeric, of which three were satellite DNAs and two were minisatellite DNAs. Monomers of 144, 148, and 168 bp were identified in MsCR-1, MsCR-2, and MsCR-3, respectively, while 15 and 39 bp monomers were identified in MsCR-4 and MsCR-5, respectively. Three repetitive sequences were characterized as subtelomeric; one repetitive sequence, MsTR-1, had a 184 bp monomer, and two repetitive sequences had fragments of 204 and 327 bp. Sequence analysis revealed homology (70–80 %) between MsTR-1 and a highly repeated sequence (C300) isolated from M. ssp. caerulea. Three identified repetitive sequences produced hybridization signals at multiple sites in a few of the chromosomes; one repetitive sequence was identified as the E180 satellite DNA previously isolated from M. sativa, while the other 163 and 227 bp fragments had distinct sequences. Physical mapping of the repetitive sequences with double-target FISH revealed different patterns. Thus, nine novel tandemly repetitive sequences that can be adopted as distinct chromosome markers in alfalfa were identified in this study. Furthermore, the chromosome distribution of each sequence was well described. Though significant chromosome variations were detected within and between cultivars, a molecular karyotype of alfalfa was suggested with the chromosome markers we identified. Therefore, these novel chromosome markers will still be a powerful tool for genome composition analysis, phylogenetic studies, and breeding applications.     

A new family of dispersed,highly repetitive sequences in bovine genome

A new family of highly repetitive sequences which are dispersed in bovine genome is described. The members of the family are visible on agarose or polyacrylamide gels as a diffused band about 510 bp in length arising after digestion with PstI restriction nuclease. This family of fragments comprises the 160 bp bovine Bsu family and is linked with bovine Alu-like sequences.