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.     

Chloroplast DNA variation within and among five Plantago species

Chloroplast DNA variation was studied within and among five Plantago species. We determined polymorphism by surveying 86% of the chloroplast genome using 9 or 11 restriction enzymes for intra or interspecific variation, respectively. All five species were polymorphic for both site and length mutations. The outcrossing P. lanceolata has six chloroplast haplotypes of which some were found in the Netherlands as well as in the United States. The highly selfing P. major had five haplotypes and Dutch and United States populations were differentiated from each other in cpDNA. Plantago species are highly differentiated from each other. Of 252 restriction-sites, 52% were variable among species. Most of this variation was localized in part of the large single copy region. We derived a molecular phylogeny of the five species from restriction-site variability using PAUP 3.0. P. coronopus and P. maritima form a group as do P. major and P. media. P. lanceolata is more distantly related to the other four species. In a consensus tree both P. major haplotypes and both P. media haplotypes were connected to one node.     

Monotelodisomics in pearl millet

Summary In the progeny of crosses between plants with the chromosome number 2n=13+2 telocentrics as the male parents and the normal diploids of Pennisetum typhoides S. & H., two plants with 2n=13+1 telocentric chromosome were located. These two plants were heterozygous for an interchange, since at diakinesis and metaphase I associations of four chromosomes were observed. These plants had a chromosome constitution of 2n=13+t (or 6+tI); one chromosome of a homologous pair was represented by a telocentric chromosome so was monosomic for one arm, that is, these plants were monotelodisomics (Kimber and Sears, 1968).     

DNA sequence organization in the genomes of three related millet plant species

Summary A major portion of the genomes of three millet species, namely, barn yard millet, fox tail millet and little millet has been shown to consist of interspersed repeat and single copy DNA sequences. The interspersed repetitive DNA sequences are both short (0.15–1.0 kilo base pairs, 62–64% and long (>1.5 kilo base pairs, 36–38%) in barn yard millet and little millet while in fox tail millet, only long interspersed repeats (>1.5 kilo base pairs) are present. The length of the interspersed single copy DNA sequences varies in the range of 1.6–2.6 kilo base pairs in all the three species. The repetitive duplexes isolated after renaturation of 1.5 kilo base pairs and 20 kilo base pairs long DNA fragments exhibit a high thermal stability with Tms either equal to or greater than the corresponding native DNAs. The S1 nuclease resistant repetitive DNA duplexes also are thermally stable and reveal the presence of only 1–2% sequence divergence.The present data on the modes of sequence arrangement in millets substantiates the proposed trend in plants, namely, plants with 1C nuclear DNA content of less than 5 picograms have diverse patterns of sequence organization while those with 1C nuclear DNA content greater than 5 picograms have predominantly a short period interspersion pattern.Abbreviations kbp kilobase pairs NCL Communication No. 3606.     

Possible causes of variation in trivalent orientation frequencies in pearl millet and rye

In pearl millet, chain trivalents composed of two telocentric and one metacentric chromosomes, showed an excess of linear orientation over the 1/3 expected with random centromere activation and inactivity of a central centromere stretched between the two outer centromeres. Chain trivalents composed of two metacentrics and one telo or of three metacentrics behaved as predicted. The difference was explained by assuming precocious activation of completely terminal centromeres as opposed to median centromeres. This early activity was reflected in precocious separation at late metaphase. In rye, all trivalents composed of two telos and one metacentric showed alternate orientation and anaphase separation did not precede that of metacentric chromosomes. It is concluded that in rye terminal centromeres are not precocious and that the spindle at meiosis is not long enough to permit stretching of the central centromere, which consequently always has the opportunity to orient and to induce the other centromeres to choose the opposite pole either directly or after reorientation, accumulating the most stable (alternate) orientation type.     

Genotype dependent variation in mycorrhizal colonization and response to inoculation of pearl millet

Summary Genotypes of pearl millet (Pennisetum americanum L. Leeke) were examined for differences in vesicular-arbuscular mycorrhizal (VAM) colonization and response to inoculation. For thirty genotypes tested across three field locations there was a range of mycorrhizal colonization intensity between 25 and 56%. In another experiment with two male-sterile lines, restorer lines and their derived crosses, grown in pots filled with non-sterilized soil there were significant differences between genotypes for colonization by mycorrhiza. This showed hostgenotype dependence for mycorrhizal colonization.Root growth rates, mycorrhizal root length, percentage root colonization and plant growth and P uptake were studied in ten genotypes. A set of 3 genotypes with similar root lengths varied significantly with regard to mycorrhizal root length and the percentage colonization. This supports the suggestion that VAM colonization and spread is dependent on the host genotype. The growth responses differed significantly between the genotypes and they also differed in their responses to P uptake and VAM inoculation. The utility of host-genotype dependent differences in VAM symbiosis in plant breeding is discussed.Journal Article No. 453     

Telo aneuploids in pearl millet

Summary Nomenclature and chromosome constitution for twenty different aneuploids with telocentrics in Pennisetum typhoides are presented.     

Drought resistance in pearl millet

The influence of wilting on the levels of free proline, soluble proteins, reducing sugars, starch and on the activities of nitrate reductase, invertase, amylase and pyrophosphatases have been studied in the leaf tissue of five cultivars of pearl millet at their vegetative stage under pot culture conditions. The metabolic changes could not be correlated with the yield behaviour of the cultivars under a drought condition in the field.     

Cytogenetics of pearl millet

Summary The somatic karyotype of pearl millet Pennisetum americanum (L.) Leeke. (2n = 14) has been studied in several cultivars, but few cytological markers have been discovered which could help in the easy identification of the chromosomes. Analysis of pachytene bivalents permits such identification but is feasible only in a few cultivars. Recently, several lines having telocentric chromosomes have been produced and classified but their potentialities as cytogenetic tools have yet to be explored. Some African populations of pearl millet carry B-chromosomes in their karyotype. Cytogenetics of B-chromosomes has been reported in great detail. Bs undergo spontaneous changes to produce deficient- and iso-chromosomes. The main effect of B-chromosomes is on chiasma frequency which is exerted by the relative amounts of chiasma promoting euchromatin and the chiasma depressing heterochromatin in the Bs. Haploid plants occur occasionally and sometimes show a low degree of seed set, offering a possibility of establishing homozygous inbred lines. Cytogenetics of several spontaneous and induced autotetraploids have been reported. In general quadrivalent formation between the seven sets of four homologues was random. Seed set of the autotetraploids could be improved by selection; improved seed fertility was found to be associated with increased chiasma frequency, increased quadrivalent frequency and regular distribution of chromosomes at anaphase I. Genes controlling morphological characters of plant phenotype segregate independent of those controlling fertility and in pearl millet polyploidy per se is not limiting to plant vigour. Primary trisomics represent the best studied among the aneuploids of pearl millet. All the seven primary trisomics have been identified and described. Some were used in assigning genes to specific chromosomes but in general trisomies have poor vigour and fertility, and show low frequency of transmission. Apart from B-chromosomes, cytogenetics of interchanges has been the best studied aspect of pearl millet. The frequency of co-orientation of an interchange complex at metaphase I, which determines the fertility or sterility of the interchange heterozygote, is influenced by the genetic background and thus is theoretically amenable for selection leading to improved fertility of the heterozygote. Interchange tester-stocks have been assembled which can be used to identify the chromosomes involved in any newly obtained interchange. A complex interchange line involving all the chromosomes of the complement has also been produced, but the ring-of-fourteen produces total male and female sterility.Genotypic control of mitosis and meiosis has been reported, with reference to chromosome numerical mosaicism, multiploid sporocytes, desynapsis and chromosome fragmentation, and male sterility. Pearl millet being a largely outbreeding species, forced inbreeding was mainly found to result in loss of morphological vigour and reduction in mean chiasma frequency per PMC. Interspecific hybrids between pearl millet and several related species have been cytologically investigated and homology of the seven chromosomes of pearl millet with seven of the fourteen chromosomes of P. purpureum has been demonstrated. Cytogenetic evidence from haploids, autopolyploids and interspecific hybrids has indications to suggest that the haploid number of x = 7 is derived from x = 5, but the evidence is inconclusive and needs critical evaluation.     

Chloroplast DNA variation and phylogeny of theRanunculaceae

Restriction site mapping of chloroplast DNA from 31 species representing 26 genera of theRanunculaceae was performed using eleven restriction endonucleases. The chloroplast genome varies in length from approximately 152 to 160 kb. Length variants are frequent in theRanunculaceae and range from usually less than 300 bp to rarely 1.5 kb. The inverted repeat is extended into the large single copy (LSC) region by 4–4.5 kb inAnemone, Clematis, Clematopsis, Hepatica, Knowltonia, andPulsatilla. Several inversions are present in the LSC-region of the cpDNA in all these genera and inAdonis. The frequency of restriction site mutations varies within the chloroplast genome in theRanunculaceae between 4 and 32 mutations per kilobase, and is lowest in the inverted repeat and the regions containing the ATPase-genes and the genespsaA, psaB, psbA, rpoB, andrbcL. A total of 547 phylogenetically informative restriction sites was utilized in cladistic analyses of the family using Wagner, Dollo, and weighted parsimony. These three parsimony analyses result in different tree topologies. Four, six, and one equally most parsimonious trees were obtained with Wagner, Dollo, and weighted parsimony, respectively. The amount of support for the monophyletic groups was evaluated using bootstrapping and decay analysis. All three parsimony methods suggest thatHydrastis is the sister group to the remainder of theRanunculaceae, and that theAnemone-Clematis group, which shares several derived cpDNA rearrangements, is monophyletic. Only a few of the traditional groups in theRanunculaceae are supported by cpDNA restriction side data. Only Dollo parsimony provides support for the hypothesis thatThalictroideae andRanunculoideae are monophyletic.     

Multiple chromosomal interchanges in pearl millet

Summary Intercrossing and irradiation were successfully used in pearl millet (Pennisetum typhoides) to develop multiple interchanges involving up to the total complement of all the chromosomes in one complex. In interchange heterozygotes showing 12 + 1 II and 14, 90.9 and 87.8 per cent of the cells, respectively, had chromosome configurations other than that of 12 and 14 chromosomes. In general, the frequency of such cells resulting from breakdown of the expected complex configuration increased with the increase in the number of translocated chromosomes in the complex. The higher the number of chromosomes involved in the interchange ring, the higher were the pollen and ovule sterility. The results indicated that meiotic instability, deficiency-duplication gametes, and unequal distribution of chromosomes account for increased sterility of multiple interchange heterozygotes. Even though interchanges in pearl millet predominantly show the alternate type of segregation, sterility seems to be the major barrier for the exploitation of the multiple interchange method for gamete selection and the establishment of homozygous lines in this plant species.     

Chloroplast DNA variation and parentage analysis in 55 apples

The chloroplastic atpB-rbcL spacer and the first 53 codons of the rbcL coding sequence was sequenced for 40 apple cultivars and 15 wild species. This chloroplast DNA region is 904 base pairs long, and only five mutations sites were found among the tested samples. Although the cpDNA variation was low, some parentages are proposed based on the maternal inheritance of plastid DNA: the male and female parents are specified, or else suggested, for Worcester, Discovery, Starking, Starkrimson, Kidd's Orange Red, Priscilla, and Gloster, as well as for the putative wild origin for Malus x domestica.     

Chloroplast DNA variation in the grass tribe Festuceae

Summary Six grasses, Hordeum sativum, Dactylis glomerata, Festuca arundinacea, F. pratensis, F. rubra and Lolium multiflorum were subjected to chloroplast DNA analysis based on restriction endonuclease digestion fragments and end labeling with ³⁵S nucleotides. This method is compared with others in general use. The results indicate that Lolium multiflorum is closely affiliated with Festuca pratensis and F. arundinacea; in fact much closer than F. rubra is to any of them.     

Characterization of pearl millet prolamins

We report the physical-chemical characterization of the major alcohol-soluble proteins present in seeds of pearl millet (Pennisetum glaucum) by SDS-PAGE, bidimensional gel electrophoresis, MALDI-TOF/MS and RP-HPLC. We demonstrate the presence of three major prolamins, called A-, B- and C-pennisetin with mass values around 27, 22 and 12 kDa, respectively. We present partial amino acid sequences of these major proteins, which should allow the posterior isolation of the respective genes.     

Chloroplast DNA variation in diploid and polyploid species of Bromus (Poaceae) subgenera Festucaria and Ceratochloa

Summary Chloroplast DNA (cpDNA) restriction endonuclease patterns are used to examine phylogenetic relationships between Bromus subgenera Festucaria and Ceratochloa. Festucaria is considered monophyletic based on the L genome, while Ceratochloa encompasses two species complexes: the B. catharticus complex, which evolved by combining three different genomes, and the B. carinatus complex, which is thought to have originated from hybridization between polyploid species of B. catharticus and diploid members of Festucaria. All species of subgenus Ceratochloa (hexaploids and octoploids) were identical in chloroplast DNA sequences. Similarly, polyploid species of subgenus Festucaria, except for B. auleticus, were identical in cpDNA sequences. In contrast, diploid species of subgenus Festucaria showed various degrees of nucleotide sequence divergence. Species of subgenus Ceratochloa appeared monophyletic and phylogenetically closely related to the diploid B. anomalus and B. auleticus of subgenus Festucaria. The remaining diploid and polyploid species of subgenus Festucaria appeared in a distinct grouping. The study suggests that the B. catharticus complex must have been the maternal parent in the proposed hybrid origin of B. carinatus complex. Although there is no direct evidence for the paternal parent of the latter complex, the cpDNA study shows the complex to be phylogenetically very related to the diploid B. anomalus of subgenus Festucaria.     

Chloroplast DNA variation and evolution in the genus Lens Mill

 Chloroplast DNA (cpDNA) restriction site diversity was assessed by 21 enzyme/probe combinations in 30 accessions of six Lens species, including the recently recognized L. lamottei and L. tomentosus. A total of 118 fragments were scored and 26 restriction site mutations were identified. The cpDNA restriction pattern supports circumscribing L. lamottei and L. tomentosus as independent species. The value of the data for reconstructing phylogeny in the genus is discussed. The cpDNA of all 13 accessions of the lentil’s wild progenitor, L. culinaris subsp. orientalis, differed from that of the single lentil cultivars used in this study. This diversity indicates that other populations of this subspecies from Turkey and Syria examined by Mayer and Soltis (1994) are potentially the founder members of lentil. Examination of L. lamottei×L. nigricans hybrids between accessions having different restriction patterns showed paternal plastid inheritance in L. nigricans. Received: 2 July 1996 / Accepted: 19 July 1996     

Mitochondrial DNA variation in Drosophila pseudoobscura and related species in Pacific northwest populations

We have analysed mitochondrial DNA (mtDNA) from Pacific Northwest populations of Drosophila pseudoobscura, D. persimilis, and D. miranda using six restriction enzymes. We find that HpaII restriction sites are hypervariable compared to the other enzymes used. This hypervariability allows construction of a maximum parsimony map linking each mtDNA genotype. Small insertions, possibly tandem duplications, appear to have arisen concomitantly with, or subsequent to, speciation events, perhaps within the A + T rich region. Convergence of mtDNA genotypes is also evident. Unlike findings for other populations of these species, we find little evidence of mitochondrial introgression between D. pseudoobscura and D. persimilis, despite their ability to produce fertile hybrid females.     

RFLP analysis of mitochondrial DNA from cytoplasmic male-sterile lines of pearl millet

Mitochondrial DNA (mtDNA) from 13 cytoplasmic male-sterile (cms) lines from diverse sources were characterized by Southern blot hybridization to pearl millet and maize mtDNA probes. Hybridization patterns of mtDNA digested with PstI, BamHI, SmaI or XhoI and probed with 13.6-, 10.9-, 9.7- or 4.7-kb pearl millet mtDNA clones revealed similarities among the cms lines 5141 A and ICMA 1 (classified as the S-A1 type of cytoplasm based on fertility restoration patterns), PMC 30A and ICMA 2. The remaining cms lines formed a distinct group, within which three subgroups were evident. Among the maize mitochondiral gene clones used, the coxI probe revealed two distinct groups of cytoplasms similar to the pearl millet mtDNA clones. The atp9 probe differentiated the cms line 81 A4, derived from P. glaucum subsp. monodii, while the coxII gene probe did not detect any polymorphism among the cms lines studied. MtDNA digested with BamHI, PstI or XhoI and hybridized to the atp6 probe revealed distinct differences among the cms lines. The maize atp6 gene clone identified four distinct cytoplasmic groups and four subgroups within a main group. The mtDNA fragments hybridized to the atp6 gene probe with differing intensities, suggesting the presence of more than one copy of the gene in different stoichiometries. Rearrangements involving the coxI and/or rrn18-rrn5 genes (mapped within the pearl millet clones) probably resulted in the S-A1 type of sterility. Rearrangements involving the atp6 gene (probably resulting in chimeric form) may be responsible for male sterility in other cms lines of pearl millet.     

Mechanisms of starvation tolerance in pearl millet

The response of pearl millet (Pennisetum glaucum [L.]) seedlings to prolonged starvation was investigated at the biochemical and ultrastructural level. After 2 days of darkness the bulk of the seedling carbohydrate reserves were depleted. After 8 days in the dark the respiratory rate had declined to less than 50% of its initial value and the plants had lost half of their total protein content. Unlike the situation with carbohydrate depletion, protein loss was restricted to specific organs. The secondary leaf and stem (including the apical meristem) showed little or no protein loss during this period. In the primary leaf, seed, and roots, protein loss was substantial. In spite of the high rate of protein degradation in the primary leaf and roots, these organs showed no ultrastructural changes suggestive of tissue, cellular, or subcellular degradation. In addition, ribulose bisphosphate carboxylase was not preferentially degraded during starvation and only a small decline in chlorophyll content was observed after 8 days in the dark. During the period from 8 to 14 days, cell death started at the tip of the primary leaf and gradually spread downward. Both shoot and root meristems remained alive up to 14 days. Consequently, the eventual death of the plant was due to the loss of the carbohydrate-producing regions rather than the meristems. We suggest that these results provide an explanation for the high degree of starvation tolerance exhibited by pearl millet.