Intraspecific evolution ofMicroseris pygmaea (Asteraceae,Lactuceae) analyzed by cosegregation of phenotypic characters (QTLs) and molecular markers (RAPDs)

The Chilean annual,Microseris pygmaea, has differentiated in distinct coastal and inland series of populations after long-distance dispersal from western North America. Two plants from the most diverse biotypes were crossed, a large F₂ was raised and analysed for segregation of 30 phenotypic characters. Segregation of molecular markers (47 RAPDs, 1 RFLP, 2 isozymes) was determined in a subpopulation of 45 plants which include all extremes for the phenotypic characters. 32 marker/character cosegregations were significant at the 1% level in t-tests between dominant and homozygous recessive marker genotypes. Considering linkage among markers and pleiotropy of certain marker loci, the number of independent quantitative trait loci (QTLs) is reduced to about 18. Interactions among 2 or 3 QTLs affecting one character have been characterized. The phenotypic differentiation ofM. pygmaea during its evolution from a single founder individual begins to be understood at the level of single-gene mutants.     

Identification of minor DNA variations in rice somaclonal variants

Some somaclonal variants derived from a landrace rice variety, Indrayani, were shown to be high yielding and resistant to multiple diseases in previous analysis carried out in our laboratory. An attempt was made to assess the effect of culturing and regeneration of rice plants on DNA variation at microsatellite loci in R₂ progeny of callus-derived rice plants. Different somaclones of the rice line Indrayani differing in yield and disease response (high, low and no change in yield, as compared to the original genotype) were used as genetic material for these analyses. Analysis of microsatellite loci was accomplished by digesting DNA from regenerated rice somaclones and assaying for polymorphisms at microsatellite loci by in-gel hybridization with synthetic oligonucleotide probes such as (GATA)₄, (CAC)₅ and (TG)₁₀. Specific variation at a PCR-amplified locus containing three internal microsatellite repeats (1E₆) using restriction site fingerprinting was also investigated. The locus-specific amplification of a sequence-tagged microsatellite marker followed by digestion with HinfI and Sau3AI restriction endonucleases showed differences in some somaclonal variants. The technique used in this study enables monitoring of DNA changes in successive generations of somaclonal variants as a measure of DNA variability and possibly to identify the regions which are responsible for specific traits. Received: 7 November 1997 / Revision received: 22 April 1997 / Accepted: 5 June 1998     

Diversity of DNA methylation pattern and total DNA restriction pattern in symbiotic Nostoc

Attempts were made to use total DNA restriction patterns and the response of purified DNA to treatment with restriction endonucleases to characterize several symbiotic Nostoc strains which had been isolated from different host plants cultivated in Italy. Among 27 restriction endonucleases tested, several did not cut any DNA and no significant variation in the susceptibility of the genomes to DNA restriction was seen among the strains. Therefore the Nostoc strains could not be separated into groups based on their different susceptibilities to the action of restriction endonucleases. However, in studies of total DNA restriction patterns, the restriction endonucleases BfrI and HpaI gave unique band patterns for each cyanobacterial isolate. Different profiles were even found in strains isolated from host plants belonging to the same species. The results do not support any definition of symbiotic Nostoc genomic groups or species and show that a tight specificity between the host plant and the cyanobacterium might not exist in the symbiotic associations involving Nostoc.     

Phylogenetic relationships among subgenera,species, and varieties of Japanese <Emphasis Type="Italic">Salvia</Emphasis> L. (Lamiaceae)

To determine evolutionary relationships among all Japanese members of the genus Salvia (Lamiaceae), we conducted molecular phylogenetic analyses of two chloroplast DNA (cpDNA) regions (rbcL and the intergenic spacer region of trnL−trnF:trnL−trnF) and one nuclear DNA (nrDNA) region (internal transcribed spacer, ITS). In cpDNA, nrDNA, and cpDNA+nrDNA trees, we found evidence that all Japanese and two Taiwanese Salvia species are included in a clade with other Asian Salvia, and Japanese Salvia species were distributed among three subclades: (1) S. plebeia (subgenus Sclarea), (2) species belonging to subg. Salvia, and (3) species belonging to subg. Allagospadonopsis. At the specific level our findings suggest: a close relationship between S. nipponica and S. glabrescens, no support for monophyly of S. lutescens and its varieties in cpDNA, nrDNA and cpDNA+nrDNA trees, and that S. pygmaea var. simplicior may be more closely related to S. japonica than to other varieties of S. pygmaea.     

Genetic linkage mapping in Acacia mangium 1. Evaluation of restriction endonucleases, inheritance of RFLP loci and their conservation across species

Random genomic probes were used to assess levels of restriction fragment length polymorphism (RFLP) in two 2-generation outbred pedigrees of Acacia mangium Willd. Probes were evaluated for their ability to detect polymorphic loci in each pedigree and to determine the relative efficiency of different restriction enzymes in revealing polymorphisms. Sixty two percent of the probes which detected single- or low-copy number sequences revealed polymorphisms with at least one restriction enyzme. HpaII was the most efficient in detecting polymorphism among first-generation individuals. The recognition sequence of HpaII contains a CpG dimer, suggesting that cytosines in the CpG sequence may be hotspots for mutation in plant genomes, as previously reported in bacterial and mammalian genomes. Mendelian inheritance of 230 loci was demonstrated based on single-locus segregation in second-generation individuals. Less than 5% of loci showed evidence of segregation distortion. The proportion of fully informative loci (15%) was lower than previously reported in eucalypts reflecting the lower level of genetic diversity in A. mangium. The RFLP probes are suitable for the construction of a high-density genetic linkage map in A. mangium. Cross-hybridisation of the A.mangium RFLPs to DNA from species representing the three subgenera of the genus Acacia indicates that these markers could be used in breeding programs of other diploid acacias, for comparative studies of genome organisation, and for phylogenetic studies. Received: 5. June 1999 / Accepted: 30 July 1999     

Detection of Phytoplasma from deferent infected hosts in Jordan based on PCR amplification of the 16S rDNA sequences

Sixteen leaf samples, both healthy and Phytoplasma diseased, were collected from different plants such as grape, peach, almond, tomato, paper, squash, apple and pear in northern Jordan. Extracted DNA from diseased grape, peach, almond, tomato, paper and squash plus from infected periwinkle (Catharanthus roseus) samples were amplified with the Phytoplasma universal 16S rDNA sequences primer pairs. Extracted DNA samples from healthy and diseased apple and pear plants were not amplified with the same primer pairs. All the PCR-amplified DNA samples show a common band with size of 558 bp, indicating Phytoplasma pathogens as a disease-causative agent for grape, peach, almond, tomato, paper and squash plants. The restriction fragment length polymorphisms of Alu1 enzyme for the amplified 16S rDNA sequences shows the same DNA fragment patterns indicating no or a limited diversity among the DNA of the detected Phytoplasma pathogens.     

Duplicated chromosome segments in maize (Zea mays L.): further evidence from hexokinase isozymes

Summary The genetic control of hexokinase isozymes (ATP: d-hexose-6-phosphotransferase, E.C. 2.7.7.1, HEX) in maize (Zea mays L.) was studied by starch gel electrophoresis. Genetic analysis of a large number of inbred lines and crosses indicates that the major isozymes observed are encoded by two nuclear loci, designated Hex1 and Hex2. Five active allozymes and one null variant are associated with Hex1, while Hex2 has nine active alleles in addition to a null variant. Alleles at both loci govern the presence of single bands, with no intragenic or intergenic heteromers visible, suggesting that maize HEX's are active as monomers. Organelle preparations demonstrate that the products of both loci are cytosolic. All alleles, including the nulls, segregate normally in crosses. Vigorous and fertile plants were synthesized that were homozygous for null alleles at both loci, suggesting that other hexosephosphorylating enzymes exist in maize that are undetected with our assay conditions. Linkage analyses and crosses with B-A translocation stocks place Hex1 on the short arm of chromosome 3, 27 centimorgans from Pgd2 (phosphogluconate dehydrogenase) and Hex2 on the long arm of chromosome 6, approximately 45 centimorgans from Pgd1. It is suggested that the parallel linkages among these two pairs of duplicated genes reflects an evolutionary history involving chromosome segment duplication or polyploidy.Paper No. 10170 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC     

Pappus part number in annual species ofMicroseris (Compositae,Cichoriaceae)

All North American annual species of the genusMicroseris have a five-part pappus, the one South American annual,M. pygmaea, has ten pappus parts. The pappus develops over a constant number of ten provascular bundles with or without inhibition between alternate sites of pappus development. Each natural population contains a predictable proportion of achenes with aberrant pappus part numbers. Hybridization betweenM. bigelovii (5 parts) andM. pygmaea results in F 1 and F 2 plants with many aberrant achenes. In each plant either five or ten can be shown to be the basic number with aberrant numbers following a Poisson distribution for numbers added to 5 or deleted from 10. Occasional plants show no basic number but have a random distribution of numbers about an intermediate mean. The evolutionary genetics of this character is discussed.     

Polymorphism and stability in the histone gene cluster of Drosophila melanogaster

Histone genes in Drosophila melanogaster are organized into repeats of 4.8 and 5.0 kb (Lifton et al., 1978). We find these repeat sizes in every one of the more than 20 Drosophila strains we have examined. Strains differ in the relative amounts of the two repeat types, with ratios varying from 11 to 14, the 5.0 kb repeat always present in equal or greater amounts than the 4.8 kb repeat. Restriction enzyme digestion and blotting analysis reveals that the strains also differ in a number of far less abundant fragments containing histone DNA sequences. In the Amherst and Samarkand strains, there are, in addition, many copies of 4.0 and 5.5 kb repeat-like fragments respectively. A series of stocks were made isogenic for single second chromosomes from the Amherst strain. The hybridization patterns of the histone DNA from these stocks containing different Amherst chromosomes are very similar but a number of differences in the minor fragments were seen. The stability of the histone locus restriction pattern was tested by following the DNA derived from a single second chromosome of the b Adhn² pr cn strain over a two year period. The restriction pattern of major and minor bands remained identical. Finally, histone loci distinguishable by their restriction pattern on blots were recombined with visible markers. These chromosomes will be useful in tracing the fate of specific histone loci during genetic manipulations.     

Identification of Brassica oleracea monosomic alien chromosome addition lines with molecular markers reveals extensive gene duplication

Summary Chromosomes of Brassica oleracea (2n=18) were dissected from the resynthesized amphidiploid B. napus Hakuran by repeated backcrosses to B. campestris (2n=20), creating a series of monosomic alien chromosome addition line plants (2n=21). Using morphological, isozyme and restriction fragment length polymorphism markers (RFLPs), 81 putative loci were identified. Of nine possible synteny groups, seven were represented in the 25 monosomic addition plants tested. Sequences homologous to 26% of the 61 DNA clones utilized (80% were cDNA clones) were found on more than one synteny group, indicating a high level of gene duplication. Anomalous synteny associations were detected in four 2n=21 plants. One of these plants showed two markers from one B. oleracea chromosome associated with a second complete B. oleracea synteny group, suggesting translocation or recombination between non-homologous chromosomes in Hakuran or the backcross derivatives. The other three 2n=21 plants each contained two or more B. oleracea synteny groups, suggesting chromosome substitution.     

2种嵩草属植物形态特征对生境干旱化的响应

该研究于西藏自治区东南部的东达山高山草甸沿生境干旱化梯度设置10个样地,采用线性回归方法分析优势种高山嵩草(Kobresia pygmaea)和矮生嵩草(K.humilis)叶片数量与其它构件数值(分株数量、茎基直径、根系数量和根系长度)之间的关系,并采用线性回归斜率测度分株功效、叶片萌生能力、根系分生功效和根系伸长功效,以探讨嵩草的分株能力、茎基生长和根系生长对生境干旱化过程的响应机制。结果显示:(1)2种嵩草植物的叶片数量与4种构件数值均为显著线性正相关关系。(2)随着生境干旱化程度增加,高山嵩草分株数量增加,矮生嵩草分株受干旱抑制程度高,分株数量呈下降趋势;2种嵩草分株功效下降,即单株叶片数量因干旱化程度增加而减少。(3)2种嵩草的茎基直径、叶片萌生能力随着生境干旱化程度增加而下降;高山嵩草叶片萌生能力的变化与生境干旱化梯度一致,具有连续性;矮生嵩草叶片萌生能力对生境干旱化的适应性弱,干旱到一定程度发生骤降。(4)随着生境干旱化,高山嵩草的根系数量和长度均增加,矮生嵩草根系长度增加,但数量却无规律变化;2种嵩草的根系分生功效和根系伸长功效均下降。研究表明:嵩草属植物分布的最适宜生境为表面稍有积水的沼生生境。为适应生境干旱化,高山嵩草降低叶片萌生率以减少蒸腾作用,增加分株数量以增强对空间的占有能力,并增加根系数量和长度来提高对土壤水分的吸收能力,因此对干旱有较强适应性且分布范围广;矮生嵩草只通过降低叶片萌生率,增加根系长度响应干旱化生境,其分布范围较窄。     

Molecular characterization of the nuclear and cytoplasmic genomes of intergeneric diploid plants from cell fusion between Microcitrus papuana and Rough lemon

Simple-sequence-repeat (SSR) and PCR-RFLP were employed to characterize the nuclear and cytoplasmic genomes of intergeneric diploid plants derived from symmetric fusion between Microcitrus papuana Swingle and Rough lemon ( Citrus jambhiri Lush). Three out of five SSR primers distinguished the fusion parents from each other and the regenerated plants showed band profiles completely identical to the leaf parent, Rough lemon. Amplified products from the intergenic regions of cpDNA between trnD -trnT were digested with HaeIII and MspI, and those between trnH -trnK were digested with HinfI, and both the regenerated plants and Rough lemon shared the same band patterns, which were different from the embryogenic parent, M. papuana. With mtDNA, only 2 out of 12 primer pair/restriction enzyme combinations ( nad4 ex 1–2/ TaqI and nad4 ex 1–2/ HindIII) revealed polymorphisms between the fusion parents. With the former combination the regenerated plants showed the same fragment distribution as that of the embryogenic parent, M. papuana, whereas with the latter, a novel band absent in the fusion parents was detected in all of the regenerated plants, suggesting a possible rearrangement. The present research indicates that the plants analyzed were putative cybrids containing nuclear DNA and cpDNA from Rough lemon and mtDNA from M. papuana. Presumed mechanisms leading to the regeneration of diploid hybrid plants following symmetric fusion are discussed herein.     

Mitochondrial DNA diversity and male sterility in natural populations of Daucus carota ssp carota

Mitochondrial variability was investigated in natural populations of wild carrot (Daucus carota ssp carota) in different regions: South of France, Greece, and various sites in the Mediterranean Basin and Asia. Total DNA was digested with two restriction endonucleases (EcoRV and HindIII) and probed with three mitochondrial DMA-specific genes (coxI, atp6, and coxII). Twenty-five different mitochondrial types were found in 80 analyzed individuals. Thirteen mitotypes were found among the 7 French populations studied. On average, 4.4 different mitotypes were observed per population, and these mitotypes were well-distributed among the populations. All of the mitochondrial types were specific to a single region. However, the proportion of shared restriction fragments between 2 mitotypes from different regions was not particularly lower than that which occurred among mitotypes from a single region. On the basis of the sexual phenotype [male-sterile (MS) or hermaphrodite] of the plants studied in situ and that of their progeny, 2 mitotypes were found to be highly associated with male sterility. Eighty percent of the plants bearing these mitotypes were MS in situ, and all of these plants produced more than 30% MS plants in their progeny. This association with male sterility was consistent in several populations, suggesting an association with a cytoplasmic male-sterility system. Moreover, these two mitotypes had very similar mitochondrial DNA restriction patterns and were well-differentiated from the other mitotypes observed in wild plants and also from those observed in the two CMS types already known in the cultivated carrot. This suggests that they correspond to a third cytoplasmic sterility.     

Variation at the Nor loci in triticale derived from tissue culture

Summary Plants derived from tissue cultures of six triticale genotypes were the subject of an analysis for changes in the rRNA genes located at the site of nucleolar organizer regions (the Nor loci) on chromosomes 1B, 6B and 1R. In addition whole plant phenotypes and the chromosomal constitutions of their progenies were examined for alterations. Following treatment of DNA with the restriction endonuclease Taq1, it was possible to assign electrophoretic bands representing rDNA spacer sequences to each of the chromosomes known to carry a major Nor locus. In general, the rRNA genes were found to be stable except in one family where a marked reduction in the number of rDNA units was observed. This reduction in 1R rDNA spacer sequences was heritable and correlated with reduced C-banding at the position of Nor-R1 on chromosome 1R. The change was clearly a consequence of tissue culture since six other plants regenerated from the same culture, and the original parent, did not carry the alteration.     

A linkage map for sugi (Cryptomeria japonica) based on RFLP,RAPD, and isozyme loci

A linkage map for sugi was constructed on the basis of restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), and isozyme loci using a three-generation pedigree prepared for genetic analysis of heartwood color. A total of 128 RFLP (123 cDNA and 5 genomic probes), 33 RAPD, 2 isozyme, and 1 morphological (dwarf) loci segregated in 73 progeny. Of the 164 segregating loci, 145 loci were distributed in 20 linkage groups. Of these loci, 91 with confirmed map positions were assigned to 13 linkage groups, covering a total of 887.3 cM. A clustering of markers with distorted segregation was observed in 6 linkage groups. In the four clusters, distortions with a reduction in the number of homozygotes from one parent only were found.Abbreviations MAS marker-assisted selection - PAGE polyacrylamide gel electrophoresis - QTL quantitative traits of loci - RAPD random amplified polymorphic DNA - RFLP restriction fragment length polymorphism This work was supported by a Grant-in-Aid from the Ministry of Agriculture, Forestry and Fisheries of Japan (Integrated Research Program for the Use of Biotechnological Procedures for Plant Breeding) and by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan (Cooperative Research, no. 04304017)     

The thioester and isotypic sites of complement component C4 in sheep and cattle

The region inclusive of the thioester and the isotype-determining sites of the sheep C4 genes from a single animal was amplified by polymerase chain reaction (PCR). Two bands, at 880 base pairs (bp) and 1000 bp, were resolved by agarose gel electrophoresis. Four different clones were obtained for the 880 bp (type 1) product and two from the 1000 bp (type 2) product. Two of the type 1 clones (type 1H) and both type 2 clones (type 2H) code for the PCPVIH sequence at the isotypic site whereas the other two type 1 clones (type 1D) code for the PFPVMD sequence. By restriction mapping and Southern blot analysis, there appears to be four C4 gene loci for the sheep: two type 1H, one type 1D, and one type 2H. The type 1H and type 2H genes are likely to code for proteins with C4B-like properties whereas the type 1D genes for proteins with C4A-like properties. The same region of the sheep C4 genes of nine other breeds of sheep are also amplified by PCR and analyzed by restriction mapping and Southern hybridization. Each of the sheep has type 1H, type 2H, and type 1D genes and appears to have four C4 gene loci except for the Orkney, which may have five. A single band of 880 bp was obtained from the PCR product from the genomic DNA of a single cow. Five different clones were identified, two of which code for the PFPVMD sequence and three for the PCPVIH sequence at the isotypic site, which is consistent with previous finding that C4 proteins with A- and B-like activities could be purified from the plasma of the same animal. Comparison of the nucleotide sequences of the isotype-determining region of the sheep and cattle C4 genes with those of the primates and mouse suggests that the C4A-like genes evolved independently in the primates and the ungulates. Correspondence to: S. K. A. Law.     

RNA polymerase V-dependent small RNAs in Arabidopsis originate from small,intergenic loci including most SINE repeats

In plants, heterochromatin is maintained by a small RNA-based gene silencing mechanism known as RNA-directed DNA methylation (RdDM). RdDM requires the non-redundant functions of two plant-specific DNA-dependent RNA polymerases (RNAP), RNAP IV and RNAP V. RNAP IV plays a major role in siRNA biogenesis, while RNAP V may recruit DNA methylation machinery to target endogenous loci for silencing. Although small RNA-generating regions that are dependent on both RNAP IV and RNAP V have been identified previously, the genomic loci targeted by RNAP V for siRNA accumulation and silencing have not been described extensively. To characterize the RNAP V-dependent, heterochromatic siRNA-generating regions in the Arabidopsis genome, we deeply sequenced the small RNA populations of wild-type and RNAP V null mutant (nrpe1) plants. Our results showed that RNAP V-dependent siRNA-generating loci are associated predominately with short repetitive sequences in intergenic regions. Suppression of small RNA production from short repetitive sequences was also prominent in RdDM mutants including dms4, drd1, dms3 and rdm1, reflecting the known association of these RdDM effectors with RNAP V. The genomic regions targeted by RNAP V were small, with an estimated average length of 238 bp. Our results suggest that RNAP V affects siRNA production from genomic loci with features dissimilar to known RNAP IV-dependent loci. RNAP V, along with RNAP IV and DRM1/2, may target and silence a set of small, intergenic transposable elements located in dispersed genomic regions for silencing. Silencing at these loci may be actively reinforced by RdDM.     

Generation of polymorphic markers tightly linked to the thymus enlargement loci by phenotype-directed representational difference analysis

To obtain genetic markers linked to a specific genetic locus, genomic subtraction with a DNA pool of backcross or F₂ intercross animals with a specific genotype at the locus is known to be effective. To determine whether the pooling strategy is also effective for isolation of genetic markers linked to a quantitative phenotype that can potentially be controlled by multiple genetic loci, we tested the ability of representational difference analysis (RDA) to isolate genetic markers linked to the thymus enlargement observed in the BUF/Mna (BUF) rat. This is known to be controlled by single major and minor genes, Ten1 and Ten2, on Chromosomes (Chrs) 1 and 13, respectively, both of which have dose effects on the normal WKY/Ncj (WKY) allele. DNA from an inbred WKY rat was used as the tester, and the driver was prepared from a DNA pool of 12 (WKY × BUF)F₁× BUF backcross rats with high thymus ratios (thymus weight/body weight), expected to have dominance of the BUF allele in the responsible loci. By two RDA series with the restriction enzymes BglII and BamHI, respectively, 28 polymorphic markers were isolated, and 8 of them were shown to be linked to Ten1, and one to Ten2. One of the 8 markers linked to Ten1 demonstrated no recombination in 18 rats with high thymus ratios. RDA with a DNA pool based on a quantitative phenotype (phenotype-directed RDA) can thus be considered an efficient approach for direct isolation of polymorphic markers linked to a quantitative trait. Received: 15 April 1997 / Accepted: 8 May 1998     

Centromere and telomere sequence alterations reflect the rapid genome evolution within the carnivorous plant genus Genlisea

Linear chromosomes of eukaryotic organisms invariably possess centromeres and telomeres to ensure proper chromosome segregation during nuclear divisions and to protect the chromosome ends from deterioration and fusion, respectively. While centromeric sequences may differ between species, with arrays of tandemly repeated sequences and retrotransposons being the most abundant sequence types in plant centromeres, telomeric sequences are usually highly conserved among plants and other organisms. The genome size of the carnivorous genus Genlisea (Lentibulariaceae) is highly variable. Here we study evolutionary sequence plasticity of these chromosomal domains at an intrageneric level. We show that Genlisea nigrocaulis (1C = 86 Mbp; 2n = 40) and G. hispidula (1C = 1550 Mbp; 2n = 40) differ as to their DNA composition at centromeres and telomeres. G. nigrocaulis and its close relative G. pygmaea revealed mainly 161 bp tandem repeats, while G. hispidula and its close relative G. subglabra displayed a combination of four retroelements at centromeric positions. G. nigrocaulis and G. pygmaea chromosome ends are characterized by the Arabidopsis‐type telomeric repeats (TTTAGGG); G. hispidula and G. subglabra instead revealed two intermingled sequence variants (TTCAGG and TTTCAGG). These differences in centromeric and, surprisingly, also in telomeric DNA sequences, uncovered between groups with on average a > 9‐fold genome size difference, emphasize the fast genome evolution within this genus. Such intrageneric evolutionary alteration of telomeric repeats with cytosine in the guanine‐rich strand, not yet known for plants, might impact the epigenetic telomere chromatin modification.