Genetic analysis of aMicroseris douglasii (Asteraceae) population polymorphic for an alien chloroplast type

Recent evidence suggests chloroplast introgression fromMicroseris bigelovii intoM. douglasii. We have examined 23 plants from a population ofM. douglasii polymorphic forM. douglasii andM. bigelovii chloroplast types. All 23 plants were completely homozygous for morphological and RAPD markers, and inbred lines derived by selfing have been used for DNA analysis. Chloroplast RFLP analysis identified 16 plants withM. bigelovii chloroplasts and seven withM. douglasii chloroplasts. The nuclear genomes of the 16 plants withM. bigelovii chloroplasts were examined with 22 primers for RAPD amplification products shared exclusively withM. bigelovii. Five of 268 markers appeared to be shared betweenM. bigelovii and one or more of these 16 plants on the basis of their position in gels. Detailed examination of these five amplification products showed that none of them are nuclear DNA fromM. bigelovii. Very little, if any, nuclear DNA fromM. bigelovii can be present inM. douglasii plants with chloroplasts typical ofM. bigelovii. The study demonstrates the usefulness of the RAPD technique for screening large numbers of markers to select a few potentially informative ones for rigorous examination.Dedicated to emer. Univ.-Prof. DrFriedrich Ehrendorfer on the occasion of his 70th birthday     

Comparison of chloroplast and nuclear phylogeny in the autogamous annualMicroseris douglasii (Asteraceae: Lactuceae)

Morphology suggests that the Californian annualMicroseris douglasii is a monophyletic sister group to the other three diploid annuals ofMicroseris. Phylogenetic analysis of 44 inbred strains ofM. douglasii derived from 23 populations with 72 RAPD markers in the nuclear DNA strongly supports this phylogeny. However, 13 chloroplast RFLPs divideM. douglasii into four distinct groups. Two of these each share one or more cpRFLPs withM. bigelovii andM. pygmaea. Several hypotheses can explain the incongruence between nuclear and chloroplast phylogeny: (1) random sorting out of chloroplasts during phylogeny from a polymorphic pool, (2) cytoplasmic introgression from the related annualM. bigelovii intoM. douglasii after hybridization followed by elimination of theM. bigelovii nuclear genome. We suggest cytoplasmic introgression as the most likely origin. Possible remnants of nuclear introgression have been found in two populations ofM. douglasii that are polymorphic for chloroplast types. In these populationsM. bigelovii type chloroplast DNA seems to be accompanied by nuclear genes for flower color and leaf shape.     

Phenotypic Similarity and Genetic Relationship Among Populations of Microseris bigelovii (Asteraceae: Lactuceae)

Twelve inbred strains have been derived from 10 representative populations of the North American annual M. bigelovii. The distribution and correlation of 21 phenotypic characters distinguishing these strains has been studied. The genetic similarity of these strains has been previously determined using nu clear DNA polymorphisms. Genetically similar strains (always geographic near neighbors) share phenotypic character states but these involve different characters in different cases. A few character state differences are correlated across all strains via early rosette growth rate, timing of reproduction, or overall capitulum size. Most character states recombine freely. This is evidence for effective gene flow (probably by achene dispersal) among these isolated populations.     

Genotype relationships inMicroseris elegans (Asteraceae,Lactuceae) revealed by DNA amplification from arbitrary primers (RAPDs)

The genetic relationships among 10 inbred lines representing 10 populations of the autogamous annualMicroseris elegans from throughout California has been determined using random amplified polymorphic DNAs (RAPDs). Seventeen arbitrary 10 base pair primers produced 134 amplification products; 81 of these were shared by two or more strains. The 3 genotypes from Northern California are closely related as are 3 genotypes from Middle Californian populations which are not nearest neighbors. DNA fingerprinting with the oligonucleotide (GATA)₄ gave compatible results, but the comparison was limited to samples run on one gel. Isoenzyme patterns are compatible with the DNA results, but limited by the very low number of informative polymorphisms. The clustered relationship among genotypes within a species and their geographic distribution suggests very restricted genetic recombination and an origin of new populations from randomly dispersed achenes within the range of the species.     

Phylogenetic relationships and genetic polymorphisms in wild Indian mice

Randomly amplified polymorphic DNA (RAPD) analysis was used to examine the extent of variability in 11 Indian wild derived commensal house mice (Mus musculus) populations and compared with inbred strains of musculus and domesticus subspecies as well as commonly used laboratory inbred strains C57BL/6J and DBA/2J. Arbitrary designed 10 mer oligonucleotide primers with 60-70% (G+C) content were used to amplify DNA template. Out of 52 primers screened initially on the laboratory strains, 20 were selected for analysis on the basis of amplification product in the size range of 200-1400 bp. Among 353 total polymorphic bands, 220 bands (64%) were found to be polymorphic in Indian wild mice, 85 bands (25%) in wild derived inbred strains and 37 bands (11%) in laboratory mice strains. The amplification patterns produced by primers were statistically analysed by Jaccard's similarity coefficient the value of which ranged from 0.56 to 0.80. High level of genetic diversity was seen in the Indian wild mice populations as compared to the controls. The UPGMA phenogram grouped mice population into two major clusters except Bikaner [BIK], Bilaspur [BIL] and Ranikhet [RK] populations which were placed outside the close-knit clusters. Inspite of low values of bootstrap estimates obtained by Wagner and Dollo parsimony analysis, the results were comparable with UPGMA phenogram when constitution of the populations in the major cluster was considered. Indian mice populations appeared to be diverse from laboratory inbred mice strains.     

Chloroplast and nuclear DNA variation among homozygous plants in a population of the autogamous annualMicroseris douglasii (Asteraceae,Lactuceae)

The autogamous diploid annualMicroseris douglasii of California occurs in many isolated populations. The populations consist of one to many highly inbred biotypes. Morphological variation among populations usually is greater than within populations. In spite of the virtual absence of gene flow even within populations, genetically determined character differences are randomly distributed and associated throughout the range of the species. Recent evidence even suggests introgression of chloroplasts from the relatedM. bigelovii. Offspring families from 25 plants of a very variable population were raised and examined for segregation of morphological and molecular (RAPD) markers. All 25 original plants were completely homozygous for all markers, but each differed from all others at least in some markers. The population consisted of two genetically isolated groups of plants: a distinct inbred line (3 plants) and 22 plants with random associations of a common set of markers and characters, possibly recombinant inbreds from a past hybridization event. One of these 22 plants contained a chloroplast genome found inM. bigelovii, the other 24 plants a chloroplast genome found only inM. douglasii.     

Random amplified polymorphic DNA (RAPD) identification of genetic variation in three species ofPorphyra (Bangiales,Rhodophyta)

The random amplified polymorphic DNA (RAPD) technique was used to characterize three species ofPorphyra from the western North Atlantic and adjacent Gulf of Mexico. Twenty 10-mer primers were screened for DNA amplification usingPorphyra template DNA. Nine of these oligonucleotide primers, all (G+C)-rich, were positive or band-producing, but yielded poor or variable band resolution. Subsequent use of the universal 20-mer M 13 primer resulted in both clear band resolution with a minimum of secondary bands and a high degree of reproducibility. Amplification products for DNA from six regional isolates ofPorphyra carolinensis Coll et Cox,P. leucosticta Thuret in Le Jolis andP. rosengurttii Coll et Cox were compared to each other and toBangia atropurpurea (Roth) C. Agardh. Results provide evidence of both genetically hetero- and homogeneous populations. Use of the RAPD method with the M 13 primer yields amplification products which can be used to fingerprint specific genotypes. This procedure could be used to discriminate between hetero- and homokaryotic fusion products from previously characterized donor strains.     

Fingerprinting genomes by use of PCR with primers that encode protein motifs or contain sequences that regulate gene expression

PCR primers of arbitrary nucleotide sequence have identified DNA polymorphisms useful for genetic mapping in a large variety of organisms. Although technically very powerful, the use of arbitrary primers for genome mapping has the disadvantage of characterizing DNA sequences of unknown function. Thus, there is no reason to anticipate that DNA fragments amplified by use of arbitrary primers will be enriched for either transcribed or promoter sequences that may be conserved in evolution. For these reasons, we modified the arbitrarily primed PCR method by using oligonucleotide primers derived from conserved promoter elements and protein motifs. Twenty-nine of these primers were tested individually and in pairwise combinations for their ability to amplify genomic DNA from a variety of species including various inbred strains of laboratory mice and Mus spretus. Using recombinant inbred strains of mice, we determined the chromosomal location of 27 polymorphic fragments in the mouse genome. The results demonstrated that motif sequence-tagged PCR products are reliable markers for mapping the mouse genome and that motif primers can also be used for genomic fingerprinting of many divergent species.     

Study of Resistance of Musa acuminata to Fusarium oxysporum using RAPD markers

Suckers collected from different populations of Musa acuminata ssp. malaccensis were found to be highly resistant to race 4 of Fusarium oxysporum f. sp. cubense (FOC) suggesting that local wild banana populations co-evolved with the pathogen. Seedlings from these wild banana plants segregated for resistance to the pathogen. The infected seedlings were characterized based on external and internal symptoms and the variable response to FOC was mainly due to the genetic factors. Using the technique of random amplified polymorphic DNA (RAPD), 96 major amplification products from 15 primers were identified. Only 10 out of 96 markers were monomorphic and shared among the seed progenies, whereas the remaining 86 were highly polymorphic. Three primers showed banding patterns specific to resistant or susceptible seedlings. These results showed the great potential of the wild Musa acuminata ssp. malaccensis as a source for banana improvement and also for the synthesis of segregating populations for linkage mapping, gene cloning and DNA markers related to FOC resistance.     

Inheritance of polymorphic markers generated by DNA amplification fingerprinting and their use as genetic markers in soybean

DNA amplification fingerprinting (DAF) using a high primer-to-template ratio and single, very short arbitrary primers, was used to generate amplified fragment length polymorphic markers (AFLP) in soybean (Glycine max (L.) Merr.). The inheritance of AFLPs was studied using a cross between the ancestral Glycine soja PI468.397 and Glycine max (L.) Merr. line nts382, F1 and F2 progeny. The amplification reaction was carried out with soybean genomic DNA and 8 base long oligounucleotide primers. Silver-stained 5% polyacrylamide gels containing 7 M urea detected from 11 to 28 DAF products with primers of varying GC content (ranging from 50 to 100% GC). Depending on their intensity, AFLPs were classified into three classes. DAF profiles were reproducible for different DNA extractions and gels. Forty AFLPs were detected by 26 primers when comparing G. soja and G. max. Most AFLPs were inherited as dominant Mendelian markers in F1 and F2 populations. However, abnormal inheritance occured with about 25% of polymorphisms. One marker was inherited as a maternal marker, presumably originating from organelle DNA while another showed apparent paternal inheritance. To confirm the nuclear origin and utility of dominant Mendelian markers, three DAF polymorphisms were mapped using a F11 mapping population of recombinant inbred lines from soybean cultivars Minsoy × Noir 1. The study showed that DAF-generated polymorphic markers occur frequently and reliably, that they are inherited as Mendelian dominant loci and that they can be used in genome mapping.     

The Evolutionary Reduction of Microsporangia in Microseris (Asteraceae): Transition Genotypes and Phenotypes

Abstract: The loss of the two inner (adaxial) microsporangia (MS) on the anthers is a shared, derived character for three species of the genus Microseris (Asteraceae). In a hybrid between M. douglasii (4 MS) and M. bigelovii (2 MS), one major gene and four modifier loci are responsible for the difference in MS number. The homozygous recessive (2 MS) genotype of the major gene is necessary but not sufficient for the reduction. In addition, at least five M. bigelovii (2 MS) alleles of the three major modifiers are needed for a stable 2‐MS phenotype in all florets of a plant. One, two or three M. bigelovii alleles of the modifiers cause the random reduction or loss of some of the adaxial MS. When the major gene and two modifiers specify 2 MS and only one modifier is homozygous for the M. douglasii (4 MS) alleles, sister plants can have any phenotype from pure 2 MS to pure 4 MS. Here, we examine the phenotypic expression of these genotypes raised under the normal winter annual conditions and under long‐day conditions. In all cases, the phenotypes vary among sister plants, but the range of variation (most notably under long‐day conditions) depends on the specific modifier gene contributing the M. douglasii alleles. The phenotypic variance in one of the genotypes was decreased by a factor of ten in the depauperate heads produced in the long‐day experiment. This effect is mediated by a dependence of the MS phenotype on the position of the floret relative to the edge of the flowering head (capitulum) and directly by the size of the capitulum. Genotypes specifying phenotypes with more or less precisely two or four MS in all florets show hardly any dependence on environmental or developmental factors. The significance of these observations lies in the non‐linear, “canalized” relationship between phenotypic expression and gene dosage, which shows how a qualitative morphological change dependent on a single major gene mutation can pass through a potentially maladaptive intermediate stage.     

Development of primer sets for PCR amplification of the PgiC gene in ferns

Polymerase chain reaction (PCR)-based nuclear DNA markers were developed for fern species. We first determined the partial nucleotide sequence of cDNA of the pgiC gene encoding cytosolic phosphoglucose isomerase from Dryopteris caudipinna, and then PCR primers for exon-primed, intron-crossing (EPIC) amplifications were designed. The EPIC primers are universally applicable to the most derived indusiate fern families such as Dryopteridaceae, Thelypteridaceae, and Woodsiaceae. The PCR products of primers 14F/16R containing two introns are moderate in size (534 bp–ca.1000 bp) and are possibly of value in phylogenetic reconstruction at specific and generic levels. Codominant nuclear DNA markers applicable to the estimation of mating systems and other population genetic studies were also developed by a combination of single-strand conformation polymorphism (SSCP) and EPIC amplification using primers 14F/15R and 15F/16R. In order to provide a case study using these markers, allelic variation of PCR products using 15F/16R was examined in populations of Arachniodes standishii (Dryopteridaceae). Received: July 4, 2001 / Accepted: September 12, 2001     

南方玉米锈病抗病基因的定位及不同遗传背景对基因标记的比较分析

玉米自交系齐319高抗南方玉米锈病。利用SSR标记技术和BSA分析对齐319抗南方玉米锈病基因进行了标记分析,结果表明SSR标记phi041和phi118与齐319抗南方锈病基因连锁,其遗传距离分别为7.69cM和8.55cM。因此南方玉米锈病抗病基因定位于玉米10号染色体短臂上。本研究进行的抗病基因标记,选择使用了两个杂交组合的3个分离群体,标记结果显示同一杂交组合的不同分离群体其标记结果是一致的,而不同组分分离群体的标记结果有显著差异,这可能与基因的遗传背景相关。因此,在进行基因标记分析时,选择合适的分离群体是至关重要的。     

PCR-analyzed microsatellites: Data concerning laboratory and wild-derived mouse inbred strains

We have investigated 67 primers designed by Dr. J. Todd and co-workers to amplify microsatellites sequences in the mouse. We report on additional polymorphisms concerning seven laboratory inbred strains, complementary to those already published. We include the survey of three independently derived strains of Mus spretus: SPE/Pas, SEG/Pas and SPR/Smh. SPE/Pas and SEG/Pas are very close (3% polymorphism), whereas the third one, (SPR/Smh), is very different from the other two strains (33% polymorphism). Seventy-four to 84% of the microsatellites analyzed in this study are polymorphic between C57BL/6Pas and Mus spretus strains. By comparison, 36–46% are polymorphic between laboratory inbred strains involved in established sets of recombinant inbred strains. A strain derived from Mus musculus musculus (PWK/Pas) was found to be very different from both C57BL/6Pas (70% polymorphism) and SPE/Pas (82% polymorphism). These results emphasize the interest of using Mus musculus musculus inbred strains to establish interspecific crosses, particularly when considering their breeding performances.     

微卫星DNA标记在近交系大鼠HFJ和MIJ遗传监测中的应用

目的 用24对引物对近交系HFJ和MIJ大鼠的微卫星位点进行多态性分析,并选用近交系Lewis和F344大鼠作为对照,进行比较分析.方法 用传统的酚-氯仿法分别提取4个近交系大鼠MIJ、HFJ、Lewis和F344 的基因组DNA,选取大鼠24个微卫星位点,通过PCR扩增,扩增产物经过非变性聚丙烯酰胺凝胶电泳和银染,根据电泳结果,比较分析4种品系近交系大鼠之间微卫星多态性.结果 4种品系及品系内不同个体的近交系大鼠在24个微卫星位点上的扩增产物均出现一个条带,MIJ和HFJ大鼠在品系间和品系内均表现为单态性,同Lewis 和F344的扩增结果比较,14个位点显示多态性,有10个位点显示单态性.结论 两个近交系大鼠品系MIJ和HFJ符合近交系要求,筛选出的14个多态性微卫星位点可用于有关近交系大鼠的遗传背景监测.     

A group-specific PCR assay for the detection of Helicobacteraceae in human gut

Background. Enterohepatic Helicobacter species are emerging pathogens, which are increasingly isolated from humans with enteric diseases. Nevertheless, current methods to detect Helicobacteraceae in the human gut have significant limitations. Methods. Based on 16S‐rRNA gene alignments and computer aided primer analysis a set of group‐specific PCR primers was designed. The evaluation of the PCR assay was performed using 36 ATCC reference strains and intestinal biopsies from 10 patients with defined gastric Helicobacter pylori status. The amplification products derived from clinical samples were cloned and subsequently analyzed by DNA sequencing. Sensitivity of the PCR‐assay was determined by spiking previously negative tested samples with decreasing amounts of Helicobacter DNA. Results. The analysis of the ATCC reference strains revealed amplification products in all 14 Helicobacter strains and Wolinella succinogenes, 21 other microorganisms representing negative controls did not produce PCR fragments. Four out of the 10 patient‐derived samples were positive. Three of them represented H. pylori‐derived DNA confirming the gastric H. pylori infection in these patients. In the fourth patient, who was suffering from Crohn's disease, H. pullorum was identified. The sensitivity of the PCR assay was 0.1 pg of Helicobacter‐derived DNA representing about 40 bacteria. Conclusion. The novel PCR assay described here is an important new tool in rapid and sensitive assessment for the presence of Helicobacteraceae in human gut.     

Random sequence oligonucleotide primers detect polymorphic DNA products which segregate in inbred strains of mice

The random amplification of polymorphic DNA (RAPD) using primers of arbitrary nucleotide sequence has been extremely valuable in identifying heritable markers in a variety of systems. The present studies examined whether the RAPD technique can identify large numbers of polymorphisms that can be used to construct genetic maps in inbred strains of mice. By screening the inbred mouse strains C57BL/6J and DBA/2J with 481 random 10-mer oligonucleotide primers, we identified 95 polymorphisms and mapped 76 of these by use of the BXD series of recombinant inbred (RI) strains. The results clearly demonstrate that the RAPD technique allows for the identification of large numbers of DNA-based polymorphisms that distinguish these two inbred strains of mice,and that such markers can readily be used to construct molecular genetic linkage maps.     

Characterization of natural populations of Nitrobacter spp. using PCR/RFLP analysis of the ribosomal intergenic spacer

DNA sequences from the intergenic spacer (IGS) region of the ribosomal operon were amplified by the polymerase chain reaction (PCR) technique using two primers derived from 16S and 23S rRNA conserved sequences. The PCR products, cleaved by 4 base cutting restriction enzymes, were used to differentiate Nitrobacter strains. This method offered a convenient alternative to serological testing for characterization of Nitrobacter isolates and enabled a large number of strains to be genotypically characterized easily and rapidly. This method was successfully used to characterize natural populations of Nitrobacter from various soils and a lake. A diversity was demonstrated in various soils, and in a lake both in freshwater and in sediments. Strains closely related to both WL and LL were found in these eco-systems. It seems that the diversity of Nitrobacter populations was not associated with global environments but may be related to the presence of locally coexisting niches.Non-commun abbreviations PCR polymerase chain reaction - RFLP restriction fragment length polymorphism - IGS intergenic spacer     

Identification of RAPD markers linked to the karnal bunt resistance genes in wheat

A set of 104 wheat recombinant inbred lines (RILs) obtained from a cross between parents resistant (HD 29) and susceptible (WH 542) to karnal bunt (KB) (caused by Neovossia indica) were screened and used to identify random amplified polymorphic DNA (RAPD) markers linked with resistance to karnal bunt as these would allow indirect marker assisted selection of KB resistant genotypes. The two parents were analysed with 92 RAPD primers. A total of 65 primers proved functional by giving scorable polymerase chain reaction (PCR) products. Of these, 21 (32 %) primers detected polymorphism between the two parental genotypes. Using these primers, bulked segregant analysis was carried out on two bulk DNAs, one obtained by pooling DNA from 10 KB resistant RILs and the other similarly derived by pooling 10 KB susceptible RILs. One marker, OPM-20 showed apparent association with resistance to KB. This was confirmed following selective genotyping of individual RILs included in the bulks.     

Assessment ofGibberella fujikuroi mating type by PCR

Mating type (MAT)-specific fragments of the two idiomorphs ofGibberella fujikuroi (anamorph,Fusarium moniliforme) were obtained by PCR amplification using primers to conserved regions ofMAT homologs from other fungal species and used to assign mating type by molecular criteria rather than the arbitrary historical designation. Mating type—strains of mating populations A-E and a mating type+strain of mating population F carry an α-box motif and should therefore be designatedMAT-1. Mating type+strains of mating populations A-E and a mating type—strain of mating population F carry an HMG-box motif and should be designatedMAT-2. Thus, assessment of mating type ofG. fujikurol strains can be easily achieved usingMAT-specific primers.