Genetic diversity of Agrobacterium vitis as determined by DNA fingerprints of the 5'-end of the 23S rRNA gene and Random Amplified Polymorphic DNA

Sixty-nine strains of Agrobacterium vitis , the causal agent of grape crown gall, originating from different geographical regions of the USA and Europe, were characterized by fingerprint analysis of the 5'-end of the 23S rRNA gene and by Random Amplified Polymorphic DNA (RAPD) markers. For 5'-end 23S fingerprinting, amplicons were digested with Taq I, Rsa I, Ava I, Cfo I and Alu I. For RAPD analysis, three 10-mer primers were used to generate PCR products. There was a high degree of correlation between strain groupings generated by the two methods. However, more diversity was identified when groupings were based on RAPDs. For example, 28 of 29 strains having nopaline type Ti plasmids generated identical 5'-end 23S patterns but formed two distinct RAPD groups that separated strains originating from the USA and Hungary. Similarly by RAPDs, one cluster of strains carrying vitopine-type Ti plasmids could be separated into those originating in the USA and Europe. The composition of strain groups generated by 5'-end 23S and RAPDs were highly correlated with a previous fingerprint analysis of the intergenic spacer region (located between the 16S and 23S rRNA genes) and with RFLP analysis for characterizing Ti plasmids. These findings show that among Ag. vitis strains there is a high level of correlation between two regions of the rRNA operon, total genomic DNA (as determined by RAPDs) and the type of Ti plasmid they carry.     

Molecular characterisation of inter and intra-specific somatic hybrids of potato using randomly amplified polymorphic DNA (RAPD) markers

Summary Protoplast fusion allows the transfer of both mono- and polygenic traits between species that are sexually incompatible. This approach has particular relevance for potato, and somatic hybridisation has been used to introduce a range of disease resistance genes from sexually incompatible wild species into the cultivated potato gene pool. In addition, protoplast fusion allows the resynthesis of tetraploid genotypes from pre-selected diploid or dihaploid donor parents. A limiting factor for the efficient exploitation of this technology in potato breeding is the difficulty of unequivocally identifying nuclear hybrids (heterokaryons). In order to facilitate the identification of hybrids at an early stage following fusion, Randomly Amplified Polymorphic DNA markers (RAPDs) have been used to characterise molecularly both inter- and intra-specific somatic hybrids of potato. RAPD markers detect naturally occurring polymorphism in the donor genotypes and utilise short oligonucleotide primers of arbitrary nucleotide sequence in combination with the polymerase chain reaction (PCR). The exploitation of RAPDs in the characterisation of both somatic and sexual hybrids is discussed.     

Positional cloning without a genome map: using 'Targeted RFLP Subtraction' to isolate dense markers tightly linked to the regA locus of Volvox carteri.

The ability to isolate genes defined by mutant phenotypes has fueled the rapid progress in understanding basic biological mechanisms and the causes of inherited diseases. Positional cloning, a commonly used method for isolating genes corresponding to mutations, is most efficiently applied to the small number of model organisms for which high resolution genetic maps exist. We demonstrate a new and generally applicable positional cloning method that obviates the need for a genetic map. The technique is based on Restriction Fragment Length Polymorphism (RFLP) Subtraction, a method that isolates RFLP markers spanning an entire genome. The new method, Targeted RFLP Subtraction (TRS), isolates markers from a specific region by combining RFLP Subtraction with a phenotypic pooling strategy. We used TRS to directly isolate dense markers tightly linked to the regA gene of the eukaryotic green alga Volvox. As a generally applicable method for saturating a small targeted region with DNA markers, TRS should facilitate gene isolation from diverse organisms and accelerate the process of physically mapping specific regions in preparation for sequence analysis.     

Development of cost-effective Hordeum chilense DNA markers: molecular aids for marker-assisted cereal breeding

Hordeum chilense is a potential source of useful genes for wheat breeding. The use of this wild species to increase genetic variation in wheat will be greatly facilitated by marker-assisted introgression. In recent years, the search for the most suitable DNA marker system for tagging H. chilense genomic regions in a wheat background has lead to the development of RAPD and SCAR markers for this species. RAPDs represent an easy way of quickly generating suitable introgression markers, but their use is limited in heterogeneous wheat genetic backgrounds. SCARs are more specific assays, suitable for automatation or multiplexing. Direct sequencing of RAPD products is a cost-effective approach that reduces labour and costs for SCAR development. The use of SSR and STS primers originally developed for wheat and barley are additional sources of genetic markers. Practical applications of the different marker approaches for obtaining derived introgression products are described.     

Use of a subset of doubled-haploid lines for RAPD interval mapping in barley.

Molecular markers have been used in barley to locate genes and quantitative trait loci. Only a few RAPD markers have been located on barley marker maps. The objectives of this study were (i) to place RAPD markers in specific intervals on the barley linkage map developed from the cross Steptoe (S) x Morex (M), (ii) to examine the distribution of RAPD markers, and (iii) to compare markers amplified by Taq DNA polymerase with those amplified by the Stoffel fragment of Taq DNA polymerase. Screening of DNA from S and M with 362 decamer primers identified 85 that amplified 127 reliable RAPDs. A subset of 15 doubled-haploid (DH) lines from the 150 DH line mapping population was used to place these RAPD markers in intervals on the SM map. This subset can be used for rapid placement of any new markers on the SM linkage map. Most of the RAPD markers were dominant but four codominant RAPDs were identified. The RAPDs were not evenly distributed, with many clustered around the centromeric region of each chromosome. Two of these clusters were located in intervals larger than 15 cM. Testing of 38 to 42 additional DH lines provided more precise placement of eight of the markers in these clusters. Reliable RAPDs were detected with 44% of the primers tested with the Stoffel fragment, but with only 17% of the primers tested with Taq DNA polymerase. These RAPDs provide additional markers for use in barley improvement.     

Identification of a codominant scar marker linked to the seedlessness character in grapevine

 The variety Vitis vinifera cv Sultanine presents a type of seedlessness in which fertilization occurs but seeds subsequently fail to develop. It has been suggested that this trait might be controlled by three complementary recessive genes regulated by a dominant gene named I. Bulk segregant analysis was used to search for random amplified polymorphic DNA (RAPD) markers linked to the I gene in progeny obtained by crossing two partially seedless genotypes. One hundred and forty decamer primers were screened using bulks obtained by pooling the DNA of extreme individuals from the phenotypic distribution. We identified two RAPD markers which appeared tightly linked to I (at 0.7 and 3.5 cM respectively). The closest marker was used to develop a codominant SCAR (sequence characterized amplified region), named SCC8. This latter marker appeared of great value either to exclude from the progeny potentially seeded individuals or to select for seedless individuals. Indeed, all the seeded individuals of the progeny were found to be homozygous scc8 ^-/scc8 ^-, and all the individuals homozygous SCC8 ⁺/SCC8 ⁺ were seedless. Moreover, this marker was successfully applied to other natural seedless varieties where codominance persisted. SCC8 was also used to dissect more precisely the genetics of seedlessness. ANOVA analysis indicated that this SCAR marker accounted for at least 64.9% of the phenotypic variation of the seed’s fresh weight and for at least 78.7% of the phenotypic variation of the seed’s dry matter. These results confirmed the presence of a major gene, and also the existence of other complementary recessive genes, controlling the expression of seedlessness. Received: 29 July 1997 / Accepted: 16 March 1998     

羊草种群遗传分化的RAPD分析Ⅱ.RAPD数据的统计分析

对松嫩草原上分布的灰绿型和黄绿型羊草9个种群进行了15个引物的RAPD分析,统计结果表明,两类种群的扩增片段数和多态位点比率明显不同,黄绿型种群低于灰绿型,其值分别＜90与＞100,＜50％与＞70％,比较了7种不同统计方法据RAPD表型或基因型频率估算的种群遗传多样性,几种统计结果都揭示,黄绿型种群低于灰绿型种群,用F1s值矫正种群对Hardy-Weinberg平衡的偏离后,估算等位基因频率,通过Shannon指数和Nei指数估计羊草种群间分化分别为37．6％和35．7％,高于等位酶的分析,讨论比较了等位酶和RAPD分析结果的异同。     

Differentiation and grouping of isolates of the Ganoderma lucidum complex by random amplified polymorphic DNA-PCR compared with grouping on the basis of internal transcribed spacer sequences.

Laccate polypores of the Ganoderma lucidum species complex are widespread white rot fungi of economic importance, but isolates cannot be identified by traditional taxonomic methods. Parsimony analysis of nucleotide sequences from the internal transcribed spacers (ITS) of the ribosomal gene (rDNA) distinguished six lineages in this species complex. Each ITS lineage may represent one or more putative species. While some isolates have identical ITS sequences, all of them could be clearly differentiated by genetic fingerprinting using random amplified polymorphic DNA (RAPD). To investigate the suitability of RAPD markers for taxonomic identification and grouping of isolates of the G. lucidum complex, RAPD fragments (RAPDs) were used as phenotypic characters in numerical and parsimony analyses. Results show that data from RAPDS do not distinguish the same clades as ITS data do. Groupings based on analysis of RAPD data were very sensitive to the choice of the grouping method used, and no consistent grouping of isolates could be proposed. However, analysis with RAPDs did resolve several robust terminal clades containing putatively conspecific isolates, suggesting that RAPDs might be helpful for systematics at the lower taxonomic levels that are unresolved by ITS sequence data. The limitations of RAPDs for systematics are briefly discussed. The conclusion of this study is that ITS sequences can be used to identify isolates of the G. lucidum complex, whereas RAPDs can be used to differentiate between isolates having identical ITS sequences. The practical implications of these results are briefly illustrated.     

Identification of the essential EPE1 gene involved in retention of secreted proteins on the cell surface of Saccharomyces cerevisiae cells.

Saccharomyces cerevisiae yeast cells secrete extracellularly low amounts of a few proteins. The reasons for retardation of secreted proteins on the cell surface remain obscure. We describe here a mutant able to export enhanced amount of proteins. Classical genetic methods, nucleic acids manipulations and cloning procedures were used to isolate and characterize the mutant and to clone and sequence the corresponding wild type gene. The isolated Saccharomyces cerevisiae mutant MW11, is temperature sensitive and exports on average twenty-fold more proteins at 37 degrees C than parental wild type strain (80 micrograms of proteins/1 x 10(8) mutant cells, SEM +/- 5, n22; versus 3 micrograms of proteins/1 x 10(8) parental cells, SEM +/- 1, n22). Protein overexport in the mutant requires a functional SEC1 pathway and is independent of cell lysis. Cloning and sequencing of the corresponding wild type gene identified an open reading frame of 786 bp coding for a hydrophilic protein with predicted molecular mass of 30 kDa and cytosolic localization. The newly identified gene, designated EPE1, is an essential gene. Its DNA and amino acids sequence showed no homology with other yeast genes and proteins. It is concluded that the function of unknown yet genes, such as EPE1 is needed for retention of secreted proteins on the surface of Saccharomyces cerevisiae cells.     

Identification of RAPD markers linked to genetic factors controlling the milling energy requirement of barley

Doubled haploid (DH) populations of barley have been used in combination with PCR-based polymorphic-assay procedures to identify molecular markers linked to genes controlling the milling energy requirement of the grain. Milling energy (ME) is a quantitative trait and locating individual quantitative trait loci (QTLs) involved the construction of bulks by combining DNA from DH families representing the extreme members of the distribution for ME. In addition, the individuals had alternative alleles at theRrn2 locus that has previously been shown to be linked to an ME QTL. The DNA bulks were screened with Randomly Amplified Polymorphic DNA (RAPD) markers and polymorphic amplification products tested for linkage to genes influencing the expression of ME in a DH population. Several markers were identified which are linked to a QTL controlling ME and the recombination fraction determined by maximum likelihood procedures. The results indicate that DHs in combination with RAPDs and bulked segregant analysis provide an efficient method for locating QTLs in barely. Furthermore, this approach is applicable to mapping other QTLs in a range of organisms from which DH or recombinant inbred lines can be extracted.     

Molecular characterization of the SCAR markers tightly linked to the Tm-2 locus of the genus Lycopersicon

The Tm-2 gene and its alleles conferring tomato mosaic virus resistance in tomato originate from Lycopersicon peruvianum, a wild relative of tomato. DNA fragments of several RAPD markers tightly linked with the Tm-2 locus in tomato were successfully cloned and sequenced. Subsequently, the 24-mer oligonucleotide primer pairs of the SCAR markers corresponding to the RAPD markers were designed based on the 5’-endmost sequences. A fragment of the same size as that of a SCAR marker was amplified in the ToMV-susceptible tomato line with no Tm-2, but the digests of the PCR fragments by AccI exhibited polymorphism in fragment length between the two lines. We chose three SCAR markers and three RAPD markers tightly linked with the Tm-2 locus, and examined whether the same-sized fragments corresponding to these markers were also present in three other lines carrying Tm-2a or one of the other Tm-2 alleles. The fragments corresponding to the three SCAR markers were present in all of the three lines, but the other markers (three RAPDs ) were absent in one or two lines, suggesting that the three SCAR markers are closer to Tm-2 than the other markers. Comparison of the nucleotide sequences of these fragments revealed that they are all homologous to the corresponding SCAR markers. Received: 8 November 1999 / Accepted: 15 November 1999     

Specific genetic markers for wheat, spelt, and four wild relatives: comparison of isozymes, RAPDs, and wheat microsatellites.

Three types of markers-isozymes, RAPDs (random amplified polymorphic DNAs), and wheat microsatellites- were tested on wheat, spelt, and four wild wheat relatives (Aegilops cylindrica, Elymus caninus, Hordeum marinum, and Agropyron junceum). The aim was to evaluate their capability to provide specific markers for differentiation of the cultivated and wild species. The markers were set up for subsequent detection of hybrids and introgression of wheat DNA into wild relatives. All markers allowed differentiation of the cultivated from the wild species. Wheat microsatellites were not amplified in all the wild relatives, whereas RAPDs and isozymes exhibited polymorphism for all species. The dendrograms obtained with RAPD and isozyme data separated Swiss wheat cultivars from those collected in Austria and England, while no difference was found between Swiss spelt and wheat. RAPD data provided a weak discrimination between English and Austrian E. caninus. The microsatellite-based dendrogram discriminated populations of Ae. cylindrica, but no clear separation of H. marinum from E. caninus was revealed. The similarity matrices based on the three different sets of data were strongly correlated. The highest value was recorded between the matrices based on RAPDs and isozymes (Mantel's test, r = 0.93). Correlations between the similarity matrix based on microsatellites and matrices based on RAPDs and isozymes were lower: 0.74 and 0.68, respectively. While microsatellites are very useful for comparisons of closely related accessions, they are less suitable for studies involving less-related taxa. Isozymes provide interesting markers for species differentiation, but their use seems less appropriate for studies of within-species genetic variation. RAPDs can produce a large set of markers, which can be used for the evaluation of both between- and within-species genetic variation, more rapidly and easily than isozymes and microsatellites.     

Analysis of population genetic structure with RAPD markers

Recent advances in the application of the polymerase chain reaction make it possible to score individuals at a large number of loci. The RAPD (random amplified polymorphic DNA) method is one such technique that has attracted widespread interest. The analysis of population structure with RAPD data is hampered by the lack of complete genotypic information resulting from dominance, since this enhances the sampling variance associated with single loci as well as induces bias in parameter estimation. We present estimators for several population-genetic parameters (gene and genotype frequencies, within- and between-population heterozygosities, degree of inbreeding and population subdivision, and degree of individual relatedness) along with expressions for their sampling variances. Although completely unbiased estimators do not appear to be possible with RAPDs, several steps are suggested that will insure that the bias in parameter estimates is negligible. To achieve the same degree of statistical power, on the order of 2 to 10 times more individuals need to be sampled per locus when dominant markers are relied upon, as compared to codominant (RFLP, isozyme) markers. Moreover, to avoid bias in parameter estimation, the marker alleles for most of these loci should be in relatively low frequency. Due to the need for pruning loci with low-frequency null alleles, more loci also need to be sampled with RAPDs than with more conventional markers, and some problems of bias cannot be completely eliminated.     

RAPD polymorphism of wild emmer wheat populations, Triticum dicoccoides, in Israel

 Genetic diversity in random amplified polymorphic DNAs (RAPDs) was studied in 110 genotypes of the tetraploid wild progenitor of wheat, Triticum dicoccoides, from 11 populations sampled in Israel and Turkey. Our results show high level of diversity of RAPD markers in wild wheat populations in Israel. The ten primers used in this study amplified 59 scorable RAPD loci of which 48 (81.4%) were polymorphic and 11 monomorphic. RAPD analysis was found to be highly effective in distinguishing genotypes of T. dicoccoides originating from diverse ecogeographical sites in Israel and Turkey, with 95.5% of the 100 genotypes correctly classified into sites of origin by discriminant analysis based on RAPD genotyping. However, interpopulation genetic distances showed no association with geographic distance between the population sites of origin, negating a simple isolation by distance model. Spatial autocorrelation of RAPD frequencies suggests that migration is not influential. Our present RAPD results are non-random and in agreement with the previously obtained allozyme patterns, although the genetic diversity values obtained with RAPDs are much higher than the allozyme values. Significant correlates of RAPD markers with various climatic and soil factors suggest that, as in the case of allozymes, natural selection causes adaptive RAPD ecogeographical differentiation. The results obtained suggest that RAPD markers are useful for the estimation of genetic diversity in wild material of T. dicoccoides and the identification of suitable parents for the development of mapping populations for the tagging of agronomically important traits derived from T. dicoccoides. Received: 13 July 1998 / Accepted: 13 August 1998     

Transformation reveals a chromosomal locus of the gene(s) for methicillin resistance in Staphylococcus aureus.

The localization of the gene(s) mediating methicillin (mecr) in Staphylococcus aureus was determined by transformation with deoxyribonucleic acid (DNA) from a natural mecr strain (DU 4916) and transformation obtained with DNA from this strain. Streptomycin resistance genes (strr) and novobiocin resistance genes (novr) were used concurrently as representatives for chromosomal genes; penicillinase (PI254) and tetracycline plasmids were used as examples of medium- and small-size extrachromosomal genes, respectively. Superinfection of the lysogenic recipients with the competence-inducing phage phi11 or 83A enhanced transformation for all markers. Phenotypic expression of cadmium (cadr), tetracycline (tetr), or methicillin resistance (mecr) did not appear to require a host recombination system since a recA1 mutant could serve as the recipient provided it was superinfected with a competence-inducing phage. There was, furthermore, no requirement for preexisting plasmids for phenotypic expression. Ultraviolet irradiation of transforming DNA enhanced at low doses the transformation frequency for chromosomal genes strr and novr but not for mecr, cadr, or tetr. The gene(s) for mecr was transformed with chromosomal DNA after sodium dodecyl sulfate-sodium chloride extraction and after neutral sucrose gradient centrifugation of bulk DNA from wild-type strain DU 4916 and the transformats. No cavalently closed circular DNA or open circular DNA carrying the methicillin resistance gene(s) could be detected in the wild type or the transformants either by ethidium bromide-cesium chloride gradient centrifugation or by zonal rate centrifugation of cells directly lysed on top of the gradients. The mecr gene(s) is thus probably of chromosomal nature but possibly under recombinational control of phage genes, since transfer of mecr is independent of the recA1 gene(s) but can be accomplished in this strain after superinfection with a competence-inducing phage. Ultraviolet light inactivation of transforming DNA shows first-order kinetics for mecr transformability similar to that observed for both transfecting and plasmid DNA.     

Polymorphism of a complex resistance gene candidate family in wild populations of common bean (Phaseolus vulgaris) in Argentina: comparison with phenotypic resistance polymorphism

Fifteen populations of wild bean (Phaseolus vulgaris), located in three provinces in Argentina, were analysed for their polymorphism for a complex resistance gene candidate (RGC) family clustered on the genome and for resistance phenotypes to strains of Colletotrichum lindemuthianum. Results indicate that RGC polymorphism is high. Population structure obtained for markers related to resistance was compared to population structure obtained for RAPD markers in order to infer the evolutionary forces driving polymorphism for resistance in wild populations at both molecular and phenotypic levels. Hierarchical analysis of differentiation showed that, within provinces, populations were differentiated for RAPD as well as for molecular and phenotypic markers of resistance. In contrast, provinces were differentiated only for RAPD and RGC markers and not for resistance phenotypes. The discrepancies found between diversity structures for molecular markers (RAPD and RGCs) and for resistance phenotypes suggest an effect of selection and indicate that diversity for resistance may not be driven by the same selective forces at the molecular and phenotypic levels. We further discuss whether specific selective forces are exerted on RGC markers and underline the importance of spatial scale of analysis for demonstrating an impact of selection.     

A genetic linkage map of cowpea (Vigna unguiculata) developed from a cross between two inbred, domesticated lines

 We have constructed a genetic linkage map within the cultivated gene pool of cowpea (2n=2x=22) from an F₈ recombinant inbred population (94 individuals) derived from a cross between the inbreds IT84S-2049 and 524B. These breeding lines, developed in Nigeria and California, show contrasting reactions against several pests and diseases and differ in several morphological traits. Parental lines were screened with 332 random RAPD decamers, 74 RFLP probes (bean, cowpea and mung bean genomic DNA clones), and 17 AFLP primer combinations. RAPD primers were twice as efficient as AFLP primers and RFLP probes in detecting polymorphisms in this cross. The map consists of 181 loci, comprising 133 RAPDs, 19 RFLPs, 25 AFLPs, three morphological/classical markers, and a biochemical marker (dehydrin). These markers identified 12 linkage groups spanning 972 cM with an average distance of 6.4 cM between markers. Linkage groups ranged from 3 to 257 cM in length and included from 2 to 41 markers, respectively. A gene for earliness was mapped on linkage group 2. Seed weight showed a significant association with a RAPD marker on linkage group 5. This map should facilitate the identification of markers that “tag” genes for pest and disease resistance and other traits in the cultivated gene pool of cowpea. Received: 16 September 1996 / Accepted: 25 April 1997     

A collection of enhancer trap insertional mutants for functional genomics in tomato

With the completion of genome sequencing projects, the next challenge is to close the gap between gene annotation and gene functional assignment. Genomic tools to identify gene functions are based on the analysis of phenotypic variations between a wild type and its mutant; hence, mutant collections are a valuable resource. In this sense, T‐DNA collections allow for an easy and straightforward identification of the tagged gene, serving as the basis of both forward and reverse genetic strategies. This study reports on the phenotypic and molecular characterization of an enhancer trap T‐DNA collection in tomato (Solanum lycopersicum L.), which has been produced by Agrobacterium‐mediated transformation using a binary vector bearing a minimal promoter fused to the uidA reporter gene. Two genes have been isolated from different T‐DNA mutants, one of these genes codes for a UTP‐glucose‐1‐phosphate uridylyltransferase involved in programmed cell death and leaf development, which means a novel gene function reported in tomato. Together, our results support that enhancer trapping is a powerful tool to identify novel genes and regulatory elements in tomato and that this T‐DNA mutant collection represents a highly valuable resource for functional analyses in this fleshy‐fruited model species.     

双孢蘑菇96．4菌株和单孢后代间的遗传变异

本文研究了野生双孢蘑菇菌株96．4（Agaricus bisporus)和它的10个单孢菌株的生长发育和遗传变异。研究表明,在菌株96．4和它的单孢菌株之间以及在10个单孢菌株之间在菌丝生速度和随机扩增多态DNA（RAPD）揭示的遗传变异等方面都存在着明显的变异。RAPD指纹揭示了96．4菌株通过有性生殖产生的丰富的生物学变异。     

Morphological and Molecular Differentiation of Sporidiobolus johnsonii ATCC 20490 and Its Coenzyme Q10 Overproducing Mutant Strain UF16

Coenzyme Q₁₀ (CoQ₁₀) is an industrially important molecule having nutraceutical and cosmeceutical applications. CoQ₁₀ is mainly produced by microbial fermentation and the process demands the use of strains with high productivity and yields of CoQ₁₀. During strain improvement program consisting of sequential induced mutagenesis, rational selection and screening process, a mutant strain UF16 was generated from Sporidiobolus johnsonii ATCC 20490 with 2.3-fold improvements in CoQ₁₀ content. EMS and UV rays were used as mutagenic agents for generating UF16 and it was rationally selected based on atorvastatin resistance as well as survival at free radicals exposure. We investigated the genotypic and phenotypic changes in UF16 in order to differentiate it from wild type strain. Morphologically it was distinct due to reduced pigmentation of colony, reduced cell size and significant reduction in mycelial growth forms with abundance of yeast forms. At molecular level, UF16 was differentiated based on PCR fingerprinting method of RAPD as well as large and small-subunit rRNA gene sequences. Rapid molecular technique of RAPD analysis using six primers showed 34 % polymorphic fragments with mean genetic distance of 0.235. The partial sequences of rRNA-gene revealed few mutation sites on nucleotide base pairs. However, the mutations detected on rRNA gene of UF16 were less than 1 % of total base pairs and its sequence showed 99 % homology with the wild type strain. These mutations in UF16 could not be linked to phenotypic or genotypic changes on CoQ₁₀ biosynthetic pathway that resulted in improved yield. Hence, investigating the mutations responsible for deregulation of CoQ₁₀ pathway is essential to understand the cause of overproduction in UF16. Phylogenetic analysis based on RAPD bands and rRNA gene sequences coupled with morphological variations, exhibited the novelty of mutant UF16 having potential for improved CoQ₁₀ production.