Sequence-tagged-site (STS) markers of arbitrary genes: the utility of black spruce-derived STS primers in other conifers

 Sequence-tagged-site primers, previously developed based upon black spruce (Picea mariana) cDNA sequences, were tested for their ability to direct specific amplification in two individuals of each of 12 additional conifer species. Nearly all (95–97%) of the primers functioned well in congeneric trials, while a lower proportion (21–33%) scored positively in other Pinaceae genera. Outside of the Pinaceae, amplification of homologous products was not achieved. Products from the various species often differed in size from their homologs in black spruce. In one case a large difference in size was due to the lack of an intron in a jack pine product while in several other cases the differences were due to the presence or absence of large direct repeats in the DNA sequences. Length polymorphism was occasionally evident between the two individuals examined of a given species. We investigated marker polymorphism in detail in a panel of 15 white spruce (Picea glauca) trees. Allelic segregation among haploid megagametophytes was revealed directly at 16 loci by standard agarose-gel electrophoresis without any additional manipulation of amplification products. Polymorphisms observed at 12 of these loci were exclusively co-dominant. For this subset of 12 loci, the average number of alleles was 3.2 and the average observed heterozygosity was 0.37. Received: 10 April 1998 / Accepted: 22 April 1998     

Characterisation of type-I thionin loci from the A, B, D and R genomes of wheat and rye

 DNA sequences encoding type-I thionins were isolated from Triticum aestivum L. cv ‘Chinese Spring’ using PCR with consensus primers. Blunt-end cloning, sequencing and PCR-based chromosome assignment of these fragments uncovered the three orthologous sequences corresponding to the single-copy genes at the Pur-1 loci on each of the group-1 chromosomes. Comparison with two previously published cDNA sequences revealed the presence of two introns that contain most of the polymorphic nucleotide sites. The observed orthologous DNA sequence variation among Pur-1 loci, encoded by each of the A, B and D genomes, enabled us to establish interlocus relationships and to construct locus-specific primer sets. Analogously, the Pur-R1 sequence from rye was isolated, and a locus-specific primer pair was constructed as well. Hence, four locus-specific primer sets are now available as molecular markers for the homoeologous 1AL, 1BL, 1DL and 1RL chromosome arms. Amplification from several diploid and tetraploid wheat species showed that the primers can be used as molecular tools for studying wheat phylogeny. Received: 30 January 1997 / Accepted: 23 June 1997     

Applicability of inter-simple sequence repeat polymorphisms in wheat for use as DNA markers in comparison to RFLP and RAPD markers

 Inter-simple sequence repeat polymorphic DNA (ISSR) was evaluated for its applicability as a genetic marker system in wheat. PCR was carried out with primers that annealed to simple sequence repeats. The resultant products were subjected to agarose-gel electrophoresis, and the banding patterns were compared among six wheat accessions containing diploid, tetraploid, and hexaploid members. Out of 100 examined, 33 primers produced distinguishable as well as polymorphic bands in each of the six accessions. Although most of the primers that gave distinct bands (30 primers out of 33) contained dinucleotide repeats, each of the primers with tri-, tetra-, and penta-nucleotide motifs also yielded discrete bands. Primers based on (AC)_n repeats gave the most polymorphic bands. In total, 224 polymorphic bands were found in the comparison between Einkorn wheats whereas, on the average, 120 polymorphic bands were detected between common wheats. ISSR primers produced several times more information than RAPD markers. The extent of band polymorphism was similar to that of RFLP markers, and greater than that of RAPDs. The genetic relationships of wheat accessions estimated by the polymorphism of ISSR markers were identical with those inferred by RFLP and RAPD markers, indicating the reliability of ISSR markers for estimation of genotypes. These polymorphic bands are potential candidates as novel markers for use in linkage-map construction in wheat. The characteristic features of ISSR markers, i.e. polymorphism, generation of information and ease of handling, suggest their applicability to the analysis of genotypes as well as to the construction of PCR-based genome maps of wheats. Received: 15 September 1996 / Accepted: 25 October 1996     

The use of real-time PCR and species-specific primers for the identification and monitoring of Paecilomyces lilacinus

The Paecilomyces lilacinus is the most widely tested fungus for the control of root-knot and cyst nematodes. The fungus has also been implicated in a number of human and animal infections, difficulties in diagnosis often result in misdiagnosis or delays in identification leading to a delay in treatment. Here, we report the development of species-specific primers for the identification of P. lilacinus based on sequence information from the ITS gene, and their use in identifying P. lilacinus isolates, including clinical isolates of the fungus. The primer set generated a single PCR fragment of 130 bp in length that was specific to P. lilacinus and was also used to detect the presence of P. lilacinus from soil, roots and nematode eggs. Real-time PCR primers and a TaqMan probe were also developed and provided quantitative data on the population size of the fungus in two field sites. PCR, bait and culture methods were combined to investigate the presence and abundance of the fungus from two field sites in the United Kingdom where potato cyst nematode populations were naturally declining, and results demonstrated the importance of using a combination of methods to investigate population size and activity of fungi.     

Development of PCR markers for the wheat leaf rust resistance gene Lr47

The leaf rust resistance gene Lr47 confers resistance to a wide spectrum of leaf rust strains. This gene was recently transferred from chromosome 7 S of Triticum speltoides to chromosome 7 A of hexaploid wheat Triticum aestivum. To facilitate the transfer of Lr47 to commercial varieties, the completely linked restriction fragment length polymorphism (RFLP) locus Xabc465 was converted into a PCR-based marker. Barley clone ABC465 is orthologous to the type-I wheat sucrose synthase gene and primers were designed for the conserved regions between the two sequences. These conserved primers were used to amplify, clone and sequence different alleles from T. speltoides and T. aestivum. This sequence information was then used to identify the T. speltoides sequence, detect allele-specific mutations, and design specific primers. Cosegregation of the PCR product of these primers and the T. speltoides chromosome segment was confirmed in four backcross-populations. To complement this dominant marker, a cleavage amplified polymorphic sequence (CAPS) was developed for the 7 A allele of Xabc465. This CAPS marker is useful to select homozygous Lr47 plants from F₂or backcross-F₂ segregating populations, and in combination with the T. speltoides-specific primers is expected to facilitate the deployment of Lr47 in new bread wheat varieties. Received: 7 June 1999 / Accepted: 30 September 1999     

Conversion of AFLP markers to sequence-specific PCR markers in barley and wheat

 Conversion of amplified fragment length polymorphisms (AFLPs) to sequence-specific PCR primers would be useful for many genetic-linkage applications. We examined 21 wheat nullitetrasomic stocks and five wheat-barley addition lines using 12 and 14 AFLP primer combinations, respectively. On average, 36.8% of the scored AFLP fragments in the wheat nullitetrasomic stocks and 22.3% in the wheat-barley addition lines could be mapped to specific chromosomes, providing approximately 461 chromosome-specific AFLP markers in the wheat nullitetrasomic stocks and 174 in the wheat-barley addition lines. Ten AFLP fragments specific to barley chromosomes and 16 AFLP fragments specific to wheat 3BS and 4BS chromosome arms were isolated from the polyacrylamide gels, re-amplified, cloned and sequenced. Primer sets were designed from these sequences. Amplification of wheat and barley genomic DNA using the barley derived primers revealed that three primer sets amplified DNA from the expected chromosome, five amplified fragments from all barley chromosomes but not from wheat, one amplified a similar-sized fragment from multiple barley chromosomes and from wheat, and one gave no amplification. Amplification of wheat genomic DNA using the wheat-derived primer sets revealed that three primer sets amplified a fragment from the expected chromosome, 11 primer sets amplified a similar-sized fragment from multiple chromosomes, and two gave no amplification. These experiments indicate that polymorphisms identified by AFLP are often not transferable to more sequence-specific PCR applications. Received: 30 June 1998 / Accepted: 26 October 1998     

Development of PCR markers for wheat leaf rust resistance gene Lr47

The leaf rust resistance gene Lr47 confers resistance to a wide spectrum of leaf rust strains. This gene was recently transferred from chromosome 7S of Triticum speltoides to chromosome 7A of hexaploid wheat Triticum aestivum. To facilitate the transfer of Lr47 to commercial varieties, the completely linked restriction fragment length polymorphism (RFLP) locus Xabc465 was converted into a PCR-based marker. Barley clone ABC465 is orthologous to the type-I wheat sucrose synthase gene and primers were designed for the conserved regions between the two sequences. These conserved primers were used to amplify, clone and sequence different alleles from T. speltoides and T. aestivum. This sequence information was used to identify the T. speltoides sequence, detect allele-specific mutations, and design specific primers. Cosegregation of the PCR product of these primers and the T. speltoides chromosome segment was confirmed in four backcross-populations. To complement this dominant marker, a cleavage amplified polymorphic sequence (CAPS) was developed for the 7A allele of Xabc465. This CAPS marker is useful to select homozygous Lr47 plants from F₂ or backcross-F₂ segregating populations, and in combination with the T- speltoides specific primers is expected to facilitate the deployment of Lr47 in new bread wheat varieties. Received: 11 October 1999 / Accepted: 30 December 1999     

Genetic linkage map of ISSR and RAPD markers in Einkorn wheat in relation to that of RFLP markers

 The potential of PCR-based markers for construction of a genetic linkage map in Einkorn wheat was investigated. From a comparison of polymorphisms between two Einkorn wheats, Triticum monococcum (Mn) and T. boeoticum (Bt), we obtained 49 polymorphic bands produced by 33 primers for inter-simple sequence repeat (ISSR) and 36 polymorphic bands shown by 25 combinations of random amplified polymorphic DNA (RAPD) primers for mapping in 66 individuals in the F₂ population. Although 44 ISSR fragments and 29 RAPD fragments statistically showed a 3 : 1 segregation ratio in the F₂ population, only 9 markers each of the ISSR and RAPD bands were able to be mapped on the RFLP linkage map of Einkorn wheat. ISSR markers were distributed throughout the chromosomes. The mapped positions of the ISSR markers seemed to be similar to those obtained by the RFLP markers. On the other hand, 4 of the 9 RAPD markers could map the RFLP marker-poor region on the short arm of 3A^m, suggesting a potential to map novel regions containing repetitive sequences. Comparisons of the genetic linkage map of Einkorn wheat to the linkage map and cytological map of common wheat revealed that the marker orders between the two maps of Einkorn wheat and common wheat coincided except for 4A, which harbors chromosome rearrangements specific for polyploid wheats, indicating a conservatism between the two genomes. Recombinations in Einkorn wheat chromosomes took place more frequently around the centromere and less at the distal part of chromosomes in comparison to those in common wheat. Nevertheless, recombinations even in Einkorn wheat chromosomes were strongly suppressed around the centromere. In fact, the markers located within 1 cM of the centromere were located almost in the central part of the chromosome arm. Received: 7 June 1997 / Accepted: 17 June 1997     

Chromosomal location of PCR fragments as a source of DNA markers linked to aluminium tolerance genes in rye

 To identify and locate rye DNA sequences homologous to three wheat c-DNAs (wali1, wali2 and wali5) whose expression is induced by aluminium (Al) stress, we designed three pairs of specific primers. They were used in the amplification of genomic DNA from wheat-rye disomic addition lines. The wali2 pair of primers amplified a 878-bp rye DNA fragment (rali2) located on chromosomes 4R and 7R that showed 79.37% homology with the corresponding wheat c-DNA. RAPD fragments were also used as genetic markers. We located 22 different RAPDs distributed on 11 different rye chromosome arms using wheat-rye disomic and ditelocentric addition lines. Thirteen of these markers were located on the chromosomes 3R, 4R and 6R, which also carry aluminium-tolerance genes. The OPA08 ₄₁₅ and OPR01 ₆₀₀ RAPD markers, located on the 6RL and 6RS chromosome arms, respectively, were converted to SCAR markers (SCA08 ₄₁₅ and SCR01 ₆₀₀ ) and linked to Alt1 gene (SCR01 ₆₀₀ -2.1 cM-Alt1-33.5 cM-SCA08 ₄₁₅ ). We propose that the chromosomal location of RAPDs and SCARs using wheat-rye addition lines is a source of DNA markers linked to aluminium-tolerance loci and offers a valuable strategy in marker-assisted selection for the introgression of tolerance genes in wheat. Received: 9 June 1997 / Accepted: 19 September 1997     

PCR primers specific for the genus Tuber reveal the presence of several truffle species in a truffle-ground

Truffles are hypogeous Ascomycete fungi belonging to the genus Tuber and forming fruiting bodies highly prized for their taste and aroma. The identification of the genus Tuber and its species is important to investigate their ecology and avoid fraud in the food market. As genus-specific primers are not available, the aims of this work were (1) to assess the usefulness of the β-tubulin gene as a DNA barcoding region for designing Tuber genus-specific primers, (2) to test the primers on a range of fruiting bodies, representing a large part of truffle biodiversity and (3) to check their ecological usefulness, applying them to truffle-ground soil. The new primers designed on the β-tubulin gene were specific to the Tuber genus in nested PCR. When applied to DNA from soils, they gave a positive signal for 23 of 32 soils. Phylogenetic analysis confirmed that the bands corresponded to Tuber and that at least five Tuber species were present in the truffle-ground. β-tubulin was found to be a good barcoding region for designing Tuber genus-specific primers, detecting a high Tuber diversity in a natural environment. These primers will be useful for understanding truffle ecology and for practical needs in plantation management.     

Universal primers for the PCR-mediated amplification of DNA 1: a satellite-like molecule associated with begomovirus-DNA beta complexes

DNA 1 is a single-stranded DNA molecule of approximately 1370 nucleotides. It is associated with monopartite geminiviruses of the genus Begomovirus, which require a DNA β component for symptomatic infection. The DNA 1 molecule requires the helper begomovirus for movement in plants, but is capable of self-replication. We designed two abutting primer pairs (DNA101/DNA102 and UN101/UN102) to conserved sequences of DNA 1. This allowed polymerase chain reaction-mediated amplification of the full-length molecule from total nucleic acid extracts produced from various host plants from geographically distinct, worldwide locations. These primers are useful both as diagnostic probes and for producing full-length infectious clones for in planta studies.     

Molecular characterization of a LMW-GS gene located on chromosome 1B and the development of primers specific for the Glu-B3 complex locus in durum wheat

 Low-molecular-weight glutenin subunits (LMW-GS) represent a specific class of wheat storage proteins encoded at the Glu-3 loci. Particularly interesting are the LMW-GS encoded at the Glu-B3 locus because they have been shown to play an important role in determining the pasta-making properties of durum wheat. Genes encoding LMW-GS have been characterized but only a few of them have been assigned to specific loci. Notably, no complete LMW-GS gene encoded at the Glu-B3 locus has yet been described. The present paper reports the isolation and characterization of a lmw-gs gene located at the Glu-B3 locus. The clone involved, designated pLDNLMW1B, contains the entire coding region and 524 bp of the 5′ upstream region. A nucleotide comparison between the pLDNLMW1B clone and other LMW-GS genes showed the presence of some peculiar structural characteristics, such as short insertions in the promoter region, the presence of a cysteine codon in the repetitive domain, and a more regular structure of this region, which could be important for its tissue-specific expression and for the functional properties of the encoded protein, respectively. Received : 30 May 1997 / Accepted : 29 July 1997     

Development of microsatellite markers for white spruce (Picea glauca) and related species

We report the development of 13 primer pairs that allow the unambiguous amplification of 15 microsatellite (SSR) loci in white spruce (Picea glauca). Fourteen of these loci were polymorphic in trees sampled at three geographically separated regions of western Canada. Segregation analysis carried out on these loci confirmed a Mendelian inheritance pattern for all except two, which showed significant segregation distortion. All of these primer pairs amplified SSR loci in at least one of the other Picea species tested [black spruce (P. mariana), red spruce (P. rubens), Norway spruce (P. abies), Colorado spruce (P. pungens), sitka spruce (P. sitchensis) and Engelmann spruce (P. engelmannii)]. Given the important commercial and ecological roles of these species, this set of markers will be invaluable for their management, the improvement of commercially important traits, and the study of their ecology and genetics. Received: 18 August 2000 / Accepted: 28 September 2000     

Genetic diversity and classification of cyanobacteria in different Azolla species by the use of PCR fingerprinting

Symbiotically associated cyanobacteria from 18 accessions within all known species in the genus Azolla were examined and classified by the use of polymerase chain reaction (PCR)-fingerprinting. A repetitive sequence specific for cyanobacteria, the short tandemly repeated repetitive (STRR) sequence, was used as a primer in the reaction. Cyanobacterial filaments isolated directly from the Azolla leaf cavity or contained within homogenised symbiotic Azolla tissue were used as templates. Based on the fingerprint pattern, distinct differences were demonstrated between cyanobacteria isolated from the Euazolla and Rhizosperma sections. In addition, individual fingerprints were obtained from all cyanobacteria isolated from the different Azolla species. The fingerprints were used to generate a phylogenetic tree. Three clusters were distinguished: one contained the four isolates from the section Euazolla, a second the isolate from Azolla filiculoides, and a third the three isolates from the section Rhizosperma. By the use of STRR-PCR fingerprinting, new data on the taxonomy of cyanobacteria in Azolla were obtained, which have been difficult to generate by other classification methods. PCR-fingerprinting may, therefore, be a valuable tool for diversity and classification studies of symbiotic cyanobateria from Azolla and, as co-evolution between the cyanobacteria and its corresponding host exists the method may also be useful for the taxonomy of Azolla. Received: 19 December 1998 / Accepted: 31 May 1999     

Identification of molecular markers for resistance to Septoria nodorum blotch in durum wheat

The development of Septoria nodorum blotch-resistant cultivars has become a high priority objective for durum wheat breeding programs. Marker-assisted selection enables breeders to improve selection efficiency. In order to develop markers for resistance to Septoria nodorum blotch, a set of F₅ recombinant inbred lines, derived from the crosses Sceptre/3–6, Sceptre/S9–10 and Sceptre/S12–1, was developed based on the F₂-derived family method. Two RAPD markers, designated UBC521₆₅₀ and RC37₅₁₀, were detected by bulked segregant analysis and located approximately 15 and 13.1 centiMorgans (cM) from the resistance gene snbTM, respectively. A SCAR marker was also successfully developed for marker-assisted selection in breeding programs based on the sequence of the RAPD marker UBC521₆₅₀. This is the first report of DNA-based markers linked to resistance for Septoria nodorum blotch in durum wheat. Received: 8 March 2000 / Accepted: 25 June 2000     

The development of oat microsatellite markers and their use in identifying relationships among Avena species and oat cultivars

Microsatellites have many desirable marker properties. There has been no report of the development and utilization of microsatellite markers in oat. The objectives of the present study were to construct oat microsatellite-enriched libraries, to isolate microsatellite sequences and evaluate their level of polymorphism in Avena species and oat cultivars. One hundred clones were isolated and sequenced from three oat microsatellite-libraries enriched for either (AC/TG) _n , (AG/TC) _n or (AAG/TTC) _n repeats. Seventy eight clones contained microsatellites. A database search showed that 42% of the microsatellite flanking sequences shared significant homology with various repetitive elements. Alu and retrotransposon sequences were the two largest groups associated with the microsatellites. Forty four primer sets were used to amplify the DNA from 12 Avena species and 20 Avena sativa cultivars. Sixty two percent of the primers revealed polymorphism among the Avena species, but only 36% among the cultivars. In the cultivars, the microsatellites associated with repetitive elements were less polymorphic than those not associated with repetitive elements. Only 25% of the microsatellites associated with repetitive elements were polymorphic, while 46% of the microsatellites not associated with repetitive elements showed polymorphism in the cultivars. An average of four alleles with a polymorphism information content (PIC) of 0.57 per primer set was detected among the Avena species, and 3.8 alleles with a PIC of 0.55 among the cultivars. In addition, 54 barley microsatellite primers were tested in Avena species and 26% of the primers amplified microsatellites from oat. Using microsatellite polymorphisms, dendrograms were constructed showing phylogenetic relationships among Avena species and genetic relationships among oat cultivars. Received: 1 November 1999 / Accepted: 14 April 2000     

Novel method for preparation of the template DNA and selection of primers to differentiate the material rice cultivars of rice wine by PCR

As many rice wine brewers label the name of the cultivar of the material rice, authentication technology is necessary. The problems are (1) decomposition of DNAs during the fermentation, (2) contamination of DNAs from microorganisms, (3) co-existence of PCR inhibitors, such as polyphenols. The present authors improved the PCR method by (1) lyophilizing and pulverizing the rice wine to concentrate DNAs, (2) decomposition of starches and proteins so as not to inhibit DNA extraction by the use of heat-resistant amylase and proteinase K, (3) purification of the template DNA by the combination of CTAB method and fractional precipitation by 70% EtOH. To prevent the amplification of microorganism's DNAs during PCR, the present authors selected the suitable plant-specific primers. It became possible to prepare the template DNAs for PCR from the rice wine. The sequences of the amplified DNAs by PCR were ascertained to be same with those of material rice. Mislabeling of material rice cultivar was detected by PCR using the commercial rice wine. It became possible to extract and purify the template DNAs for PCR from the rice wine and to differentiate the material rice cultivars by the PCR using the rice wine as a sample.     

Development of RAPD and SCAR markers linked to the Russian wheat aphid resistance gene Dn2 in wheat

 RAPD (random amplified polymorphic DNA) analysis was used to identify molecular markers linked to the Dn2 gene conferring resistance to the Russian wheat aphid (Diuraphis noxia Mordvilko). A set of near-isogenic lines (NILs) was screened with 300 RAPD primers for polymorphisms linked to the Dn2 gene. A total of 2700 RAPD loci were screened for linkage to the resistance locus. Four polymorphic RAPD fragments, two in coupling phase and two in repulsion phase, were identified as putative RAPD markers for the Dn2 gene. Segregation analysis of these markers in an F₂ population segregating for the resistance gene revealed that all four markers were closely linked to the Dn2 locus. Linkage distances ranged from 3.3 cM to 4.4 cM. Southern analysis of the RAPD products using the cloned RAPD markers as probes confirmed the homology of the RAPD amplification products. The coupling-phase marker OPB10_880c and the repulsion-phase marker OPN1_400r were converted to sequence characterized amplified region (SCAR) markers. SCAR analysis of the F₂ population and other resistant and susceptible South African wheat cultivars corroborated the observed linkage of the RAPD markers to the Dn2 resistance locus. These markers will be useful for marker-assisted selection of the Dn2 gene for resistance breeding and gene pyramiding. Received: 1 July 1997 / Accepted: 20 October 1997     

The use of microsatellites for detecting DNA polymorphism, genotype identification and genetic diversity in wheat

A set of 20 wheat microsatellite markers was used with 55 elite wheat genotypes to examine their utility (1) in detecting DNA polymorphism, (2)in the identifying genotypes and (3) in estimating genetic diversity among wheat genotypes. The 55 elite genotypes of wheat used in this study originated in 29 countries representing six continents. A total of 155 alleles were detected at 21 loci using the above microsatellite primer pairs (only 1 primer amplified 2 loci; all other primers amplified 1 locus each). Of the 20 primers amplifying 21 loci, 17 primers and their corresponding 18 loci were assigned to 13 different chromosomes (6 chromosomes of the A genome, 5 chromosomes of the B genome and 2 chromosomes of the D genome). The number of alleles per locus ranged from 1 to 13, with an average of 7.4 alleles per locus. The values of average polymorphic information content (PIC) and the marker index (MI) for these markers were estimated to be 0.71 and 0.70, respectively. The (GT)_n microsatellites were found to be the most polymorphic. The genetic similarity (GS) coefficient for all possible 1485 pairs of genotypes ranged from 0.05 to 0.88 with an average of 0.23. The dendrogram, prepared on the basis of similarity matrix using the UPGMA algorithm, delineated the above genotypes into two major clusters (I and II), each with two subclusters (Ia, Ib and IIa, IIb). One of these subclusters (Ib) consisted of a solitary genotype (E3111) from Portugal, so that it was unique and diverse with respect to all other genotypes belonging to cluster I and placed in subcluster Ia. Using a set of only 12 primer pairs, we were able to distinguish a maximum of 48 of the above 55 wheat genotypes. The results demonstrate the utility of microsatellite markers for detecting polymorphism leading to genotype identification and for estimating genetic diversity. Received: 15 May 1999 / Accepted: 27 July 1999