PCR assay based on a microsatellite-containing locus for detection and quantification of Epichloë endophytes in grass tissue.

A PCR assay which allows detection and quantification of Epichloë endophytes in tissues of the grass Bromus erectus is described. PCR with specific primers flanking a microsatellite-containing locus (MS primers) amplified fragments 300 to 400 bp in length from as little as 1.0 pg of fungal genomic DNA in 100 ng of DNA from infected plant material. When annealing temperatures were optimized, all Epichloë and Acremonium strains tested, representing many of the known taxonomic groups, yielded an amplification product, indicating that the MS primers may be useful for in planta detection of a variety of related species, including agronomically important Acremonium coenophialum and Acremonium lolii. No fragments were generated from DNA isolates from uninfected plant material or from unrelated fungi isolated from B. erectus. For diagnostic applications, a B. erectus-specific primer pair was designed for use in multiplex PCR to allow simultaneous amplification of plant and fungal DNA sequences, providing an internal control for PCR failure caused by inhibitory plant compounds present in DNA extracts. For quantitative applications, a heterologous control template in primer binding sites complementary to the MS primers was constructed for use in competitive PCR, allowing direct quantification of Epichloë in plant DNA extracts. The fungal DNA present in infected leaves of B. erectus between 1 and 20 pg per 100 ng of leaf DNA, but the amounts of fungal DNA present in the sheath and blade of a given leaf were correlated, indicating that the degree of infection varied between plant individuals but that leaves were colonized in a uniform way.     

IRAP and REMAP for retrotransposon-based genotyping and fingerprinting

Retrotransposons can be used as markers because their integration creates new joints between genomic DNA and their conserved ends. To detect polymorphisms for retrotransposon insertion, marker systems generally rely on PCR amplification between these ends and some component of flanking genomic DNA. We have developed two methods, retrotransposon-microsatellite amplified polymorphism (REMAP) analysis and inter-retrotransposon amplified polymorphism (IRAP) analysis, that require neither restriction enzyme digestion nor ligation to generate the marker bands. The IRAP products are generated from two nearby retrotransposons using outward-facing primers. In REMAP, amplification between retrotransposons proximal to simple sequence repeats (microsatellites) produces the marker bands. Here, we describe protocols for the IRAP and REMAP techniques, including methods for PCR amplification with a single primer or with two primers and for agarose gel electrophoresis of the product using optimal electrophoresis buffers and conditions. This protocol can be completed in 1-2 d.     

Disparate Independent Genetic Events Disrupt the Secondary Metabolism Gene perA in Certain Symbiotic Epichlo? Species

Peramine is an insect-feeding deterrent produced by Epichloë species in symbiotic association with C₃ grasses. The perA gene responsible for peramine synthesis encodes a two-module nonribosomal peptide synthetase. Alleles of perA are found in most Epichloë species; however, peramine is not produced by many perA-containing Epichloë isolates. The genetic basis of these peramine-negative chemotypes is often unknown. Using PCR and DNA sequencing, we analyzed the perA genes from 72 Epichloë isolates and identified causative mutations of perA null alleles. We found nonfunctional perA-ΔR* alleles, which contain a transposon-associated deletion of the perA region encoding the C-terminal reductase domain, are widespread within the Epichloë genus and represent a prevalent mutation found in nonhybrid species. Disparate phylogenies of adjacent A2 and T2 domains indicated that the deletion of the reductase domain (R*) likely occurred once and early in the evolution of the genus, and subsequently there have been several recombinations between those domains. A number of novel point, deletion, and insertion mutations responsible for abolishing peramine production in full-length perA alleles were also identified. The regions encoding the first and second adenylation domains (A1 and A2, respectively) were common sites for such mutations. Using this information, a method was developed to predict peramine chemotypes by combining PCR product size polymorphism analysis with sequencing of the perA adenylation domains.     

Characteristics of single- and multi-copy microsatellites from Pinus radiata

 Dinucleotide microsatellites were isolated from Pinus radiata using both a standard genomic library and libraries enriched for microsatellites. Locus-specific primers were designed to amplify 43 unique microsatellites. Thirty two of these loci had interpretable PCR patterns, 11 of which were polymorphic in a screen of 19 P. radiata individuals; all 11 polymorphic loci contained at least 17 repeats in the sequenced plasmid. Six of the eleven primer pairs amplified multiple fragments per individual (3–8), suggesting that these loci were present in multiple copies in the genome. Genotyping a 48-tree P. radiata production population with seven of the most polymorphic microsatellites revealed an average of 17 bands per locus (the multi-copy microsatellites were treated as one locus). When tested on known pedigrees, both single and multi-copy microsatellites exhibited co-dominant inheritance and Mendelian segregation. Two loci had null alleles and one locus had a high frequency of non-parental alleles, suggesting a high mutation rate. Eight of these microsatellites, including five multi-copy loci, were placed on a partially constructed P. radiata genetic map. Four of the five multi-copy microsatellites had two or more sets of alleles that mapped to the same locus, and the fifth mapped to two unlinked loci. All seven tested primer pairs amplified PCR products from other species of hard pine, three amplified products from soft-pine species, and one amplified bands in other conifers. Received: 10 November 1997 / Accepted: 5 January 1998     

<Emphasis Type="Italic">Prunus</Emphasis> microsatellite marker transferability across rosaceous crops

A total of 145 microsatellite primer pairs from Prunus DNA sequences were studied for transferability in a set of eight cultivars from nine rosaceous species (almond, peach, apricot, Japanese plum, European plum, cherry, apple, pear, and strawberry), 25 each of almond genomic, peach genomic, peach expressed sequence tags (EST), and Japanese plum genomic, 22 of almond EST, and 23 of apricot (13 EST and 10 genomic), all known to produce single-locus and polymorphic simple-sequence repeats in the species where they were developed. Most primer pairs (83.6%) amplified bands of the expected size range in other Prunus. Transferability, i.e., the proportion of microsatellites that amplified and were polymorphic, was also high in Prunus (63.9%). Almond and Japanese plum were the most variable among the diploid species (all but the hexaploid European plum) and peach the least polymorphic. Thirty-one microsatellites amplified and were polymorphic in all Prunus species studied, 12 of which, covering its whole genome, are proposed as the “universal Prunus set”. In contrast, only 16.3% were transferable in species of other Rosaceae genera (apple, pear, and strawberry). Polymorphic Prunus microsatellites also detected lower levels of variability in the non-congeneric species. No significant differences were detected in transferability and the ability to detect variability between microsatellites of EST and genomic origin.     

Origin,Divergence, and Phylogeny of Asexual Epichlo? Endophyte in Elymus Species from Western China

Asexual Epichloë species are likely derived directly from sexual Epichloë species that then lost their capacity for sexual reproduction or lost sexual reproduction because of interspecific hybridization between distinct lineages of sexual Epichloë and/or asexual Epichloë species. In this study we isolated asexual Epichloë endophytes from Elymus species in western China and sequenced intron-rich regions in the genes encoding β-tubulin (tubB) and translation elongation factor 1-α (tefA). Our results showed that there are no gene copies of tubB and tefA in any of the isolates. Phylogenetic analysis showed that sequences in this study formed a single clade with asexual Epichloë bromicola from Hordeum brevisubulatum, which implies asexual Epichloë endophytes that are symbionts in a western Chinese Elymus species likely share a common ancestor with asexual E. bromicola from European H. brevisubulatum. In addition, our results revealed that asexual E. bromicola isolates that are symbionts in a western Chinese Elymus species and sexual Epichloë species that are symbionts in a North American Elymus species have a different origin. Further analysis found that Epichloë species likely originated in Eurasia. In addition, the results support the hypothesis that migratory birds or humans might have aided the dispersal of these fungal endophytes to other continents.     

Identification of abundant and informative microsatellites from shrimp (Penaeus monodon) genome

Microsatellites were isolated from P. monodon genomic libraries by direct sequencing of recombinant clones without probe screening. Forty-nine out of 83 clones sequenced contained 99 microsatellite arrays of three or more repeats. When five or more and ten or more repeats were considered, 28 and 14 microsatellites were detected, respectively. The 99 microsatellites were classified as perfect (75%), imperfect (6%), compound perfect (3%) and compound imperfect (16%). The abundance of di-, tri-, tetra- and hexanucleotide repeats were 67%, 20%, 9% and 3%, respectively. The dinucleotide repeats included 36 (CT)n, 31 (GT)n, 17(AT)n and 3 (CG)n. One octanucleotide repeat (ATTTATTC)5 was found within a large repeat sequence. Optimal annealing temperatures were determined for PCR using 11 primer sets encompassing 15 microsatellites. Ten primer sets provided successful amplifications with allele sizes generally ranging from 139 to 410 bp. All these primers amplified polymorphic loci with PIC values ranging from 0.63 to 0.96. Two primer sets amplified additional bands which can easily be distinguished from the bands of the main locus. Three out of 10 P. monodon microsatellites also amplified alleles in P. vannamei. The abundance and informative nature of P. monodon microsatellites and their potential for cross-species amplification make them useful for genetic studies.     

Amplification of DNA markers from evolutionarily diverse genomes using single primers of simple-sequence repeats

The abundance and scattered distribution of simple-sequence repeats (SSR) in eukaryotic genomes prompted us to explore the use of SSR-based oligonucleotide primers in single primer amplification reactions. In a pilot experiment, 23 primers were used across a panel of evolutionarily diverse eukaryotic genomes, including grapes, lettuce, tomato, pine, maize, salmon, chicken, Holstein cows and humans. The primers were 16–20 bases in length and represented SSRs of di-, tri-, tetra-, and pentanucleotide repeats. The results showed that tetranucleotide repeat primers were most effective in amplifying polymorphic patterns. Of 11 such primers tested, 70% produced polymorphic patterns from the DNA of one or more species. Primers representing a combination of two tetranucleotide repeats, or compound microsatellites, were equally effective. The polymorphisms contained in such fingerprints were able to identify individuals of vertebrate species as well as lines or varieties of plants. Inheritance of the polymorphic bands was studied in a maize recombinant inbred population, DE811 x B73. Thirty-two polymorphic bands, derived from two amplification patterns, were mapped as dominant markers on an existing RFLP map of the same population. The bands were distributed across nine of the ten chromosomes.     

Isolation and sequencing of CA/GT repeat microsatellites from chromosomal libraries without subcloning.

A method for the isolation of (CA)n microsatellites from chromosome-specific genomic libraries is described. Clones were first screened using a polynucleotide CA/GT probe. Those shown to contain CA repeats were plaque purified and either subcloned or the insert amplified directly using vector primers. Polymerase chain reaction products were then used to directly sequence the regions flanking CA repeats by using biotinylated primers that amplify cloned inserts outward from CA repeat containing regions of DNA to vector primers. This method provides rapid access to microsatellites from chromosomes or chromosome regions of interest.     

Molecular characterization and genetic mapping of random amplified microsatellite polymorphism in barley

 This study has analyzed the molecular basis and genetic behaviour of the polymorphism generated by the amplification of barley genomic DNA with primers complementary to microsatellites. Primers anchored at the 5′ end, used alone or in combination with arbitrary sequence primers, generated random amplified microsatellite polymorphisms (RAMPs). Unanchored primers were also used as single primers in a microsatellite primed-PCR (MP-PCR). Twenty six randomly selected RAMP DNA fragments which showed polymorphism between the cultivars Steptoe and Morex were cloned and sequenced. All sequences showed the expected repeated motif at the end of the insert, with the number of repeats ranging from five to ten. Genomic sequences containing low numbers of microsatellite motifs were preferentially amplified; therefore, only a fraction of the polymorphism could be attributed to variation in the number of microsatellite motifs at the priming site. Some sequences contained either cryptic simple sequences or members of families of repeated DNA. Polymorphism at the internal cryptic simple sequences was detected by RAMP bands inherited as co-dominant markers. Four MP-PCR bands were cloned and sequenced. A number of repeats identical to the primer itself were found at each end of the insert. Two allelic bands were polymorphic for an internal microsatellite. The potential use of cloned bands as fingerprinting tools was investigated by employing them as hybridization probes in Southern blots containing digested barley DNA from a sample of cultivars. RAMP probes produced complex hybridization band patterns. MP-PCR probes produced either a highly variable single locus or low-copy number loci. Segregations for 31 RAMPs and three MP-PCR bands were studied in a population of 70 doubled-haploids from the Steptoe/Morex cross. One third of all markers were co-dominantly inherited. Markers were positioned on an RFLP map and found to be distributed in all barley chromosomes. The new markers enlarged the overall length of the map to 1408 cM. Received: 6 May 1998 / Accepted: 20 July 1998     

Male-specific DNA sequences in pigs

One hundred primers (Operon kits OPAA, OPAO, OPAV, OPC, and OPE series) were used for random amplified polymorphic DNA (RAPD) fingerprinting to determine male-specific fragments. Seventy-four percent of the primers yielded Yorkshire polymorphic fragments. One of these primers, OPAV-18, produced a novel 1098-bp DNA fragment found only in tested males. This male-specific fragment was isolated and constructed into plasmids for nucleotide sequencing. Two primers (5'-TTGCTCACGG TAGATAACAA GAGAG-3' and 5'-TTGCTCACGG ACCAGGTAGG GAATG-3') were designed according to the cloned male-specific sequence to amplify the male-specific band using polymerase chain reaction (PCR) for pig sexing. Sex-specific bands in the PCR gel products were represented in males but none were found in females when Yorkshire, Duroc, and Landrace genomic DNA samples were amplified with these two primers by PCR. The PCR products in the gel were transferred to nylon membranes and hybridized with a 32P-dCTP labeled probe of the cloned male-specific DNA fragment. There was a clear hybridization signal in samples from all of the male pigs, but not from those of female pigs. Male and female genomic DNA samples from these pigs were spotted onto nylon membranes and hybridized with the male-specific probe. The probe hybridized strongly to males only. A high degree of sequence homology was found among the novel male-specific DNA sequences in Yorkshire, Duroc and Landrace. The sex of these three breeds of pigs could be easily and effectively determined using these two primers.     

A Comparative Analysis of ISSR and RAPD Markers for Study of Genetic Diversity in Shisham (<Emphasis Type="Italic">Dalbergia sissoo</Emphasis>)

Shisham (Dalbergia sissoo) is one of the most preferred timber tree species of South Asia. Two DNA-based molecular marker techniques, intersimple sequence repeat (ISSR) and random amplified polymorphism DNA (RAPD), were compared to study the genetic diversity in this species. A total of 30 polymorphic primers (15 ISSR and 15 random) were used. Amplification of genomic DNA of 22 genotypes, using ISSR analysis, yielded 117 fragments, of which 64 were polymorphic. Number of amplified fragments with ISSR primers ranged from five to ten and varied in size from 180 to 1,900 bp. Percentage polymorphism ranged from 0 to 87.5. The 15 RAPD primers produced 144 bands across 22 genotypes, of which 84 were polymorphic. The number of amplified bands varied from five to 13, with size range from 180 to 2,400 bp. Percentage polymorphism ranged from 0 to 100, with an average of 58.3 across. RAPD markers were relatively more efficient than the ISSR assay. The mental test between two Jaccard’s similarity matrices gave r ≥ 0.90, showing very good fit correlation in between ISSR- and RAPD-based similarities. Clustering of isolates remained more or less the same in RAPD and combined data of RAPD and ISSR. The similarity coefficient ranged from 0.734 to 0.939, 0.563 to 0.946, and 0.648 to 0.920 with ISSR, RAPD, and combined dendrogram, respectively.     

Characteristics and a comparison of three classes of microsatellite-based markers and their application in plants

Microsatellites (SSR--simple sequence repeats, STR--short tandem repeats, SSLP--simple sequence length polymorphism, VNTR--variable number of tandem repeats) are the class of repetitive DNA sequences present in all living organisms. Particular characteristics of microsatellites, such as their presence in the genomes of all living organisms, high level of allelic variation, co-dominant mode of inheritance and potential for automated analysis make them an excellent tool for a number of approaches like genotyping, mapping and positional cloning of genes. The three most popular types of markers containing microsatellite sequences that are presently used are: (1) SSR (simple sequence repeats), generated by amplifying in a PCR reaction with the use of primers complementary to flanking regions; (2) ISSR (inter-simple sequence repeats), based on the amplification of regions between inversely oriented closely spaced microsatellites; and (3) SAMPL (selective amplification of microsatellite polymorphic loci), which utilises AFLP (amplified fragment-length polymorphism) methodology, with one exception--for the second amplification, one of the starters is complementary to the microsatellite sequence. The usefulness of the three above-mentioned markers for numerous purposes has been well documented for plants.     

Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce

Summary Sequence characterized amplified regions (SCARs) were derived from eight random amplified polymorphic DNA (RAPD) markers linked to disease resistance genes in lettuce. SCARs are PCR-based markers that represent single, genetically defined loci that are identified by PCR amplification of genomic DNA with pairs of specific oligonucleotide primers; they may contain high-copy, dispersed genomic sequences within the amplified region. Amplified RAPD products were cloned and sequenced. The sequence was used to design 24-mer oligonucleotide primers for each end. All pairs of SCAR primers resulted in the amplification of single major bands the same size as the RAPD fragment cloned. Polymorphism was either retained as the presence or absence of amplification of the band or appeared as length polymorphisms that converted dominant RAPD loci into codominant SCAR markers. This study provided information on the molecular basis of RAPD markers. The amplified fragment contained no obvious repeated sequences beyond the primer sequence. Five out of eight pairs of SCAR primers amplified an alternate allele from both parents of the mapping population; therefore, the original RAPD polymorphism was likely due to mismatch at the primer sites.     

Evaluation of genetic diversity of <Emphasis Type="Italic">Clinacanthus nutans</Emphasis> (Acanthaceaea) using RAPD,ISSR and RAMP markers

Three polymerase chain reaction (PCR) techniques were compared to analyse the genetic diversity of Clinacanthus nutans eight populations in the northern region of Peninsular Malaysia. The PCR techniques were random amplified polymorphic deoxyribonucleic acids (RAPD), inter-simple sequence repeats (ISSR) and random amplified microsatellite polymorphisms (RAMP). Leaf genomic DNA was PCR amplified using 17 RAPD, 8 ISSR and 136 RAMP primers . However, only 10 RAPD primers, 5 ISSR primers and 37 RAMP primers produced reproducible bands. The results were evaluated for polymorphic information content (PIC), marker index (MI) and resolving power (RP). The RAMP marker was the most useful marker compared to RAPD and ISSR markers because it showed the highest average value of PIC (0.25), MI (11.36) and RP (2.86). The genetic diversity showed a high percentage of polymorphism at the species level compared to the population level. Furthermore, analysis of molecular variance revealed that the genetic diversity was higher within populations, as compared to among populations of C. nutans. From the results, the RAMP technique was recommended for the analysis of genetic diversity of C. nutans.     

茶树EST-SSRs分布特征及引物开发

为了在茶树中开发EST-SSRs功能性标记,利用生物信息学方法对NCBI网上公开的3288奈茶树（Camellia subebsus）ESTs序列进行EST-SSRs特征分析。剔除冗余序列,得到非冗余序列2083条。在非冗余序列中发现含不同重复基元SSRs的EST序列有385条,共486个EST-SSRs,平均相隔2．10kb出现1个SSR。在2～6bp的重复基元中,二核苷酸重复基元的SSRs出现频率最高（51．97％）,其次是三核苷酸（19．55％）。对所有的重复基元类型进行统计分析发现,所占比例最高的是AG／CT（47．74％）,其次分别是AT／TA（4．73％）和AAG／CTT（4．73％）。利用Prime5软件,设计了206对EST-SSRs引物,随机选用72对引物进行SSR扩增,发现31对引物可以扩增出条带,其中29对引物具有多态性,多态性比率为93．5％。这些EST-SSRs将有助于茶树基因组学方面的研究。     

A PCR Assay to Identify and Distinguish Single Juveniles of Meloidogyne hapla and M. chitwoodi

Random amplified polymorphic DNA (RAPD) bands that distinguish Meloidogyne hapla and M. chitwoodi from each other, and from other root-knot nematode species, were identified using a series of random octamer primers. The species-specific amplified DNA fragments were cloned and sequenced, and then the sequences were used to design 20-mer primer pairs that specifically amplified a DNA fragment from each species. Using the primer pairs, successful amplifications from single juveniles were readily attained. A mixture of four primers in a single PCR reaction mixture was shown to identify single juveniles of M. hapla and M. chitwoodi. To confirm specificity, the primers were used to amplify DNA from several isolates of M. hapla that originated from different crops and locations in North America and also from isolates of M. chitwoodi that differed in host range. In characterizing the M. hapla isolates, it was noted that there was a mitochondrial DNA polymorphism among isolates for cleavage by the restriction endonuclease DraI.     

Creation of a SINE enriched library for the isolation of polymorphic (AGC)n microsatellite markers in the bovine genome

Trinucleotide (AGC)_n microsatellites are found as 3′ tails of the artiodactyl short interspersed nuclear element (SINE) A-dimer. We describe a polymerase chain reaction (PCR)-based method for the construction of a plasmid library enriched for SINE (AGC)_n microsatellites. By amplifying Sau3AI inserts with a conserved SINE primer and a flanking vector primer, a 35-fold enrichment of (AGC)_n microsatellites over a conventional genomic library was obtained. The SINE primer was used for both sequencing of AGC-containing inserts and analysis of polymorphism. Twenty-three unique reverse primers were synthesized and used on bovine genomic DNA, 21 producing PCR products of expected size. Five polymorphic (AGC)_n microsatellites with 2–4 alleles each were characterized. Allele sizes differed by a 3 bp motif and lacked the stutter bands associated with dinucleotide repeats. A tendency of increased polymorphism for longer AGC repeat arrays was observed. High stringency selection for positive clones containing eight or more AGC repeats can thus facilitate the isolation of polymorphic (AGC)_n microsatellites, Enrichment for (AGC), microsatellites by SINE-vector PCR can be applied to other bovidae species, such as sheep or goat, containing the artiodactyl SINE elements.     

鲸类微卫星引物对长江江豚的适用性研究

微卫星在长江江豚 (Neophocaenaphocaenoidesasiaeorientalis)中的应用研究还未见报道。本研究采用已发表的来自 6个鲸种的 2 3对微卫星引物对一个长江江豚群体DNA样本进行了微卫星扩增。结果表明其中有 7对引物在此群体中的扩增产物是稳定且多态的 ,序列分析结果表明这 7对引物的扩增产物都具有AC或GT两碱基重复单元 ,从而证明了扩增的有效性。研究结果表明用从其他鲸类分离出的微卫星引物可以快速筛选到适用于长江江豚指纹分析的引物     

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