Identification of a male-specific amplified fragment length polymorphism (AFLP) marker in Broussonetia papyrifera
24 Apr 2012-African Journal of Biotechnology (Academic Journals (Kenya))-Vol. 11, Iss: 33, pp 8196-8201
TL;DR: The present study exhibits amplified fragment length polymorphism (AFLP) molecular marker for sex identification in Broussonetia papyrifera based on nine selective amplification primer combinations and indicates that common homology sequence is existed in both male and female plants.
Abstract: The present study exhibits amplified fragment length polymorphism (AFLP) molecular marker for sex identification in Broussonetia papyrifera . Based on nine selective amplification primer combinations, 230 bands were produced and the E -AGG/M-CAA combination was found to be a male-specific AFLP marker. Subsequently, this male-specific AFLP fragment was sequenced and converted into a sequence tagged site (STS) marker. Based on STS sequence, two primers, MADB-1 and MADB-2 (Male-Associated DNA from B. papyrifera ), were designed to verify the specificity of the fragment. The results indicate that common homology sequence is existed in both male and female plants while one of the bands amplified via MADB-2 primer was solely present in male individuals at high annealing temperature up to 66°C. Finally, MADB-2 primer was introduced to amplify another 16 plants and it revealed that this primer could be used as a convenient, efficient, reliable, and low-cost molecular marker for sex identification in B. papyrifera . Keywords: Broussonetia papyrifera , AFLP, STS, male-specific marker
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TL;DR: This is the first study of a commensal species to show genetic structuring within Remote Oceania, and in spite of the genetic bottleneck, the presence of only one sex, a timespan of less than 5000 years, and asexual propagation of this crop in Remote OCEania, the genetic diversity and regional structuring are detected.
Abstract: Paper mulberry, Broussonetia papyrifera (L.) L'Her. ex Vent. (Moraceae), a dioecious species, was transported by humans from Taiwan to the islands of Remote Oceania. Its introduction and cultivation in Remote Oceania was intentional due to its cultural importance as a fiber source for barkcloth textiles. The aim of this study was to explore the genetic diversity and structure of paper mulberry populations within Remote Oceania in order to infer dispersal patterns that may reflect past human interaction among island groups. We present the integrated analysis of 380 samples (313 contemporary and 67 herbarium specimens) collected in Near and Remote Oceania. Genetic characterization was based on a set of ten microsatellites developed for B. papyrifera and complemented with the analysis of the ribosomal internal transcribed spacer ITS-1 sequence, a sex marker and the chloroplast ndhF-rpl32 intergenic spacer. Microsatellite data identify a total of 64 genotypes, despite this being a clonally propagated crop, and show three major dispersal hubs within Remote Oceania, centered on the islands of Fiji, Tonga, and Pitcairn. Of 64 genotypes identified, 55 correspond to genotypes associated to female-sexed plants that probably descend from plants introduced by the prehistoric Austronesian-speaking voyagers. The ratio of accessions to genotypes between herbarium and contemporary samples, suggests recent loss of genetic diversity. In addition to the chloroplast haplotypes described previously, we detected two new haplotypes within Remote Oceania both originating in Taiwan. This is the first study of a commensal species to show genetic structuring within Remote Oceania. In spite of the genetic bottleneck, the presence of only one sex, a timespan of less than 5000 years, and asexual propagation of this crop in Remote Oceania, we detect genetic diversity and regional structuring. These observations suggest specific migration routes between island groups within Remote Oceania.
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TL;DR: The AFLP technique provides a novel and very powerful DNA fingerprinting technique for DNAs of any origin or complexity that allows the specific co-amplification of high numbers of restriction fragments.
Abstract: A novel DNA fingerprinting technique called AFLP is described. The AFLP technique is based on the selective PCR amplification of restriction fragments from a total digest of genomic DNA. The technique involves three steps: (i) restriction of the DNA and ligation of oligonucleotide adapters, (ii) selective amplification of sets of restriction fragments, and (iii) gel analysis of the amplified fragments. PCR amplification of restriction fragments is achieved by using the adapter and restriction site sequence as target sites for primer annealing. The selective amplification is achieved by the use of primers that extend into the restriction fragments, amplifying only those fragments in which the primer extensions match the nucleotides flanking the restriction sites. Using this method, sets of restriction fragments may be visualized by PCR without knowledge of nucleotide sequence. The method allows the specific co-amplification of high numbers of restriction fragments. The number of fragments that can be analyzed simultaneously, however, is dependent on the resolution of the detection system. Typically 50-100 restriction fragments are amplified and detected on denaturing polyacrylamide gels. The AFLP technique provides a novel and very powerful DNA fingerprinting technique for DNAs of any origin or complexity.
12,960 citations
"Identification of a male-specific a..." refers methods in this paper
...Amplified fragment length polymorphism (AFLP), based on the selective PCR amplification technology was firstly developed by Vos et al. (1995)....
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TL;DR: A synthesis of areas of AFLP technique, including comparison to other genotyping methods, assessment of errors, homoplasy, phylogenetic signal and appropriate analysis techniques are provided, with the aim of providing a review that will be applicable to all AFLP-based studies.
631 citations
"Identification of a male-specific a..." refers background in this paper
...After the initial finding, this efficient, high-thought technology has been thought to be an important molecular marker, and was widely applied in a variety of organisms from bacteria and fungi to plants and animals (Bensch and Akesson, 2005; Hua et al., 2009; Meudt and Clarke, 2007)....
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TL;DR: A review of research areas in the study of wild species of animals where the AFLP method should be a very valuable tool to help molecular ecologists to identify when AFLP is likely to be superior to other more established methods, such as microsatellites, SNP (single nucleotide polymorphism) analyses and multigene DNA sequencing.
Abstract: Researchers in the field of molecular ecology and evolution require versatile and low-cost genetic typing methods. The AFLP (amplified fragment length polymorphism) method was introduced 10 years ago and shows many features that fulfil these requirements. With good quality genomic DNA at hand, it is relatively easy to generate anonymous multilocus DNA profiles in most species and the start-up time before data can be generated is often less than a week. Built-in dynamic, yet simple modifications make it possible to find a protocol suitable to the genome size of the species and to screen thousands of loci in hundreds of individuals for a relatively low cost. Until now, the method has primarily been applied in studies of plants, bacteria and fungi, with a strong bias towards economically important cultivated species and their pests. In this review we identify a number of research areas in the study of wild species of animals where the AFLP method, presently very much underused, should be a very valuable tool. These aspects include classical problems such as studies of population genetic structure and phylogenetic reconstructions, and also new challenges such as finding markers for genes governing adaptations in wild populations and modifications of the protocol that makes it possible to measure expression variation of multiple genes (cDNA-AFLP) and the distribution of DNA methylation. We hope this review will help molecular ecologists to identify when AFLP is likely to be superior to other more established methods, such as microsatellites, SNP (single nucleotide polymorphism) analyses and multigene DNA sequencing.
491 citations
"Identification of a male-specific a..." refers background in this paper
...After the initial finding, this efficient, high-thought technology has been thought to be an important molecular marker, and was widely applied in a variety of organisms from bacteria and fungi to plants and animals (Bensch and Akesson, 2005; Hua et al., 2009; Meudt and Clarke, 2007)....
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TL;DR: The conversion process of AFLP fragments to STS markers was technically difficult, mainly because of the presence of contaminating fragments, but a general verification strategy was formed prior to clone sequencing to reduce the frequency of false positives and to identify the correct clone.
Abstract: Amplified fragment length polymorphism (AFLP) markers were used to enrich the map of the wheat chromosomal region containing the Thinopyrum-derived Lr19 leaf rust resistance gene. The region closest to Lr19 was targeted through the use of deletion and recombinant lines of the translocated segment. One of the AFLP bands thus identified was converted into a sequence-tagged-site (STS) marker. This assay generated a 130-bp PCR fragment in all Lr19-carrying lines tested, except for one deletion mutant, while non-carrier template failed to amplify any product. This sequence represents the first marker to map on the distal side of Lr19 on chromosome 7el1. The conversion process of AFLP fragments to STS markers was technically difficult, mainly because of the presence of contaminating fragments. Various approaches were taken to reduce the frequency of false positives and to identify the correct clone. We were able to formulate a general verification strategy prior to clone sequencing. Various other factors causing problems with converting AFLP bands to an STS assays are also discussed.
188 citations
"Identification of a male-specific a..." refers background in this paper
...It has been demonstrated that the majority of AFLP fragments were caused by single nucleotide polymorphisms (SNPs), insertion/deletion (indels) or point mutation at/within the restriction sites (Brugmans et al., 2003; Prins et al., 2001; von et al., 2003)....
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TL;DR: Molecular markers and bulked segregant analysis will enable the rapid transfer of pm42 to wheat breeding populations thus adding to their genetic diversity.
Abstract: Powdery mildew, caused by Blumeria graminis f. sp. tritici, is one of the most important wheat diseases worldwide in areas with cool or maritime climates. Wild emmer (Triticum turgidum var. dicoccoides) is an important potential donor of disease resistances and other traits for common wheat improvement. A powdery mildew resistance gene was transferred from wild emmer accession G-303-1M to susceptible common wheat by crossing and backcrossing, resulting in inbred line P63 (Yanda1817/G-303-1 M//3*Jing411, BC2F6). Genetic analysis of an F2 population and the F2:3 families developed from a cross of P63 and a susceptible common wheat line Xuezao showed that the powdery mildew resistance in P63 was controlled by a single recessive gene. Molecular markers and bulked segregant analysis were used to characterize and map the powdery mildew resistance gene. Nine genomic SSR markers (Xbarc7, Xbarc55, Xgwm148, Xgwm257, Xwmc35, Xwmc154, Xwmc257, Xwmc382, Xwmc477), five AFLP-derived SCAR markers (XcauG3, XcauG6, XcauG10, XcauG20, XcauG22), three EST–STS markers (BQ160080, BQ160588, BF146221) and one RFLP-derived STS marker (Xcau516) were linked to the resistance gene, designated pm42, in P63. pm42 was physically mapped on chromosome 2BS bin 0.75–0.84 using Chinese Spring nullisomic-tetrasomic, ditelosomic and deletion lines, and was estimated to be more than 30 cM proximal to Xcau516, a RFLP-derived STS marker that co-segregated with the wild emmer-derived Pm26 which should be physically located in 2BS distal bin 0.84–1.00. pm42 was highly effective against 18 of 21 differential Chinese isolates of B. graminis f. sp. tritici. The closely linked molecular markers will enable the rapid transfer of pm42 to wheat breeding populations thus adding to their genetic diversity.
145 citations
"Identification of a male-specific a..." refers background in this paper
...After the initial finding, this efficient, high-thought technology has been thought to be an important molecular marker, and was widely applied in a variety of organisms from bacteria and fungi to plants and animals (Bensch and Akesson, 2005; Hua et al., 2009; Meudt and Clarke, 2007)....
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...Consideration of laborious, time-consuming during its utilization for routine sex identification and mapping, AFLP markers were frequently converted into easy-operation approaches, that is, sequence tagged site (STS) and sequencecharacterized amplified region (SCAR) markers (Hua et al., 2009)....
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