Amplification Of Specific Sequences Research Articles

Application of iPBS in high-throughput sequencing for the development of retrotransposon-based molecular markers

Retrotransposons are major components of higher plant genomes, and long terminal repeat (LTR) retrotransposons are especially predominant. Thus, numerous LTR retrotransposon families with high copy numbers exist in most plant genomes. As the integrated copies of these retrotransposons are genetically inherited, their insertion polymorphisms among crop cultivars have been used as functional molecular markers such as inter-retrotransposon amplification polymorphism (IRAP), retrotransposon microsatellite amplification polymorphism (REMAP), retrotransposon-based insertion polymorphism (RBIP) and sequence-specific amplification polymorphism (S-SAP). However, the effective use of these methods requires suitable LTR sequences showing high insertion polymorphism among crop cultivars. Recently, we conducted an efficient screening of LTR retrotransposon families that showed high insertion polymorphism among closely related strawberry cultivars using a next-generation sequencing platform. This method focuses on the primer binding site (PBS), which is adjacent to the 5′ LTR sequence and is conserved among different LTR retrotransposon families. Construction of a sequencing library using the PBS motif allowed us to identify a large number of LTR sequences and their insertion sites throughout the genome. The LTR sequences identified by our method showed high insertion polymorphism among closely related strawberry cultivars, and these families should thus be useful in the development of molecular markers for phylogenetic and genetic diversity studies. This article briefly describes the general aspects of retrotransposon-based molecular markers and also outlines our method for screening LTR sequences suitable for genetic analyses.(http://www.ddbj.nig.ac.jp/).

Loop‐Mediated Isothermal Amplification for Detection of Nucleic Acids

Sequence-specific isothermal nucleic acid amplification techniques are ideally suited for use in molecular diagnostic applications because they do not require thermal cycling equipment and the reactions are typically fast. One of the most widely cited isothermal techniques is termed loop-mediated isothermal amplification (LAMP). This protocol allows amplification times as fast as 5 to 10 min. Furthermore, various methodologies to detect amplification have been applied to LAMP to increase its utility for the point-of-care market. Basic LAMP protocols are provided herein for detection of specific DNA and RNA targets, along with a method to perform multiplex LAMP reactions, permitting even greater flexibility from this powerful technique.

Clinical Interpretation and Implications of Whole Genome Sequencing

In 2013, 60 years after the discovery of the double-helical structure of the DNA by James Watson and Francis Crick and 10 years after the completion of the sequence of the human genome, the first market authorization of a high throughput (‘next-generation’) sequencer (Illumina MiSeqDx) was granted by the American Food and Drug Administration. Next-generation sequencing (NGS) allows to effectively performing whole genome sequencing (WGS) that may lead to the introduction of new genome-based analyses into clinical practice [1]. WGS is one of very few recent developments in medicine which are characterized by the attributes ‘better’, ‘faster’ and ‘cheaper’. With the most recent development of NGS technologies (i.e., Illumina HiSeqX-Ten), complete sequencing of the human genome is feasible within a day for less than US $ 1'000 (not including costs for curation). Several companies offer NGS solutions and compete for a market share. Technologies differ with respect to their miniaturization (e.g., beads, DNA clusters, DNA nanoballs, single molecule sequencing, nanopores). Most still involve sequence-specific nucleic acid hybridization and amplification. Differences exist in the average reading length as well as in the rate, reproducibility, and cost of sequencing. NGS always involves non-directed mass sequencing. Restriction of sequencing on a genomic region, a panel of genes, or the exome (all protein-coding exons), for instance, can be achieved by a capturing tool based on hybridizing oligonucleotides that define the sequence area. The application of NGS for the analysis of RNAs (‘transcriptome’) or epigenetic modifications (‘methylome’) is straightforward. For biomedical scientists as well as for clinicians NGS promises nearly unlimited possibilities to search for genetic variants with clinical relevance. This includes the detection of constitutional as well as of somatic mutations as causes of monogenic diseases, the genome-wide assignment of risk scores for polygenic diseases, the identification of tumoror metastasis-specific mutations in individual tumor patients as well as the determination of gene expression profiles (signatures’) with prognostic and/or therapeutic relevance [2,3]. Another example is pharmacogenomics that may contribute to a personalized therapy, allowing choosing for the individual patient the appropriate drug and/or the appropriate dose.

Detection of Transposable Element Insertion Site Polymorphisms by Sequence-Specific Amplification Polymorphism (SSAP)

Transposable elements represent a major part of any eukaryotic genomes. Notably in plants they can account for more than 80% of the whole genomic sequence (such as in maize). Due to their mobility across the genome, they can act as mutagens but can also be considered as an important source of genetic diversity. It has been shown that they may be activated following various stresses, and it has been assumed that they may contribute to genome evolution and adaptation. Molecular methods have thus been proposed to allow identification of new transposition events, or more generally to tag transposable element insertion site polymorphisms. Sequence-Specific Amplification Polymorphism (SSAP) is a high throughput method derived from AFLP, which has been first tested on the barley genome (Waugh et al., 1997). Its efficiency in tagging TEs in comparison to AFLP is based on the use of specific primers anchored in the TE sequences of interest, requiring the TEs under survey to be previously characterized. SSAP can thus be used to identify any genomic reorganization in the vicinity of TE insertion sites, and still represents an efficient approach to analyse evolutionary dynamics of TEs.

LINE class retroposon is a component of DNA polymorphic fragments of trematode Himasthla elongata parthenitae

We found that the S-SAP (Sequence-Specific Amplification Polymorphism) method revealed clonal variability in the genomes of flatworm Himasthla elongata (Trematoda, Echinostomatidae) larvae. The larvae are parthenogenetic and have been considered genetically homogeneous. We performed cloning and sequencing of an about 500-bp conservative fragment (B1). B1 sequence analysis showed that this fragment had maximal homology with LINE elements from the CR1 family of Hydra and sparrow. In situ hybridization (FISH) revealed B1 dispersed distribution. Several other fragments cloned from the same agarose electrophoresis band correspond to the conservative domain of reverse transcriptase (RT) from CR1 family. Thus, it has been shown that (1) cercariae of trematode H. elongata have clonal variability, (2) the S-SAP method allows patterns to be obtained of fragment distribution characteristic for individual cercariae, and (3) RT conservative domain of the CR1 family participates in the pattern of polymorphic fragments generation. Identification of the CR1 transcripts in H. elongata cercariae transcriptom will be the aim of a future work. Cloning of variable fragments from the pattern of fragments is in progress.

S-SAP MARKERS - A VALUABLE TOOL FOR GENETIC DISTANCE ANALYSIS IN BRASSICA RAPA L.

Because transposable elements contribute significantly to diversification between and within plant species, transposon-based molecular markers could have special benefits for phylogenetic studies. Sequence-specific amplification polymorph-ism (S-SAP) technique uses transposon-related primers combined with AFLP primers. Among the Class II TEs CACTA elements are of special interest, having highly conserved ends and highly variable internal sequences. For our analysis Bot1-related TIR sequences were used. Combinations with different AFLP primers were tested for number of bands, polymorphism degree and reproducibility. With selected primer combinations the VIR Brassica rapa core collection of 96 accessions encompassing a wide range of morphological types and geographical origins was investigated. Twelve primer combinations generated 123 polymorphic markers. The mean PIC value of these markers was 1.47-fold that of 149 SSR alleles tested in the same core collection. The set of S-SAP markers was used for distance analysis to generate a phylogenetic tree as basis for association studies with morphological traits, metabolite content and resistance traits.

Isolation and characterization of RARE-1, a Ty1/copia-like retrotransposon domesticated in the genome of Rhizophora apiculata

Abstract Long terminal repeat (LTR) retrotransposons are predominant mobile elements that play important roles in plant genome evolution. Here, we isolated the first putative complete Ty1/ copia -like retrotransposon of 6303 bp in mangrove Rhizophora apiculata , named RARE-1 . RARE-1 was homologous to the soybean retroelement 1 ( SORE-1 ) and exhibited abundant cis -regulatory motifs involved in various stress responses in its LTRs. Using the sequence-specific amplification polymorphism (S-SAP) technique, we obtained a total of 112 bands for two R. apiculata populations from Hainan, China and Ranong, Thailand. The Hainan population showed slightly higher S-SAP polymorphism but fewer unique bands than the Ranong population. Moreover, the Hainan population also had significantly more copies of RARE-1 than the Ranong population as revealed by quantitative real-time PCR (qPCR). Our results suggested that RARE-1 might have been domesticated in the R. apiculata genome, as a result of the long-term evolution of mangroves under the extreme environment.

Genomic change, retrotransposon mobilization and extensive cytosine methylation alteration in Brassica napus introgressions from two intertribal hybridizations.

Hybridization and introgression represent important means for the transfer and/or de novo origination of traits and play an important role in facilitating speciation and plant breeding. Two sets of introgression lines in Brassica napus L. were previously established by its intertribal hybridizations with two wild species and long-term selection. In this study, the methods of amplified fragment length polymorphisms (AFLP), sequence-specific amplification polymorphism (SSAP) and methylation-sensitive amplified polymorphism (MSAP) were used to determine their genomic change, retrotransposon mobilization and cytosine methylation alteration in these lines. The genomic change revealed by the loss or gain of AFLP bands occurred for ∼10% of the total bands amplified in the two sets of introgressions, while no bands specific for wild species were detected. The new and absent SSAP bands appeared for 9 out of 11 retrotransposons analyzed, with low frequency of new bands and their total percentage of about 5% in both sets. MSAP analysis indicated that methylation changes were common in these lines (33.4–39.8%) and the hypermethylation was more frequent than hypomethylation. Our results suggested that certain extents of genetic and epigenetic alterations were induced by hybridization and alien DNA introgression. The cryptic mechanism of these changes and potential application of these lines in breeding were also discussed.

Cytosine Methylation Alteration in Natural Populations of Leymus chinensis Induced by Multiple Abiotic Stresses

BackgroundHuman activity has a profound effect on the global environment and caused frequent occurrence of climatic fluctuations. To survive, plants need to adapt to the changing environmental conditions through altering their morphological and physiological traits. One known mechanism for phenotypic innovation to be achieved is environment-induced rapid yet inheritable epigenetic changes. Therefore, the use of molecular techniques to address the epigenetic mechanisms underpinning stress adaptation in plants is an important and challenging topic in biological research. In this study, we investigated the impact of warming, nitrogen (N) addition, and warming+nitrogen (N) addition stresses on the cytosine methylation status of Leymus chinensis Tzvel. at the population level by using the amplified fragment length polymorphism (AFLP), methylation-sensitive amplified polymorphism (MSAP) and retrotransposon based sequence-specific amplification polymorphism (SSAP) techniques.Methodology/Principal FindingsOur results showed that, although the percentages of cytosine methylation changes in SSAP are significantly higher than those in MSAP, all the treatment groups showed similar alteration patterns of hypermethylation and hypomethylation. It meant that the abiotic stresses have induced the alterations in cytosine methylation patterns, and the levels of cytosine methylation changes around the transposable element are higher than the other genomic regions. In addition, the identification and analysis of differentially methylated loci (DML) indicated that the abiotic stresses have also caused targeted methylation changes at specific loci and these DML might have contributed to the capability of plants in adaptation to the abiotic stresses.Conclusions/SignificanceOur results demonstrated that abiotic stresses related to global warming and nitrogen deposition readily evoke alterations of cytosine methylation, and which may provide a molecular basis for rapid adaptation by the affected plant populations to the changed environments.

Allopolyploidy has a moderate impact on restructuring at three contrasting transposable element insertion sites in resynthesized Brassica napus allotetraploids

The role played by whole-genome duplication (WGD) in evolution and adaptation is particularly well illustrated in allopolyploids, where WGD is concomitant with interspecific hybridization. This 'Genome Shock', usually accompanied by structural and functional modifications, has been associated with the activation of transposable elements (TEs). However, the impact of allopolyploidy on TEs has been studied in only a few polyploid species, and not in Brassica, which has been marked by recurrent polyploidy events. Here, we developed sequence-specific amplification polymorphism (SSAP) markers for three contrasting TEs, and compared profiles between resynthesized Brassica napus allotetraploids and their diploid Brassica progenitors. To evaluate restructuring at TE insertion sites, we scored changes in SSAP profiles and analysed a large set of differentially amplified SSAP bands. No massive structural changes associated with the three TEs surveyed were detected. However, several transposition events, specific to the youngest TE originating from the B. oleracea genome, were identified. Our study supports the hypothesis that TE responses to allopolyploidy are highly specific. The changes observed in SSAP profiles lead us to hypothesize that they may partly result from changes in DNA methylation, questioning the role of epigenetics during the formation of a new allopolyploid genome.

Phylogenetic analysis of pigeonpea (<i>Cajanus cajan</i>) genotypes using <i>Panzee </i>retrotransposon based SSAP markers

In the present study, twenty one pigeonpea [Cajanus cajan (L.) Millspaugh] genotypes representing three different gene pools were analysed for phylogenetic relationships using sequence specific amplification polymorphism markers. Four primers were screened involving selective AFLP primers in combination with primer specific to 5'LTR region of Panzee retrotransposon. A total of 183 bands were scored, out of which 166 (90.71%) bands were polymorphic with an average of 41.5 polymorphic bands per primer combination. Polymorphism information content (PIC) values ranged from 0.15 [Panzee-LTR/ EcoRI (+AAC)] to 0.26 [Panzee-LTR/ EcoRI (+AAG)] for four primer combinations. Resolving power (RP) values ranged from 10.14 [Panzee-LTR/ EcoRI (+AAC)] to 24.92 [Panzee-LTR/ MseI (+CAC)], whereas marker index (MI) values ranged from 5.25 [Panzee-LTR/ EcoRI (+AAC)] to 14.25 [Panzee-LTR/ MseI (+CAC)] exhibiting positive correlation (r2=0.99 P<0.05). Primer Panzee-LTR/ MseI (+CAC) with higher values of RP (24.92) and MI (14.25) was found most informative for analysing the phylogenetic relationships. NJ tree based on 166 polymorphic SSAP markers exhibited concordant relationships among pigeonpea genotypes supported by high bootstrap values. The genotypes belonging to tertiary gene pools and primary gene pool were placed at extremes, whereas one accession of C. cajanifolius (ICP 15632) was placed closer to primary gene pool, confirming C. cajanifolius is the closest progenitor of cultivated pigeonpea. SSAP markers produced high rate of polymorphism and revealed phylogenetic relationships among pigeonpea genotypes.

Simultaneous Multiple Target Detection in Real-Time Loop-Mediated Isothermal Amplification

Loop-mediated isothermal amplification (LAMP) is a rapid and reliable sequence-specific isothermal nucleic acid amplification technique. To date, all reported real-time detection methods for LAMP have been restricted to single targets, limiting the utility of this technique. Here, we adapted standard LAMP primers to contain a quencher-fluorophore duplex region that upon strand separation results in a gain of fluorescent signal. This approach permitted the real-time detection of 1-4 target sequences in a single LAMP reaction tube utilizing a standard real-time fluorimeter. The methodology was highly reproducible and sensitive, detecting below 100 copies of human genomic DNA. It was also robust, with a 7-order of magnitude dynamic range of detectable targets. Furthermore, using a new strand-displacing DNA polymerase or its warm-start version, Bst 2.0 or Bst 2.0 WarmStart DNA polymerases, resulted in 50% faster amplification signals than wild-type Bst DNA polymerase, large fragment in this new multiplex LAMP procedure. The coupling of this new multiplex technique with next generation isothermal DNA polymerases should increase the utility of the LAMP method for molecular diagnostics.

The αIIb p.Leu841Met (Cab3a+) polymorphism results in a new human platelet alloantigen involved in neonatal alloimmune thrombocytopenia

Fetal-neonatal alloimmune thrombocytopenia (FNAIT) diagnosis relies on maternofetal incompatibility and alloantibody identification. Genotyping for rare platelet (PLT) polymorphisms allowed the identification of three families with suspected or confirmed maternofetal incompatibility for the αIIb-c.2614C>A mutation (Halle et al., Transfusion 2008;48:14-15). A polymerase chain reaction-sequence-specific primers amplification assay was designed to genotype the αIIb-c.2614C>A mutation. HEK293 cells expressing αIIb-Leu841 or αIIb-Met841 αIIbβ3 forms were used to probe the reactivity of maternal sera from these families and to study the effects of the substitution on αIIbβ3 expression and functions. Tested by flow cytometry (FCM), one serum sample specifically reacted with αIIb-Met841 but not with αIIb-Leu841 αIIbβ3. This specificity revealed the αIIb-Leu841 polymorphism as a new alloantigen named Cab3(a+) . Cross-match testing using FCM also showed the Cab3(a+) antigen to be expressed at the PLT surface. As for anti-human PLT alloantigen (HPA)-3a (or -3b) and anti-HPA-9bw, detection of anti-Cab3(a+) alloantibodies appeared difficult and required whole PLT assays when classical monoclonal antibody-specific immobilization of PLT antigen test failed. In our FNAIT set, the immune response to Cab3(a+) maternofetal incompatibility could induce severe thrombocytopenias and life-threatening hemorrhages. The p.Leu841Met substitution has limited effects, if any, on local αIIb structure, preserving both αIIbβ3 expression and functions. The Cab3(a+) polymorphism is a new rare alloantigen (allelic frequency <1%) carried by αIIb that might result in severe life-threatening thrombocytopenias. In Sub-Saharan African populations, higher Cab3(a+) gene frequencies (up to 8.2%; Halle et al., Transfusion 2008;48:14-15) and homozygous people are observed.

P-0232 Colorectal Cancer Screening by Human DNA Stool Quantification

ABSTRACT Introduction The colorectal cancer (CRC) tends to increase with population aging reaching currently 1 million cases with 500.000 deaths around the world. In Brazil, the estimation for this year is 30.140 new cases with 12.471 deaths that make this neoplasia the fourth cause of death in the country. Colonoscopy and fecal occult blood (FOBT) test are largely used for colorectal cancer screening. However, the FOBT had a low sensibility and the colonoscopy is expensive and brings great inconvenience to the subjects. New methods involving the DNA of the stools are being studied to increase the diagnosis of cancer with a noninvasive method. Aim: Quantification of the human DNA of the stools of patients with colorectal cancer. Methods We analyzed 97 stool samples of patients, divided in two groups: 51 individuals without CRC that form the control group and 46 patients with colorectal cancer, which form the case group. All the patients from the control group had been submitted to colonoscopy. PCR Real Time was utilized, through the quantification method by amplification of specific sequences of DNA (Quantifier Human Standard - Applied Systems) and by comparison between these groups. Also to compare the quantification by amplification, we used the fluorescent spectrophotometry. The total DNA of the stools had also been quantified. Results The mean total DNA from the stools of the CRC patients was 91.1ng/µL (SD=57.8) and of the control group was 109.7ng/µL (SD=95.3), (p>0.05). The mean human DNA quantification of the case group was 15.03ng/µL (SD=30.7) and for the control group 0.47ng/µL (SD=1.02). This difference between both groups was significant (p Conclusion No difference on total DNA stool quantification was found between the groups. Patients with colorectal cancer had significant more quantities of human DNA compared to patients without cancer. Higher quantities of human DNA were found in patients with CRC localized in descendent, sigmoid and rectal cancer.

SSAP markers based on a novelTy1-copialike element are a powerful tool for the assessment of genetic diversity in rye (Secale cerealeL.) inbred lines

A partial sequence of a Ty1-copia retrotransposon was isolated from the genome of the rye (Secale cereale L.) inbred line L318 using degenerate primers targeted at the RNaseH sequence. Its putative 3’LTR region exhibited similarity to LTRs of known plant retrotransposons such as BARE-1, while no significant similarity to known rye retrotransposon sequences was found. A Sequence Specific Amplification Polymorphism (SSAP) protocol employing primer designed to correspond to the putative polypurine tract was developed and used for analysis of 30 rye inbred lines. Values of the parameters calculated to evaluate the efficiency of the method as well as the results of cluster analysis demonstrate that it is a powerful tool for genetic diversity analyses of rye.

Genetic assessment of the genus Pisum L. based on sequence specific amplification polymorphism data

The present study was conducted to analyze genetic diversity among 56 accessions of Pisum sativum, Pisum elatius, Pisum pumilio, Pisum fulvum and Podandrogyne brevipedunculata from Syria (42) and a set of 16 reference accessions from Israel (3), Ethiopia (2), Greece (2), Italy (2), Turkey (3), Tunisia (1) and Georgia (1). The study was based on the analysis of multiple data sets generated using sequence specific amplification polymorphism (SSAP) and associated morphological and ecogeographic data. The diversity analysis based on SSAP markers resulted in 83 scorable fragments in P. sativum, P. elatius (63), P. fulvum (62), P. pumilio (53), P. brevipedunculata (31) and P. abyssinicum (29). Genetic variability was measured as Nei’s gene diversity and maximum polymorphism was found in P. elatius (33.87%) and P. fulvum (32.74%) whereas minimum diversity was observed in P. brevipedunculata (12.48%). Pair wise band sharing among Pisum species was also calculated, significant band sharing was observed between P. fulvum and P. elatius, P. elatius and P. sativum and P. pumilio and P. elatius. The relationship among species as revealed by SSAP data could not significantly be correlated with those based on the agro-morphologic characters, suggesting that the two inferences are independent and estimates of genetic relations differently among Pisum taxa. The ecogeographic data associated with Pisum species (latitude, longitude) were also plotted using Arc View GIS 3.2 in order to show the distribution of species in Syria.

Development of Ty1-copia retrotransposon-based S-SAP markers in strawberry (Fragaria × ananassa Duch.)

The most popular retrotransposon-based molecular marker system at the present time is the sequence-specific amplification polymorphism (S-SAP) system, which is widely used for various applications including phylogenetic analysis. In this study, we successfully developed a new S-SAP marker system based on FaRE1 retrotransposon in strawberry genome. Among 34 primer combinations used to develop the S-SAP marker system, seven primer combinations produced high quality and reliability banding patterns. A total of 288 polymorphic bands from 722 amplified bands were detected in 13 strawberry accessions, with a polymorphic ratio of 39.8%. Phylogenetic analysis showed that genetic coefficient ranged from 0.67 to 1.00 for 13 strawberry accessions. An 187bp S-SAP band was detected from 50 strawberry plantlets regenerated from leaves which were cultured on MS media supplemented with 1.0mg/l thidiazuron (TDZ) and 0.1mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). The available evidences proved S-SAP marker had high level of polymorphism in strawberry accessions.

Clinical Phenotype and Mutation Spectrum of the CYP21A2 Gene in Patients with Steroid 21-Hydroxylase Deficiency

Steroid 21-hydroxylase deficiency is caused by inactivating mutations in the CYP21A2 gene. This paper reports on the mutation spectrum and the genotype-phenotype correlation of 21-hydroxylase deficiency. 72 unrelated patients with congenital adrenal hyperplasia (CAH) were included. Molecular analysis of CYP21A2 was performed, via the multiplex ligation-dependent probe amplification (MLPA) analysis and sequence-specific differenzial PCR amplification of the CYP21A2 and CYP21A1P genes, using 4 pair-wise sequence-specific primers, followed by sequencing of the entire CYP21A2 gene. Large gene deletions were identified in 45 (31.3%) of the 144 unrelated CAH alleles, whereas the most frequent point mutations were intron 2 splice mutations (c.293-13A>G) (41/144, 28.5%). The MLPA analysis successfully identified 23 of 72 patients (31.9%) with single copy deletion in CYP21A2. This paper describes a rapid and accurate method for the molecular diagnosis of 21-hydroxylase deficiency, which relies on the identification of point mutations and structural rearrangements within the CYP21A2 gene.

English

Apple (malus domestica&nbsp;Borkh.), which is a widely cultivated, important and economic fruit crop with nutritive and medicinal importance, has emerged as a model horticultural crop in this post-genomic era. Wild apple resources are important and they develop gradually in apple industry and genetic diversity. In this study, two molecular markers (inter-simple sequence repeats, ISSR and sequence-specific amplification polymorphism, S-SAP) were evaluated for genetic analysis of 31 wild apple resources in Shandong. A total of 20 ISSR primers were used and 110 polymorphic bands were amplified. Five S-SAP primer sets yielded a total of 496 bands, of which 201 (40.52%) were polymorphic. The similarity coefficient between resources ranged from 0.70 to 0.94 for ISSR analysis and from 0.66 to 0.95 using the S-SAP methodology. This study indicates&nbsp;that the results obtained based on the dendrograms constructed using unweighted pair-group using arithmetic average (UPGMA) cluster analysis were significantly correlated. The ISSR and S-SAP markers were found to be useful for wild apple resources identification and assessment of phonetic relationships. &nbsp; Key words:&nbsp;Apple, wild resource, inter-simple sequence repeats (ISSR), sequence-specific amplification polymorphism (S-SAP).

Two novel Ty1-copia retrotransposons isolated from coffee trees can effectively reveal evolutionary relationships in the Coffea genus (Rubiaceae)

In the study, we developed new markers for phylogenetic relationships and intraspecies differentiation in Coffea. Nana and Divo, two novel Ty1-copia LTR-retrotransposon families, were isolated through C. canephora BAC clone sequencing. Nana- and Divo-based markers were used to test their: (1) ability to resolve recent phylogenetic relationships; (2) efficiency in detecting intra-species differentiation. Sequence-specific amplification polymorphism (SSAP), retrotransposon-microsatellite amplified polymorphism (REMAP) and retrotransposon-based insertion polymorphism (RBIP) approaches were applied to 182 accessions (31 Coffea species and one Psilanthus accession). Nana- and Divo-based markers revealed contrasted transpositional histories. At the BAC clone locus, RBIP results on C. canephora demonstrated that Nana insertion took place prior to C. canephora differentiation, while Divo insertion occurred after differentiation. Combined SSAP and REMAP data showed that Nana could resolve Coffea lineages, while Divo was efficient at a lower taxonomic level. The combined results indicated that the retrotransposon-based markers were useful in highlighting Coffea genetic diversity and the chronological pattern of speciation/differentiation events. Ongoing complete sequencing of the C. canephora genome will soon enable exhaustive identification of LTR-RTN families, as well as more precise in-depth analyses on contributions to genome size variation and Coffea evolution.