Applicability of inter-primer binding site iPBS- retrotransposon marker system for the assessment of genetic diversity and population structure of Peruvian rosewood (Aniba rosaeodora Ducke) germplasm.

BACKGROUND: Rosewood (Aniba rosaeodora Ducke), which has a great demand due to its essential oil globally, is an evergreen tree of the Amazon forests. Rosewood natural stands have been depleted through deforestation and the destruction of habitat. Currently, rosewood is included in the ICUN red list of endangered species. METHODS AND RESULTS: The 11 highly polymorphic primers amplified total 305 bands of which 301 (98.69%) were polymorphic. The number of effective alleles (Ne), Shannon's information index (I), overall gene diversity (Ht), gene diversity (h), and polymorphism information content (PIC) were (1.562), (0.505), (0.330), (0.337) and (0.343), respectively. These diversity indices explored high genetic diversity in rosewood germplasm. Among studied germplasm, the Santa Marta population was found most diverse by reflecting higher values of diversity indices while the Zungarococha population was found least diverse. The analysis of molecular variance (AMOVA) revealed that 79% of the genetic variation was within the populations. The STRUCTURE algorithm, unweighted pair group with arithmetic mean (UPGMA), and principal coordinate's analysis (PCoA) separated all germplasms into different population groups according to their geographic locations. Santa Marta population was found more diverse by reflecting higher values of diversity indices. The maximum genetic distance (0.868) was found between the Huajoya-10 and Nanay-3. In this investigation, iPBS- retrotransposon marker system was used to explore the genetic diversity of Peruvian rosewood germplasm. CONCLUSIONS: The results in this study such as higher genetic diversity indices, AMOVA (79%) within population and PIC value (0.343) showed the utility and reproducibility of iPBS-retrotransposons in this species successfully. The STRUCTURE algorithm separated the germplasms into six population groups according to their geographic locations. These results have valuable information for the conservation, management strategies and future breeding activities of rosewood.

Assessment of genetic diversity and variety identification based on developed retrotransposon-based insertion polymorphism (RBIP) markers in sweet potato (Ipomoea batatas (L.) Lam.).

Sweet potato, a dicotyledonous and perennial plant, is the third tuber/root crop species behind potato and cassava in terms of production. Long terminal repeat (LTR) retrotransposons are highly abundant in sweet potato, contributing to genetic diversity. Retrotransposon-based insertion polymorphism (RBIP) is a high-throughput marker system to study the genetic diversity of plant species. To date, there have been no transposon marker-based genetic diversity analyses of sweet potato. Here, we reported a structure-based analysis of the sweet potato genome, a total of 21555 LTR retrotransposons, which belonged to the main LTR-retrotransposon subfamilies Ty3-gypsy and Ty1-copia were identified. After searching and selecting using Hidden Markov Models (HMMs), 1616 LTR retrotransposon sequences containing at least two models were screened. A total of 48 RBIP primers were synthesized based on the high copy numbers of conserved LTR sequences. Fifty-six amplicons with an average polymorphism of 91.07% were generated in 105 sweet potato germplasm resources based on RBIP markers. A Unweighted Pair Group Method with Arithmatic Mean (UPGMA) dendrogram, a model-based genetic structure and principal component analysis divided the sweet potato germplasms into 3 groups containing 8, 53, and 44 germplasms. All the three analyses produced significant groupwise consensus. However, almost all the germplasms contained only one primary locus. The analysis of molecular variance (AMOVA) among the groups indicated higher intergroup genetic variation (53%) than intrapopulation genetic variation. In addition, long-term self-retention may cause some germplasm resources to exhibit variable segregation. These results suggest that these sweet potato germplasms are not well evolutionarily diversified, although geographic speciation could have occurred at a limited level. This study highlights the utility of RBIP markers for determining the intraspecies variability of sweet potato and have the potential to be used as core primer pairs for variety identification, genetic diversity assessment and linkage map construction. The results could provide a good theoretical reference and guidance for germplasm research and breeding.

Genomic re-assessment of the transposable element landscape of the potato genome.

KEY MESSAGE: We provide a comprehensive and reliable potato TE landscape, based on a wide variety of identification tools and integrative approaches, producing clear and ready-to-use outputs for the scientific community. Transposable elements (TEs) are DNA sequences with the ability to autoreplicate and move throughout the host genome. TEs are major drivers in stress response and genome evolution. Given their significance, the development of clear and efficient TE annotation pipelines has become essential for many species. The latest de novo TE discovery tools, along with available TEs from Repbase and sRNA-seq data, allowed us to perform a reliable potato TEs detection, classification and annotation through an open-source and freely available pipeline ( https://github.com/DiegoZavallo/TE_Discovery ). Using a variety of tools, approaches and rules, we were able to provide a clearly annotated of characterized TEs landscape. Additionally, we described the distribution of the different types of TEs across the genome, where LTRs and MITEs present a clear clustering pattern in pericentromeric and subtelomeric/telomeric regions respectively. Finally, we analyzed the insertion age and distribution of LTR retrotransposon families which display a distinct pattern between the two major superfamilies. While older Gypsy elements concentrated around heterochromatic regions, younger Copia elements located predominantly on euchromatic regions. Overall, we delivered not only a reliable, ready-to-use potato TE annotation files, but also all the necessary steps to perform de novo detection for other species.

High-throughput retrotransposon-based genetic diversity of maize germplasm assessment and analysis.

Maize is one of the world's most important crops and a model for grass genome research. Long terminal repeat (LTR) retrotransposons comprise most of the maize genome; their ability to produce new copies makes them efficient high-throughput genetic markers. Inter-retrotransposon-amplified polymorphisms (IRAPs) were used to study the genetic diversity of maize germplasm. Five LTR retrotransposons (Huck, Tekay, Opie, Ji, and Grande) were chosen, based on their large number of copies in the maize genome, whereas polymerase chain reaction primers were designed based on consensus LTR sequences. The LTR primers showed high quality and reproducible DNA fingerprints, with a total of 677 bands including 392 polymorphic bands showing 58% polymorphism between maize hybrid lines. These markers were used to identify genetic similarities among all lines of maize. Analysis of genetic similarity was carried out based on polymorphic amplicon profiles and genetic similarity phylogeny analysis. This diversity was expected to display ecogeographical patterns of variation and local adaptation. The clustering method showed that the varieties were grouped into three clusters differing in ecogeographical origin. Each of these clusters comprised divergent hybrids with convergent characters. The clusters reflected the differences among maize hybrids and were in accordance with their pedigree. The IRAP technique is an efficient high-throughput genetic marker-generating method.

Development and validation of InnoQuant® HY, a system for quantitation and quality assessment of total human and male DNA using high copy targets.

The development and validation of InnoQuant® HY, a real-time PCR system containing four DNA targets-two RE autosomal targets of different sizes, male specific targets, and an internal positive control target-are described herein. The ratio of the two autosomal targets provides a Degradation Index, or a quantitative value of a sample's degradation state. The male specific targets are multi-copy targets located on the Y chromosome, which provides information about a sample's male DNA composition. The experimental results demonstrate InnoQuant HY as a robust qPCR method producing accurate DNA quantitation results even at low dynamic ranges, with reproducibility among population groups. The system is human specific with low level higher primate cross reactivity and is able to consistently and reproducibly detect sub-picogram concentrations of human and human male DNA. The use of high copy number Alu and SVA (>1000 copies per genome) retrotransposable elements as the two autosomal targets significantly enhances both sensitivity and reproducibility of determination of DNA quantitation as well as DNA degradation in forensic samples. The inclusion of a sensitive multi-copy Y-chromosome specific target provides accurate quantitation of DNA from a male in challenging male-female mixtures (i.e. sexual assault samples). Even in the presence of a large excess of DNA from a female, accurate quantitation was achieved with a male to female ratio of 1:128,000. Population database studies reveal an average Short/Y target ratio of the quantification values across all four populations tested was 1.124±0.282, exhibiting the system's reproducibility across multiple populations. The results from InnoQuant HY provide a tool equipping a forensic analyst with crucial data about a sample's DNA quantitation, male:female ratio, degradation state, and the presence or absence of PCR inhibitors. With the information gained from the InnoQuant HY kit, a more streamlined and efficient workflow can be created that minimizes unnecessary sample processing and retesting while maximizing recovery of probative DNA profiles from challenging biological evidence.

Terminal-repeat retrotransposons with GAG domain in plant genomes: a new testimony on the complex world of transposable elements.

A novel structure of nonautonomous long terminal repeat (LTR) retrotransposons called terminal repeat with GAG domain (TR-GAG) has been described in plants, both in monocotyledonous, dicotyledonous and basal angiosperm genomes. TR-GAGs are relatively short elements in length (<4 kb) showing the typical features of LTR-retrotransposons. However, they carry only one open reading frame coding for the GAG precursor protein involved for instance in transposition, the assembly, and the packaging of the element into the virus-like particle. GAG precursors show similarities with both Copia and Gypsy GAG proteins, suggesting evolutionary relationships of TR-GAG elements with both families. Despite the lack of the enzymatic machinery required for their mobility, strong evidences suggest that TR-GAGs are still active. TR-GAGs represent ubiquitous nonautonomous structures that could be involved in the molecular diversities of plant genomes.

Prediction of retrotransposons and assessment of genetic variability based on developed retrotransposon-based insertion polymorphism (RBIP) markers in Pyrus L.

Interspecific hybridization has been considered the major mode of evolution in Pyrus (pear), and thus, the genetic relationships within this genus have not been well documented. Retrotransposons are ubiquitous components of plant genomes and 42.4 % of the pear genome was reported to be long terminal repeat (LTR) retrotransposons, implying that retrotransposons might be significant in the evolution of Pyrus. In this study, 1,836 putative full-length LTR retrotransposons were isolated and 196 retrotransposon-based insertion polymorphism (RBIP) primers were developed, of which 24 pairs to the Ppcr1 subfamily of copia retrotransposons were used to analyze genetic diversity among 110 Pyrus accessions from Eurasia. Our results showed that Ppcr1 replicated many times in the development of cultivated Asian pears. The genetic structure analysis and the unweighted pair group method with arithmetic mean (UPGMA) dendrogram indicated that all accessions could be divided into Oriental and Occidental groups. In Oriental pears, wild pea pears clustered separately into independent groups in accordance with their morphological classifications. Cultivars of P. ussuriensis Maxim, P. pyrifolia Nakai, and P. pyrifolia Chinese white pear were mingled together, which inferred that hybridization events occurred during the development of the cultivated Asian pears. In Occidental pears, two clades were obtained in the UPGMA dendrogram in accordance with their geographical distribution; one contained the European species and the other included species from North Africa and West Asia. New findings in this study will be important to further understand the phylogeny of Pyrus and origins of cultivated pears.

Development and validation of InnoQuant™, a sensitive human DNA quantitation and degradation assessment method for forensic samples using high copy number mobile elements Alu and SVA.

There is a constant need in forensic casework laboratories for an improved way to increase the first-pass success rate of forensic samples. The recent advances in mini STR analysis, SNP, and Alu marker systems have now made it possible to analyze highly compromised samples, yet few tools are available that can simultaneously provide an assessment of quantity, inhibition, and degradation in a sample prior to genotyping. Currently there are several different approaches used for fluorescence-based quantification assays which provide a measure of quantity and inhibition. However, a system which can also assess the extent of degradation in a forensic sample will be a useful tool for DNA analysts. Possessing this information prior to genotyping will allow an analyst to more informatively make downstream decisions for the successful typing of a forensic sample without unnecessarily consuming DNA extract. Real-time PCR provides a reliable method for determining the amount and quality of amplifiable DNA in a biological sample. Alu are Short Interspersed Elements (SINE), approximately 300bp insertions which are distributed throughout the human genome in large copy number. The use of an internal primer to amplify a segment of an Alu element allows for human specificity as well as high sensitivity when compared to a single copy target. The advantage of an Alu system is the presence of a large number (>1000) of fixed insertions in every human genome, which minimizes the individual specific variation possible when using a multi-copy target quantification system. This study utilizes two independent retrotransposon genomic targets to obtain quantification of an 80bp "short" DNA fragment and a 207bp "long" DNA fragment in a degraded DNA sample in the multiplex system InnoQuant™. The ratio of the two quantitation values provides a "Degradation Index", or a qualitative measure of a sample's extent of degradation. The Degradation Index was found to be predictive of the observed loss of STR markers and alleles as degradation increases. Use of a synthetic target as an internal positive control (IPC) provides an additional assessment for the presence of PCR inhibitors in the test sample. In conclusion, a DNA based qualitative/quantitative/inhibition assessment system that accurately predicts the status of a biological sample, will be a valuable tool for deciding which DNA test kit to utilize and how much target DNA to use, when processing compromised forensic samples for DNA testing.

Assessment of genetic diversity among Indian potato (Solanum tuberosum L.) collection using microsatellite and retrotransposon based marker systems.

Potato (Solanum tuberosum) is an important non-cereal crop throughout the world and is highly recommended for ensuring global food security. Owing to the complexities in genetics and inheritance pattern of potato, the conventional method of cross breeding for developing improved varieties has been difficult. Identification and tagging of desirable traits with informative molecular markers would aid in the development of improved varieties. Insertional polymorphism of copia-like and gypsy-like long terminal repeat retrotransposons (RTN) were investigated among 47 potato varieties from India using Inter-Retrotransposon Amplified Polymorphism (IRAP) and Retrotransposon Microsatellite Amplified Polymorphism (REMAP) marker techniques and were compared with the DNA profiles obtained with simple sequence repeats (SSRs). The genetic polymorphism, efficiency of polymorphism and effectiveness of marker systems were evaluated to assess the extent of genetic diversity among Indian potato varieties. A total of 139 polymorphic SSR alleles, 270 IRAP and 98 REMAP polymorphic bands, showing polymorphism of 100%, 87.9% and 68.5%, respectively, were used for detailed characterization of the genetic relationships among potato varieties by using cluster analysis and principal coordinate analysis (PCoA). IRAP analysis resulted in the highest number of polymorphic bands with an average of 15 polymorphic bands per assay unit when compared to the other two marker systems. Based on pair-wise comparison, the genetic similarity was calculated using Dice similarity coefficient. The SSRs showed a wide range in genetic similarity values (0.485-0.971) as compared to IRAP (0.69-0.911) and REMAP (0.713-0.947). A Mantel's matrix correspondence test showed a high positive correlation (r=0.6) between IRAP and REMAP, an intermediate value (r=0.58) for IRAP and SSR and the lowest value (r=0.17) for SSR and REMAP. Statistically significant cophenetic correlation coefficient values, of 0.961, 0.941 and 0.905 were observed for REMAP, IRAP and SSR, respectively. The widespread presence and distinct DNA profiles for copia-like and gypsy-like RTNs in the examined genotypes indicate that these elements are active in the genome and may have even contributed to the potato genome organization. Although the three marker systems were capable of distinguishing all the 47 varieties; high reproducibility, low cost and ease of DNA profiling data collection make IRAP and REMAP markers highly efficient whole-genome scanning molecular probes for population genetic studies. Information obtained from the present study regarding the genetic association and distinctiveness provides an useful guide for selection of germplasm for plant breeding and conservation efforts.

Footprint of APOBEC3 on the genome of human retroelements.

Almost half of the human genome is composed of transposable elements. The genomic structures and life cycles of some of these elements suggest they are a result of waves of retroviral infection and transposition over millions of years. The reduction of retrotransposition activity in primates compared to that in nonprimates, such as mice, has been attributed to the positive selection of several antiretroviral factors, such as apolipoprotein B mRNA editing enzymes. Among these, APOBEC3G is known to mutate G to A within the context of GG in the genome of endogenous as well as several exogenous retroelements (the underlining marks the G that is mutated). On the other hand, APOBEC3F and to a lesser extent other APOBEC3 members induce G-to-A changes within the nucleotide GA. It is known that these enzymes can induce deleterious mutations in the genome of retroviral sequences, but the evolution and/or inactivation of retroelements as a result of mutation by these proteins is not clear. Here, we analyze the mutation signatures of these proteins on large populations of long interspersed nuclear element (LINE), short interspersed nuclear element (SINE), and endogenous retrovirus (ERV) families in the human genome to infer possible evolutionary pressure and/or hypermutation events. Sequence context dependency of mutation by APOBEC3 allows investigation of the changes in the genome of retroelements by inspecting the depletion of G and enrichment of A within the APOBEC3 target and product motifs, respectively. Analysis of approximately 22,000 LINE-1 (L1), 24,000 SINE Alu, and 3,000 ERV sequences showed a footprint of GG→AG mutation by APOBEC3G and GA→AA mutation by other members of the APOBEC3 family (e.g., APOBEC3F) on the genome of ERV-K and ERV-1 elements but not on those of ERV-L, LINE, or SINE.

Assessment of genetic and epigenetic stability in long-term in vitro shoot culture of pea (Pisum sativum L.).

In vitro clonal propagation of plants should generate identical copies of the selected genotype. However, associated stress might result in a breakdown of control mechanisms and consequent instability of the genome. We have used several molecular methods to assess the genetic stability of long-term propagated (24 years) multiple shoot in vitro culture of pea (Pisum sativum L.). We focused on assessing the stability of repetitive sequences, such as simple sequence repeats (SSR) and retrotransposons, both comprising a large part of genome. No differences were found when seedlings (Co-2004) or original seed (Co-1982) controls and long-term or newly established in vitro (one subculture cycle) samples were investigated by the SSR, inter-repeats (ISSR) or inter-retrotransposon amplified polymorphism (IRAP) method. However, the more global amplified fragment length polymorphism (AFLP) and particularly the methylation sensitive MSAP methods detected 11 and 18% polymorphism among samples, respectively. Interestingly, investigation of the global cytosine methylation status by HPCE measurement revealed no statistically significant differences. Some evidence of retrotransposon re-arrangement was observed by sequence-specific amplification polymorphism. This occurred mostly in the abundant Ty3-gypsy type Cyclop element and to a smaller extent in the Ogre element. Alternatively, no polymorphism was detected among the PDR-1 element of the Ty1-copia type retrotransposon. Based on these results, multiple shoot culture of pea maintained over a long period may be considered as a true to type multiplication method of the original genotype.

Retrotransposon-microsatellite amplified polymorphism (REMAP) markers for genetic diversity assessment of the rice blast pathogen (Magnaporthe grisea).

This present study is the first report of the application of the retrotransposon-microsatellite amplified polymorphism (REMAP) technique in fungi. Genome fingerprinting has a major role in the characterization of population structure and in the analysis of the variability in fungi. Retrotransposon-microsatellite amplified polymorphism assay was used in virulent isolates of a rice blast pathogen (Magnaporthe grisea) as a new assay system for genetic variability studies that overcomes the limitations of previous techniques. The high polymorphism observed in REMAP could be due to past or recent actions of retrotransposon in M. grisea. Retrotransposon-microsatellite amplified polymorphism, with its superior marker utility, was concluded to be the marker of choice for characterizing M. grisea isolates.

Extensive gene traffic on the mammalian X chromosome.

Mammalian sex chromosomes have undergone profound changes since evolving from ancestral autosomes. By examining retroposed genes in the human and mouse genomes, we demonstrate that, during evolution, the mammalian X chromosome has generated and recruited a disproportionately high number of functional retroposed genes, whereas the autosomes experienced lower gene turnover. Most autosomal copies originating from X-linked genes exhibited testis-biased expression. Such export is incompatible with mutational bias and is likely driven by natural selection to attain male germline function. However, the excess recruitment is consistent with a combination of both natural selection and mutational bias.