Development and Deployment of High-Throughput Retrotransposon-Based Markers Reveal Genetic Diversity and Population Structure of Asian Bamboo

Bamboo, a non-timber grass species, known for exceptionally fast growth is a commercially viable crop. Long terminal repeat (LTR) retrotransposons, the main class I mobile genetic elements in plant genomes, are highly abundant (46%) in bamboo, contributing to genome diversity. They play significant roles in the regulation of gene expression, chromosome size and structure as well as in genome integrity. Due to their random insertion behavior, interspaces of retrotransposons can vary significantly among bamboo genotypes. Capitalizing this feature, inter-retrotransposon amplified polymorphism (IRAP) is a high-throughput marker system to study the genetic diversity of plant species. To date, there are no transposon based markers reported from the bamboo genome and particularly using IRAP markers on genetic diversity. Phyllostachys genus of Asian bamboo is the largest of the Bambusoideae subfamily, with great economic importance. We report structure-based analysis of bamboo genome for the LTR-retrotransposon superfamilies, Ty3-gypsy and Ty1-copia, which revealed a total of 98,850 retrotransposons with intact LTR sequences at both the ends. Grouped into 64,281 clusters/scaffold using CD-HIT-EST software, only 13 clusters of retroelements were found with more than 30 LTR sequences and with at least one copy having all intact protein domains such as gag and polyprotein. A total of 16 IRAP primers were synthesized, based on the high copy numbers of conserved LTR sequences. A study using these IRAP markers on genetic diversity and population structure of 58 Asian bamboo accessions belonging to the genus Phyllostachys revealed 3340 amplicons with an average of 98% polymorphism. The bamboo accessions were collected from nine different provinces of China, as well as from Italy and America. A three phased approach using hierarchical clustering, principal components and a model based population structure divided the bamboo accessions into four sub-populations, PhSP1, PhSP2, PhSP3 and PhSP4. All the three analyses produced significant sub-population wise consensus. Further, all the sub-populations revealed admixture of alleles. The analysis of molecular variance (AMOVA) among the sub-populations revealed high intra-population genetic variation (75%) than inter-population. The results suggest that Phyllostachys bamboos are not well evolutionarily diversified, although geographic speciation could have occurred at a limited level. This study highlights the usability of IRAP markers in determining the inter-species variability of Asian bamboos

Affinities of Terminal Inverted Repeats to DNA Binding Domain of Transposase Affect the Transposition Activity of Bamboo Ppmar2 Mariner-Like Element

Mariner-like elements (MLE) are a super-family of DNA transposons widespread in animal and plant genomes. Based on their transposition characteristics, such as random insertions and high-frequency heterogeneous transpositions, several MLEs have been developed to be used as tools in gene tagging and gene therapy. Two active MLEs, Ppmar1 and Ppmar2, have previously been identified in moso bamboo (Phyllostachys edulis). Both of these have a preferential insertion affinity to AT-rich region and their insertion sites are close to random in the host genome. In Ppmar2 element, we studied the affinities of terminal inverted repeats (TIRs) to DNA binding domain (DBD) and their influence on the transposition activity. We could identify two putative boxes in the TIRs which play a significant role in defining the TIR’s affinities to the DBD. Seven mutated TIRs were constructed, differing in affinities based on similarities with those of other plant MLEs. Gel mobility shift assays showed that the TIR mutants with mutation sites G669A-C671A had significantly higher affinities than the mutants with mutation sites C657T-A660T. The high-affinity TIRs indicated that their transposition frequency was 1.5–2.0 times higher than that of the wild type TIRs in yeast transposition assays. The MLE mutants with low-affinity TIRs had relatively lower transposition frequency from that of wild types. We conclude that TIR affinity to DBD significantly affects the transposition activity of Ppmar2. The mutant MLEs highly active TIRs constructed in this study can be used as a tool for bamboo genetic studies

Development and Deployment of High-Throughput Retrotransposon-Based Markers Reveal Genetic Diversity and Population Structure of Asian Bamboo

Bamboo, a non-timber grass species, known for exceptionally fast growth is a commercially viable crop. Long terminal repeat (LTR) retrotransposons, the main class I mobile genetic elements in plant genomes, are highly abundant (46%) in bamboo, contributing to genome diversity. They play significant roles in the regulation of gene expression, chromosome size and structure as well as in genome integrity. Due to their random insertion behavior, interspaces of retrotransposons can vary significantly among bamboo genotypes. Capitalizing this feature, inter-retrotransposon amplified polymorphism (IRAP) is a high-throughput marker system to study the genetic diversity of plant species. To date, there are no transposon based markers reported from the bamboo genome and particularly using IRAP markers on genetic diversity. Phyllostachys genus of Asian bamboo is the largest of the Bambusoideae subfamily, with great economic importance. We report structure-based analysis of bamboo genome for the LTR-retrotransposon superfamilies, Ty3-gypsy and Ty1-copia, which revealed a total of 98,850 retrotransposons with intact LTR sequences at both the ends. Grouped into 64,281 clusters/scaffold using CD-HIT-EST software, only 13 clusters of retroelements were found with more than 30 LTR sequences and with at least one copy having all intact protein domains such as gag and polyprotein. A total of 16 IRAP primers were synthesized, based on the high copy numbers of conserved LTR sequences. A study using these IRAP markers on genetic diversity and population structure of 58 Asian bamboo accessions belonging to the genus Phyllostachys revealed 3340 amplicons with an average of 98% polymorphism. The bamboo accessions were collected from nine different provinces of China, as well as from Italy and America. A three phased approach using hierarchical clustering, principal components and a model based population structure divided the bamboo accessions into four sub-populations, PhSP1, PhSP2, PhSP3 and PhSP4. All the three analyses produced significant sub-population wise consensus. Further, all the sub-populations revealed admixture of alleles. The analysis of molecular variance (AMOVA) among the sub-populations revealed high intra-population genetic variation (75%) than inter-population. The results suggest that Phyllostachys bamboos are not well evolutionarily diversified, although geographic speciation could have occurred at a limited level. This study highlights the usability of IRAP markers in determining the inter-species variability of Asian bamboos

Genetics and genomics of moso bamboo (Phyllostachys edulis) : Current status, future challenges, and biotechnological opportunities toward a sustainable bamboo industry

Sustainable goals for contemporary world seek viable solutions for interconnected challenges, particularly in the fields of food and energy security and climate change. We present bamboo, one of the versatile plant species on earth, as an ideal candidate for bioeconomy for meeting some of these challenges. With its potential realized, particularly in the industrial sector, countries such as China are going extensive with bamboo development and cultivation to support a myriad of industrial uses. These include timber, fiber, biofuel, paper, food, and medicinal industries. Bamboo is an ecologically viable choice, having better adaptation to wider environments than do other grasses, and can help to restore degraded lands and mitigate climate change. Bamboo, as a crop species, has not become amenable to genetic improvement, due to its long breeding cycle, perennial nature, and monocarpic behavior. One of the commonly used species, moso bamboo (Phyllostachys edulis) is a potential candidate that qualifies as industrial bamboo. With its whole-genome information released, genetic manipulations of moso bamboo offer tremendous potential to meet the industrial expectations either in quality or in quantity. Further, bamboo cultivation can expect several natural hindrances through biotic and abiotic stresses, which needs viable solutions such as genetic resistance. Taking a pragmatic view of these future requirements, we have compiled the present status of bamboo physiology, genetics, genomics, and biotechnology, particularly of moso bamboo, to drive various implications in meeting industrial and cultivation requirements. We also discuss challenges underway, caveats, and contextual opportunities concerning sustainable development.Peer reviewe

Affinities of Terminal Inverted Repeats to DNA Binding Domain of Transposase Affect the Transposition Activity of Bamboo Ppmar2 Mariner-Like Element

Mariner-like elements (MLE) are a super-family of DNA transposons widespread in animal and plant genomes. Based on their transposition characteristics, such as random insertions and high-frequency heterogeneous transpositions, several MLEs have been developed to be used as tools in gene tagging and gene therapy. Two active MLEs, Ppmar1 and Ppmar2, have previously been identified in moso bamboo (Phyllostachys edulis). Both of these have a preferential insertion affinity to AT-rich region and their insertion sites are close to random in the host genome. In Ppmar2 element, we studied the affinities of terminal inverted repeats (TIRs) to DNA binding domain (DBD) and their influence on the transposition activity. We could identify two putative boxes in the TIRs which play a significant role in defining the TIR’s affinities to the DBD. Seven mutated TIRs were constructed, differing in affinities based on similarities with those of other plant MLEs. Gel mobility shift assays showed that the TIR mutants with mutation sites G669A-C671A had significantly higher affinities than the mutants with mutation sites C657T-A660T. The high-affinity TIRs indicated that their transposition frequency was 1.5–2.0 times higher than that of the wild type TIRs in yeast transposition assays. The MLE mutants with low-affinity TIRs had relatively lower transposition frequency from that of wild types. We conclude that TIR affinity to DBD significantly affects the transposition activity of Ppmar2. The mutant MLEs highly active TIRs constructed in this study can be used as a tool for bamboo genetic studies

Long terminal repeats (LTR) and transcription factors regulate PHRE1 and PHRE2 activity in Moso bamboo under heat stress

Background LTR retrotransposons play a significant role in plant growth, genome evolution, and environmental stress response, but their regulatory response to heat stress remains unclear. We have investigated the activities of two LTR retrotransposons, PHRE1 and PHRE2, of moso bamboo (Phyllostachys edulis) in response to heat stress. Results The differential overexpression of PHRE1 and PHRE2 with or without CaMV35s promoter showed enhanced expression under heat stress in transgenic plants. The transcriptional activity studies showed an increase in transposition activity and copy number among moso bamboo wild type and Arabidopsis transgenic plants under heat stress. Comparison of promoter activity in transgenic plants indicated that 5'LTR promoter activity was higher than CaMV35s promoter. Additionally, yeast one-hybrid (Y1H) system and in planta biomolecular fluorescence complementation (BiFC) assay revealed interactions of heat-dependent transcription factors (TFs) with 5'LTR sequence and direct interactions of TFs with pol and gag. Conclusions Our results conclude that the 5'LTR acts as a promoter and could regulate the LTR retrotransposons in moso bamboo under heat stress.Peer reviewe

Retrotransposons: How the continuous evolutionary front shapes plant genomes for response to heat stress

Long terminal repeat retrotransposons (LTR retrotransposons) are the most abundant group of mobile genetic elements in eukaryotic genomes and are essential in organizing genomic architecture and phenotypic variations. The diverse families of retrotransposons are related to retroviruses. As retrotransposable elements are dispersed and ubiquitous, their “copy-out and paste-in” life cycle of replicative transposition leads to new genome insertions without the excision of the original element. The overall structure of retrotransposons and the domains responsible for the various phases of their replication is highly conserved in all eukaryotes. The two major superfamilies of LTR retrotransposons, Ty1/Copia and Ty3/Gypsy, are distinguished and dispersed across the chromosomes of higher plants. Members of these superfamilies can increase in copy number and are often activated by various biotic and abiotic stresses due to retrotransposition bursts. LTR retrotransposons are important drivers of species diversity and exhibit great variety in structure, size, and mechanisms of transposition, making them important putative actors in genome evolution. Additionally, LTR retrotransposons influence the gene expression patterns of adjacent genes by modulating potential small interfering RNA (siRNA) and RNA-directed DNA methylation (RdDM) pathways. Furthermore, comparative and evolutionary analysis of the most important crop genome sequences and advanced technologies have elucidated the epigenetics and structural and functional modifications driven by LTR retrotransposon during speciation. However, mechanistic insights into LTR retrotransposons remain obscure in plant development due to a lack of advancement in high throughput technologies. In this review, we focus on the key role of LTR retrotransposons response in plants during heat stress, the role of centromeric LTR retrotransposons, and the role of LTR retrotransposon markers in genome expression and evolution.Peer reviewe

Implications of tolerance to iron toxicity on root system architecture changes in rice (Oryza sativa L.)

IntroductionToxicity due to excess soil iron (Fe) is a significant concern for rice cultivation in lowland areas with acidic soils. Toxic levels of Fe adversely affect plant growth by disrupting the absorption of essential macronutrients, and by causing cellular damage. To understand the responses to excess Fe, particularly on seedling root system, this study evaluated rice genotypes under varying Fe levels.MethodsSixteen diverse rice genotypes were hydroponically screened under induced Fe levels, ranging from normal to excess. Morphological and root system characteristics were observed. The onset of leaf bronzing was monitored to identify the toxic response to the excess Fe. Additionally, agronomic and root characteristics were measured to classify genotypes into tolerant and sensitive categories by computing a response stability index.ResultsOur results revealed that 460 ppm of Fe in the nutrient solution served as a critical threshold for screening genotypes during the seedling stage. Fe toxicity significantly affected root system traits, emphasizing the consequential impact on aerial biomass and nutrient deprivation. To classify genotypes into tolerant and sensitive categories, leaf bronzing score was used as a major indicator of Fe stress. However, the response stability index provided a robust basis for classification for the growth performance. Apart from the established tolerant varieties, we could identify a previously unrecognized tolerant variety, ILS 12–5 in this study. Some of the popular mega varieties, including BPT 5204 and Pusa 44, were found to be highly sensitive.DiscussionOur findings suggest that root system damage, particularly in root length, surface area, and root volume, is the key factor contributing to the sensitivity responses under Fe toxicity. Tolerant genotypes were found to retain more healthy roots than the sensitive ones. Fe exclusion, by reducing Fe2+ uptake, may be a major mechanism for tolerance among these genotypes. Further field evaluations are necessary to confirm the behavior of identified tolerant and sensitive lines under natural conditions. Insights from the study provide potential scope for enhancement of tolerance through breeding programs as well as throw light on the role root system in conferring tolerance

Genome-wide association study reveals novel genomic regions governing agronomic and grain quality traits and superior allelic combinations for Basmati rice improvement

BackgroundBasmati is a speciality segment in the rice genepool characterised by explicit grain quality. For the want of suitable populations, genome-wide association study (GWAS) in Basmati rice has not been attempted.MaterialsTo address this gap, we have performed a GWAS on a panel of 172 elite Basmati multiparent population comprising of potential restorers and maintainers. Phenotypic data was generated for various agronomic and grain quality traits across seven different environments during two consecutive crop seasons. Based on the observed phenotypic variation, three agronomic traits namely, days to fifty per cent flowering, plant height and panicle length, and three grain quality traits namely, kernel length before cooking, length breadth ratio and kernel length after cooking were subjected to GWAS. Genotyped with 80K SNP array, the population was subjected to principal component analysis to stratify the underlying substructure and subjected to the association analysis using Bayesian-information and Linkage-disequilibrium Iteratively Nested Keyway (BLINK) model.ResultsWe identified 32 unique MTAs including 11 robust MTAs for the agronomic traits and 25 unique MTAs including two robust MTAs for the grain quality traits. Six out of 13 robust MTAs were novel. By genome annotation, six candidate genes associated with the robust MTAs were identified. Further analysis of the allelic combinations of the robust MTAs enabled the identification of superior allelic combinations in the population. This information was utilized in selecting 77 elite Basmati rice genotypes from the panel.ConclusionThis is the first ever GWAS study in Basmati rice which could generate valuable information usable for further breeding through marker assisted selection, including enhancing of heterosis

Development and Deployment of High-Throughput Retrotransposon-Based Markers Reveal Genetic Diversity and Population Structure of Asian Bamboo

Bamboo, a non-timber grass species, known for exceptionally fast growth is a commercially viable crop. Long terminal repeat (LTR) retrotransposons, the main class I mobile genetic elements in plant genomes, are highly abundant (46%) in bamboo, contributing to genome diversity. They play significant roles in the regulation of gene expression, chromosome size and structure as well as in genome integrity. Due to their random insertion behavior, interspaces of retrotransposons can vary significantly among bamboo genotypes. Capitalizing this feature, inter-retrotransposon amplified polymorphism (IRAP) is a high-throughput marker system to study the genetic diversity of plant species. To date, there are no transposon based markers reported from the bamboo genome and particularly using IRAP markers on genetic diversity. Phyllostachys genus of Asian bamboo is the largest of the Bambusoideae subfamily, with great economic importance. We report structure-based analysis of bamboo genome for the LTR-retrotransposon superfamilies, Ty3-gypsy and Ty1-copia, which revealed a total of 98,850 retrotransposons with intact LTR sequences at both the ends. Grouped into 64,281 clusters/scaffold using CD-HIT-EST software, only 13 clusters of retroelements were found with more than 30 LTR sequences and with at least one copy having all intact protein domains such as gag and polyprotein. A total of 16 IRAP primers were synthesized, based on the high copy numbers of conserved LTR sequences. A study using these IRAP markers on genetic diversity and population structure of 58 Asian bamboo accessions belonging to the genus Phyllostachys revealed 3340 amplicons with an average of 98% polymorphism. The bamboo accessions were collected from nine different provinces of China, as well as from Italy and America. A three phased approach using hierarchical clustering, principal components and a model based population structure divided the bamboo accessions into four sub-populations, PhSP1, PhSP2, PhSP3 and PhSP4. All the three analyses produced significant sub-population wise consensus. Further, all the sub-populations revealed admixture of alleles. The analysis of molecular variance (AMOVA) among the sub-populations revealed high intra-population genetic variation (75%) than inter-population. The results suggest that Phyllostachys bamboos are not well evolutionarily diversified, although geographic speciation could have occurred at a limited level. This study highlights the usability of IRAP markers in determining the inter-species variability of Asian bamboos