Clonal Generations Research Articles

Association study of yield and its component traits in sugarcane seedlings

An experiment was carried out to investigate the relationship between specific quantitative characters and sugarcane yield at the Sugarcane Research Institute, Pusa, Bihar, in February 2022. Thirty sugarcane seedlings obtained from three different crosses along with check were subjected to estimation trait association by correlation and path analyses. Cane yield showed strong positive and highly significant correlation with single cane weight, number of millable canes per clump and brix percent at 12th month. There was also positive significant correlation of number of shoots per clump with cane yield. Path analysis revealed the highest positive direct effect of brix percent at 12th month on cane yield followed by brix percent at 10th month and cane diameter. However, number of shoots per clump had considerable negative direct effects and indirect positive effects through single cane weight and number of millable canes per clump on cane yield. The traits that provide considerable positive correlation and positive direct effects to cane yield could be used as selection indices for yield improvement in for further clonal generations.

Colour change in potato (Solanum tuberosum L.) tubers by disruption of the anthocyanin pathway via ribonucleoprotein complex delivery of the CRISPR/Cas9 system

Potato is an important part of the traditional Norwegian diet, and the crop faces several challenges with respect to pests and diseases, as well as the increasingly challenging changes in climate. Genome editing may provide tools to improve the resilience of Norwegian potato cultivars to new climate challenges. We have altered the skin colour of two potato cultivars, ‘Desirée’ and ‘Nansen’ from red to yellow, as a proof-of-concept for the use of CRISPR/Cas9 in a Norwegian cultivar. Our method has involved the use of protoplasts and we have grown the regenerants for three successive clonal tuber generations to evaluate the stability of the edited plants over time and under varying temperature conditions in contained rooms in a greenhouse. We found that the protoplast method is well suited to achieving CRISPR/Cas9 applications. The results show that the yellow skin is consistent over the three generations of tuber propagation. We found some suspected somaclonal variation in the protoplast regenerants. Some of the variation which we observed under high temperatures (up to nearly 40ºC) during the second growth cycle, disappeared when cultivated under lower temperatures in the third cultivation cycle.

DNA methylation in the wild: epigenetic transgenerational inheritance can mediate adaptation in clones of wild strawberry (Fragaria vesca).

Due to the accelerating climate change, it is crucial to understand how plants adapt to rapid environmental changes. Such adaptation may be mediated by epigenetic mechanisms like DNA methylation, which could heritably alter phenotypes without changing the DNA sequence, especially across clonal generations. However, we are still missing robust evidence of the adaptive potential of DNA methylation in wild clonal populations. Here, we studied genetic, epigenetic and transcriptomic variation of Fragaria vesca, a predominantly clonally reproducing herb. We examined samples from 21 natural populations across three climatically distinct geographic regions, as well as clones of the same individuals grown in a common garden. We found that epigenetic variation was partly associated with climate of origin, particularly in non-CG contexts. Importantly, a large proportion of this variation was heritable across clonal generations. Additionally, a subset of these epigenetic changes affected the expression of genes mainly involved in plant growth and responses to pathogen and abiotic stress. These findings highlight the potential influence of epigenetic changes on phenotypic traits. Our findings indicate that variation in DNA methylation, which can be environmentally inducible and heritable, may enable clonal plant populations to adjust to their environmental conditions even in the absence of genetic adaptation.

Identifying the Principal Contributing Traits for Commercial Cane Sugar Yield at First Clonal Selection Stage in Sugarcane (Saccharum spp.)

Commercial cane sugar (CCS) yield is the main target product in sugarcane, which depends on the sucrose content and cane yield related traits. Understanding the factors influencing CCS yield would help us to enhance the selection efficiency for sugar yield. A population of 1,080 seedlings derived from nine bi-parental crosses involving Indian Co canes and Co allied hybrids were evaluated in seedling stage. A subset comprised of 270 clones selected from the single stool stage were evaluated in the first clonal trial to identify major component traits of CCS yield in the early stages of selection. Investigations were undertaken in the population to explore the feasibility of employing PCA and regression analyses to provide a basis for selection criteria for identification of sugarcane genotypes that are higher in CCS yield. Comparative performance of families at ground nursery indicated that screening of seedling progenies should be towards the crosses with higher mean value of H.R. brix% and single cane weight to enhance the frequency of superior individuals in subsequent clonal generations. The results of correlation analysis at first clonal stage suggested that yield related traits including tillers at 120 days, single cane weight, number of millable stalks and cane yield are the most important characters that significantly influences the CCS yield. Principal component analysis revealed that four factors could explain approximately 80.40% of the total variation for CCS yield in the population studied. The first factor which accounted for 31.19% of the variation was strongly associated with juice brix%, sucrose%, H.R. brix%, purity% and CCS%, whereas the second component had a positive effect from single cane weight, juice weight and cane diameter explaining 21.74% of total variability. The third factor accounted for 18.97% of variability and had contribution from tillers at 120 days, number of millable stalks and cane yield. Stalk height was independent from other traits which accounted for 8.48% of variation in component 4. Stepwise regression analysis demonstrated that CCS%, cane yield and sucrose % had considerable high effects on commercial cane sugar yield which explained 85.53% (expressed as R2) of variability in clones derived from bi-parental crosses between Indian Co canes.

DNA methylation in clonal duckweed (Lemna minor L.) lineages reflects current and historical environmental exposures.

Environmentally induced DNA methylation variants may mediate gene expression responses to environmental changes. If such induced variants are transgenerationally stable, there is potential for expression responses to persist over multiple generations. Our current knowledge in plants, however, is almost exclusively based on studies conducted in sexually reproducing species where the majority of DNA methylation changes are subject to resetting in germlines, limiting the potential for transgenerational epigenetics stress memory. Asexual reproduction circumvents germlines, and may therefore be more conducive to long-term inheritance of epigenetic marks. Taking advantage of the rapid clonal reproduction of the common duckweed Lemna minor, we hypothesize that long-term, transgenerational stress memory from exposure to high temperature can be detected in DNA methylation profiles. Using a reduced representation bisulphite sequencing approach (epiGBS), we show that temperature stress induces DNA hypermethylation at many CG and CHG cytosine contexts but not CHH. Additionally, differential methylation in CHG context that was observed was still detected in a subset of cytosines, even after 3-12 generations of culturing in a common environment. This demonstrates a memory effect of stress reflected in the methylome and that persists over multiple clonal generations. Structural annotation revealed that this memory effect in CHG methylation was enriched in transposable elements. The observed epigenetic stress memory is probably caused by stable transgenerational persistence of temperature-induced DNA methylation variants across clonal generations. To the extent that such epigenetic memory has functional consequences for gene expression and phenotypes, this result suggests potential for long-term modulation of stress responses in asexual plants.

Spontaneous rate of clonal single nucleotide mutations in Daphnia galeata.

Mutations are the ultimate source of heritable variation and therefore the fuel for evolution, but direct estimates of mutation rates exist only for few species. We estimated the spontaneous single nucleotide mutation rate among clonal generations in the waterflea Daphnia galeata with a short-term mutation accumulation approach. Individuals from eighteen mutation accumulation lines over five generations were deep sequenced to count de novo mutations that were not present in a pool of F1 individuals, representing the parental genotype. We identified 12 new nucleotide mutations in 90 clonal generational passages. This resulted in an estimated single nucleotide mutation rate of 0.745 x 10−9 (95% c.f. 0.39 x 10−9–1.26 x 10−9), which is slightly lower than recent estimates for other Daphnia species. We discuss the implications for the population genetics of Cladocerans.

The power of genomic estimated breeding values for selection when using a finite population size in genetic improvement of tetraploid potato.

Potato breeding relies heavily on visual phenotypic scoring for clonal selection. Obtaining robust phenotypic data can be labor intensive and expensive, especially in the early cycles of a potato breeding program where the number of genotypes is very large. We have investigated the power of genomic estimated breeding values (GEBVs) for selection from a limited population size in potato breeding. We collected genotypic data from 669 tetraploid potato clones from all cycles of a potato breeding program, as well as phenotypic data for eight important breeding traits. The genotypes were partitioned into a training and a test population distinguished by cycle of selection in the breeding program. GEBVs for seven traits were predicted for individuals from the first stage of the breeding program (T1) which had not undergone any selection, or individuals selected at least once in the field (T2). An additional approach in which GEBVs were predicted within and across full-sib families from unselected material (T1) was tested for four breeding traits. GEBVs were obtained by using a Bayesian Ridge Regression model estimating single marker effects and phenotypic data from individuals at later stages of selection of the breeding program. Our results suggest that, for most traits included in this study, information from individuals from later stages of selection cannot be utilized to make selections based on GEBVs in earlier clonal generations. Predictions of GEBVs across full-sib families yielded similarly low prediction accuracies as across generations. The most promising approach for selection using GEBVs was found to be making predictions within full-sib families.

Screening and Evaluation of Initial Clonal Generations of Potato Hybrids for Yield and Yield Attributing Characters

A field experiment was conducted during Rabi 2015-16 and 2016-17 at ICAR-Central Potato Research Station, Patna Bihar. In this experiment F1 with initial clonal generations viz F1C1, F1C2 and F1C3 were evaluated. The screening and selection of these generations were done in randomized block design for the character like early bulking, tuber size, tuber shape, depth of eye, tuber colour and resistance to late blight of potato etc. A total of 12,360 True Potato Seeds of 17 successful crosses and 7,322 True Potato Seeds of 23 successful crosses were sown and seedlings were transplanted, out of these 610 and 187 clones were selected during 2015-16 and 2016-17 respectively. Large number of potato cultures were planted for further evaluation in next generations. In F1C3 the cultures were planted along with more than six check varieties of potato. Cultures were evaluated and selected considering total tuber yield, shape, colour and disease reactions for further evaluation in PYT.

Previous exposure mediates the response of eelgrass to future warming via clonal transgenerational plasticity.

Mortality and shifts in species distributions are among the most obvious consequences of extreme climatic events. However, the sublethal effects of an extreme event can have persistent impacts throughout an individual's lifetime and into future generations via within-generation and transgenerational phenotypic plasticity. These changes can either confer resilience or increase susceptibility to subsequent stressful events, with impacts on population, community, and potentially ecosystem processes. Here, we show how a simulated extreme warming event causes persistent changes in the morphology and growth of a foundation species (eelgrass, Zostera marina) across multiple clonal generations and multiple years. The effect of previous parental exposure to warming increased aboveground biomass, shoot length, and aboveground-belowground biomass ratios while also greatly decreasing leaf growth rates. Long-term increases in aboveground-belowground biomass ratios could indicate an adaptive clonal transgenerational response to warmer climates that reduces the burden of increased respiration in belowground biomass. These transgenerational responses were likely decoupled from clonal parent provisioning as rhizome size of clonal offspring was standardized at planting and rhizome starch reserves were not impacted by warming treatments. Future investigations into potential epigenetic mechanisms underpinning such clonal transgenerational plasticity will be necessary to understand the resilience of asexual foundation species to repeated extreme climatic events.

Tropical seagrass Halophila stipulacea shifts thermal tolerance during Mediterranean invasion.

Exotic species often face new environmental conditions that are different from those that they are adapted to. The tropical seagrass Halophila stipulacea is a Lessepsian migrant that colonized the Mediterranean Sea around 100 years ago, where at present the minimum seawater temperature is cooler than in its native range in the Red Sea. Here, we tested if the temperature range in which H. stipulacea can exist is conserved within the species or if the exotic populations have shifted their thermal breadth and optimum due to the cooler conditions in the Mediterranean. We did so by comparing the thermal niche (e.g. optimal temperatures, and upper and lower thermal limits) of native (Saudi Arabia in the Red Sea) and exotic (Greece and Cyprus in the Mediterranean Sea) populations of H. stipulacea. We exposed plants to 12 temperature treatments ranging from 8 to 40°C for 7 days. At the end of the incubation period, we measured survival, rhizome elongation, shoot recruitment, net population growth and metabolic rates. Upper and lower lethal thermal thresholds (indicated by 50% plant mortality) were conserved across populations, but minimum and optimal temperatures for growth and oxygen production were lower for Mediterranean populations than for the Red Sea one. The displacement of the thermal niche of exotic populations towards the colder Mediterranean Sea regime could have occurred within 175 clonal generations.

Genetic variability and transgenerational regulation of investment in sex in the monogonont rotifer Brachionus plicatilis

In cyclical parthenogens such as aphids, cladocerans and rotifers, the coupling between sexual reproduction and the production of resting stages (diapausing eggs) imposes strong constraints on the timing of sex. Whereas induction of sex is generally triggered by environmental cues, the response to such cues may vary across individuals according to genetic and nongenetic factors. In this study, we explored genetic and epigenetic causes of variation for the propensity for sex using a collection of strains from a Spanish population of monogonont rotifers (Brachionus plicatilis) in which variation for the threshold population density at which sex is induced (mixis threshold) had been documented previously. Our results show significant variation for the mixis threshold among 20 clones maintained under controlled conditions for 15 asexual generations. The effect of the number of clonal generations since hatching of the diapausing egg on the mixis ratio (proportion of sexual offspring produced) was tested on 4 clones with contrasted mixis thresholds. The results show a negative correlation between the mixis threshold and mixis ratio, as well as a significant effect of the number of clonal generations since fertilization, sex being repressed during the first few generations after hatching of the diapausing egg.

Clonal Plants as Meta-Holobionts.

The holobiont concept defines a given organism and its associated symbionts as a potential level of selection over evolutionary time. In clonal plants, recent experiments demonstrated vertical transmission of part of the microbiota from one ramet (i.e., potentially autonomous individual) to another within the clonal network (i.e., connections by modified stems present in ∼35% of all plants). Because of this heritability, and potentially reciprocal exchange of microbes between generations of ramets, we propose to extend the existing holobiont framework to the concept of meta-holobiont. A meta-holobiont is a network of holobionts that can exchange biomolecules and microbiota across generations, thus impacting the fitness of both biological scales: holobionts and meta-holobionts. Specifically, meta-holobiont dynamics can result in sharing, specialization, and division of labor across plant clonal generations. This paper, which coins the meta-holobiont concept, is expected to stimulate discussion and to be applied beyond the context of networked clonal plants (e.g., to social insects).

Variable Inheritance of AmplifiedEPSPSGene Copies in Glyphosate-Resistant Palmer Amaranth (Amaranthus palmeri)

AbstractGlyphosate-resistant (GR) Palmer amaranth (Amaranthus palmeriS. Watson) is considered one of the most troublesome weeds in the southern and central United States, but results of previous research to determine the mode of inheritance of this trait have been conflicting and inconclusive. In this study, we examined segregation patterns ofEPSPSgene-copy numbers in F1and F2generations ofA. palmeriand found no evidence of a Mendelian single-gene pattern of inheritance. Transgressive segregation for copy number was exhibited by several F1and all of the F2families, most likely the product ofEPSPScopy-number variation within each plant. This variation was confirmed by assaying gene-copy number across clonal generations and among individual shoots on the same plant, demonstrating thatEPSPSamplification levels vary significantly within a single plant. Increases and decreases in copy number occurred in a controlled, stress-free environment in the absence of glyphosate, indicating thatEPSPSgene amplification is a random and variable process within the plant. The ability ofA. palmerito gain or loseEPSPSgene copies is a valuable adaptive trait, allowing this species to respond rapidly to selection pressures and changing environments.

Genome Editing in Trees: From Multiple Repair Pathways to Long-Term Stability.

The CRISPR technology continues to diversify with a broadening array of applications that touch all kingdoms of life. The simplicity, versatility and species-independent nature of the CRISPR system offers researchers a previously unattainable level of precision and control over genomic modifications. Successful applications in forest, fruit and nut trees have demonstrated the efficacy of CRISPR technology at generating null mutations in the first generation. This eliminates the lengthy process of multigenerational crosses to obtain homozygous knockouts (KO). The high degree of genome heterozygosity in outcrossing trees is both a challenge and an opportunity for genome editing: a challenge because sequence polymorphisms at the target site can render CRISPR editing ineffective; yet an opportunity because the power and specificity of CRISPR can be harnessed for allele-specific editing. Examination of CRISPR/Cas9-induced mutational profiles from published tree studies reveals the potential involvement of multiple DNA repair pathways, suggesting that the influence of sequence context at or near the target sites can define mutagenesis outcomes. For commercial production of elite trees that rely on vegetative propagation, available data suggest an excellent outlook for stable CRISPR-induced mutations and associated phenotypes over multiple clonal generations.

Transgenerational Effects and Epigenetic Memory in the Clonal Plant Trifolium repens.

Transgenerational effects (TGE) can modify phenotypes of offspring generations playing thus a potentially important role in ecology and evolution of many plant species. These effects have been studied mostly across generations of sexually reproducing species. A substantial proportion of plant species are however reproducing asexually, for instance via clonal growth. TGE are thought to be enabled by heritable epigenetic modification of DNA, although unambiguous evidence is still scarce. On the clonal herb white clover (Trifolium repens), we tested the generality of clonal TGE across five genotypes and five parental environments including soil contamination and above-ground competition. Moreover, by genome wide-methylation variation analysis we explored the role of drought, one of the parental environments that triggered the strongest TGE. We tested the induction of epigenetic changes in offspring generations using several intensities and durations of drought stress. We found that TGE of different environments were highly genotype specific and all tested environments triggered TGE at least in some genotypes. In addition, parental drought stresses triggered epigenetic change in T. repens and most of the induced epigenetic change was maintained across several clonal offspring generations. We conclude that TGE are common and genotype specific in clonal plant T. repens and potentially under epigenetic control.

Tropical root and tuber crops breeding in the Pacific: a review of 35 years of efforts

The tropical root and tuber crops (aroids, cassava, sweet potato and yams) represent major contributions to food security in the Pacific Islands Countries (PICs) but are also cash crops for local and export markets. Conventional breeding is complex as the flowering ability of cultivars is erratic; they are predominantly allogamous, highly heterozygous with variable ploidy levels. Breeding is based on phenotypic recurrent selection with comprehensive characterisation and elimination of numerous hybrids evaluated across successive clonal generations for major agronomic traits. Over the last three decades, the PICs breeding programmes have been project-driven but have successfully delivered improved clones. The results obtained for Colocasia esculenta, Xanthosoma sagittifolium, Ipomoea batatas, Dioscorea spp., and Manihot esculenta, are reviewed. For each species, the technical bottlenecks and research priorities are identified and perspectives for further genetic improvement are discussed.

In vitro, induction of salt tolerant potato (solanum tuberosum l.) Plants with gamma irradiation and characterization of genetic variations through sds-page and issr-pcr analysis

Salt tolerant mutants of potato (Solanum tuberosum L. ‘Sponta’) were obtained via gamma irradiation. bud explants of two strains of potato (Sponta and lady rosetta were treated with various dosages of gamma irradiation, and the clonal generations were developed. Selection of salt-tolerant mutants was accomplished by in vitro selection media containing 30, 60, 90 and 120 mM NaCl. Molecular-level differences between the control and mutant plants were elucidated using ISSR technique, and the polymorphism rate according to the selected primers was calculated as 89.66%. Genetic distances between the controls and mutants were also calculated, and related dendrograms were produced. On average the mutants were genetically 27.5% different from the control plants. The greatest difference encountered between the control and mutants was 47%, which was detected in mutant plants produced by 20 or 30 Gy gamma irradiation and regenerated in selection medium containing 100 mM NaCl.

Dating the Cryptococcus gattii Dispersal to the North American Pacific Northwest.

The emergence of Cryptococcus gattii, previously regarded as a predominantly tropical pathogen, in the temperate climate of the North American Pacific Northwest (PNW) in 1999 prompted several questions. The most prevalent among these was the timing of the introduction of this pathogen to this novel environment. Here, we infer tip-dated timing estimates for the three clonal C.gattii populations observed in the PNW, VGIIa, VGIIb, and VGIIc, based on whole-genome sequencing of 134 C.gattii isolates and using Bayesian evolutionary analysis by sampling trees (BEAST). We estimated the nucleotide substitution rate for each lineage (1.59 × 10-8, 1.59 × 10-8, and 2.70 × 10-8, respectively) to be an order of magnitude higher than common neutral fungal mutation rates (2.0 × 10-9), indicating a microevolutionary rate (e.g., successive clonal generations in a laboratory) in comparison to a species' slower, macroevolutionary rate (e.g., when using fossil records). The clonal nature of the PNW C.gattii emergence over a narrow number of years would therefore possibly explain our higher mutation rates. Our results suggest that the mean time to most recent common ancestor for all three sublineages occurred within the last 60 to 100years. While the cause of C.gattii dispersal to the PNW is still unclear, our research estimates that the arrival is neither ancient nor very recent (i.e., <25 years ago), making a strong case for an anthropogenic introduction. IMPORTANCE The recent emergence of the pathogenic fungus Cryptococcus gattii in the Pacific Northwest (PNW) resulted in numerous investigations into the epidemiological and enzootic impacts, as well as multiple genomic explorations of the three primary molecular subtypes of the fungus that were discovered. These studies lead to the general conclusion that the subtypes identified likely emerged out of Brazil. Here, we conducted genomic dating analyses to determine the ages of the various lineages seen in the PNW and propose hypothetical causes for the dispersal events. Bayesian evolutionary analysis strongly suggests that these independent fungal populations in the PNW are all 60 to 100 years old, providing a timing that is subsequent to the opening of the Panama Canal, which allowed for more direct shipping between Brazil and the western North American coastline, a possible driving event for these fungal translocation events.

Increased pregnancy losses following serial somatic cell nuclear transfer in goats.

Serial cloning by somatic cell nuclear transfer (SCNT) is a critical tool for the expansion of precious transgenic lines or resetting the lifespan of primary transgenic cells for multiple genetic modifications. We successfully produced second-generation cloned goats using donor neonatal fibroblasts from first-generation clones. However, our attempts to produce any third-generation clones failed. SCNT efficiency decreased progressively with the clonal generations. The rate of pregnancy loss was significantly greater in recloning groups (P<0.05). While no pregnancy loss was observed during the first round of SCNT, 14 out of 21 pregnancies aborted in the second round of SCNT and all pregnancies aborted in the third round of SCNT. In this retrospective study, we also investigated the expression of 21 developmentally important genes in muscle tissue of cloned (G1) and recloned (G2) offspring. The expression of most of these genes in live clones was found to be largely comparable to naturally reproduced control goats, but fibroblast growth factor 10 (FGF10), methyl CpG binding protein 2 (MECP2) and growth factor receptor bound protein 10 (GRB10) were differentially expressed (P<0.05) in G2 goats compared with G1 and controls. To study the effects of serial cloning on DNA methylation, the methylation pattern of differentially methylated regions in imprinted genes H19 and insulin like growth factor 2 receptor (IGF2R) were also analysed. Aberrant H19 DNA methylation patterns were detected in G1 and G2 clones.

Evaluation and selection of true potato (Solanum tuberosum) seed families in North-Central plains of India

Segregating progenies from 19 crosses of potato (Solanum tuberosum L.) developed at the International Potato Center (CIP), Peru were tested during 2008-2013 for adaptation and yield attributes in the sub-tropical plains of India i.e. Modipuram, Uttar Pradesh. The progenitors of the crosses showed early foliage maturity, tolerance to biotic stresses and processing attributes. In initial clonal generations, selection was based on desirable tuber attributes and tuber yield components. Clones showing viral symptoms, producing long stolons, irregular tuber shape, russetting, cracking or deep eyes were eliminated. From the initial population of 17,300 true seeds, 13 advanced clones of fivecrosses were subjected to evaluation in replicated trials and nine better performing clones were selected. Critical evaluation of these nine clones resulted in the identification of five promising clones (305069.701, 305111.701, 305138.702, 397186.703 and 397186.704) with high tuber yield (43 to 50 t/ha) and desirable tuber attributes. These advanced clones hold promise of becoming new potato cultivars and may be used in breeding programmes aiming at high productivity in the region for improving the livelihood of farmers.