Insertional Mutagenesis Using Tnt1 Retrotransposon in Potato

Insertional mutagenesis using transfer DNA or transposable elements, which is an important tool in functional genomics and is well established in several crops, has not been developed in potato (Solanum tuberosum). Here, we report the application of the tobacco (Nicotiana tabacum) Tnt1 retrotransposon as an insertional mutagen in potato. The Tnt1 retrotransposon was introduced into a highly homozygous and self-compatible clone, 523-3, of the diploid wild potato species Solanum chacoense. Transposition of the Tnt1 elements introduced into 523-3 can be efficiently induced by tissue culture. Tnt1 preferentially inserted into genic regions in the potato genome and the insertions were stable during sexual reproduction, making Tnt1 an ideal mutagen in potato. Several distinct phenotypes associated with plant stature and leaf morphology were discovered in mutation screening from a total of 38 families derived from Tnt1-containing lines. We demonstrate that the insertional mutagenesis system based on Tnt1 and the 523-3 clone can be expanded to the genome-wide level to potentially tag every gene in the potato genome.Insertional mutagenesis is one of the most important tools in plant functional genomics. Application of T-DNA, the transfer DNA of the Ti plasmid of Agrobacterium tumefaciens, was the first and the most successful methodology of genome-wide insertional mutagenesis in plants. Many T-DNA lines were developed in two of the most important model plant species, Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa; Krysan et al., 1999; Jeon et al., 2000; Alonso et al., 2003; Sallaud et al., 2004). These T-DNA stocks have served as the foundation for the identification and characterization of numerous genes in these two species. A major limitation of the T-DNA-based technique is the requirement for a highly efficient transformation system to generate a large number of transgenic lines. Unfortunately, in many plant species, A. tumefaciens-based transformation is either not yet developed or is not efficient enough to produce a sufficient number of T-DNA lines that would allow a genome-wide gene tagging.Several transposable elements (TEs) have been used for insertional mutagenesis in plants, including the Activator and Mutator transposons in maize (Zea mays; Walbot, 1992), the Tam3 transposon in Antirrhinum majus (Luo et al., 1991), the Tos17 retrotransposon in rice (Hirochika, 2010), and the LORE1 retrotransposon in Lotus japonicus (Fukai et al., 2012). Most remarkably, the Tnt1 retrotransposon, originally identified in tobacco (Nicotiana tabacum; Grandbastien et al., 1989), has been successfully used in insertional mutagenesis in several heterologous species, including Arabidopsis (Courtial et al., 2001), Medicago truncatula (d’Erfurth et al., 2003), lettuce (Lactuca sativa; Mazier et al., 2007), and soybean (Glycine max; Cui et al., 2013). Tnt1 was used to generate approximately 12,000 independent lines that represent over 300,000 insertions in M. truncatula (Tadege et al., 2008; Cheng et al., 2011). Many M. truncatula genes have been identified through forward and reverse genetics approaches using the Tnt1 retrotransposon insertion population (Pang et al., 2009; Zhao et al., 2010; Laurie et al., 2011; Tadege et al., 2011; Zhou et al., 2011; Cheng et al., 2012; Bourcy et al., 2013).Potato (Solanum tuberosum) is one of the most important food crops in the world. Cultivated potato is an autotetraploid (2n = 4x = 48) with a highly heterozygous genome. These characteristics make potato a poor model for both forward and reverse genetics research. Very few potato genes have been cloned using the traditional map-based cloning strategy. The lack of fertile homozygous clones makes insertional mutagenesis infeasible in potato. However, the recent sequencing of the potato genome (Xu et al., 2011) has significantly changed the status of potato genetics and genomics research. Highly efficient genotyping systems based on DNA microarrays or DNA sequencing will make genetic mapping and association mapping much more efficient in potato (Hamilton et al., 2011; Felcher et al., 2012). We expect an acceleration of forward genetics-based gene identification in potato. A genome-wide mutagenesis system is urgently needed for the potato research community. Here, we report the development of an insertional mutagenesis system in potato using the Tnt1 retrotransposon. This system is built on a highly homozygous and self-compatible clone (523-3) of the diploid potato species Solanum chacoense (2n = 2x = 24), one of the most widespread wild Solanum species (Miller and Spooner, 1996). S. chacoense is sexually compatible with diploid cultivated potato (Leue and Peloquin, 1980; Hermundstad and Peloquin, 1985). It has been an important germplasm source for the improvement of potato cultivars, especially those for use in the processing market (Love et al., 1998). We demonstrate that this system can be used to potentially tag every potato gene and serve as an important foundation for potato functional genomics research.     

Facilitation among plants can accelerate density‐dependent mortality and steepen self‐thinning lines in stressful environments

The speed and slope of plant self‐thinning are all affected by plant–plant interactions across environmental gradients. Possible mechanisms driving the self‐thinning dynamics include the relative strength of root versus shoot competition, and the interplay between competition and facilitation. Although these mechanisms often act in concert, their relative importance has not yet been fully explored. We used both a one‐layer and a two‐layer zone‐of‐influence (ZOI) model to examine how competition and facilitation drive self‐thinning across stress gradients. As a development of the traditional ZOI model, the two‐layer version explicitly models shoot and root growth and neighbor interactions, and thus the overall size‐symmetry of competition is regulated by the relative strength of root versus shoot competition. One‐layer model simulations revealed that increasingly asymmetric competition accelerated thinning, and steepened (slope ranged from about –1 to –4/3) and lowered self‐thinning lines. Stress slowed down density‐dependent mortality considerably when competition was not completely symmetric. Stress significantly decreased the self‐thinning intercept, while facilitation simply counteracted stress effects. Both stress and facilitation showed little effect on the slope. In the two‐layer model, both stress and facilitation affected mortality in the same way as in the one‐layer version when competition was not completely symmetric. Different from the one‐layer model, the two‐layer version showed that the effects of stress and facilitation on the self‐thinning slope were mediated by the asymmetry of competition. As stress increased, the overall asymmetry of competition shifted from asymmetric to symmetric due to increased relative strength of root competition. High stress thus dramatically flattened self‐thinning lines, whereas the inclusion of facilitation counteracted stress and led to steeper self‐thinning lines. Our two‐layer model is based on the current knowledge of plant–plant interactions, and better represents ecological realities. It can help elaborate experiments for testing the role of competition and facilitation in driving plant population dynamics.     

AFLP analysis to assess genomic stability in Solanum regenerants derived from wild and cultivated species

The cultivated potato as well as its tuber-bearing relatives are considered model plants for cell and tissue culture, and therefore for exploiting the genetic variation induced by in vitro culture. The association between molecular stability and tissue culture in different genetic backgrounds and ploidy levels has already been explored. However, it still remains to be ascertained whether somaclonal variation differs between callus-derived chromosome-doubled and undoubled regenerants. Our research aimed at investigating, through amplified fragment length polymorphism (AFLP) markers, the genetic changes in marker-banding patterns of diploid and tetraploid regenerants obtained from one clone each of Solanum bulbocastanum Dunal and S. cardiophyllum Lindl (both 2n = 2x = 24) and tetraploids from cultivated S. tuberosum L. (2n = 4x = 48). Pairwise comparisons between the banding patterns of regenerants and parents allowed detecting considerable changes associated to in vitro culture both at diploid and tetraploid level. The percentages of polymorphic bands between diploid and tetraploid regenerants were, respectively, 57 and 69% in S. bulbocastanum and 58 and 63% in S. cardiophyllum. On average, the frequencies of lost parental fragments in regenerants were significantly higher than novel bands both in S. bulbocastanum (48 vs. 22%) and S. tuberosum (36 vs. 18%) regenerants. By contrast, in S. cardiophyllum, a similar incidence of the two events was detected (32 vs. 29%). Our results revealed that structural changes after tissue culture process strongly affected the genome of the species studied, but diploid and tetraploids regenerated plants responded equally.     

Highly asymmetric intergeneric nuclear hybrids between Nicotiana and Petunia: evidence for recombinogenic and translocation events in somatic hybrid plants after “gamma”-fusion

Summary Extremely asymmetric nuclear hybrids have been obtained via protoplast fusion in an intergeneric combination. Irradiated (cobalt⁶⁰,100 krad) kanamycinresistant Petunia hybrida mesophyll protoplasts were chemically fused with wild-type mesophyll protoplasts of Nicotiana plumbaginifolia. Eighty-six hybrid colonies were selected on kanamycin-containing medium, and twenty-four of these could be induced to regenerate numerous shoots. Cytological analysis of the regenerants showed the presence of a few chromosome fragments in some lines, and even a metacentric chromosome in yet another line. Besides additional chromosome fragments some lines only possessed typical Nicotiana chromosomes, and this at the diploid (2n = 2X = 20) as well as the tetraploid (2n = 2X = 40) level. Biochemical analysis showed that all regenerants had neomycin phosphotransferase activity (NPTII), which suggests that intergenomic recombination and or translocation events took place at least in those lines where no additional chromosome fragments could be detected. The presence of the NPTII gene was shown by Southern hybridization. All regenerants tested were fertile, and the segregation ratios for the kanamycin gene (for self and backcross pollinations to the recipient partner) for some of the regenerants correspond with Mendelian rules for a monogenic dominant marker. Most of the regenerants showed abnormal segregation ratios; in this case, no correlation could be made between segregation ratio and chromosome composition.Our results demonstrate the existence of intergenomic recombination and translocations evens in nuclear somatic hybrid plants obtained via gamma-fusion.     

Anther culture as a breeding tool in rape

Summary Progeny analysis of androgenetic plants from inbred rape-seed (Brassica napus) shows that selective growth of microspores can occur in cultured anthers. The property of privileged growth in culture seems to be linked to such characters as flowering time and seed glucosinolate content which can be analyzed in regenerated plants. This type of selection and the fact that more variability is visible in regenerants from different microspores than in the progeny of the highly inbred anther donor line, demonstrates the higher degree of homozygosity in the doubled amphihaploids of B. napus. Furthermore, it is shown that haploid genomes of rape may be mutable. Thus it is possible to obtain several different homozygous lines from a single microspore. A system of haploid embryoids arising from single cells of the primary microspore regenerant has also been used to produce experimentally induced mutants. It is demonstrated that recessive mutations can be obtained in a homozygous state in doubled haploid regenerants from mutagenized haploid single cells.Part of the material analyzed in this work was produced under the guidance of Emrys Thomas when he was one of the project leaders of the Projektgruppen Haploide in der Pflanzenzüchtung at the Max-Planck-Institut für Pflanzengenetik in Ladenburg. His untimely death at the age of 36 (May 23, 1981), which overtook him at his new place of work at Harpenden, prevented him seeing the completion of this paper. It is dedicated to the memory of this distinguished and restless researcher and highly-valued colleague     

Effect of ambiol on stem growth in regenerants of source and transgenic potato plants

The effects of ambiol, a new growth regulator, on stem growth and morphological features of stem development have been compared in regenerants of potato (Solanum tuberosum L., var. Desire) plants transgenic for a defensin gene and in original potato plants. The original and transgenic plants exhibited differences in shoot development, which were observed both in control settings (no ambiol) and in the presence of various ambiol concentrations. In addition to normal plants of both forms, plant regenerants with morphological deviations were present in ambiol-treated groups. It is suggested that the abnormal shoot development observed in original and transgenic potato plants treated with ambiol is associated with (a) hormonal changes caused by expression of the defensin gene in the transgenic plants and (b) effects of ambiol on the hormonal balance of the plants.     

Somaclonal variation in wheat: genetic and cytogenetic characterisation of alcohol dehydrogenase 1 mutants

Summary The progeny of 551 regenerants of the hexaploid wheat cultivar Millewa were analysed for somaclonal mutants at the threeAdh-1 loci in hexaploid wheat. Seventeen regenerants gave rise to progeny having altered ADH1 zymograms. Progeny with altered zymograms in 13 of these regenerants were aneuploid. The remaining 4 regenerants gave rise to euploid progeny with altered ADH1 zymograms. The genetics of three of these somaclonal mutants is described in detail. These regenerants were interpreted to possess a 4A isochromosome, a 3BS/4A translocation and a 7BS/4A translocation, respectively.This research was partly supported by the Rockefeller Foundation and a Reserve Bank of Australia Rural Credit Development Fund research grant     

Effect of Ambiol on Stem Growth in Regenerants of Source and Transgenic Potato Plants

The effects of ambiol, a new growth regulator, on stem growth and morphological features of stem development have been compared in regenerants of potato (Solanum tuberosum L., var. Desire) plants transgenic for a defensin gene and in original potato plants. The original and transgenic plants exhibited differences in shoot development, which were observed both in control settings (no ambiol) and in the presence of various ambiol concentrations. In addition to normal plants of both forms, plant regenerants with morphological deviations were present in ambiol-treated groups. It is suggested that the abnormal shoot development observed in original and transgenic potato plants treated with ambiol is associated with (a) hormonal changes caused by expression of the defensin gene in the transgenic plants and (b) effects of ambiol on the hormonal balance of the plants.     

Haploid technology allows for the efficient and rapid generation of homozygous antibody-accumulating transgenic tobacco plants

The large-scale production of plant-derived recombinant proteins requires the breeding of lines homozygous for the transgene(s). These can be selected by progeny testing over multiple sexual generations, but a more efficient means is to fix homozygosity in a single generation using doubled haploid technology. In this study, transgenic tobacco plants, hemizygous for both of the independently inherited genes encoding the light and heavy chains of the anti-human immunodeficiency virus monoclonal antibody 2F5, were used to establish embryogenic pollen cultures. The improved protocol employed in this study guaranteed a very high regeneration efficiency, with more than 50% of the regenerants being spontaneously doubled haploids. Hence, there was no requirement to chemically induce chromosome doubling to recover sufficient entirely homozygous recombinants. As expected, approximately 25% of the regenerants were homozygous for both transgenes. Thus, the employment of haploid technology allowed for the efficient and rapid generation of true-breeding tobacco lines accumulating functional immunoglobulins.     

An Assay for Express Screening of Potato Transformants by GFP Fluorescence

An express assay for screening of potato transformants by their GFP fluorescence intensities is developed. In comparison to the widely used methods of transgenic plant screening by PCR, Real-Time RTPCR or Northern-blotting, the GFP fluorescence assay needs no expensive reagents and takes less time. This approach may also be used for nondestructive screening of the T0 transgenic regenerants which can be further grown and used. To prove this assay reliability, the expression of the hGFP gene in the leaves of transgenic potato (cv. Skoroplodny) plants, determined by its mRNA accumulation, was compared to GFP fluorescence intensity in the micro-samples of aseptic plant leaves. The strong correlation between the results of these two methods is the evidence of positive dependence of GFP fluorescence intensity on the target mRNA content.     

TEMPO AND MODE IN PLANT BREEDING SYSTEM EVOLUTION

Classic questions about trait evolution—including the directionality of character change and its interactions with lineage diversification—intersect in the study of plant breeding systems. Transitions from self‐incompatibility to self‐compatibility are frequent, and they may proceed within a species (“anagenetic” mode of breeding system change) or in conjunction with speciation events (“cladogenetic” mode of change). We apply a recently developed phylogenetic model to the nightshade family Solanaceae, quantifying the relative contributions of these two modes of evolution along with the tempo of breeding system change, speciation, and extinction. We find that self‐incompatibility, a genetic mechanism that prevents self‐fertilization, is lost largely by the cladogenetic mode. Self‐compatible species are thus more likely to arise from the isolation of a newly self‐compatible population than from species‐wide fixation of self‐compatible mutants. Shared polymorphism at the locus that governs self‐incompatibility shows it to be ancestral and not regained within this family. We demonstrate that failing to account for cladogenetic character change misleads phylogenetic tests of evolutionary irreversibility, both for breeding system in Solanaceae and on simulated trees.     

In vitro physical mutagenesis of giant reed (Arundo donax L.)

Giant reed (Arundo donax L.) is a C₃ perennial, warm‐season, rhizomatous grass of emerging interest for bioenergy and biomass derivatives production, and for phytoremediation. It only propagates vegetatively and very little genetic variation is found among ecotypes, basically precluding breeding efforts. With the objective to increase the genetic variation in this species, we developed and applied a mutagenesis protocol based on γ‐irradiation of in vitro cell cultures from which regenerants were obtained. Based on a radiosensitivity test, the irradiation dose reducing to 50% the number of regenerants per callus (RD₅₀) was estimated at 35 Gy. A large mutagenic experiment was carried out by irradiating a total of 3120 calli with approx. 1×, 1.5× and 2× RD₅₀. A total of 1004 regenerants from irradiated calli were hardened in pots and transplanted to the field. Initial phenotypic characterization of the collection showed correlated responses of biomass‐related quantitative traits to irradiation doses. Approx. 10% of field‐grown clones showed remarkable morphological aberrations including dwarfism, altered tillering, abnormal inflorescence, leaf variegation and others, which were tested for stability over generations. Clone lethality reached 0.4%. Our results show for the first time that physical mutagenesis can efficiently induce new genetic and phenotypic variation of agronomic and prospective industrial value in giant reed. The methodology and the plant materials described here may contribute to the domestication and the genetic improvement of this important biomass species.     

Alleles conferring improved fiber quality from EMS mutagenesis of elite cotton genotypes

Key message

Genetic improvements for many fiber traits are obtained by mutagenesis of elite cottons, mitigating genetic uniformity in this inbred polyploid by contributing novel alleles important to ongoing crop improvement.

Abstract

The elite gene pool of cotton (Gossypium spp.) has less diversity than those of most other major crops, making identification of novel alleles important to ongoing crop improvement. A total of 3,164 M₅ lines resulting from ethyl methanesulfonate (EMS) mutagenesis of two G. hirsutum breeding lines, TAM 94L-25 and Acala 1517-99, were characterized for basic components of fiber quality and selected yield components. Across all measured traits, the ranges of phenotypic values among the mutant lines were consistently larger than could be explained by chance (5.27–10.1 for TAM 94 L-25 and 5.29–7.94 standard deviations for Acala 1517-99-derived lines). Multi-year replicated studies confirmed a genetic basis for these differences, showing significant correlations between lines across years and environments. A subset of 157 lines selected for superior fiber qualities, including fiber elongation (22 lines), length (22), lint percent (17), fineness (23), Rd value (21), strength (19), uniformity (21) and multiple attributes in a selection index (26) were compared to 55 control lines in replicated trials in both Texas and Georgia. For all traits, mutant lines showing substantial and statistically significant improvements over control lines were found, in most cases from each of the two genetic backgrounds. This indicates that genetic improvements for a wide range of fiber traits may be obtained from mutagenesis of elite cottons. Indeed, lines selected for one fiber trait sometimes conferred additional attributes, suggesting pleiotropic effects of some mutations and offering multiple benefits for the incorporation of some alleles into mainstream breeding programs.     

Genetic regulation of self‐incompatibility

Self‐incompatibility is a cell‐cell recognition system in higher plants that is based on the ability of the pistil to discriminate “self‐pollen from “non‐self"‐pollen. In the simplest systems, this recognition response is controlled by a single locus — the S‐locus — with multiple alleles. Pollination of a pistil with pollen bearing an S‐allele recognition factor identical to that expressed in the host plant stigma or style results in rejection of the “self"‐pollen. Most of the studies on the molecular genetics of self‐incompatibility that are summarized in this review have had as their goal the identification and characterization of the gene product(s) associated with the self‐incompatibility response. These studies have provided a great deal of new and important information about self‐incompatibility — despite the fact that many critical questions remain unresolved. Taken together, the present evidence from these studies indicates that the self‐incompatibility response is likely to be far more complex than suggested by historical models.