A Comparison of Methods to Estimate Additive-by-Additive-by-Additive of QTL×QTL×QTL Interaction Effects by Monte Carlo Simulation Studies.

The goal of the breeding process is to obtain new genotypes with traits improved over the parental forms. Parameters related to the additive effect of genes as well as their interactions (such as epistasis of gene-by-gene interaction effect and additive-by-additive-by-additive of gene-by-gene-by-gene interaction effect) can influence decisions on the suitability of breeding material for this purpose. Understanding the genetic architecture of complex traits is a major challenge in the post-genomic era, especially for quantitative trait locus (QTL) effects, QTL-by-QTL interactions and QTL-by-QTL-by-QTL interactions. With regards to the comparing methods for estimating additive-by-additive-by-additive of QTL×QTL×QTL interaction effects by Monte Carlo simulation studies, there are no publications in the open literature. The parameter combinations assumed in the presented simulation studies represented 84 different experimental situations. The use of weighted regression may be the preferred method for estimating additive-by-additive-by-additive of QTL-QTL-QTL triples interaction effects, as it provides results closer to the true values of total additive-by-additive-by-additive interaction effects than using unweighted regression. This is also indicated by the obtained values of the determination coefficients of the proposed models.

Engineering tropane alkaloid production and glyphosate resistance by overexpressing AbCaM1 and G2-EPSPS in Atropa belladonna.

Atropa belladonna is an important industrial crop for producing anticholinergic tropane alkaloids (TAs). Using glyphosate as selection pressure, transgenic homozygous plants of A. belladonna are generated, in which a novel calmodulin gene (AbCaM1) and a reported EPSPS gene (G2-EPSPS) are co-overexpressed. AbCaM1 is highly expressed in secondary roots of A. belladonna and has calcium-binding activity. Three transgenic homozygous lines were generated and their glyphosate tolerance and TAs' production were evaluated in the field. Transgenic homozygous lines produced TAs at much higher levels than wild-type plants. In the leaves of T2GC02, T2GC05, and T2GC06, the hyoscyamine content was 8.95-, 10.61-, and 9.96 mg/g DW, the scopolamine content was 1.34-, 1.50- and 0.86 mg/g DW, respectively. Wild-type plants of A. belladonna produced hyoscyamine and scopolamine respectively at the levels of 2.45 mg/g DW and 0.30 mg/g DW in leaves. Gene expression analysis indicated that AbCaM1 significantly up-regulated seven key TA biosynthesis genes. Transgenic homozygous lines could tolerate a commercial recommended dose of glyphosate in the field. In summary, new varieties of A. belladonna not only produce pharmaceutical TAs at high levels but tolerate glyphosate, facilitating industrial production of TAs and weed management at a much lower cost.

Anther-derived microspore embryogenesis in pepper hybrids orobelle and Bomby.

BACKGROUND: Traditional breeding methods have long been employed worldwide for the evaluation and development of pepper cultivars. However, these methods necessitate multiple generations of screening, line development, evaluation, recognition, and crossing to obtain highly homozygous lines. In contrast, in vitro anther-derived microspore culture represents a rapid method to generate homozygous lines within a single generation. In the present study, we have optimized a protocol for microspore embryogenesis from anther cultures of pepper hybrids Orobelle and Bomby. RESULTS: We achieved early and successful embryo formation from both genotypes by subjecting the buds to a cold pretreatment at 4 °C for 4 days. Our optimized culture medium, comprised of MS medium supplemented with 4 mg/L NAA, 1 mg/L BAP, 0.25% activated charcoal, 2.6 g/L gelrite, 30 g/L sucrose, and 15 mg/L silver nitrate, exhibited the highest efficiency in embryo formation (1.85% and 1.46%) for Orobelle and Bomby, respectively. Furthermore, successful plant regeneration from the anther derived microspore embryos was accomplished using half-strength MS medium fortified with 2% sucrose and 0.1 mg/L 6-benzylaminopurine (BA), solidified with 2.6 g/L gelrite. The ploidy status of the microspore-derived plantlets was analyzed using flow cytometry technique. Notably, the haploid plants exhibited distinct characteristics such as reduced plant height, leaf length, leaf width, and shorter internode length when compared to their diploid counterparts derived from seeds. CONCLUSION: Our findings highlight the potential of anther culture and microspore embryogenesis as an advanced method for accelerating pepper breeding programs, enabling the rapid production of superior homozygous lines.

Molecular characterization of doubled haploid lines derived from different cycles of the Iowa Stiff Stalk Synthetic (BSSS) maize population.

Molecular characterization of a given set of maize germplasm could be useful for understanding the use of the assembled germplasm for further improvement in a breeding program, such as analyzing genetic diversity, selecting a parental line, assigning heterotic groups, creating a core set of germplasm and/or performing association analysis for traits of interest. In this study, we used single nucleotide polymorphism (SNP) markers to assess the genetic variability in a set of doubled haploid (DH) lines derived from the unselected Iowa Stiff Stalk Synthetic (BSSS) maize population, denoted as C0 (BSSS(R)C0), the seventeenth cycle of reciprocal recurrent selection in BSSS (BSSS(R)C17), denoted as C17 and the cross between BSSS(R)C0 and BSSS(R)C17 denoted as C0/C17. With the aim to explore if we have potentially lost diversity from C0 to C17 derived DH lines and observe whether useful genetic variation in C0 was left behind during the selection process since C0 could be a reservoir of genetic diversity that could be untapped using DH technology. Additionally, we quantify the contribution of the BSSS progenitors in each set of DH lines. The molecular characterization analysis confirmed the apparent separation and the loss of genetic variability from C0 to C17 through the recurrent selection process. Which was observed by the degree of differentiation between the C0_DHL versus C17_DHL groups by Wright's F-statistics (FST). Similarly for the population structure based on principal component analysis (PCA) revealed a clear separation among groups of DH lines. Some of the progenitors had a higher genetic contribution in C0 compared with C0/C17 and C17 derived DH lines. Although genetic drift can explain most of the genetic structure genome-wide, phenotypic data provide evidence that selection has altered favorable allele frequencies in the BSSS maize population through the reciprocal recurrent selection program.

The use of weighted multiple linear regression to estimate QTL-by-QTL epistatic effects.

Knowledge of the nature and magnitude of gene effects, as well as their contribution to the control of metric traits, is important in formulating efficient breeding programs for the improvement of plant genetics. Information concerning a genetic parameter such as the additive-by-additive epistatic effect can be useful in traditional breeding. This report describes the results obtained by applying weighted multiple linear regression to estimate the parameter connected with an additive-by-additive epistatic interaction. Three weight variants were used: (1) standard weights based on estimated variances, (2) different weights for minimal, maximal and other lines, and (3) different weights for extreme and other lines. The approach described here combines two methods of estimation, one based on phenotypic observations and the other using molecular marker data. The comparison was done using Monte Carlo simulations. The results show that the application of weighted regression to the marker data yielded estimates similar to those obtained by phenotypic methods.

Measuring the frequency and distribution of meiotic crossovers in homozygous barley inbred lines.

We report a novel approach for establishing the number and position of CO events in individual homozygous inbred plants by combining low level EMS mutagenesis, speed breeding, whole genome shotgun sequencing and sliding window analysis of the induced molecular variant data. We demonstrate the approach by exploring CO frequency and distribution in self-fertilised progeny of the inbred barley cultivar Bowman and compare these observations to similar data obtained from a Bowman nearly isogenic line (BW230 Hvmlh3) containing a mutation in the DNA mismatch repair gene HvMLH3. We have previously shown that Hvmlh3 decreases both plant fertility and recombination by ~50%. We compare our results to those from previously published traditional genetic analysis of F3 families derived from multiple F2 lines containing WT or mutant alleles of HvMLH3, revealing a high level of correspondence between analyses. We discuss possible applications of the approach in streamlining the assessment of recombination in plant meiosis research.

Production of Wheat Doubled Haploids Through Intergeneric Hybridization with Maize.

The intergeneric hybridization of wheat (Triticum aestivum L.) with maize (Zea mays L.) enables the production of doubled haploids (DHs) of wheat from all wheat hybrids with high efficiencies. Wheat and maize donor plants are raised in environmentally controlled greenhouses until crossing. Before anthesis, wheat spikes are emasculated and then pollinated with maize. Auxin is applied to each individual wheat floret 1 day after pollination. About 2 weeks after crossing, in vitro embryo culture is performed, enabling the regeneration of haploid wheat plantlets after maize chromosome elimination. Haploid plantlets are transferred to the greenhouse and after recovery, their genome is doubled with colchicine. Haploid plantlets can be sampled for DNA extractions and molecular analyses to aid the rapid discard of undesirable plantlets. Doubled haploid plants are raised in a greenhouse until maturity. Seeds of each fertile DH are harvested and often sown the same year. Several cycles of multiplication and evaluation in replicated plot trials and different geographical locations are then done to select the best candidate(s) for varietal registration.

Anther Culture in Coconut (Cocos nucifera L.).

Doubled haploids have a high impact on the improvement of heterozygous crops through hybridization. Anther culture is a doubled haploid technique for producing homozygous lines. In coconut, a tree species reported to be recalcitrant for tissue culture, a successful doubled haploid protocol was established through anther culture. All the factors affecting androgenesis induction have been optimized. In this chapter, a stepwise protocol, from doubled haploid induction including palm selection, anther isolation, pretreatment, and culture initiation, up to plant regeneration and thereafter acclimatization of the regenerated plants, is described. Furthermore, the protocol for testing the anther-derived plants for the ploidy level is also presented.

Androgenesis of Red Cabbage in Isolated Microspore Culture In Vitro.

Red cabbage belongs to the economically important group of vegetable crops of the Brassicaceae family. A unique feature of this vegetable crop that distinguishes it from other members of the family is its unique biochemical composition characterized by high anthocyanin content, which gives it antioxidant properties. The production mainly uses F1 hybrids, which require constant parental lines, requiring 6-7 generations of inbreeding. Culture of isolated microspores in vitro is currently one of the promising methods for the accelerated production of pure lines with 100% homozygosity. The aim of this study is to investigate the factors and select optimal parameters for successful induction of red cabbage embryogenesis in isolated microspore culture in vitro and subsequent regeneration of DH plants. As a result of research, for the first time, it was possible to carry out the full cycle of obtaining DH plants of red cabbage from the induction of embryogenesis to their inclusion in the breeding process. The size of buds containing predominantly microspores at the late vacuolated stage and pollen at the early bi-cellular stage has to be selected individually for each genotype, because the embryoid yield will be determined by the interaction of these two factors. In the six samples studied, the maximum embryoid yield was obtained from buds 4.1-4.4 mm and 4.5-5.0 mm long, depending on the genotype. Cultivation of microspores was carried out on liquid NLN culture medium with 13% sucrose. The maximum number of embryoids (173.5 ± 7.5 pcs./Petri dish) was obtained on culture medium with pH 5.8 and heat shock at 32 °C for 48 h. Successful embryoid development and plant regeneration by direct germination from shoot apical meristem were achieved on MS culture medium with 2% sucrose and 0.7% agar, supplemented with 6-benzylaminopurine at a concentration of 1 mg/L. Analysis of the obtained regenerated plants, which successfully passed the stage of adaptation to ex vitro conditions by flow cytometry, showed that most of them were doubled haploids (up to 90.9%). A low number of seeds produced by self-fertilization in DH plants was observed.

Production of Gynogenic Plants of Red Beet (Beta vulgaris L.) in Unpollinated Ovule Culture In Vitro.

The unique and balanced components of the biochemical composition, together with high antioxidant activity, make the red beet necessary a dietary vegetable crop, much contributing to healthy food ration. The application of the technology for producing gynogenic plants in vitro increases the genetic diversity and significantly reduces the period of time required to obtain the appropriate homozygous lines used to create the F1 hybrids that are demanded in the market. For induction of gynogenesis, we used IMB medium developed by us with the addition of 55 g/L sucrose, 3 g/L phytogel, 200 mg/L ampicillin, and 0.4 mg/L thidiazuron (TDZ) and cultured at 28 °C in the dark for 4-6 weeks. Shoot regeneration from embryoids and callus was performed on MS medium with 20 g/L sucrose, 3 g/L phytogel, 1 mg/L 6-benzylaminopurine (BAP), and 0.1 mg/L gibberellic acid (GA3). Immersion of the obtained microshoots with 5-7 well-developed leaves for 10-15 s into concentrated sterile indole-3-butyric acid (IBA) solution (50 mg/L) followed by their cultivation on solid medium ½ IMB with 2% sucrose and 3 g/L phytogel was the most efficient method for root formation. The addition of silver nitrate (22 mg/L) to the nutrient medium provoked an increase in the number of induced ovules up to nine per Petri dish (up to 25% of induced ovules). Gynogenic development was produced in six out of 11 genotypes studied, and the plants that were then acclimatized to ex vitro conditions were obtained in three genotypes (Nezhnost', Dobrynya, b/a 128). The evaluation of ploidy of gynogenic plants that was carried out by flow cytometry and direct counting of chromosomes stained with propion-lacmoide revealed that all obtained gynogenic plants were haploids (2n = x = 9).