Haploid biotechnology as a tool for creating a selection material for sugar beets

Since the discovery of the phenomenon of haploidy, biotechnology has become an integral part in the successful creation of new varieties and hybrids of various plant species. In particular, these technologies are actively used in agriculture, which is concerned with increasing the volume and improving the quality of products. The integration of haploid production techniques together with other available biotechnological tools such as marker selection (MAS), induced mutagenesis and genetic engineering technologies can significantly accelerate crop breeding. This article shows the main stages in the development of biotechnology since 1921. Now they are successfully used to create doubled haploids to accelerate the selection process of various plants and, in particular, sugar beet, which is the most important sugar crop in regions with a temperate climate. There are several methods for obtaining forms with a single set of chromosomes. For sugar beets, the use of gynogenesis turned out to be expedient, since in this case the other methods turned out to be ineffective in the mass production of haploids. The article considers the stages of obtaining the H and DH lines of Beta vulgaris L., as well as the main stages of biotechnological production of homozygous breeding material of this culture. These stages include selecting parental forms – donor explants, sterilizing buds and introducing non-pollinated ovules in vitro, obtaining haploids, doubling their chromosome set, creating doubled haploids, determining ploidy at different stages, relocating the obtained plants to greenhouses and growing stecklings. A number of advantages that the technology of creating doubled haploids in vitro has in comparison with traditional methods of selection are described. It has been shown that the use of these approaches is relevant when obtaining new highly productive hybrids and varieties of agricultural plants; however, the methods for the production of homozygous forms in sugar beet still require additional research aimed at increasing the efficiency and reproducibility of each stage of the process.

Analytical and numerical comparisons of two methods of estimation of additive × additive × additive interaction of QTL effects

This paper presents the analytical and numerical comparison of two methods of estimation of additive × additive × additive (aaa) interaction of QTL effects. The first method takes into account only the plant phenotype, while in the second we also included genotypic information from molecular marker observation. Analysis was made on 150 doubled haploid (DH) lines of barley derived from cross Steptoe × Morex and 145 DH lines from Harrington × TR306 cross. In total, 153 sets of observation was analyzed. In most cases, aaa interactions were found with an exert effect on QTL. Results also show that with molecular marker observations, obtained estimators had smaller absolute values than phenotypic estimators.

Genomic Regions Influencing Preharvest Sprouting Tolerance in Two Doubled-Haploid Wheat Populations (Triticum aestivum L.)

The current and projected climate change that is represented by increasing temperatures, humidity levels and irregular rainfall patterns, promotes the occurrence of preharvest sprouting (PHS) in wheat. PHS results in significant economic losses, globally, which necessitates the need for high-yielding cultivars with increased PHS tolerance, hence this study was conducted. The current study evaluated two doubled-haploid (DH) wheat populations of Tugela-Dn × Elands and Elands × Flamink across six environments in the Free State Province of South Africa to select genotypes with increased PHS tolerance and further map the underlying loci. Significant effects of DH lines (194) and environments (6) were observed for PHS tolerance. The results of this study validate previous findings that PHS is only expressed when environmental conditions are conducive. Quantitative trait loci (QTL) mapping using single nucleotide polymorphism (SNP) and silicoDArT markers revealed three additive QTL with major effects on chromosomes 5B and 7B, and these QTL were detected more than once, when conditions were favourable. These QTL explained a phenotypic variation (PVE) varying between 10.08% and 20.30% (LOD = 2.73 – 3.11). About 16.50% of DH lines performed to the level of Elands (the PHS-tolerant parent) and are recommended for further selection in a pre-breeding or breeding programme. The findings of the study are expected to facilitate the on-going breeding efforts for PHS tolerance in winter wheat.

A novel locus (Bnsdt2) in a TFL1 homologue sustaining determinate growth in Brassica napus

Background The determinate growth habits is beneficial for plant architecture modification and the development of crops cultivars suited to mechanized production systems. Which play an important role in the genetic improvement of crops. In Brassica napus, a determinate inflorescence strain (4769) has been discovered among doubled haploid (DH) lines obtained from a spring B. napus × winter B. napus cross, but there are few reports on it. We fine mapped a determinate inflorescence locus, and evaluated the effect of the determinate growth habit on agronomic traits. Results In this study, we assessed the effect of the determinate growth habit on agronomic traits. The results showed that determinacy is beneficial for reducing plant height and flowering time, advancing maturity, enhancing lodging resistance, increasing plant branches and maintaining productivity. Genetic analysis in the determinate (4769) and indeterminate (2982) genotypes revealed that two independently inherited recessive genes (Bnsdt1, Bnsdt2) are responsible for this determinate growth trait. Bnsdt2 was subsequently mapped in BC2 and BC3 populations derived from the combination 2982 × 4769. Bnsdt2 could be delimited to an approximately 122.9 kb region between 68,586.2 kb and 68,709.1 kb on C09. BLAST analysis of these candidate intervals showed that chrC09g006434 (BnaC09.TFL1) is homologous to TFL1 of A. thaliana. Sequence analysis of two alleles identified two non-synonymous SNPs (T136C, G141C) in the first exon of BnaC09.TFL1, resulting in two amino acid substitutions (Phe46Leu, Leu47Phe). Subsequently, qRT-PCR revealed that BnaC09.TFL1 expression in shoot apexes was significantly higher in NIL-4769 than in 4769, suggesting its essential role in sustaining the indeterminate growth habit. Conclusions In this study, the novel locus Bnsdt2, a recessive genes for determinate inflorescence in B. napus, was fine-mapped to a 68,586.2 kb - 68,709.1 kb interval on C09. The annotated genes chrC09g006434 (BnaC09.TFL1) that may be responsible for inflorescence traits were found.

Comparative proteomic analysis provides new insights into regulation of microspore embryogenesis induction in winter triticale (× Triticosecale Wittm.) after 5-azacytidine treatment

Effective microspore embryogenesis (ME) requires substantial modifications in gene expression pattern, followed by changes in the cell proteome and its metabolism. Recent studies have awakened also interest in the role of epigenetic factors in microspore de-differentiation and reprogramming. Therefore, demethylating agent (2.5–10 μM 5-azacytidine, AC) together with low temperature (3 weeks at 4 °C) were used as ME-inducing tiller treatment in two doubled haploid (DH) lines of triticale and its effect was analyzed in respect of anther protein profiles, expression of selected genes (TAPETUM DETERMINANT1 (TaTPD1-like), SOMATIC EMBRYOGENESIS RECEPTOR KINASE 2 (SERK2) and GLUTATHIONE S-TRANSFERASE (GSTF2)) and ME efficiency. Tiller treatment with 5.0 µM AC was the most effective in ME induction; it was associated with (1) suppression of intensive anabolic processes-mainly photosynthesis and light-dependent reactions, (2) transition to effective catabolism and mobilization of carbohydrate reserve to meet the high energy demand of cells during microspore reprograming and (3) effective defense against stress-inducing treatment, i.e. protection of proper folding during protein biosynthesis and effective degradation of dysfunctional or damaged proteins. Additionally, 5.0 µM AC enhanced the expression of all genes previously identified as being associated with embryogenic potential of microspores (TaTPD1-like, SERK and GSTF2).

Aromatic Rice with Multiple Disease Resistance Developed through Anther Culture

The experiments were conducted during kharif (June to September) 2018 and 2019 to screen 7 aromatic rice double haploid (DH) lines developed against Bacterial Leaf Bligh (BLB) and blast disease. Three genes Xa21, xa13, xa5 for BLB resistance and two genes Pi1, Pi2 for blast resistance was introgressed into a local aromatic rice variety Dubraj from Chhattisgarh State of India. Dubraj is extremely popular aromatic short medium grain variety in Chhattisgarh and Madhya Pradesh region and hence used in this study. RPBIO-226 and PR122 rice variety is used as donor for BLB and blast resistance respectively. 7 DH lines (L-1 to L-7) were developed from the crosses (Dubraj X RPBIO-226 X PR122) in the year 2017. Phenotypic evaluation for BLB diseases resistance has shown that L-1, L-6 and L-7 (score 1) has a similar resistance as the control check RPBIO-226(score-1) and 3 lines with moderate resistance (L-3 score 2) (L-4 score 3) (L-5 score 3). For blast disease, three lines (L-4, L-6 and L-7) were identified as resistance with the disease score of 1, whereas three were grouped under the section moderately resistant (L-1, L-3 and L- 5) with a disease score of 3. Sensory test (1.7% KOH) was conducted for the estimation of presence / absence of aroma. Moderately strong aroma was present in four lines (L-1, L-3, L-6 and L-7). On the basis of yield and presence of aroma in grain, L-3 has been selected with moderately resistant to BLB and blast for further advancement and subsequently evaluated under State Initial Evaluation Trial (SIET) during kharif 2020. Within 3 years of development of DH line, L-3 is under varietal developmental trail, which itself indicate the efficacy of anther culture in fixing homozygosity and speedy development of desired variety within short period of time.

Impact of Selected PSII Parameters on Barley DH Lines Biomass and Yield Elements

In our study, we focused on the link among various parameters of chlorophyll a fluorescence and yield elements in the barley doubled haploid (DH) lines. There were significant differences in all studied DH lines, both in yield components and parameters of chlorophyll a fluorescence. The most variable parameter was overall performance index of PSII (PI) while the least was the amount of energy trapped in PSII reaction centers (TRo/CS). Considering yield components, high variation was also observed in the subsequent order from highest to lowest variation: biomass, thousand-grain weight (TGW) and grain number per plant (GNP). Significant negative correlation was found among the following fluorescence parameters: PI and light energy absorption (ABS/CS), as well as between maximum photochemical efficiency (Fv/Fm) and TGW, and between biomass and electron acceptors pool size from PSII (Area). Conversely, significant positive correlation was found between: Area and PI, Area and energy used for electron transport (ETo/CS), Area and GNP, PI and ETo/CS, PI and GNP, ABS/CS and TRo/CS, as well as between ETo/CS and GNP. Yield components combined with fluorescence parameters of chlorophyll a expressed with canonical variate analysis did not clearly distinguish the barley DH lines into hulled and hull-less groups. The mean value for these groups significantly differs only for ETo/CS and TGW values. The other parameters are distributed almost uniformly in hulled and hull-less lines. However, certain hull-less DH lines possess higher yield parameters compared to parental forms, which suggests a possibility of occurrence of transgression effects. The results suggest the chance to find valuable hull-less forms that are desired by breeders and plant producers, since these forms possess favorable functional features.

SNP-based assessment of genetic purity and diversity in maize hybrid breeding

Assessment of genetic purity of parental inbred lines and their resultant F1 hybrids is an essential quality control check in maize hybrid breeding, variety release and seed production. In this study, genetic purity, parent-offspring relationship and diversity among the inbred lines were assessed using 92 single-nucleotide polymorphism (SNP) markers. A total of 188 maize genotypes, comprising of 26 inbred lines, four doubled haploid (DH) lines and 158 single-cross maize hybrids were investigated in this study using Kompetitive Allele Specific Polymerase Chain Reaction (KASP) genotyping assays. The bi-allelic data was analyzed for genetic purity and diversity parameters using GenAlex software. The SNP markers were highly polymorphic and 90% had polymorphic information content (PIC) values of > 0.3. Pairwise genetic distances among the lines ranged from 0.05 to 0.56, indicating a high level of dissimilarity among the inbred lines. A maximum genetic distance of (0.56) was observed between inbred lines CKDHL0089 and CML443 while the lowest (0.05) was between I-42 and I-40. The majority (67%) of the inbred lines studied were genetically pure with residual heterozygosity of <5%, while only 33% had heterozygosity levels of >5%. Inbred lines, which were not pure, require purification through further inbreeding. Cluster analysis partitioned the lines into three distinct genetic clusters with the potential to contribute new beneficial alleles to the maize breeding program. Out of the 68 hybrids (43%) that passed the parent-offspring test, seven hybrids namely; SCHP29, SCHP95, SCHP94, SCHP134, SCHP44, SCHP114 and SCHP126, were selected as potential candidates for further evaluation and release due to their outstanding yield performance.

Clubroot resistance derived from the European Brassica napus cv. ‘Tosca’ is not effective against virulent Plasmodiophora brassicae isolates from Alberta, Canada

In this study, clubroot resistance in the resynthesized European winter Brassica napus cv. ‘Tosca’ was introgressed into a Canadian spring canola line ‘11SR0099’, which was then crossed with the clubroot susceptible spring line ‘12DH0001’ to produce F1 seeds. The F1 plants were used to develop a doubled haploid (DH) mapping population. The parents and the DH lines were screened against ‘old’ pathotypes 2F, 3H, 5I, 6M and 8N of the clubroot pathogen, Plasmodiophora brassicae, as well as against the ‘new’ pathotypes 5X, 5L, 2B, 3A, 3D, 5G, 8E, 5C, 8J, 5K, 3O and 8P. Genotyping was conducted using a Brassica 15K SNP array. The clubroot screening showed that ‘Tosca, ‘11SR0099’ and the resistant DH lines were resistant to three (2F, 3H and 5I) of the five ‘old’ pathotypes and four (2B, 3O, 8E and 8P) of the 12 ‘new’ pathotypes, while being moderately resistant to the ‘old’ pathotype 8N and the ‘new’ pathotypes 3D and 5G. ‘Tosca’ was susceptible to isolates representing pathotype 3A (the most common among the ‘new’ pathotypes) as well as pathotypes 6M, 5X, 5L, 5K and 8J. Linkage analysis and QTL mapping identified a ca. 0.88–0.95 Mb genomic region on the A03 chromosome of ‘Tosca’ as conferring resistance to pathotypes 2F, 3H, 5I, 2B, 3D, 5G, 8E, 3O and 8P. The identified QTL genomic region housed the CRk, Crr3 and CRd gene(s). However, the susceptibility of ‘Tosca’ to most of the common virulent pathotypes makes it unattractive as a sole CR donor in the breeding of commercial canola varieties in western Canada.

Inheritance of seed quality and seed germination in two doubled haploid populations of oilseed rape segregating for acid detergent lignin (ADL) content

The high lignin content of the seed hull of oilseed rape leads to its black seed color compared to yellow seeded soybean. Reducing lignin and increasing oil and protein content is an important breeding aim in oilseed rape. The objective of the present study was to analyze the inheritance of two rapeseed genetic resources with reduced lignin content in the seeds and to elucidate the effects on seed germination and composition. These resources were crossed to black seeded Express 617 and F1 plants were used to develop two doubled haploid (DH) populations. The two DH populations were tested in field experiments in three to five environments. Significant genetic variation for lignin content and bimodal frequency distributions were found in both populations. SNP marker segregation in contrasting bulks revealed for both populations overlapping narrow genomic regions on chromosome C03, responsible for reduced lignin content. The low lignin bulks had a significantly higher percentage of seeds showing bacterial and fungal growth, but germination and vigor was not affected. Non-targeted metabolome analysis of immature green seeds showed exclusively a depletion of metabolites of the proanthocyanidin pathway in DH lines with a reduced lignin content. In these DH lines, a cinnamate-4-hydroxylase gene copy was identified as candidate gene in the overlapping genomic region of both populations. The newly identified QTL for reduced lignin content on chromosome C03 is valuable for studying additive and epistatic effects in combination with other low lignin genotypes.