Theoretical and experimental assessment of genome-based prediction in landraces of allogamous crops.

Discovery and enrichment of favorable alleles in landraces are key to making them accessible for crop improvement. Here, we present two fundamentally different concepts for genome-based selection in landrace-derived maize populations, one based on doubled-haploid (DH) lines derived directly from individual landrace plants and the other based on crossing landrace plants to a capture line. For both types of populations, we show theoretically how allele frequencies of the ancestral landrace and the capture line translate into expectations for molecular and genetic variances. We show that the DH approach has clear advantages over gamete capture with generally higher prediction accuracies and no risk of masking valuable variation of the landrace. Prediction accuracies as high as 0.58 for dry matter yield in the DH population indicate high potential of genome-based selection. Based on a comparison among traits, we show that the genetic makeup of the capture line has great influence on the success of genome-based selection and that confounding effects between the alleles of the landrace and the capture line are best controlled for traits for which the capture line does not outperform the ancestral population per se or in testcrosses. Our results will guide the optimization of genome-enabled prebreeding schemes.

Novel resistance to the Bymovirus BaMMV established by targeted mutagenesis of the PDIL5-1 susceptibility gene in barley.

The Potyviridae are the largest family of plant-pathogenic viruses. Members of this family are the soil-borne bymoviruses barley yellow mosaic virus (BaYMV) and barley mild mosaic virus (BaMMV), which, upon infection of young winter barley seedlings in autumn, can cause yield losses as high as 50%. Resistance breeding plays a major role in coping with these pathogens. However, some viral strains have overcome the most widely used resistance. Thus, there is a need for novel sources of resistance. In ancient landraces and wild relatives of cultivated barley, alleles of the susceptibility factor PROTEIN DISULFIDE ISOMERASE LIKE 5-1 (PDIL5-1) were identified to confer resistance to all known strains of BaYMV and BaMMV. Although the gene is highly conserved throughout all eukaryotes, barley is thus far the only species for which PDIL5-1-based virus resistance has been reported. Whereas introgression by crossing to the European winter barley breeding pool is tedious, time-consuming and additionally associated with unwanted linkage drag, the present study exemplifies an approach to targeted mutagenesis of two barley cultivars employing CRISPR-associated endonuclease technology to induce site-directed mutations similar to those described for PDIL5-1 alleles that render certain landraces resistant. Homozygous primary mutants were produced in winter barley, and transgene-free homozygous M2 mutants were produced in spring barley. A variety of mutants carrying novel PDIL5-1 alleles were mechanically inoculated with BaMMV, by which all frameshift mutations and certain in-frame mutations were demonstrated to confer resistance to this virus. Under greenhouse conditions, virus-resistant mutants showed no adverse effects in terms of growth and yield.

Combining multiplex gene editing and doubled haploid technology in maize.

A major advantage of using CRISPR/Cas9 for gene editing is multiplexing, that is, the simultaneous targeting of many genes. However, primary transformants typically contain hetero-allelic mutations or are genetic mosaic, while genetically stable lines that are homozygous are desired for functional analysis. Currently, a dedicated and labor-intensive effort is required to obtain such higher-order mutants through several generations of genetic crosses and genotyping. We describe the design and validation of a rapid and efficient strategy to produce lines of genetically identical plants carrying various combinations of homozygous edits, suitable for replicated analysis of phenotypical differences. This approach was achieved by combining highly multiplex gene editing in Zea mays (maize) with in vivo haploid induction and efficient in vitro generation of doubled haploid plants using embryo rescue doubling. By combining three CRISPR/Cas9 constructs that target in total 36 genes potentially involved in leaf growth, we generated an array of homozygous lines with various combinations of edits within three generations. Several genotypes show a reproducible 10% increase in leaf size, including a septuple mutant combination. We anticipate that our strategy will facilitate the study of gene families via multiplex CRISPR mutagenesis and the identification of allele combinations to improve quantitative crop traits.

Rapid Generation of Barley Homozygous Transgenic Lines Based on Microspore Culture: HvPR1 Overexpression as an Example.

Obtaining homozygous lines from transgenic plants is an important step for phenotypic evaluations, but the selection of homozygous plants is time-consuming and laborious. The process would be significantly shortened if anther or microspore culture could be completed in one generation. In this study, we obtained 24 homozygous doubled haploid (DH) transgenic plants entirely by microspore culture from one T0 transgenic plant overexpressing the gene HvPR1 (pathogenesis-related-1). Nine of the doubled haploids grew to maturity and produced seeds. qRCR (quantitative real-time PCR) validation showed that the HvPR1 gene was expressed differentially even among different DH1 plants (T2) from the same DH0 line (T1). Phenotyping analysis suggested that the overexpression of HvPR1 inhibited nitrogen use efficiency (NUE) only under low nitrogen treatment. The established method of producing homozygous transgenic lines will enable the rapid evaluation of transgenic lines for gene function studies and trait evaluation. As an example, the HvPR1 overexpression of DH lines also could be used for further analysis of NUE-related research in barley.

Genome wide association mapping for heat tolerance in sub-tropical maize.

BACKGROUND: Heat tolerance is becoming increasingly important where maize is grown under spring season in India which coincide with grain filling stage of crop resulting in tassel blast, reduced pollen viability, pollination failure and barren ears that causes devastating yield losses. So, there is need to identify the genomic regions associated with heat tolerance component traits which could be further employed in maize breeding program. RESULTS: An association mapping panel, consisting of 662 doubled haploid (DH) lines, was evaluated for yield contributing traits under normal and natural heat stress conditions. Genome wide association studies (GWAS) carried out using 187,000 SNPs and 130 SNPs significantly associated for grain yield (GY), days to 50% anthesis (AD), days to 50% silking (SD), anthesis-silking interval (ASI), plant height (PH), ear height (EH) and ear position (EPO) were identified under normal conditions. A total of 46 SNPs strongly associated with GY, ASI, EH and EPO were detected under heat stress conditions. Fifteen of the SNPs was found to have common association with more than one trait such as two SNPs viz. S10_1,905,273 and S10_1,905,274 showed colocalization with GY, PH and EH whereas S10_7,132,845 SNP associated with GY, AD and SD under normal conditions. No such colocalization of SNP markers with multiple traits was observed under heat stress conditions. Haplotypes trend regression analysis revealed 122 and 85 haplotype blocks, out of which, 20 and 6 haplotype blocks were associated with more than one trait under normal and heat stress conditions, respectively. Based on SNP association and haplotype mapping, nine and seven candidate genes were identified respectively, which belongs to different gene models having different biological functions in stress biology. CONCLUSIONS: The present study identified significant SNPs and haplotype blocks associated with yield contributing traits that help in selection of donor lines with favorable alleles for multiple traits. These results provided insights of genetics of heat stress tolerance. The genomic regions detected in the present study need further validation before being applied in the breeding pipelines.

Changes in plastid biogenesis leading to the formation of albino regenerants in barley microspore culture.

BACKGROUND: Microspore embryogenesis is potentially the most effective method of obtaining doubled haploids (DH) which are utilized in breeding programs to accelerate production of new cultivars. However, the regeneration of albino plants significantly limits the exploitation of androgenesis for DH production in cereals. Despite many efforts, the precise mechanisms leading to development of albino regenerants have not yet been elucidated. The objective of this study was to reveal the genotype-dependent molecular differences in chloroplast differentiation that lead to the formation of green and albino regenerants in microspore culture of barley. RESULTS: We performed a detailed analysis of plastid differentiation at successive stages of androgenesis in two barley cultivars, 'Jersey' and 'Mercada' that differed in their ability to produce green regenerants. We demonstrated the lack of transition from the NEP-dependent to PEP-dependent transcription in plastids of cv. 'Mercada' that produced mostly albino regenerants in microspore culture. The failed NEP-to-PEP transition was associated with the lack of activity of Sig2 gene encoding a sigma factor necessary for transcription of plastid rRNA genes. A very low level of 16S and 23S rRNA transcripts and impaired plastid translation machinery resulted in the inhibition of photomorphogenesis in regenerating embryos and albino regenerants. Furthermore, the plastids present in differentiating 'Mercada' embryos contained a low number of plastome copies whose replication was not always completed. Contrary to 'Mercada', cv. 'Jersey' that produced 90% green regenerants, showed the high activity of PEP polymerase, the highly increased expression of Sig2, plastid rRNAs and tRNAGlu, which indicated the NEP inhibition. The increased expression of GLKs genes encoding transcription factors required for induction of photomorphogenesis was also observed in 'Jersey' regenerants. CONCLUSIONS: Proplastids present in microspore-derived embryos of albino-producing genotypes did not pass the early checkpoints of their development that are required for induction of further light-dependent differentiation of chloroplasts. The failed activation of plastid-encoded RNA polymerase during differentiation of embryos was associated with the genotype-dependent inability to regenerate green plants in barley microspore culture. The better understanding of molecular mechanisms underlying formation of albino regenerants may be helpful in overcoming the problem of albinism in cereal androgenesis.

Transcriptomic diversity in seedling roots of European flint maize in response to cold.

BACKGROUND: Low temperatures decrease the capacity for biomass production and lead to growth retardation up to irreversible cellular damage in modern maize cultivars. European flint landraces are an untapped genetic resource for genes and alleles conferring cold tolerance which they acquired during their adaptation to the agroecological conditions in Europe. RESULTS: Based on a phenotyping experiment of 276 doubled haploid lines derived from the European flint landrace "Petkuser Ferdinand Rot" diverging for cold tolerance, we selected 21 of these lines for an RNA-seq experiment. The different genotypes showed highly variable transcriptomic responses to cold. We identified 148, 3254 and 563 genes differentially expressed with respect to cold treatment, cold tolerance and growth rate at cold, respectively. Gene ontology (GO) term enrichment demonstrated that the detoxification of reactive oxygen species is associated with cold tolerance, whereas amino acids might play a crucial role as antioxidant precursors and signaling molecules. CONCLUSION: Doubled haploids representing a European maize flint landrace display genotype-specific transcriptome patterns associated with cold response, cold tolerance and seedling growth rate at cold. Identification of cold regulated genes in European flint germplasm, could be a starting point for introgressing such alleles in modern breeding material for maize improvement.

Production and verification of the first Atlantic salmon (Salmo salar L.) clonal lines.

BACKGROUND: In several fish species homozygous and heterozygous clonal lines have been produced using gynogenetic and androgenetic techniques. These lines are standardized and can be reproduced over generations. In rainbow trout such lines have existed for decades and has become important research tools in genome studies as well as in studies of commercially important traits. The Atlantic salmon is one of the best studied fish species globally, but all experiments are done on fish of wild or domesticated origin and access to standardized immortal fish lines would be of great benefit. Here, we describe the protocols developed to produce mitotic gynogenes, and from these the first clonal lines in Atlantic salmon. RESULTS: Atlantic salmon eggs fertilized with UV irradiated sperm combined with a pressure shock applied at 4700-4800 minC at 8 °C gave all homozygous (doubled haploid) gynogenetic progeny with high survival. From the six first maturing females, five all homozygous clonal lines were produced by meiotic gynogenesis and were verified as clonal and identical to their mother with microsatellite markers. CONCLUSIONS: We have now produced the first documented cloned Atlantic salmon lines. This work demonstrates the potential for production of further Atlantic salmon clonal lines, potentially with distinct characteristics. Such lines will provide an important resource for further elucidation of phenotypic and genetic traits in this globally important species.

Embryogenic competence of microspores is associated with their ability to form a callosic, osmoprotective subintinal layer.

Microspore embryogenesis is an experimental morphogenic pathway with important applications in basic research and applied plant breeding, but its genetic, cellular, and molecular bases are poorly understood. We applied a multidisciplinary approach using confocal and electron microscopy, detection of Ca2+, callose, and cellulose, treatments with caffeine, digitonin, and endosidin7, morphometry, qPCR, osmometry, and viability assays in order to study the dynamics of cell wall formation during embryogenesis induction in a high-response rapeseed (Brassica napus) line and two recalcitrant rapeseed and eggplant (Solanum melongena) lines. Formation of a callose-rich subintinal layer (SL) was common to microspore embryogenesis in the different genotypes. However, this process was directly related to embryogenic response, being greater in high-response genotypes. A link could be established between Ca2+ influx, abnormal callose/cellulose deposition, and the genotype-specific embryogenic competence. Callose deposition in inner walls and SLs are independent processes, regulated by different callose synthases. Viability and control of internal osmolality are also related to SL formation. In summary, we identified one of the causes of recalcitrance to embryogenesis induction: a reduced or absent protective SL. In responding genotypes, SLs are markers for changes in cell fate and serve as osmoprotective barriers to increase viability in imbalanced in vitro environments. Genotype-specific differences relate to different responses against abiotic (heat/osmotic) stresses.

Developmental competence of eggs produced by rainbow trout Doubled Haploids (DHs) and generation of the clonal lines.

Poor quality eggs produced by the fully homozygous doubled haploids (DHs) may impair generation of clonal lines in fish species. In the present research, gynogenetic development of rainbow trout (Oncorhynchus mykiss) was induced in eggs originated from the DH females. Eggs were activated with the UV-irradiated grayling (Thymallus thymallus) spermatozoa and subjected to the high hydrostatic pressure (HHP) shock to provide diploid clonal individuals. Only two of four DH females produced eggs that were successfully activated by the irradiated spermatozoa and subsequently developed into the gynogenetic embryos. Survival rates of rainbow trout from the clonal lines equalled 21.5% and 19.8% during embryogenesis and decreased after hatching to 18.6% and 14.9%, respectively. Some of the dead rainbow trout clones collected between hatching and swim-up stage were emaciated and exhibited spinal deformities including scoliosis. Provided results confirmed limited developmental competences of eggs produced by rainbow trout DH females. Clonal rainbow trout developing in such eggs exhibited reduced survival and increased frequency of the body abnormalities.

Enhancing genetic gain in the era of molecular breeding.

As one of the important concepts in conventional quantitative genetics and breeding, genetic gain can be defined as the amount of increase in performance that is achieved annually through artificial selection. To develop pro ducts that meet the increasing demand of mankind, especially for food and feed, in addition to various industrial uses, breeders are challenged to enhance the potential of genetic gain continuously, at ever higher rates, while they close the gaps that remain between the yield potential in breeders' demonstration trials and the actual yield in farmers' fields. Factors affecting genetic gain include genetic variation available in breeding materials, heritability for traits of interest, selection intensity, and the time required to complete a breeding cycle. Genetic gain can be improved through enhancing the potential and closing the gaps, which has been evolving and complemented with modern breeding techniques and platforms, mainly driven by molecular and genomic tools, combined with improved agronomic practice. Several key strategies are reviewed in this article. Favorable genetic variation can be unlocked and created through molecular and genomic approaches including mutation, gene mapping and discovery, and transgene and genome editing. Estimation of heritability can be improved by refining field experiments through well-controlled and precisely assayed environmental factors or envirotyping, particularly for understanding and controlling spatial heterogeneity at the field level. Selection intensity can be significantly heightened through improvements in the scale and precision of genotyping and phenotyping. The breeding cycle time can be shortened by accelerating breeding procedures through integrated breeding approaches such as marker-assisted selection and doubled haploid development. All the strategies can be integrated with other widely used conventional approaches in breeding programs to enhance genetic gain. More transdisciplinary approaches, team breeding, will be required to address the challenge of maintaining a plentiful and safe food supply for future generations. New opportunities for enhancing genetic gain, a high efficiency breeding pipeline, and broad-sense genetic gain are also discussed prospectively.

Graft-transmissible movement of inverted-repeat-induced siRNA signals into flowers.

In plants, small interfering RNAs (siRNA) and microRNAs move to distant tissues where they control numerous developmental and physiological processes such as morphogenesis and stress responses. Grafting techniques and transient expression systems have been employed to show that sequence-specific siRNAs with a size of 21-24 nucleotides traffic to distant organs. We used inverted-repeat constructs producing siRNA targeting the meiosis factor DISRUPTED MEIOTIC cDNA 1 (DMC1) and GFP to test whether silencing signals move into meiotically active tissues. In grafted Nicotiana tabacum, a transgenic DMC1 siRNA signal made in source tissues preferably entered the anthers formed in the first flowers. Here, the DMC1 siRNA interfered with meiotic progression and, consequently, the flowers were at least partially sterile. In agro-infiltrated N. benthamiana plants, a GFP siRNA signal produced in leaves was allocated and active in most flower tissues including anthers. In hypocotyl-grafted Arabidopsis thaliana plants, the DMC1 silencing signal consistently appeared in leaves, petioles, and stem, and only a small number of plants displayed DMC1 siRNA signals in flowers. In all three tested plant species the systemic silencing signal penetrated male sporogenic tissues suggesting that plants harbour an endogenous long-distance small RNA transport pathway facilitating siRNA signalling into meiotically active cells.

Metabolic changes during wheat microspore embryogenesis induction using the highly responsive cultivar Svilena.

Androgenetically-derived haploids can be obtained by inducing embryogenesis in microspores. Thus, full homozygosity is achieved in a single generation, oppositely to conventional plant breeding programs. Here, the metabolite profile of embryogenic microspores of Triticum aestivum was acquired and integrated with transcriptomic existing data from the same samples in an effort to identify the key metabolic processes occurring during the early stages of microspore embryogenesis. Primary metabolites and transcription profiles were identified at three time points: prior to and immediately following a low temperature pre-treatment given to uninuclear microspores, and after the first nuclear division. This is the first time an integrative -omics analysis is reported in microspore embryogenesis in T. aestivum. The key findings were that the energy produced during the pre-treatment was obtained from the tricarboxylic acid (TCA) cycle and from starch degradation, while starch storage resumed after the first nuclear division. Intermediates of the TCA cycle were highly demanded from a very active amino acid metabolism. The transcription profiles of genes encoding enzymes involved in amino acid synthesis differed from the metabolite profiles. The abundance of glutamine synthetase was correlated with that of glutamine. Cytosolic glutamine synthetase isoform 1 was found predominantly after the nuclear division. Overall, energy production was shown to represent a major component of the de-differentiation process induced by the pre-treatment, supporting a highly active amino acid metabolism.

The effect of phytosulfokine alpha on haploid embryogenesis and gene expression of Brassica napus microspore cultures.

Microspore embryogenesis (ME) is the most powerful tool for creating homozygous lines in plant breeding and molecular biology research. It is still based mainly on the reprogramming of microspores by temperature, osmotic and/or nutrient stress. New compounds are being sought that could increase the efficiency of microspore embryogenesis or even induce the formation of haploid embryos from recalcitrant genotypes. Among these, the mitogenic factor phytosulfokine alpha (PSK-α) is promising due to its broad spectrum of activity in vivo and in vitro. The aim of our study was to investigate the effect of PSK-α on haploid embryogenesis from microspores of oilseed rape (Brassica napus L., DH4079), one of the most important oil crops and a model plant for studying the molecular mechanisms controlling embryo formation. We tested different concentrations (0, 0.01, 0.1 and 1 µM) of the peptide and evaluated its effect on microspore viability and embryo regeneration after four weeks of culture. Our results showed a positive correlation between addition of PSK-α and cultured microspore viability and a positive effect also on the number of developed embryos. The analysis of transcriptomes across three time points (day 0, 2 and 4) with or without PSK-α supplementation (15 RNA libraries in total) unveiled differentially expressed genes pivotal in cell division, microspore embryogenesis, and subsequent regeneration. PCA grouped transcriptomes by RNA sampling time, with the first two principal components explaining 56.8% variability. On day 2 with PSK, 45 genes (15 up- and 30 down-regulated) were differentially expressed when PSK-α was added and their number increased to 304 by day 4 (30 up- and 274 down-regulated). PSK, PSKR, and PSI gene expression analysis revealed dynamic patterns, with PSK2 displaying the highest increase and overall expression during microspore culture at days 2 and 4. Despite some variations, only PSK1 showed significant differential expression upon PSK-α addition. Of 16 ME-related molecular markers, 3 and 15 exhibited significant differential expression in PSK-supplemented cultures at days 2 and 4, respectively. Embryo-specific markers predominantly expressed after 4 days of culture, with higher expression in medium without PSK, while on day 0, numerous sporophyte-specific markers were highly expressed.

Novel features of Brassica napus embryogenic microspores revealed by high pressure freezing and freeze substitution: evidence for massive autophagy and excretion-based cytoplasmic cleaning.

Induction of embryogenesis from isolated microspore cultures is a complex experimental system where microspores undergo dramatic changes in developmental fate. After ~40 years of application of electron microscopy to the study of the ultrastructural changes undergone by the induced microspore, there is still room for new discoveries. In this work, high pressure freezing and freeze substitution (HPF/FS), the best procedures known to date for ultrastructural preservation, were used to process Brassica napus microspore cultures covering all the stages of microspore embryogenesis. Analysis of these cultures by electron microscopy revealed massive processes of autophagy exclusively in embryogenic microspores, but not in other microspore-derived structures also present in cultures. However, a significant part of the autophagosomal cargo was not recycled. Instead, it was transported out of the cell, producing numerous deposits of extracytoplasmic fibrillar and membranous material. It was shown that commitment of microspores to embryogenesis is associated with both massive autophagy and excretion of the removed material. It is hypothesized that autophagy would be related to the need for a profound cytoplasmic cleaning, and excretion would be a mechanism to avoid excessive growth of the vacuolar system. Together, the results also demonstrate that the application of HPF/FS to the study of the androgenic switch is the best option currently available to identify the complex and dramatic ultrastructural changes undergone by the induced microspore. In addition, they provide significant insights to understand the cellular basis of induction of microspore embryogenesis, and open a new door for the investigation of this intriguing developmental pathway.

Haploid Induction in Indica Rice: Exploring New Opportunities.

Haploid plants are of significant interest to crop breeders due to their ability to expedite the development of inbred lines. Chromosome-doubling of haploids, produced by either in vitro or in vivo methods, results in fully homozygous doubled haploids. For nearly five decades, in vitro methods of anther and microspore culture have been attempted in many crops. In rice, in vitro methods are used with some success in japonica cultivars, although indica types have remained recalcitrant to a large extent. This review aims to explore the reasons for the lack of success of in vitro methods in indica rice and discuss new advancements in in vivo haploid induction protocols in other cereals and their relevance to rice. In particular, the current level of understanding of in vivo haploid inducer systems that utilize MTL and CENH3 mutants is analyzed in detail. One notable advantage of in vivo haploid induction systems is that they do not require tissue culture competence. This makes these methods more accessible and potentially transformative for research, offering a pragmatic approach to improving indica rice cultivars. By embracing these in vivo methods and harnessing the power of gene editing technologies like CRISPR/Cas9 systems, breeders can reshape their approach to indica rice improvement.

Evaluation of Resistance of Oilseed Rape Genotypes to Turnip Yellows Virus.

Turnip yellows virus (TuYV), is one of the most important pathogens of oilseed rape, which has caused enormous yield losses in all growing regions of the world in recent years. Therefore, there is a need for resistant varieties for sustainable crop protection. We have investigated the resistance of known varieties and newly developed advanced-breeding lines of oilseed rape to TuYV in greenhouse and field trials. We have analysed the TuYV titre of individual genotypes inoculated with the virus using viruliferous aphids Myzus persicae. The genotypes 'DK Temptation' and 'Rescator' had the lowest and highest virus titres, respectively, and were used as resistant and susceptible models for comparative analyses with other genotypes. In the greenhouse, the best results were obtained with the genotypes 'OP-8143 DH' (2.94 × 105 copies), OP-BN-72 (3.29 × 105 copies), 'Navajo' (3.58 × 105 copies) and 'SG-C 21215' (4.09 × 105 copies), which reached virus titres about 2 times higher than the minimum virus concentration measured in 'DK Temptation' (1.80 × 105 copies). In the field trials, the genotypes 'Navajo' (3.39 × 105 copies), 'OP-8148 DH' (4.44 × 105 copies), 'SG-C 21215' (6.80 × 105 copies) and OP-8480 (7.19 × 105 copies) had the lowest virus titres and reached about 3 times the virus titre of DK Temptation (2.54 × 105 copies). Both trials showed that at least two commercial varieties (e.g., DK Temptation, Navajo) and three advanced breeding lines (e.g., OP-8143 DH, OP-BN-72, SG-C 21215) had low titres of the virus after TuYV infection. This indicates a high level of resistance to TuYV in 'Navajo' or the newly developed breeding lines and the basis of resistance is probably different from R54 (as in 'DK Temptation'). Furthermore, the greenhouse trials together with RT -qPCR-based virus titre analysis could be a cost-effective and efficient method to assess the level of resistance of a given genotype to TuYV infection compared to the field trials. However, further research is needed to identify the underlying mechanisms causing this difference in susceptibility.

Study of wheat (Triticum aestivum L.) breeding material potential for in vitro androgenesis.

Doubled haploid technology is a valuable biotechnological approach in plant breeding that enables one to quickly create new varieties through the single-stage production of homozygous lines. The aim of this study was to assess the indicators of in vitro androgenesis in the anther culture of the initial breeding material of varieties and combinations of F1 and F2 and to identify promising accessions with good responsiveness. For that purpose, the plant material that proved promising for the breeding programs of Siberian Research Institute of Plant Production and Breeding (SibRIPP

Development of doubled haploid inducer lines facilitates selection of superior haploid inducers in maize.

Haploid inducers are key components of doubled haploid (DH) technology in maize. Robust agronomic performance and better haploid induction ability of inducers are persistently sought through genetic improvement. We herein developed C1-I inducers enabling large-scale in vivo haploid induction of inducers and discovered superior inducers from the DH progenies. The haploid induction rate (HIR) of C1-I inducers ranged between 5.8% and 12.0%. Overall, the success rate of DH production was 13% on average across the 23 different inducer crosses. The anthesis-silking interval and days to flowering of inducer F1s are significantly correlated with the success rate of DH production (r = -0.48 and 0.47, respectively). Transgressive segregants in DH inducers (DHIs) were found for the traits (days to flowering, HIR, plant height, and total primary branch length). Moreover, the best HIR in DHIs exceeded 23%. Parental genome contributions to DHI progenies ranged between 0.40 and 0.55, respectively, in 25 and 75 percentage quantiles, and the mean and median were 0.48. The allele frequency of the four traits from inducer parents to DHI progenies did not correspond with the phenotypic difference between superior and inferior individuals in the DH populations by genome-wide Fst analysis. This study demonstrated that the recombinant DHIs can be accessed on a large scale and used as materials to facilitate the genetic improvement of maternal haploid inducers by in vivo DH technology.

Effectiveness of R1-nj Anthocyanin Marker in the Identification of In Vivo Induced Maize Haploid Embryos.

Doubled haploid (DH) technology has become integral to maize breeding programs to expedite inbred line development and increase the efficiency of breeding operations. Unlike many other plant species that use in vitro methods, DH production in maize uses a relatively simple and efficient in vivo haploid induction method. However, it takes two complete crop cycles for DH line generation, one for haploid induction and the other one for chromosome doubling and seed production. Rescuing in vivo induced haploid embryos has the potential to reduce the time for DH line development and improve the efficiency of DH line production. However, the identification of a few haploid embryos (~10%) resulting from an induction cross from the rest of the diploid embryos is a challenge. In this study, we demonstrated that an anthocyanin marker, namely R1-nj, which is integrated into most haploid inducers, can aid in distinguishing haploid and diploid embryos. Further, we tested conditions that enhance R1-nj anthocyanin marker expression in embryos and found that light and sucrose enhance anthocyanin expression, while phosphorous deprivation in the media had no affect. Validating the use of the R1-nj marker for haploid and diploid embryo identification using a gold standard classification based on visual differences among haploids and diploids for characteristics such as seedling vigor, erectness of leaves, tassel fertility, etc., indicated that the R1-nj marker could lead to significantly high false positives, necessitating the use of additional markers for increased accuracy and reliability of haploid embryo identification.