Genome-Wide Identification and Analysis of the EIN3/EIL Transcription Factor Gene Family in Doubled Haploid (DH) Poplar.

Ethylene (ET) is an important phytohormone that regulates plant growth, development and stress responses. The ethylene-insensitive3/ethylene-insensitive3-like (EIN3/EIL) transcription factor family, as a key regulator of the ET signal transduction pathway, plays an important role in regulating the expression of ET-responsive genes. Although studies of EIN3/EIL family members have been completed in many species, their role in doubled haploid (DH) poplar derived from another culture of diploid Populus simonii × P. nigra (donor tree, DT) remains ambiguous. In this study, a total of seven EIN3/EIL gene family members in the DH poplar genome were identified. Basic physical and chemical property analyses of these genes were performed, and these proteins were predicted to be localized to the nucleus. According to the phylogenetic relationship, EIN3/EIL genes were divided into two groups, and the genes in the same group had a similar gene structure and conserved motifs. The expression patterns of EIN3/EIL genes in the apical buds of different DH poplar plants were analyzed based on transcriptome data. At the same time, the expression patterns of PsnEIL1, PsnEIN3, PsnEIL4 and PsnEIL5 genes in different tissues of different DH plants were detected via RT-qPCR, including the apical buds, young leaves, functional leaves, xylem, cambium and roots. The findings presented above indicate notable variations in the expression levels of PsnEIL genes across various tissues of distinct DH plants. Finally, the PsnEIL1 gene was overexpressed in DT, and the transgenic plants showed a dwarf phenotype, indicating that the PsnEIL1 gene was involved in regulating the growth and development of poplar. In this study, the EIN3/EIL gene family of DH poplar was analyzed and functionally characterized, which provides a theoretical basis for the future exploration of the EIN3/EIL gene function.

Co-expression of transcription factors ZmC1 and ZmR2 establishes an efficient and accurate haploid embryo identification system in maize.

Because of their high efficiency during chromosome doubling, immature haploid maize (Zea mays L.) embryos are useful for doubled haploid production. The R1-nj marker is commonly used in doubled haploid breeding and has improved the efficiency of haploid identification. However, its effectiveness is limited by genetic background and environmental factors. We addressed this technical challenge by developing an efficient and accurate haploid embryo identification marker through co-expression of two transcription factor genes (ZmC1 and ZmR2) driven by the embryo-aleurone-specific bidirectional promoter PZmBD1 ; these factors can activate anthocyanin biosynthesis in the embryo and aleurone layer during early seed development. We developed a new haploid inducer, Maize Anthocyanin Gene InduCer 1 (MAGIC1), by introducing the transgenes into the haploid inducer line CAU6. MAGIC1 could identify haploids at 12 days after pollination, which is nine days earlier than CAU6. Importantly, MAGIC1 increased haploid identification accuracy to 99.1%, compared with 88.3% for CAU6. In addition, MAGIC1 could effectively overcome the inhibition of anthocyanin synthesis in some germplasms. Furthermore, an upgraded anthocyanin marker was developed from ZmC1 and ZmR2 to generate MAGIC2, which could identify haploids from diploids due to differential anthocyanin accumulation in immature embryos, coleoptiles, sheaths, roots, leaves, and dry seeds. This haploid identification system is more efficient and accurate than the conventional R1-nj-based method, and it simplifies the haploid identification process. Therefore, this system provides technical support for large-scale doubled haploid line production.

Characterization of QTL and eQTL controlling early Fusarium graminearum infection and deoxynivalenol levels in a Wuhan 1 x Nyubai doubled haploid wheat population.

BACKGROUND: Fusarium head blight (FHB) is a major disease of cereal crops, caused by the fungal pathogen Fusarium graminearum and related species. Breeding wheat for FHB resistance contributes to increase yields and grain quality and to reduce the use of fungicides. The identification of genes and markers for FHB resistance in different wheat genotypes has nevertheless proven challenging. RESULTS: In this study, early infection by F. graminearum was analyzed in a doubled haploid population derived from the cross of the moderately resistant wheat genotypes Wuhan 1 and Nyubai. Three quantitative trait loci (QTL) were identified: 1AL was associated with lower deoxynivalenol content, and 4BS and 5A were associated with reduced F. graminearum infection at 2 days post inoculation. Early resistance alleles were inherited from Wuhan 1 for QTL 1AL and 4BS and inherited from Nyubai for the 5A QTL. Cis and trans expression QTL (eQTL) were identified using RNA-seq data from infected head samples. Hotspots for trans eQTL were identified in the vicinity of the 1AL and 4BS QTL peaks. Among differentially expressed genes with cis eQTL within the QTL support intervals, nine genes had higher expression associated with FHB early resistance, and four genes had higher expression associated with FHB early susceptibility. CONCLUSIONS: Our analysis of genotype and gene expression data of wheat infected by F. graminearum identified three QTL associated with FHB early resistance, and linked genes with eQTL and differential expression patterns to those QTL. These findings may have applications in breeding wheat for early resistance to FHB.

Heritability of meiotic restitution and fertility restoration in haploid triticale.

KEY MESSAGE: A single division meiosis mechanism of meiotic restitution is incompletely penetrant but significantly associated with restored fertility in triticale haploids (n = 21, genome formula ABR). Meiotic restitution, or failure of meiosis to produce gametes with a reduced chromosome number, can lead to the restoration of fertility in allohaploids. Meiotic restitution is of major interest for producing doubled haploids, as haploid plants undergoing meiotic restitution can often form seeds without the need to apply mitosis inhibitors to double chromosome number. We aimed to characterize meiotic restitution in a population of 183 haploids (n = 21, genome formula ABR) derived from an F1 wheat-rye hybrid where one parent was known to carry factors responsible for restoration of fertility in wide-cross haploids. Based on cytological analysis, approximately half of the plants analyzed were characterized by normal meiosis, while half showed at least some cytological evidence of meiotic restitution. However, this mechanism was incompletely penetrant in the population, with no individual plant showing 100% unreduced gamete formation: restitution occurred sectorially within each anther and was not observed in all the anthers of a given plant. Hence, the absence of meiotic restitution could not be confirmed conclusively for any individual plant, confounding this analysis. However, cytological observation of meiotic restitution was significantly associated with seed set, further confirming the role of meiotic restitution in fertility restoration. Our results provide insight into this mechanism of unreduced gamete formation, and provide a basis for future work identifying the genetic factors responsible for this trait.

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.

QTL Mapping of Adult Plant Resistance to Stripe Rust in a Doubled Haploid Wheat Population.

Stripe rust caused by Puccinia striiformis Westend. f. sp. tritici. is a major bread wheat disease worldwide with yield losses of up to 100% under severe disease pressure. The deployment of resistant cultivars with adult plant resistance to the disease provides a long-term solution to stripe rust of wheat. An advanced line from the International Winter Wheat Improvement Program (IWWIP) 130675 (Avd/Vee#1//1-27-6275/Cf 1770/3/MV171-C-17466) showed a high level of adult plant resistance to stripe rust in the field. To identify the adult plant resistance genes in this elite line, a mapping population of 190 doubled haploid (DH) lines was developed from a cross between line 130675 and the universal stripe rust-susceptible variety Avocet S. The DH population was evaluated at precision wheat stripe rust phenotyping platform, in Izmir during 2019, 2020, and 2021 cropping seasons under artificial inoculations. Composite interval mapping (CIM) identified two stable QTLs QYr.rcrrc-3B.1, and QYr.rcrrc-3B.2, which were detected in multiple years. In addition to these two QTLs, five more QTLs, QYr.rcrrc-1B, QYr.rcrrc-2A, QYr.rcrrc-3A, QYr.rcrrc-5A, and QYr.rcrrc-7D, were identified, which were specific to the cropping year (environment). All QTLs were derived from the resistant parent, except QYr.rcrrc-3A. The significant QTLs explained 3.4-20.6% of the phenotypic variance. SNP markers flanking the QTL regions can be amenable to marker-assisted selection. The best DH lines with high yield, end-use quality, and stripe rust resistance can be used for further selection for improved germplasm. SNP markers flanking the QTL regions can aid in identifying such lines.

Callus Induction from Diverse Explants and Genotypes Enables Robust Transformation of Perennial Ryegrass (Lolium perenne L.).

Genetic transformation of perennial ryegrass (Lolium perenne L.) is critical for fundamental and translational research in this important grass species. It often relies on Agrobacterium-mediated transformation of callus tissue. However, callus induction is restricted to a few genotypes that respond well to tissue culture. Here, we report callus induction from different perennial ryegrass genotypes and explants, such as shoot tips, seeds, and anthers, which were transformed with several plasmids for functional genomics. ß-glucuronidase (GUS) histochemical staining showed the LmdsRNAbp promoter sequence was active in stigmas, spikelets, anthers, and leaves. We also transformed calli with plasmids allowing gene silencing and gene knock-out using RNA interference and CRISPR/Cas9, respectively, for which genotypic and phenotypic investigations are ongoing. Using 19 different constructs, 262 transgenic events were regenerated. Moreover, the protocol regenerated a doubled haploid transgenic event from anther-derived calli. This work provides a proof-of-concept method for expanding the range of genotypes amenable to transformation, thus, serving research and breeding initiatives to improve this important grass crop for forage and recreation.

The effect of 2,4-dichlorophenoxyacetic acid on the production of oat (Avena sativa L.) doubled haploid lines through wide hybridization.

BACKGROUND: Development of new cultivars is one of the vital options for adapting agriculture to climate change, and the production of doubled haploid (DH) plants can make a significant contribution to accelerating the breeding process. Oat is one of the cereals with particular health benefits, but it unfortunately still remains recalcitrant to haploidization. Our previous studies have clearly demonstrated that post-pollination with hormone treatment is a key step in haploid production through wide hybridization and indicated it as the most effective method for this species. Therefore, we subsequently addressed the problem of the influence of 2,4-dichlorophenoxyacetic acid (2,4-D) concentration on consecutive stages of DH production. METHODS: Twenty-nine genotypes were tested, 9,465 florets were pollinated with maize pollen 2 days after emasculation and then treated with 2,4-D at 50 mg/L and 100 mg/L. RESULTS: The applied treatments did not reveal any differences in the number of obtained haploid embryos. However, almost twice as many haploid plants formed on MS medium after applying a higher auxin concentration and 20% more successfully acclimatized. Moreover, 100 mg/L 2,4-D treatment resulted in twice as many DH lines that produced almost three times more seeds compared to 50 mg/L treatment. Nevertheless, the results have confirmed the existence of strong genotypic variation, which may significantly limit the development of an effective and economically feasible method that could be incorporated into breeding programs.

Mapping of QTL and identification of candidate genes conferring spontaneous haploid genome doubling in maize (Zea mays L.).

In vivo doubled haploid (DH) technology is widely used in commercial maize (Zea mays L.) breeding. Haploid genome doubling is a critical step in DH breeding. In this study, inbred lines GF1 (0.65), GF3(0.29), and GF5 (0) with high, moderate, and poor spontaneous haploid genome doubling (SHGD), respectively, were selected to develop mapping populations for SHGD. Three QTL, qshgd1, qshgd2, and qshgd3, related to SHGD were identified by selective genotyping. With the exception of qshgd3, the source of haploid genome doubling alleles were derived from GF1. Furthermore, RNA-Seq was conducted to identify putative candidate genes between GF1 and GF5 within the qshgd1 region. A differentially expressed formin-like protein 5 transcript was identified within the qshgd1 region.

Trichostatin A Affects Developmental Reprogramming of Bread Wheat Microspores towards an Embryogenic Route.

Microspores can be developmentally reprogrammed by the application of different stress treatments to initiate an embryogenic pathway leading to the production of doubled haploid (DH) plants. Epigenetic modifications are involved in cell reprogramming and totipotency in response to stress. To increase microspore embryogenesis (ME) efficiency in bread wheat, the effect of the histone deacetylase inhibitor trichostatin A (TSA) has been examined in two cultivars of wheat with different microspore embryogenesis response. Diverse strategies were assayed using 0-0.4 µM TSA as a single induction treatment and after or simultaneously with cold or mannitol stresses. The highest efficiency was achieved when 0.4 µM TSA was applied to anthers for 5 days simultaneously with a 0.7 M mannitol treatment, producing a four times greater number of green DH plants than mannitol. Ultrastructural studies by transmission electron microscopy indicated that mannitol with TSA and mannitol treatments induced similar morphological changes in early stages of microspore reprogramming, although TSA increased the number of microspores with 'star-like' morphology and symmetric divisions. The effect of TSA on the transcript level of four ME marker genes indicated that the early signaling pathways in ME, involving the TaTDP1 and TAA1b genes, may be mediated by changes in acetylation patterns of histones and/or other proteins.

Genome-wide association study to identify genomic regions influencing spontaneous fertility in maize haploids.

Efficient production and use of doubled haploid lines can greatly accelerate genetic gains in maize breeding programs. One of the critical steps in standard doubled haploid line production is doubling the haploid genome using toxic and costly mitosis-inhibiting chemicals to achieve fertility in haploids. Alternatively, fertility may be spontaneously restored by natural chromosomal doubling, although generally at a rate too low for practical applications in most germplasm. This is the first large-scale genome-wise association study to analyze spontaneous chromosome doubling in haploids derived from tropical maize inbred lines. Induction crosses between tropicalized haploid inducers and 400 inbred lines were made, and the resulting haploid plants were assessed for haploid male fertility which refers to pollen production and haploid fertility which refers to seed production upon self-fertilization. A small number of genotypes were highly fertile and these fertility traits were highly heritable. Agronomic traits like plant height, ear height and tassel branch number were positively correlated with fertility traits. In contrast, haploid induction rate of the source germplasm and plant aspect were not correlated to fertility traits. Several genomic regions and candidate genes were identified that may control spontaneous fertility restoration. Overall, the study revealed the presence of large variation for both haploid male fertility and haploid fertility which can be potentially exploited for improving the efficiency of doubled haploid derivation in tropical maize germplasm.

Genetic Loci Governing Androgenic Capacity in Perennial Ryegrass (Lolium perenne L.).

Immature pollen can be induced to switch developmental pathways from gametogenesis to embryogenesis and subsequently regenerate into homozygous, diploid plants. Such androgenic production of doubled haploids is particularly useful for species where inbreeding is hampered by effective self-incompatibility systems. Therefore, increasing the generally low androgenic capacity of perennial ryegrass (Lolium perenne L.) germplasm would enable the efficient production of homozygous plant material, so that a more effective exploitation of heterosis through hybrid breeding schemes can be realized. Here, we present the results of a genome-wide association study in a heterozygous, multiparental population of perennial ryegrass (n = 391) segregating for androgenic capacity. Genotyping-by-sequencing was used to interrogate gene- dense genomic regions and revealed over 1,100 polymorphic sites. Between one and 10 quantitative trait loci (QTL) were identified for anther response, embryo and total plant production, green and albino plant production and regeneration. Most traits were under polygenic control, although a major QTL on linkage group 5 was associated with green plant regeneration. Distinct genetic factors seem to affect green and albino plant recovery. Two intriguing candidate genes, encoding chromatin binding domains of the developmental phase transition regulator, Polycomb Repressive Complex 2, were identified. Our results shed the first light on the molecular mechanisms behind perennial ryegrass microspore embryogenesis and enable marker-assisted introgression of androgenic capacity into recalcitrant germplasm of this forage crop of global significance.

Accelerated Generation of Selfed Pure Line Plants for Gene Identification and Crop Breeding.

Production of pure lines is an important step in biological studies and breeding of many crop plants. The major types of pure lines for biological studies and breeding include doubled haploid (DH) lines, recombinant inbred lines (RILs), and near isogenic lines (NILs). DH lines can be produced through microspore and megaspore culture followed by chromosome doubling while RILs and NILs can be produced through introgressions or repeated selfing of hybrids. DH approach was developed as a quicker method than conventional method to produce pure lines. However, its drawbacks of genotype-dependency and only a single chance of recombination limited its wider application. A recently developed fast generation cycling system (FGCS) achieved similar times to those of DH for the production of selfed pure lines but is more versatile as it is much less genotype-dependent than DH technology and does not restrict recombination to a single event. The advantages and disadvantages of the technologies and their produced pure line populations for different purposes of biological research and breeding are discussed. The development of a concept of complete in vitro meiosis and mitosis system is also proposed. This could integrate with the recently developed technologies of single cell genomic sequencing and genome wide selection, leading to a complete laboratory based pre-breeding scheme.

Effects of Heat Shock and 2,4-D Treatment on Morphological and Physiological Characteristics of Microspores and Microspore-Derived Doubled Haploid Plants in Brassica napus L.

BACKGROUND: Stresses such as heat shock, starvation, or osmotic is essential to lead isolated microspores towards embryogenesis. Despite the effectiveness of stresses in embryogenesis, they exert adverse effects on metabolism and growth of the regenerated plants. OBJECTIVES: The effects of heat shock and 2,4-D treatment on total protein content of treated microspores, morphological and physiological characteristics of the doubled haploid (DH) plants were assessed. MATERIALS AND METHODS: Buds containing mid- to late- uninucleate microspores were used for microspore culture. Microspores were isolated and cultured in NLN-13 medium and incubated at 30ºC for 14 days or treated with 2,4-D (35 mg.L-1) for 30 min to induce embryogenesis. Microspore-derived embryos were transferred onto B5 medium for plantlet regeneration. Ploidy level of the regenerated plantlets was determined using Partec flow cytometry. Spectrophotometric readings were carried out at 490, 663 and 645 nm to determine Chl a-b and carotenoids contents. TRIzol and cetyl-threeethyl-ammonium bromide (CTAB) were used for protein extraction from microspores and leaves. Length and width of stomata and pollen grains were also photographed using light microscope (Olympus). RESULTS: Applied stressors significantly reduced total protein content of treated microspores however, protein content and concentration of chlorophyll a and b of the DH plants were only increased by heat shock treatment when compared with the donor plant 'Hyola 420'. In contrast, carotenoids were not affected by applied stressors. Longer and wider stomata were observed by 2,4-D treatment but, the length of pollen grains was significantly decreased following heat shock and 2,4-D treatment. CONCLUSIONS: Total protein content of cultured microspores, concentration of chlorophyll a and b, length and width of stomata of microspore-derived doubled haploid plants were significantly affected by the type of inductive stresses. However, carotenoids were more stable and not affected by applied stressors.