Homozygous Lines Research Articles

Nitrogen deficiency tolerance conferred by introgression of a QTL derived from wild emmer into bread wheat

Key messageGenetic dissection of a QTL from wild emmer wheat, QGpc.huj.uh-5B.2, introgressed into bread wheat, identified candidate genes associated with tolerance to nitrogen deficiency, and potentially useful for improving nitrogen-use efficiency.Nitrogen (N) is an important macronutrient critical to wheat growth and development; its deficiency is one of the main factors causing reductions in grain yield and quality. N availability is significantly affected by drought or flooding, that are dependent on additional factors including soil type or duration and severity of stress. In a previous study, we identified a high grain protein content QTL (QGpc.huj.uh-5B.2) derived from the 5B chromosome of wild emmer wheat, that showed a higher proportion of explained variation under water-stress conditions. We hypothesized that this QTL is associated with tolerance to N deficiency as a possible mechanism underlying the higher effect under stress. To validate this hypothesis, we introgressed the QTL into the elite bread wheat var. Ruta, and showed that under N-deficient field conditions the introgression IL99 had a 33% increase in GPC (p < 0.05) compared to the recipient parent. Furthermore, evaluation of IL99 response to severe N deficiency (10% N) for 14 days, applied using a semi-hydroponic system under controlled conditions, confirmed its tolerance to N deficiency. Fine-mapping of the QTL resulted in 26 homozygous near-isogenic lines (BC4F5) segregating to N-deficiency tolerance. The QTL was delimited from − 28.28 to − 1.29 Mb and included 13 candidate genes, most associated with N-stress response, N transport, and abiotic stress responses. These genes may improve N-use efficiency under severely N-deficient environments. Our study demonstrates the importance of WEW as a source of novel candidate genes for sustainable improvement in tolerance to N deficiency in wheat.

Prospects for combining the methods of haploid biotechnology and genome editing to improve spiked grains of the &lt;i&gt;Triticeae&lt;/i&gt; family (review)

Over the past few decades, haploid biotechnologies have become an integral part of breeding programs for many crops. Using the strategy of doubling haploids induced in the culture of gametic cells and tissues in vitro, through androgenesis, gynogenesis and distant hybridization, it became possible to significantly reduce the time for developing new varieties. Using the technology of doubled haploids, within one or two generations, it is possible to obtain aligned homozygous lines, which can both help speed up the breeding process and study several scientific and practical issues. Another promising tool for developing lines and samples with specified traits within several generations is genome editing (engineering) using various nuclease-based engineering complexes. The CRISPR/Cas9 genome editing technology, which came into use ten years ago, allows solving a wide variety of problems in plant functional genomics, including engineering resistance to biotic and abiotic stresses, improving productivity and product quality. The technology is better than the most known methods for improving varieties for the traits which have mono- or polygenic control, since it allows changing several genes simultaneously, which is important for polyploid species. An integral part of plant genome editing, as well as haploidogenesis technologies, is cell and tissue culture in vitro, which gives possibility for their combination. The combination of technologies allows producing homozygous plants with new gene-specific mutations, which improves genetic diversity and accelerates the selection of linear material with new economically valuable traits. The current review has summarized the experience of combining haploidy and genome editing methods in spiked grains of the Triticeae family. In addition to analyzing the current state, there have been considered the prospects for further development of technologies for obtaining haploids of wheat, barley, triticale, and rye with an edited genome.

Nanobody Mediated Atrazine Resistance in Plants.

In planta expression of recombinant antibodies has been proposed as a strategy for herbicide resistance but is not well advanced yet. Here, an atrazine nanobody gene fused with a green fluorescent protein tag was transformed to Arabidopsis thaliana, which was confirmed with PCR, ELISA, and immunoblotting. High levels of nanobody accumulation were observed in the nucleus, cytoderm, and cytosol. The nanobody expressed in the plant had similar affinity, sensitivity, and selectivity as that expressed in Escherichia coli. The T3 homozygous line showed resistance in a dose-dependent manner up to 380 g ai/ha of atrazine, which is approximately one-third of the recommended field application rate. This is the first report of utilizing a nanobody in plants against herbicides. The results suggest that utilizing a high-affinity herbicide nanobody gene rather than increasing the expression of nanobodies in plants may be a technically viable approach to acquire commercial herbicide-resistant crops and could be a useful tool to study plant physiology.

Over-expression of a plant-type phosphoenolpyruvate carboxylase derails Arabidopsis stamen formation

We examined whether plant-type phosphoenolpyruvate carboxylase (PEPC) is involved in flower organ formation or not by over-expression in Arabidopsis. A wheat PEPC isogene Tappc3A, belonging to the ppc3 group, was targeted due to its preferential expression pattern in pistils and stamens. Transgenic Arabidopsis over-expressing Tappc3A exhibited irregular stamen formation, i.e., a lesser number of stamens per flower and shorter filaments in T2 and T3 generations. Irregular stamens were frequently observed in homozygous T4 lines, but no morphological change was observed in other floral organs. High-degree gene co-expression of Tappc3 isogenes with wheat SEEDSTICKs but not with other homeotic transcription factor genes for flower formation implicates that Tappc3 is under control by the class D genes of the ABCDE model to flower development. In addition, the conservation of CArG box sequences on the Tappc3 promoters supported the developmentally programmed gene expression of ppc3 in wheat flowering organs. Thus, this study provides the first experimental evidence for the critical regulation of plant-type PEPC for flower formation.

DNA structural features and variability of complete MHC locus sequences.

The major histocompatibility (MHC) locus, also known as the Human Leukocyte Antigen (HLA) genes, is located on the short arm of chromosome 6, and contains three regions (Class I, Class II and Class III). This 5Mbp locus is one of the most variable regions of the human genome, yet it also encodes a set of highly conserved and important proteins related to immunological response. Genetic variations in this region are responsible for more diseases than in the entire rest of the human genome. However, information on local structural features of the DNA is largely ignored. With recent advances in long-read sequencing technology, it is now becoming possible to sequence the entire 5Mbp MHC locus, producing complete diploid haplotypes of the whole region. Here, we describe structural maps based on the complete sequences from six different homozygous HLA cell lines. We find long-range structural variability in the different sequences for DNA stacking energy, position preference and curvature, variation in repeats, as well as more local changes in regions forming open chromatin structures, likely to influence gene expression levels. These structural maps can be useful in visualizing large scale structural variation across HLA types, in particular when this can be complemented with epigenetic signals.

Mutation of mpv17 results in loss of iridophores due to mitochondrial dysfunction in tilapia

Abstract Mpv17 (mitochondrial inner membrane protein MPV17) deficiency causes severe mitochondrial DNA depletion syndrome in mammals and loss of pigmentation of iridophores and a significant decrease of melanophores in zebrafish. The reasons for this are still unclear. In this study, we established an mpv17 homozygous mutant line in Nile tilapia. The developing mutants are transparent due to the loss of iridophores and aggregation of pigment granules in the melanophores and disappearance of the vertical pigment bars on the side of the fish. Transcriptome analysis using the skin of fish at 30 dpf (days post fertilization) revealed that the genes related to purine (especially pnp4a) and melanin synthesis were significantly downregulated. However, administration of guanine diets failed to rescue the phenotype of the mutants. In addition, no obvious apoptosis signals were observed in the iris of the mutants by TUNEL staining. Significant downregulation of genes related to iridophore differentiation was detected by qPCR. Insufficient ATP, as revealed by ATP assay, α-MSH treatment, and adcy5 mutational analysis, might account for the defects of melanophores in mpv17 mutants. Several tissues displayed less mtDNA and decreased ATP levels. Taken together, these results indicated that mutation of mpv17 led to mitochondrial dTMP deficiency, followed by impaired mtDNA content and mitochondrial function, which in turn, led to loss of iridophores and a transparent body color in tilapia.

Using molecular markers to assist in the selection of the rice blast resistance genes Pi1, Pi2, and Pi-ta in rice wide compatibility restorer lines

Hybridization was performed between the rice materials carrying the rice blast resistance genes Pi1, Pi2, and Pi-ta (R283) as the maternal parent and the broad compatibility restorer line R110 as the paternal parent. Molecular marker-assisted breeding techniques were used to detect the target genes in segregating generations. Through field selection and resistance identification, three stable lines with multiple resistance, strong restorer ability, and excellent agronomic traits were bred. Their agronomic and economic traits were analyzed along with their resistance to different physiological races of rice blast. These three stable lines were then crossed with seven different sterile lines (Chunjiang 23A, FA1, FA3, FA4, 81, 57, and 212A) to analyze the agronomic traits and resistance to rice blast pathogens in the hybrid combinations. JR11, JR12, and JR13 were stable, homozygous polymerized lines carrying Pi1, Pi2, and Pi-ta resistance genes and exhibited resistance frequencies ranging from 90.19 to 96.08% against different physiological races of rice blast disease. This indicates the excellent resistance of these three stable lines to rice blast disease. The seed-set frequencies in the hybrid combinations of the seven sterile lines with the three stable lines ranged from 66.4 to 84.1%, showing strong recovery ability. The yield of the selected combination of 23 A/JR11, 81 A/JR11, and 212 A/JR13 was 2.2–2.3% higher than that of the control Yongyou 1540. The research results showed that the three stable aggregated rice blast resistance genes Pi1, Pi2, and Pi-ta rice restorer lines created have good practical value in production.

Androgenesis in Hot Pepper by Chilli Anther Culture

In vitro androgenesis techniques play a key role in the quick generation of fully homozygous lines. This work's goal was to put the procedure for producing androgenic embryos in hot pepper populations into practice. A number of variables can affect androgenetic success, including the types and quantities of carbohydrate sources and the nature of the nutritional medium. Different sugar types were tested at different doses in anther culture of pepper to elucidate their influence on the yield of androgenesis. In this study, sucrose was used as a source of sugar, Murashige &amp; Skoog (1962-MS) medium, silver nitrate (AgNO3), naphthaleneacetic acid (NAA), BAP and one breeding lines of pepper were used to know the genotypic effects on haploid embryo formation. It was found that one of the key elements influencing pepper anther and microspore culture success is genotype. Also, microspore stage and the cold treatment of flower bud responsible for the success of embryo formation. This embryo formation frequency has been calculated according to the results that were generated in one month and the frequency of embryo formation is 0.7%.

RNAi-mediated downregulation of AcCENH3 can induce in vivo haploids in onion (Allium cepa L.)

Haploid induction (HI) holds great promise in expediting the breeding process in onion, a biennial cross-pollinated crop. We used the CENH3-based genome elimination technique in producing a HI line in onion. Here, we downregulated AcCENH3 using the RNAi approach without complementation in five independent lines. Out of five events, only three could produce seeds upon selfing. The progenies showed poor seed set and segregation distortion, and we were unable to recover homozygous knockdown lines. The knockdown lines showed a decrease in accumulation of AcCENH3 transcript and protein in leaf tissue. The decrease in protein content in transgenic plants was correlated with poor seed set. When the heterozygous knockdown lines were crossed with wild-type plants, progenies showed HI by genome elimination of the parental chromosomes from AcCENH3 knockdown lines. The HI efficiency observed was between 0 and 4.63% in the three events, and it was the highest (4.63%) when E1 line was crossed with wildtype. Given the importance of doubled haploids in breeding programmes, the findings from our study are poised to significantly impact onion breeding.

Unraveling the complexities of drought stress in cotton: a multifaceted analysis of selection criteria and breeding approaches

Abiotic stress tolerance breeding programs present a spectrum of perspectives, yet definitive solutions remain elusive, with each approach carrying its own set of advantages and disadvantages. This study systematically evaluates extant methodologies, comparing plant performance across varied genotypes and selection traits under optimal and stress conditions. The objective is to elucidate prevailing ambiguities. Ten homozygous lines (F8 generation) were assessed using a randomized block design alongside five control varieties, with four replicates cultivated under well-watered and deficit water conditions. It is noteworthy that six of the ten homozygous lines were cultivated exclusively under well-watered conditions (F3 to F7), while four lines experienced deficit water conditions (F3 to F7). All five control varieties underwent cultivation under both conditions. These findings underscore the necessity for tailored breeding programs attuned to specific environmental exigencies, recognizing that individual traits manifest divergent responses to varying conditions. It is evident that certain traits exhibit marked disparities under well-watered conditions, while others evince heightened differentiation under water deficit conditions. Significantly, our analysis reveals a pronounced interaction between irrigation regimes and selection traits, which serves to underscore the nuanced interplay between genotype and environmental stress.

Generation of New β-Conglycinin-Deficient Soybean Lines by Editing the lincRNA lincCG1 Using the CRISPR/Cas9 System.

Soybean β-conglycinin is a major allergen that adversely affects the nutritional properties of soybean. Soybean deficient in β-conglycinin is associated with low allergenicity and high nutritional value. Long intergenic noncoding RNAs (lincRNAs) regulate gene expression and are considered important regulators of essential biological processes. Despite increasing knowledge of the functions of lincRNAs, relatively little is known about the effects of lincRNAs on the accumulation of soybean β-conglycinin. The current study presents the identification of a lincRNA lincCG1 that was mapped to the intergenic noncoding region of the β-conglycinin α-subunit locus. The full-length lincCG1 sequence was cloned and found to regulate the expression of soybean seed storage protein (SSP) genes via both cis- and trans-acting regulatory mechanisms. Loss-of-function lincCG1 mutations generated using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system led to the deficiency of the allergenic α'-, α-, and β-subunits of soybean β-conglycinin as well as higher content of proteins, sulfur-containing amino acids, and free arginine. The dominant null allele LincCG1, and consequently, the β-conglycinin-deficient phenotype associated with the lincCG1-gene-edited line was stably inherited by the progenies in a Mendelian fashion. The dominant null allele LincCG1 may therefore be exploited for engineering/developing novel hypoallergenic soybean varieties. Furthermore, Cas9-free and β-conglycinin-deficient homozygous mutant lines were obtained in the T1 generation. This study is the first to employ the CRISPR/Cas9 technology for editing a lincRNA gene associated with the soybean allergenic protein β-conglycinin. Moreover, this study reveals that lincCG1 plays a crucial role in regulating the expression of the β-conglycinin subunit gene cluster, besides highlighting the efficiency of employing the CRISPR/Cas9 system for modulating lincRNAs, and thereby regulating soybean seed components.

CRISPR/Cas9-induced knockout of an amino acid permease gene (AAP6) reduced Arabidopsis thaliana susceptibility to Meloidogyne incognita

BackgroundPlant-parasitic root-knot nematode (Meloidogyne incognita) causes global yield loss in agri- and horticultural crops. Nematode management options rely on chemical method. However, only a handful of nematicides are commercially available. Resistance breeding efforts are not sustainable because R gene sources are limited and nematodes have developed resistance-breaking populations against the commercially available Mi-1.2 gene-expressing tomatoes. RNAi crops that manage nematode infection are yet to be commercialized because of the regulatory hurdles associated with transgenic crops. The deployment of the CRISPR/Cas9 system to improve nematode tolerance (by knocking out the susceptibility factors) in plants has emerged as a feasible alternative lately.ResultsIn the present study, a M. incognita-responsive susceptibility (S) gene, amino acid permease (AAP6), was characterized from the model plant Arabidodpsis thaliana by generating the AtAAP6 overexpression line, followed by performing the GUS reporter assay by fusing the promoter of AtAAP6 with the β-glucuronidase (GUS) gene. Upon challenge inoculation with M. incognita, overexpression lines supported greater nematode multiplication, and AtAAP6 expression was inducible to the early stage of nematode infection. Next, using CRISPR/Cas9, AtAAP6 was selectively knocked out without incurring any growth penalty in the host plant. The ‘Cas9-free’ homozygous T3 line was challenge inoculated with M. incognita, and CRISPR-edited A. thaliana plants exhibited considerably reduced susceptibility to nematode infection compared to the non-edited plants. Additionally, host defense response genes were unaltered between edited and non-edited plants, implicating the direct role of AtAAP6 towards nematode susceptibility.ConclusionThe present findings enrich the existing literature on CRISPR/Cas9 research in plant-nematode interactions, which is quite limited currently while compared with the other plant-pathogen interaction systems.

Genomic-wide analysis reveals seven in absentia genes regulating grain development in wheat (Triticum aestivum L.).

Seven in absentia proteins, which contain a conserved SINA domain, are involved in regulating various aspects of wheat (Triticum aestivum L.) growth and development, especially in response to environmental stresses. However, it is unclear whether TaSINA family members are involved in regulating grain development until now. In this study, the expression pattern, genomic polymorphism, and relationship with grain-related traits were analyzed for all TaSINA members. Most of the TaSINA genes identified showed higher expression levels in young wheat spikes or grains than other organs. The genomic polymorphism analysis revealed that at least 62 TaSINA genes had different haplotypes, where the haplotypes of five genes were significantly correlated with grain-related traits. Kompetitive allele-specific PCR markers were developed to confirm the single nucleotide polymorphisms in TaSINA101 and TaSINA109 among the five selected genes in a set of 292 wheat accessions. The TaSINA101-Hap II and TaSINA109-Hap II haplotypes had higher grain weight and width compared to TaSINA101-Hap I and TaSINA109-Hap I in at least three environments, respectively. The qRT-PCR assays revealed that TaSINA101 was highly expressed in the palea shell, seed coat, and embryo in young wheat grains. The TaSINA101 protein was unevenly distributed in the nucleus when transiently expressed in the protoplast of wheat. Three homozygous TaSINA101 transgenic lines in rice (Oryza sativa L.) showed higher grain weight and size compared to the wild type. These findings provide valuable insight into the biological function and elite haplotype of TaSINA family genes in wheat grain development at a genomic-wide level.

Reaction of Tomato Lineages and Hybrids to Xanthomonas euvesicatoria pv. perforans

The use of resistant varieties is an important strategy for managing tomato bacterial spot. The objective of this study was to evaluate the reaction of tomato genotypes to Xanthomonas euvesicatoria pv. perforans. The experiment was conducted in a randomized block design with 10 genotypes and four repetitions. The genotypes consisted of hybrids (UFU-1, UFU-2, UFU-3), wild resistant accession Solanum pennellii, commercial susceptible cultivar Santa Clara and homozygous lines (UFU-5, UFU-6, UFU-11, UFU-12, UFU-15). The UFU B8 isolate of X. euvesicatoria pv. perforans was used. The bacterial suspension was prepared and adjusted in a spectrophotometer OD550 = 0.5 (1 × 109 CFU mL−1). Inoculation occurred 10 days after transplantation. Disease severity was assessed at 3, 6, 9, 12 and 15 days after inoculation, and the area under the disease progress curve (AUDPC) was calculated. There was a significant difference between the genotypes regarding the severity of the disease at 3, 6 and 12 days after inoculation. Lineages 5, 6 and 12, the hybrid UFU-1 and the wild accession S. pennellii showed the lowest severity of the disease, being promising for promoting genetic improvement programs aimed at resistance to the bacteria.

GhATL68b regulates cotton fiber cell development by ubiquitinating the enzyme required for β-oxidation of polyunsaturated fatty acids

E3 ligases are key enzymes required for protein degradation. Here, we identified a C3H2C3 RING domain-containing E3 ubiquitin ligase gene named GhATL68b. It is preferentially and highly expressed in developing cotton fiber cells and shows greater conservation in plants than in animals or archaea. The four orthologous copies of this gene in various diploid cottons and eight in the allotetraploid G. hirsutum were found to have originated from a single common ancestor that can be traced back to Chlamydomonas reinhardtii at about 992 million years ago. Structural variations in the GhATL68b promoter regions of G. hirsutum, G. herbaceum, G. arboreum, and G. raimondii are correlated with significantly different methylation patterns. Homozygous CRISPR-Cas9 knockout cotton lines exhibit significant reductions in fiber quality traits, including upper-half mean length, elongation at break, uniformity, and mature fiber weight. In vitro ubiquitination and cell-free protein degradation assays revealed that GhATL68b modulates the homeostasis of 2,4-dienoyl-CoA reductase, a rate-limiting enzyme for the β-oxidation of polyunsaturated fatty acids (PUFAs), via the ubiquitin proteasome pathway. Fiber cells harvested from these knockout mutants contain significantly lower levels of PUFAs important for production of glycerophospholipids and regulation of plasma membrane fluidity. The fiber growth defects of the mutant can be fully rescued by the addition of linolenic acid (C18:3), the most abundant type of PUFA, to the ovule culture medium. This experimentally characterized C3H2C3 type E3 ubiquitin ligase involved in regulating fiber cell elongation may provide us with a new genetic target for improved cotton lint production.

Introgression of the Self-Pruning Gene into Dwarf Tomatoes to Obtain Salad-Type Determinate Growth Lines.

The use of dwarf plants in tomato breeding has provided several advantages. However, there are no identified dwarf plants (dd) containing the self-pruning habit (spsp). The aim of this work was to obtain future generations, characterize the germplasm, and select potential dwarf plants with a determinate growth habit to obtain Salad-type lines. The work was started by carrying out hybridization, followed by the first, second, and third backcrosses. Once F2BC3 seeds became available, the introgression of the self-pruning gene (spsp) into dwarf plants (dd) began. Three strains of normal architecture and a determinate growth habit were hybridized with two strains of dwarf size and an indeterminate growth habit, thus yielding four hybrids. Additionally, donor genotype UFU MC TOM1, the commercial cultivar Santa Clara, and the wild accession Solanum pennellii were used in the experiment. Agronomic traits, fruit quality, metabolomics, and acylsugars content were evaluated, and dwarf plants with a determinate growth habit were selected. Hybrid 3 exhibited the highest yields. Visual differences between determinate and indeterminate dwarf plant seedlings were observed. It is suggested to carry out five self-pollinations of the best dwarf plant determined and subsequent hybridization with homozygous lines of normal plant architecture and determinate growth habit to obtain hybrids.

Pygo-F773W Mutation Reveals Novel Functions beyond Wnt Signaling in Drosophila.

Pygopus (Pygo) has been identified as a specific nuclear co-activator of the canonical Wingless (Wg)/Wnt signaling pathway in Drosophila melanogaster. Pygo proteins consist of two conserved domains: an N-terminal homologous domain (NHD) and a C-terminal plant homologous domain (PHD). The PHD's ability to bind to di- and trimethylated lysine 4 of histone H3 (H3K4me2/3) appears to be independent of Wnt signaling. There is ongoing debate regarding the significance of Pygo's histone-binding capacity. Drosophila Pygo orthologs have a tryptophan (W) > phenylalanine (F) substitution in their histone pocket-divider compared to vertebrates, leading to reduced histone affinity. In this research, we utilized CRISPR/Cas9 technology to introduce the Pygo-F773W point mutation in Drosophila, successfully establishing a viable homozygous Pygo mutant line for the first time. Adult mutant flies displayed noticeable abnormalities in reproduction, locomotion, heart function, and lifespan. RNA-seq and cluster analysis indicated that the mutation primarily affected pathways related to immunity, metabolism, and posttranslational modification in adult flies rather than the Wnt signaling pathway. Additionally, a reduction in H3K9 acetylation levels during the embryonic stage was observed in the mutant strains. These findings support the notion that Pygo plays a wider role in chromatin remodeling, with its involvement in Wnt signaling representing only a specific aspect of its chromatin-related functions.

Human induced pluripotent cardiomyocytes as a model for cardiomyopathy with a Myozap gene mutation

Abstract Funding Acknowledgements Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): 1- Central Department of Missions, Egyptian Ministry of Higher education and Scientific Research. 2- small fund from Tampere University. The contractility of the heart is based on cardiomyocytes (CM). These cardiomyocytes are connected through a complex network of proteins forming the intercalated disc (ID) which is crucial to ensure mechanical and electrochemical coupling. Dilated cardiomyopathy (DCM) is a disease of the myocardium that can lead to heart failure and life-threatening arrhythmias. Up to 30% of DCM cases are caused by mutations in genes encoding proteins of the sarcomere (the basic contractile unit) or desmosome (a cell-to-cell adhesion structure) and the structural cytoskeleton. Myozap (Myocardium-Enriched Zonula Occludens-1-Associated Protein) is a distinctive cardiac-enriched ID protein. It is crucial for biomechanical stress adaption in CMs due to its outstanding role in the Rho/SRF (Serum Response Factor) signalling pathway. In this study, we have identified two Finnish families with DCM and carrying a novel variant in myozap. Our aim is to characterize myozap gene mutation-both heterozygous and homozygous truncating variants-as well as its implication in DCM. We have established patient-specific human induced pluripotent stem-cell lines and derived cardiomyocyte (hiPSC-CM) to study the disease at the cellular level. Myozap expression, compared to the control cell line, was evaluated by immunohistochemistry. Electrophysiological methods, including microelectrode array (MEA) and patch clamping, were implemented to measure various parameters with emphasis on those ones related to cell-cell connection e.g., the conduction velocity. Moreover, metabolic stress indicators, including oxygen consumption rate (OCR), were estimated by Seahorse. Additionally, qPCR and ELISA were performed to measure the colocalised and closely related genes to ID and protein level of troponin, respectively. Immunohistochemistry revealed downregulation and absence of myozap expression in heterozygous and homozygous cell lines, respectively. Compared to the control one, differences in BPM, FPD were observed. Metabolic analysis of the myozap mutant elucidated mitochondrial impairment compared to the control. Some SRF and sarcomeric target genes, including MYH7, MYH6 and Myl-2 were downregulated. On the contrary, apoptotic genes (e.g., caspase 3) were upregulated. Tight junction protein (TJP) was upregulated, and this is very importance since it is closely related to the myozap in the ID proteins network. Moreover, troponin protein level, measured by ELISA, showed upregulation in mutant myozap. In conclusion, our study provides a new insight into understanding myozap’s significance in cardiac biology and in cardiomyopathy pathophysiology.

Developmental Delay and Male-Biased Sex Ratio in esr2b Knockout Zebrafish.

The estrogen receptor signaling pathway plays an important role in vertebrate embryonic development and sexual differentiation. There are four major estrogen receptors in zebrafish: esr1, esr2a, esr2b and gper. However, the specific role of different estrogen receptors in zebrafish is not clear. To investigate the role of esr2b in zebrafish development and reproduction, this study utilized TALENs technology to generate an esr2b knockout homozygous zebrafish line. The number of eggs laid by esr2b knockout female zebrafish did not differ significantly from that of wild zebrafish. The embryonic development process of wild-type and esr2b knockout zebrafish was observed, revealing a significant developmental delay in the esr2b knockout zebrafish. Additionally, mortality rates were significantly higher in esr2b knockout zebrafish than in their wild-type counterparts at 24 hpf. The reciprocal cross experiment between esr2b knockout zebrafish and wild-type zebrafish revealed that the absence of esr2b resulted in a decline in the quality of zebrafish oocytes, while having no impact on sperm cells. The knockout of esr2b also led to an abnormal sex ratio in the adult zebrafish population, with a female-to-male ratio of approximately 1:7. The quantitative PCR (qPCR) and in situ hybridization results demonstrated a significant downregulation of cyp19ab1b expression in esr2b knockout embryos compared to wild-type embryos throughout development (at 2 dpf, 3 dpf and 4 dpf). Additionally, the estrogen-mediated induction expression of cyp19ab1b was attenuated, while the estradiol-induced upregulated expression of vtg1 was disrupted. These results suggest that esr2b is involved in regulating zebrafish oocyte development and sex differentiation.

Hypogonadotropic hypogonadism in male tilapia lacking a functional rln3b gene

Relaxin 3 is a neuropeptide that plays a crucial role in reproductive functions of mammals. Previous studies have confirmed that rln3a plays an important role in the male reproduction of tilapia. To further understand the significance of its paralogous gene rln3b in male fertility, we generated a homozygous mutant line of rln3b in Nile tilapia. Our findings indicated that rln3b mutation delayed spermatogenesis and led to abnormal testes structure. Knocking out rln3b gene resulted in a decrease in sperm count, sperm motility and male fish fertility. TUNEL detection revealed a small amount of apoptosis in the testes of rln3b−/− male fish at 390 days after hatching (dah). RT-qPCR analysis demonstrated that mutation of rln3b gene caused a significant downregulation of steroid synthesis-related genes such as cyp17a1, cyp11b2, germ cell marker gene, Vasa, and gonadal somatic cell marker genes of amh and amhr2. Furthermore, we found a significant down-regulation of hypothalamic-pituitary-gonadal (HPG) axis-related genes, while a significantly up-regulation of the dopamine synthetase gene in the rln3b−/− male fish. Taken together, our data strongly suggested that Rln3b played a crucial role in the fertility of XY tilapia by regulating HPG axis genes.