Fertility Restoration Research Articles

The Alpha Subunit of Mitochondrial Processing Peptidase Participated in Fertility Restoration in Honglian-CMS Rice

The cytoplasmic male sterility (CMS) and nuclear-controlled fertility restoration system is a favorable tool for the utilization of heterosis in plant hybrid breeding. Many restorer-of-fertility (Rf) genes have been characterized in various species over the decades, but more detailed work is needed to investigate the fertility restoration mechanism. Here, we identified an alpha subunit of mitochondrial processing peptidase (MPPA) that is involved in the fertility restoration process in Honglian-CMS rice. MPPA is a mitochondrial localized protein and interacted with the RF6 protein encoded by the Rf6. MPPA indirectly interacted with hexokinase 6, namely another partner of RF6, to form a protein complex with the same molecular weight as the mitochondrial F1F0-ATP synthase in processing the CMS transcript. Loss-of-function of MPPA resulted in a defect in pollen fertility, the mppa+/- heterozygotes showed semi-sterility phenotype and the accumulation of CMS-associated protein ORFH79, showing restrained processing of the CMS-associated atp6-OrfH79 in the mutant plant. Taken together, these results threw new light on the process of fertility restoration by investigating the RF6 fertility restoration complex. They also reveal the connections between signal peptide cleavage and the fertility restoration process in Honglian-CMS rice.

The results of studying the productivity indicators of three-line sunflower hybrids and their parental forms under the influence of weather conditions

Topicality. At the present stage, the basis of the Ukrainian economy is agricultural production. Agriculture is the only sphere that is as dependent on weather fluctuations as any other. And it constantly has to adapt to the ongoing climate change. Climate changes, which cause an increase in temperatures, changes in the amount of precipitation and its distribution during the growth season, unstable weather conditions, the spread of pests and diseases, threaten the production of sunflower hybrids. The question about the influence of weather factors on the formation of productive traits of both hybrids and their parental forms, the dependence of agricultural crops on weather factors (temperature and precipitation, both for the entire growth season and for several months) is relevant. Purpose. To study of the influence of weather conditions on the formation of productive traits of hybrids and their parental components. Materials and Methods. The triple hybrids of sunflower (Helianthus annuus L.) Ahent, Ahronomicnyi, Kameniar, Marshal, Zaporizkyi 28, their parental components – single-cross sterile hybrids ZL(22х102)A, ZL(42х46)A, ZL(42х58)A and the restorers of fertility ZL512Rf, ZL678Rf and ZL7034Rf were studied. Results. Over the years of research, yield fluctuations reached to 0.90 t/ha for Ahent hybrid, 1.66 t/ha – Agronomichnyi, 0.13 t/ha – Marshal, 0.96 t/ha – Kameniar and 0.58 t/ha – Zaporizkyi 28. The highest yields and oil yield per unit area were in 2018. According to the results of three-year trials on hybrids and their parental forms, the Marshal hybrid had the highest yield – 2.64 t/ha with seed oil content of 50.78 % in 2016. Also in 2016, all pollen fertility restorers had the highest yields and oil yields. In 2018, four out of five hybrids showed the highest yields and oil yield per unit area. In 2017, the lowest oil content in the seeds of the tested hybrids was in the range of 45.16–48.49 % for Ahent and Zaporizkyi 28, respectively. Conclusions. It was established that Marshal is the most productive hybrid for sunflower cultivation in the southern Steppe of Ukraine, its average yield was 2.56 t/ha that exceeded by 0.27–0.48 t/ha this indicator in other hybrids. Key words: sunflower, hybrid, yield, oil content, 1000 seeds weight, oil yield, weather conditions, precipitation, temperature, sum of active temperatures, correlation coefficient

Bioelimination of Phytotoxic Hydrocarbons by Biostimulation and Phytoremediation of Soil Polluted by Waste Motor Oil

Soils contaminated by waste motor oil (WMO) affect their fertility, so it is necessary to recover them by means of an efficient and safe bioremediation technique for agricultural production. The objectives were: (a) to biostimulate the soil impacted by WMO by applying crude fungal extract (CFE) and Cicer arietinum as a green manure (GM), and (b) phytoremediation using Sorghum vulgare with Rhizophagus irregularis and/or Rhizobium etli to reduce the WMO below the maximum value according to NOM-138 SEMARNAT/SS or the naturally detected one. Soil impacted by WMO was biostimulated with CFE and GM and then phytoremediated by S. vulgare with R. irregularis and R. etli. The initial and final concentrations of WMO were analyzed. The phenology of S. vulgare and colonization of S. vulgaris roots by R. irregularis were measured. The results were statistically analyzed by ANOVA/Tukey's HSD test. The WMO in soil that was biostimulated with CFE and GM, after 60 days, was reduced from 34,500 to 2066 ppm, and the mineralization of hydrocarbons from 12 to 27 carbons was detected. Subsequently, phytoremediation with S. vulgare and R. irregularis reduced the WMO to 86.9 ppm after 120 days, which is a concentration that guarantees the restoration of soil fertility for safe agricultural production for human and animal consumption.

The Effect of Digestate from Liquid Cow Manure on Spring Wheat Chlorophyll Content, Soil Properties, and Risk of Leaching

Biogas production from manure and other organic matter, or combinations thereof, is part of the circular economy, and the use of the digestate from biogas production for plant nutrition and thus for soil fertility restoration and organic carbon (C) sequestration completes the circular economy cycle. The use of digestate from biogas production in agriculture is one of the sustainable ways to manage manure or organic waste, an alternative to conventional chemical fertilizers, and a means to achieve the objectives of the European Green Deal. To this end, two two-factor pot experiments were carried out in 2019–2020. Factor A—1. Control (without fertilization), 2. Liquid cow manure 170 kg ha−1 N (N170), 3. Digested manure 170 kg ha−1 N (N170), 4. Digested manure 140 kg ha−1 N (N140), 5. Digested manure 110 kg ha−1 N (N110); Factor B—1. Plants not cultivated, 2. Plants cultivated. The experiments determined the effect of digestate on the changes in soil nitrate (NO3-N), ammonium (NH4-N) and mineral (NO3-N + NH4-N) nitrogen, and available phosphorus (P2O5) and potassium (K2O) in the soil without plants as well as with plants cultivated and evaluated the risk of migration of macronutrients into deeper soil layers. The results showed that the application of the highest allowed rates under the Nitrates Directive (N170) and N140 digestate is accompanied by the addition of high levels of ammonium (NH4-N) nitrogen, which alters the balance of ammonium (NH4-N) and nitrate (NO3-N) nitrogen in the soil, and the extent of their migration to the deeper layers. These results suggest that the application of digestate to agricultural land enhances the migration of nitrogen and, in phosphorus-rich soils, of phosphorus (PO4-P) compounds to deeper layers (>25 cm). In order to achieve environmental objectives, digestate rates should be based on the agrochemical properties of the soil and the needs of the plants and should not exceed 65% of the nitrogen needed by the plants from mineral fertilizers.

Ex vivo purging of cancer cells from ovarian tissue using photodynamic therapy: a novel strategy to restore fertility in leukemia patients.

Is it possible to purge leukemia cells from ovarian tissue (OT) fragments before transplantation? Our photodynamic therapy (PDT) approach has been shown to efficiently destroy leukemia cells from tumor-infiltration mimicking models (TIMs), indicating the feasibility of this technique to purge OT samples. Autotransplantation of cryopreserved OT is the most suitable option to preserve fertility for prepubertal girls and women who require immediate cancer treatment. Up until now, more than 200 live births have already been reported after OT cryopreservation and transplantation. Leukemia is the 12th most common cancer in Europe among prepubertal girls and women of reproductive age and in 2020, the estimated number of new leukemia cases was higher than 33000 in girls between 0 and 19 years old. Unfortunately, once their health has been restored, autotransplantation of cryopreserved OT for leukemia patients is not advised due to the high risk of transferring malignant cells back to the patient leading to leukemia recurrence. To safely transplant the OT from leukemia patients and restore their fertility, our goal was to develop a PDT strategy to eliminate leukemia ex vivo. To this end, we designed OR141-loaded niosomes (ORN) to create the most effective formulation for ex vivo purging of acute myelogenous leukemia cells from OT fragments (n = 4). Moreover, to ensure that such treatments are not harmful to follicle survival and development so they can be deemed a potential fertility restoration alternative, the effect of the ORN-based PDT purging procedure on follicles was assessed after xenografting the photodynamic-treated OT in SCID mice (n = 5). The work was carried out between September 2020 and April 2022 at the Catholic University of Louvain. After establishing the best ORN formulation, our PDT approach was used to eradicate HL60 cells from ex vivo TIMs prepared by microinjection of a cancer cell suspension into OT fragments. The purging efficiency was analyzed by droplet digital polymerase chain reaction and immunohistochemical analyses. Additionally, we evaluated the effect of ORN-based PDT on follicle density, survival and development, and tissue quality in terms of fibrotic areas and vascularization after 7-day xenotransplantation to immunodeficient mice. The ex vivo purging of TIMs demonstrated that our PDT strategy could selectively eradicate the malignant cells from tissue fragments without affecting OT normal cells, as evidenced by PCR and immunohistochemical analysis. Regarding the effect of our PDT approach on follicle population and OT quality, our results after xenotransplantation revealed no significant difference between the follicle density of control (non-treated, grafted OT) and PDT-treated groups (2.38 ± 0.63 and 3.21 ± 1.94 morphologically normal follicles/mm2, respectively). In addition, our findings showed that the control and PDT-treated OT could be equally vascularized (7.65 ± 1.45% and 9.89 ± 2.21%, respectively). Similarly, the proportions of fibrotic area did not differ between the control (15.96 ± 5.94%) and PDT-treated groups (13.32 ± 3.05%). N/A. This study did not use OT fragments from leukemia patients, but TIMs created after injection of HL60 cells into OT from healthy patients. Therefore, while the results are promising, whether our PDT approach will be equally successful in eliminating malignant cells from leukemia patients remains to be assessed. Our results showed that the purging procedure causes no significant impairment effect on follicle development and tissue quality, suggesting that our novel PDT procedure could be a promising strategy to destroy leukemia cells in fragments of OT, allowing safe transplantation in cancer survivors. This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS-PDR Convention grant number T.0004.20 awarded to C.A.A.); Fondation Louvain (awarded to C.A.A.; a Ph.D. scholarship awarded to S.M., as part of a legacy from Mr Frans Heyes, and a Ph.D. scholarship awarded to A.D. as part of a legacy from Mrs. Ilse Schirmer); and Foundation Against Cancer (grant number 2018-042 awarded to A.C.). The authors declare no competing interests.

Effect of Inorganic Fertilizers on the Clover Growth in Gray-Brown Soils

The presented article presents materials on the effect of mineral fertilizers on the height of clover on irrigated gray-brown soils of the Ganja-Gazakh region. Due to the biological nitrogen collected by clover in the soil, more than twice the annual rate of nitrogen is saved in one cycle, thereby supplying other agricultural fields with nitrogen. Therefore, given the role of cotton as a strategically important plant, and alfalfa as a strong feed for livestock and restoration of soil fertility, the study of the fertilizer system of both plants in the cotton-alfalfa crop rotation is one of the most urgent tasks. The application of mineral fertilizers to alfalfa plants on irrigated gray-brown (chestnut) soils had a significant impact on alfalfa growth. High altitude means high yield. During the years of research, the highest height was observed in the N60P90K60 variant on average over 3 years. Depending on the norms of mineral fertilizers, the height of alfalfa increased by 6.3-29.5 cm compared to the variant without fertilizers.

Comparative analysis of prospect corn varieties productivity under high-risk farming Amur river region area

The article presents the results of the results of the study of corn hybrids of various ecological and geographical origin according to economic traits. The research was carried out during 2019-2021 in the nursery of the competitive variety trial of the fodder production department on the experimental plot of vegetable rotation of the Far Eastern Agricultural Research Institute (FEARI) by the method of agro-climatic analogues as applicable to the extreme conditions of the Khabarovsk territory. The targets of research were 28 maize hybrids, 16 of which were selected by FEARI. The goal of research is сomparative assessment of the productivity of promising corn hybrids in conditions of risk farming zone in the Amur river region. The studied hybrids were divided into 2 groups: short-season hybrids (FAO - 150-199), 13 samples. The standard is the local selection Birsu hybrid. 2. middle-early hybrids (FAO - 200-299), 15 samples. The standard is released in the Khabarovsk territory Moldavian 215 SV hybrid. On average during the research period the grain productivity of most of the samples in the group of short-season hybrids didn’t exceed the characteristics of standard hybrid. Only Birsu (2-cob) hybrid stood out (+12.4 dt/ha). In terms of herbage and cob productivity almost all hybrids exceed the standard values (Birsu - 194,8; 106,6 dt/ha) by 9.7-126.2 and 9.0-29.49 dt/ha respectively. In the group of middle-early hybrids the Porumben 295 ASV x fertile restorer hybrid stood out. In terms of grain, herbage and cob productivity it exceeded the standard hybrid Moldavian 215 (59.7, 256.9, 110.4 dt/ha) by 31.7, 113.7 and 59.2 dt/ha respectively. Based on the research findings the selected by FEARI Alitet 2 advantageous hybrid was marked out. In terms of herbage productivity it exceed Birsu hybrid by 54.3% and in terms of cob productivity - by 26,1%. The hybrid is transferred to the state variety testing.

DNA marker-assisted identification of promising exotic advanced breeding lines as pollen fertility restorers and their validation based on field and laboratory assayable traits in chilli (Capsicum annuum L.)

DNA marker-assisted identification of promising exotic advanced breeding lines as pollen fertility restorers and their validation based on field and laboratory assayable traits in chilli (Capsicum annuum L.)

Improving blast resistance of maintainer line DRR 9B by transferring broad spectrum resistance gene Pi2 by marker assisted selection in rice.

Hybrid rice technology offers great promise to further enhance rice production and productivity for global food security. Improving hybrid rice parental lines is the first step in developing heterotic rice hybrids. To improve resistance against blast disease, a maintainer line DRR 9B was fortified with a major broad-spectrum blast resistance gene Pi2 through marker-assisted selection. The rice blast caused by Magnaporthe oryzae is a major disease and can cause severe yield losses upto 100%. The NILs of Samba Mahsuri namely BA-23-11-89-12-168 possessing Pi2 was utilized as a donor parent. The PCR-based molecular marker tightly linked to Pi2 gene was used for the foreground selection at BC1F1 generation. The molecular marker tightly linked to the major fertility restorer gene Rf4 was used for negative selection (i.e., selection of plants possessing non fertility restoring alleles) at BC1F1 generation to identify maintainer lines. The positive plants with Rf4 gene were added to the restorer pool for restorer line development. At each stage, MAS for Pi2 coupled with stringent phenotypic selection for agro-morphological and grain quality traits were exercised. At BC1F3 generation, one hundred families were screened against blast disease at uniform blast nursery (UBN) and selected resistant lines were advanced to next generations. In the BC1F5 generation plants were subjected to agro-morphological evaluation for yield and yield-contributing traits. The selected plants at BC1F5 generation were crossed with DRR 9A to assess the maintainer ability of blast resistance lines and for further CMS line conversion for hybrid rice breeding for developing blast resistance rice hybrids.

Spontaneous reduction in macroprolactinoma volume during pregnancy and after birth: A case report

Prolactinoma, the most common secretory pituitary tumor, is found in 100 patients per million. It is responsible for impaired reproductive function in both sexes. Its treatment in the context of fertility restoration gives very satisfactory results. The natural history of these tumors in the gestational period is variable depending on their size with a clinically significative risk ranging from 5 to 30%. Their management is complex, requiring evaluation of the effects of pregnancy on tumor growth and the effects of therapy on embryonic and fetal development. We report the case of 23years patient with a macroprolactinoma and the successful outcome of this tumor during the pregnancy.

Multiomics approach to profiling Sertoli cell maturation during development of the spermatogonial stem cell niche.

Spermatogonial stem cells (SSCs) are the basis of spermatogenesis, a complex process supported by a specialized microenvironment, called the SSC niche. Postnatal development of SSCs is characterized by distinct metabolic transitions from prepubertal to adult stages. An understanding of the niche factors that regulate these maturational events is critical for the clinical application of SSCs in fertility preservation. To investigate the niche maturation events that take place during SSC maturation, we combined different '-omics' technologies. Serial single cell RNA sequencing analysis revealed changes in the transcriptomes indicative of niche maturation that was initiated at 11 years of age in humans and at 8 weeks of age in pigs, as evident by Monocle analysis of Sertoli cells and peritubular myoid cell (PMC) development in humans and Sertoli cell analysis in pigs. Morphological niche maturation was associated with lipid droplet accumulation, a characteristic that was conserved between species. Lipidomic profiling revealed an increase in triglycerides and a decrease in sphingolipids with Sertoli cell maturation in the pig model. Quantitative (phospho-) proteomics analysis detected the activation of distinct pathways with porcine Sertoli cell maturation. We show here that the main aspects of niche maturation coincide with the morphological maturation of SSCs, which is followed by their metabolic maturation. The main aspects are also conserved between the species and can be predicted by changes in the niche lipidome. Overall, this knowledge is pivotal to establishing cell/tissue-based biomarkers that could gauge stem cell maturation to facilitate laboratory techniques that allow for SSC transplantation for restoration of fertility.

Abrin inactivating mitochondrial ribosome induced anther abortion in cotton CMS line LD6A by damaging mitochondrial membrane structure

Cytoplasmic male sterile (CMS) system has extensively been exploited for hybrid vigor in plant breeding programs. However, its application in many crops is limited due to poor understanding of molecular mechanism of fertility restoration. Using advanced analytical approaches, we aimed to elucidate the molecular pathways, regulating CMS induction and fertility restoration in cotton. Reproductive structures of a novel CMS (LD6A) and its maintainer (LD6B) lines were analyzed for physiological and proteomic changes during the development process. Transmission electron micrographs of anther tapetum showed a relatively indistinct inner ridge of CMS than of LD6B mitochondria at tetrad stage. Furthermore, at pollen mother cell and tetrad stages, CMS anthers accumulated a significantly higher reactive oxygen species than the maintainer line. At the protein level, CMS anthers induced significantly higher Abrin (XP_016705148.2), lower tricarboxylic acid cycle enzyme (malate dehydrogenase, malic enzyme, isocitrate dehydrogenase), and histone acetyltransferase expression than its maintainer line. Suggesting that abnormal sequence of mitochondrial ribosome gene rps4 and rpl10 and high expression of ribosome-inactivating protein Abrin in CMS line damaged mitochondrial membrane. This inhibited energy supply from the non-degraded tapetum, and consequently induced pollen sterility in LD6A. These data provide new insights into CMS mechanism in cotton crops that can be used for developing new CMS germplasm resources.

Recombinant humanized collagen remodels endometrial immune microenvironment of chronic endometritis through macrophage immunomodulation.

Recently, evidence has suggested that chronic endometritis (CE) is a crucial factor associated with infertility and failure of assisted reproductive techniques, prompting concern in the reproductive field. Studies have shown that persistent infiltered immune cells stimulation result in the disturbance of endometrial immune microenvironment could lead to the infertility of CE patients finally. Conventional treatments are limited because they lack immune regulation, so it is urgent to develop a novel approach to treat CE and promote embryo implantation in patients with CE. Herein, we prepared recombinant humanized type III collagen (rhCol III) with high cell adhesion activity to regulate macrophages and repair the endometrium. In this study, M1 macrophages and M1 macrophages cultured medium and lipopolysaccharide (LPS) co-stimulated inflammatory endometrium stromal cells (ESCs) were established in vitro to mimic CE condition. rhCol III promoted M1 macrophages toward M2 phenotype, improved cell migration, viability and collagen components of inflammatory ESCs. Also, the inflammatory response of inflammatory ESCs was downregulated after rhCol III treatment. Subsequently, LPS was used for CE rat model and a 28-day observation was performed; inflammatory cells' infiltration, endometrium repair, extracellular matrix (ECM) remodeling and pregnancy outcomes were promoted after rhCol III endometrial infusion. In conclusion, rhCol III promoted (i) macrophage polarization toward M2 macrophages, (ii) pro-inflammatory cytokine production and anti-inflammatory cytokine reduction, (iii) ECM remodeling and (iv) fertility restoration. Meanwhile, rhCol III enhanced cell biological functions by interacting with discoidin domain receptors, regulated cell metabolism and reduced the inflammatory response through the inhibition of the NF-κB/YAP signaling pathway. Overall, the results illustrated the potential therapeutic prospects of rhCol III for CE treatment.

A construct of adipose-derived mesenchymal stem cells-laden collagen scaffold for fertility restoration by inhibiting fibrosis in a rat model of endometrial injury.

Severe endometrium damage causes pathological conditions such as thin endometrium and intrauterine adhesion, resulting in uterine factor infertility. Mesenchymal stem cell (MSC) therapy is a promising strategy in endometrial repair; yet, exogenous MSCs still raise concerns for safety and ethical issues. Human adipose-derived mesenchymal stem cells (ADMSCs) residing in adipose tissue have high translational potentials due to their autologous origin. To harness the high translation potentials of ADMSC in clinical endometrium regeneration, here we constructed an ADMSCs composited porous scaffold (CS/ADMSC) and evaluated its effectiveness on endometrial regeneration in a rat endometrium-injury model. We found that CS/ADMSC intrauterine implantation (i) promoted endometrial thickness and gland number, (ii) enhanced tissue angiogenesis, (iii) reduced fibrosis and (iv) restored fertility. We ascertained the pro-proliferation, pro-angiogenesis, immunomodulating and anti-fibrotic effects of CS/ADMSC in vitro and revealed that the CS/ADMSC influenced extracellular matrix composition and organization by a transcriptomic analysis. Our results demonstrated the effectiveness of CS/ADMSC for endometrial regeneration and provided solid proof for our future clinical study.

Successful fertility restoration in male-sterile barnase line by optimal expression of barstar gene for hybrid-rice seed production

ABSTRACT The basic requirement for the barnase-barstar-based hybrid-seed production technology is the availability of stable male-sterile barnase and restorer barstar lines. Development of two rice (Oryza sativa) male-sterile barnase lines has been earlier reported from our lab. Complete restoration of male fertility in transgenic male-sterile lines has always been a challenging task for molecular breeders. In the present study, we have reported the successful fertility restoration in male-sterile barnase lines using barstar restorer lines generated through two completely different tapetum-specific promoters. The barstar lines were not able to restore male fertility in one barnase line but did restore it in the other barnase line. The male-sterile barnase line and its corresponding restorer line could be diversified into appropriate combiners for heterosis rice breeding.

Identification and characterization of a fertility restorer line in Bhut Jolokia

Identification and characterization of a fertility restorer line in Bhut Jolokia

Evaluation of Salinity-Tolerant Backcrossed Inbre Lines (BILs) For Fertility Restoration Using Molecula Markers

Fertility restoration is a very important trait for the WA-CMS system in hybrid rice. Fifty-five BILs derived from KMR3 and FL478 cross combination possessing Saltol1 generated through Marker Assisted Backcross Breeding (MABB) approach were screened using Rf4 and Rf3 markers during Rabi 2021. The use of molecular markers tightly linked with fertility restoration aided in identifying promising groups of restorers, maintainers, partial restorers, and partial maintainers. The presence of both Rf4 and Rf3 alleles among the nineteen BILs was 35% which showed a clear difference in the marker distribution among the BILs. The BILs namely TCP32, TCP34, TCP35, TCP36, TCP36-3, TCP37-2, TCP38, TCP13, TCP18, TCP19, TCP45, TCP46, TCP48, TCP54, TCP56, TCP57-1, TCP60, TCP61, TCP61-3 were identified as promising restorers possessing both Rf4 and Rf3 genes. The current study reflects that Rf4 and Rf3 genes in combination help in the breeding of WA-CMS-based hybrids in Saltol introgressed restorers with better heterosis under various ecologies, especially coastal saline areas.

The transcription factor PbrbZIP52 positively affects pear pollen tube longevity by promoting callose synthesis.

In pear (Pyrus bretschneideri), pollen tube growth is critical for the double fertilization associated with seed setting, which in turn affects fruit yield. The normal deposition of callose mediates the polar growth of pollen tubes. However, the mechanism regulating callose synthesis in pollen tubes remains relatively uncharacterized. In this study, we revealed that the typical pear pollen tube lifecycle has a semi-growth duration (GD50) of 16.16 h under in vitro culture conditions. Moreover, callose plugs were deposited throughout the pollen tube lifecycle. The formation of callose plugs was inhibited by 2-deoxy-D-glucose, which also accelerated the senescence of pear pollen tubes. Additionally, PbrCalS1B.1, which encodes a plasma membrane-localized callose synthase, was expressed specifically in pollen tubes and restored the fertility of the Arabidopsis (Arabidopsis thaliana) cals5 mutant, in which callose synthesis is inhibited. However, this restoration of fertility was impaired by the transient silencing of PbrCalS1B.1, which restricts callose plug formation and shortens the pear pollen tube lifecycle. More specifically, PbrbZIP52 regulated PbrCalS1B.1 transcription by binding to promoter A-box elements to maintain the periodic formation of callose plugs and normal pollen tube growth, ultimately leading to double fertilization. This study confirmed that PbrbZIP52 positively affects pear pollen tube longevity by promoting callose synthesis. This finding may be useful for breeding high-yielding pear cultivars and stabilizing fruit setting in commercial orchards.

CRISPR/Cas9-editing of KISS1 to generate pigs with hypogonadotropic hypogonadism as a castration free trait.

Introduction: Most male pigs are surgically castrated to avoid puberty-derived boar taint and aggressiveness. However, this surgical intervention represents a welfare concern in swine production. Disrupting porcine KISS1 is hypothesized to delay or abolish puberty by inducing variable hypogonadotropism and thus preventing the need for castration. Methods: To test this hypothesis, we generated the first KISS1-edited large animal using CRISPR/Cas9-ribonucleoproteins and single-stranded donor oligonucleotides. The targeted region preceded the sequence encoding a conserved core motif of kisspeptin. Genome editors were intracytoplasmically injected into 684 swine zygotes and transferred to 19 hormonally synchronized surrogate sows. In nine litters, 49 American Yorkshire and 20 Duroc liveborn piglets were naturally farrowed. Results: Thirty-five of these pigs bore KISS1-disruptive alleles ranging in frequency from 5% to 97% and did not phenotypically differ from their wild-type counterparts. In contrast, four KISS1-edited pigs (two boars and two gilts) with disruptive allele frequencies of 96% and 100% demonstrated full hypogonadotropism, infantile reproductive tracts, and failed to reach sexual maturity. Change in body weight during development was unaffected by editing KISS1. Founder pigs partially carrying KISS1-disruptive alleles were bred resulting in a total of 53 KISS1 +/+, 60 KISS1 +/-, and 34 KISS1 -/- F1 liveborn piglets, confirming germline transmission. Discussion: Results demonstrate that a high proportion of KISS1 alleles in pigs must be disrupted before variation in gonadotropin secretion is observed, suggesting that even a small amount of kisspeptin ligand is sufficient to confer proper sexual development and puberty in pigs. Follow-on studies will evaluate fertility restoration in KISS1 KO breeding stock to fully realize the potential of KISS1 gene edits to eliminate the need for surgical castration.

Population structure and genetic diversity of a germplasm for hybrid breeding in rye (Secale cereale L.) using high-density DArTseq-based silicoDArT and SNP markers

Investigating genetic structure and diversity is crucial for the rye hybrid breeding strategy, leading to improved plant productivity and adaptation. The present study elucidated the population structure and genetic diversity of 188 rye accessions, comprising 94 pollen fertility restoration lines (RF) and 94 cytoplasmic male-sterile (CMS) lines with Pampa sterilizing cytoplasm using SNP and silicoDArT markers from the diversity array technology (DArT)-based sequencing platform (DArTseq). Expected heterozygosity (He) and Shanon’s diversity (I) indexes varied slightly between marker systems and groups of germplasms (He = 0.34, I = 0.51 for RF and CMS lines genotyped using SNPs; He = 0.31, I = 0.48, and He = 0.35, I = 0.53 for RF and CMS using silicoDArTs, respectively). ANOVA indicated moderate variation (7%) between RF and CMS breeding materials. The same parameter varied when chromosome-assigned markers were used and ranged from 5.8% for 5R to 7.4% for 4R. However, when silicoDArT markers were applied, the respective values varied from 6.4% (1R) to 8.2% (3R and 4R). The model-based (Bayesian) population structure analysis based on the total marker pool identified two major subpopulations for the studied rye germplasm. The first one (P1) encompasses 93 RF accessions, and the second one (P2) encompasses 94 CMS and one RF accession. However, a similar analysis related to markers assigned to selected chromosomes failed to put plant materials into any of the populations in the same way as the total marker pool. Furthermore, the differences in grouping depended on marker types used for analysis.