Parental Lines Research Articles

A study of genetic diversity of Brassica rapa L. accessions from the VIR collection by analyzing DNA polymorphism in microsatellite loci

The species Brassica rapa L. includes leafy and root crops grown in many countries of the world. Clarification of genetic relationships and population structure allows for a more accurate parental line selection for further breeding using genetically distinct forms. The B. rapa collection maintained at VIR contains 1750 accessions representing different subspecies and countries of origin, and it is a good tool for studying the diversity of cultivated forms using molecular genetic methods. The aim of the study was to investigate the genetic diversity and clarify the relationships within the B. rapa species using molecular markers. Molecular screening of 80 accessions using 16 microsatellite markers revealed alleles ranging in size from 85 to 460 bp with an average of 7.8 alleles per locus. The average polymorphic index content (PIC) was 0.278, and the expected heterozygosity (H) averaged was 0.35. Rare and unique alleles were identified for Beijing cabbage (k-63 and k-108), Japanese leafy vegetables (k-217 and k-335) and turnip (k-738) samples. Alleles of SSR loci BRMS-007 (123 bp) and BRMS-034 (136 bp) were identified, which are characteristic only of accessions of turnips, rapeseed and sarsons. In silico analysis of primer pairs was performed to clarify the sizes of expected fragments relative to the reference genome CAAS_Brap_v3.01 of Chinese cabbage line Chiifu-401-42. Phylogenetic analysis was performed using the STRUCTURE program and resulted in the distribution of accessions into four clusters according to botanical classification: Chinese cabbage; pakchoi, tatsoi; Japanese leafy vegetables, wutacai, tsoisum, mizuna and mibuna (kyona) and hybrid forms; turnip and rape accessions.

Estimation of Combining Ability Effect for Hybrid Breeding in Bottle Gourd [Lagenaria siceraria (Molina) Standl.] Using Diallel Mating Design

A half diallel mating design was employed to cross eight parental lines of bottle gourd, resulting in 28 F1 hybrids (excluding reciprocals). These F1 hybrids, along with the parental lines and a commercial check (Pant Lauki-3), were evaluated during the summer of 2024 at Govind Ballabh Pant University of Agriculture and Technology, Pantnagar. The study aimed to assess the combining ability for yield and yield-related traits using a randomized complete block design with three replications. The genetic variance ratio being less than unity suggested that non-additive gene action was dominant for all the characters. The analysis indicated that none of the parents consistently exhibited good general combining ability across all traits. However, the cross combination NDBG-104 × Pusa Sandesh followed by Pant Lauki-4 × Kashi Kirti emerged as the best for most of the traits analyzed. The results suggest that it may be possible to further enhance fruit yield and earliness through hybridization and selection in transgressive segregants.

The interaction between abiotic and biotic soil factors drive heterosis expression in maize.

Heterosis or hybrid vigor refers to the superior phenotypes of hybrids relative to their parental inbred lines. Recently, soil microbes were identified as an environmental driver of maize heterosis. While manipulation of the soil microbial community consistently altered heterosis, the direction of the effect appeared to be dependent on the microbiome composition, environment, or both. Abiotic factors are well-known modifiers of heterosis expression, however, how the interactive effects between the soil microbial community and abiotic factors contribute to heterosis are poorly understood. To disentangle the proposed mechanisms by which microbes influence heterosis, we characterize the variation in heterosis expression when maize was grown in soil inocula derived from active maize farms or prairies. While we did not observe consistent differences in heterosis among plants grown in these inocula, our observations reaffirm that microbial effects on heterosis are likely specific to the local microbial community. The introduction of a nutrient amendment resulted in greater heterosis expression in the presence of an agricultural inoculum but not a prairie inoculum. We also observed an effect of soil inocula and nutrient treatment on the composition of bacterial and fungal communities in the root endosphere. In addition, the interaction between soil and nutrient treatment significantly affected bacterial community composition, whereas fungal community composition was only marginally affected by this interaction. These results further suggest that the soil microbial community plays a role in maize heterosis expression but that the abiotic environment is likely a larger driver.

Strategies for enhancing productivity, resilience, nutritional quality, and consumption of pearl millet [Pennisetum glaucum (L.) R. Br.] for food and nutritional security in India

AbstractPearl millet [Pennisetum glaucum (L.) R. Br.] is an important component of agri‐food system in areas experiencing drought and high temperature and for increasing the resilience to climatic stresses and addressing malnutrition. The purpose of this review is to examine strategies for improving productivity, stress resilience, and nutritional quality of pearl millet and to understand its consumption pattern. Genetic diversification of hybrid parental lines remains strategically important to breed diverse, disease‐resistant and drought‐tolerant hybrids. Resistance to diseases, tolerance to drought, and high temperature and greater contents of iron and zinc are targeted in improving hybrid parental lines. Lodging resistance, compact panicles, panicle exertion, and improved seed set are universal traits, whereas duration, tillering ability, seed color, and seed size have a strong regional preference. The strategy of developing high‐yielding and disease‐resistant hybrids with adaptation to challenged agro‐ecologies has led to increase in yield from 303 to 1219 kg/ha between 1960 and 2020. Yield and stress resilience are to be increased further using conventional breeding and new tools like genomic selection, speed breeding, genome editing, and precision phenotyping. Mainstreaming grain nutritional traits, viz., iron and zinc contents in genetic improvement are essential to develop high‐yielding and nutrient‐rich pearl millet. There is need to enhance the consumption of pearl millet by strengthening existing value‐chain, providing consumer a choice of diverse range of food products, creating awareness about its health benefits and promotion through government schemes.

Acquired Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) Resistance of Human Colorectal Cancer Cells Is Linked to Histone Acetylation and Is Synergistically Ameliorated by Combination with HDAC Inhibitors.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an attractive target for the treatment of various malignancies; however, its therapeutic potential is limited because of the frequent occurrence of tumor cell resistance. In this study, we determined whether TRAIL resistance acquired by repeated administration could be overcome by HDAC inhibition in human colorectal cancer cells. TRAIL-resistant HCT116 human colorectal cancer cells (HCT116-TR) were generated by repeated treatment with 10 and 25 ng/mL TRAIL twice weekly for 28 days. The resulting TRAIL-resistant cells were noncross-resistant to other chemotherapeutic agents. The levels of histone acetylation-related proteins, such as ac-histone H4 and HDAC1, were altered in HCT116-TR cells compared with the parental HCT116 cell line. The combined treatment with TRAIL and HDAC inhibitors significantly increased apoptosis in HCT116-TR cells and indicated a synergistic effect. The mechanism by which HDAC inhibition sensitizes HCT116-TR cells to TRAIL is dependent on the intrinsic pathway. In addition, we found that HDAC inhibition enhanced the sensitivity of cells to TRAIL through mitogen-activated protein kinases/CCAAT/enhancer-binding protein homologs of protein-dependent upregulation of death receptor 5. These results suggest that histone acetylation is responsible for acquired TRAIL resistance after repeated exposure and acquired resistance to TRAIL may be overcome by combination therapies with HDAC inhibitors.

Progress in the development of anthracnose-resistant F1 hybrid strawberries using self-pollinated parental lines generated by recurrent selection

Progress in the development of anthracnose-resistant F₁ hybrid strawberries using self-pollinated parental lines generated by recurrent selection

CD133+/ABCC5+ cervical cancer cells exhibit cancer stem cell properties

ObjectiveThis study explores the correlation between Forkhead box M1 (FOXM1) and ATP-binding cassette subfamily C member 5 (ABCC5) in relation to paclitaxel resistance in cervical cancer. It aims to identify potential cervical cancer stem cell markers, offering fresh perspectives for developing therapeutic strategies to overcome paclitaxel chemoresistance in cervical cancer. MethodsPaclitaxel-resistant Hela cells (Hela/Taxol) were developed by intermittently exposing Hela cells to progressively increasing concentrations of paclitaxel. We assessed the biological properties of both Hela and Hela/Taxol cells using various assays: cell proliferation, clonogenic, cell cycle, apoptosis, scratch, and transwell. To determine which markers better represent tumor stem cells, we analyzed various known and potential stem cell markers in combination. Flow cytometry was employed to measure the proportion of positive markers in both parental and drug-resistant cell lines. Following statistical analysis to establish relative stability, CD133+ABCC5+ cells were sorted for further examination. Subsequent tests included sphere-forming assays and Western blot analysis to detect the presence of the stem cell-specific protein Sox2, aiding in the identification of viable cervical cancer stem cell markers. ResultsThe Hela/Taxol cell line exhibited significantly enhanced proliferation, migration, and invasion capabilities compared to the Hela cell line, alongside a marked reduction in apoptosis rates (P < 0.01). Notably, proportions of CD44+, CD24+CD44+, ABCC5+, CD24+CD44+ABCC5+, CD44+ABCC5+, CD24+CD44+FOXM1+, CD44+FOXM1+, CD133+ABCC5+, and CD133+FOXM1+ were significantly higher (P < 0.05). Furthermore, the size and number of spheres formed byCD133+ABCC5+ cells were greater in the sorted Hela/Taxol line (P < 0.01), with increased expression of the stem cell marker Sox2 (P < 0.001). ConclusionThe Hela/Taxol cells demonstrate increased tumoral stemness, suggesting that CD133+ABCC5+ may serve as a novel marker for cervical cancer stem cells.

Multiomics of a rice population identifies genes and genomic regions that bestow low glycemic index and high protein content

To counter the rising incidence of diabetes and to meet the daily protein needs, we created low glycemic index (GI) rice varieties with protein content (PC) surpassing 14%. In the development of recombinant inbred lines using Samba Mahsuri and IR36 amylose extender (IR36ae) as parental lines, we identified quantitative trait loci and genes associated with low GI, high amylose content (AC), and high PC. By integrating genetic techniques with classification models, this comprehensive approach identified candidate genes on chromosome 2 (qGI2.1/qAC2.1 spanning the region from 18.62 Mb to 19.95 Mb), exerting influence on low GI and high amylose. Notably, the phenotypic variant with high value was associated with the recessive allele of the starch branching enzyme 2b (sbeIIb). The genome-edited sbeIIb line confirmed low GI phenotype in milled rice grains. Further, combinations of alleles created by the highly significant SNPs from the targeted associations and epistatically interacting genes showed ultralow GI phenotypes with high amylose and high protein. Metabolomics analysis of rice with varying AC, PC, and GI revealed that the superior lines of high AC and PC, and low GI were preferentially enriched in glycolytic and amino acid metabolisms, whereas the inferior lines of low AC and PC and high GI were enriched with fatty acid metabolism. The high amylose high protein recombinant inbred line (HAHP_101) was enriched in essential amino acids like lysine. Such lines may be highly relevant for food product development to address diabetes and malnutrition.

Development of Iron and Zinc Biofortified Rice Variety for Nutrition Improvement in Bangladesh

Depleting micronutrients such as iron and zinc poses a serious risk to a population’s health in regions where nutrient-rich meals are unavailable, such as Bangladesh. We can engross the essential minerals using biofortified rice varieties enriched with essential minerals. We experimented to develop and evaluate a new high-yielding rice variety, Advanced Chemical Industries (ACI) dhan1, specifically for the rainy season in Bangladesh. We used high-yielding rice parent lines with high iron and zinc in the hybridization process, then grew and selected the resulting breeding lines during the rainy season of Bangladesh to identify the most suitable rice lines for the rainy season. The plant height and days to maturity traits were the same as shown in both cultivars, ACI dhan1 and Bangladesh Rice Research Institute (BRRI) dhan62 (standard check), which are 104 cm and 103 days, respectively. On the other hand, ACI dhan1 produced 6.05 tons per hectare, and BRRI dhan62 yielded 4.33 tons per hectare. A quality analysis of the grain quantified that ACI dhan1 contained 8.21% protein and 25.12% apparent amylose. Similarly, ACI dhan1 had very high levels of Zn (24.8 ppm) and Fe (9 ppm). We observed a more than 70% improvement in milling capabilities and head rice recovery. In proposed variety trials, the Seed Certification Agency of Bangladesh identified an average yield advantage of more than 10% for ACI dhan1 in 10 locations. National Seed Board, Bangladesh, released it as a candidate for high-iron and zinc-enriched rice variety and approved its extensive cultivation in Bangladesh. Due to its high micronutrient content, excellent agronomic performance, and non-sticky cooking quality, ACI dhan1 presents itself as a variety of choice in Bangladesh’s pursuit of food and nutrition security. Thus, the present study demonstrates the large achievement of biofortification by ACI dhan1 and hence promises to be a strong weapon in the battle against malnutrition.

Identification of a Major QTL for Seed Protein Content in Cultivated Peanut (Arachis hypogaea L.) Using QTL-Seq.

Peanut (Arachis hypogaea L.) is a great plant protein source for human diet since it has high protein content in the kernel. Therefore, seed protein content (SPC) is considered a major agronomic and quality trait in peanut breeding. However, few genetic loci underlying SPC have been identified in peanuts, and the underlying regulatory mechanisms remain unknown, limiting the effectiveness of breeding for high-SPC peanut varieties. In this study, a major QTL (qSPCB10.1) controlling peanut SPC was identified within a 2.3 Mb interval in chromosome B10 by QTL-seq using a recombinant inbred line population derived from parental lines with high and low SPCs, respectively. Sequence comparison, transcriptomic analysis, and annotation analysis of the qSPCB10.1 locus were performed. Six differentially expressed genes with sequence variations between two parents were identified as candidate genes underlying qSPCB10.1. Further locus interaction analysis revealed that qSPCB10.1 could not affect the seed oil accumulation unless qOCA08.1XH13 was present, a high seed oil content (SOC) allele for a major QTL underlying SOC. In summary, our study provides a basis for future investigation of the genetic basis of seed protein accumulation and facilitates marker-assisted selection for developing high-SPC peanut genotypes.

Assessment of Heterosis in Inter-varietal Hybrids of Mangalore Melon (Cucumismelo var. acidulus) for Yield and Quality Traits

Mangalore melon is one of the distinct class of Cucurbitaceae family widely grown in southern part of India. Despite of having many nutritional values, there is scope for systematic hybridization study to improve the many qualitative and quantitative traits. An investigation was undertaken to develop inter-varietal hybrids and their evaluation to assess the extent of heterosis in newly developed hybrids between selected genotypes of Mangalore melon during kharif, 2019 and 2020 at College of Horticulture, Sirsi, Karnataka, India. A total of 16 intervarietal hybrids were developed by crossing 15 parental lines. Hybrids were developed using hand emasculation and hand pollination technique and the developed hybrids were evaluated using RCBD design with two replications. The results showed significant positive heterosis over mid parent and better parent for all the hybrids. The hybrids Soubhagya×RNMS-1 exhibited highest and significant heterosis over both mid parent (88.05%) and better parent (72.69%) followed by MS-19×MS-5 over mid parent (73.09%) and better parent (61.10%). From this study, it was concluded that hybrid Soubhagya X RNMS-1 was superior over their parents for most of the traits with the total yield of 8.44 kg plant-1. These best performing hybrids are need to be studied for their stability and commercial exploitation, resistance of pest and diseases, keeping quality of hybrids (storability) and experimentation on hybrid seed production technologies as well as storability of seeds should be done.

Pathological Role of High Sugar in Mitochondrial Respiratory Chain Defect-Augmented Mitochondrial Stress.

According to many research groups, high glucose induces the overproduction of superoxide anions, with reactive oxygen species (ROS) generally being considered the link between high glucose levels and the toxicity seen at cellular levels. Respiratory complex anomalies can lead to the production of ROS. Calcium [Ca2+] at physiological levels serves as a second messenger in many physiological functions. Accordingly, mitochondrial calcium [Ca2+]m overload leads to ROS production, which can be lethal to the mitochondria through various mechanisms. F1F0-ATPase (ATP synthase or complex V) is the enzyme responsible for catalyzing the final step of oxidative phosphorylation. This is achieved by F1F0-ATPase coupling the translocation of protons in the mitochondrial intermembrane space and shuttling them to the mitochondrial matrix for ATP synthesis to take place. Mitochondrial complex V T8993G mutation specifically blocks the translocation of protons across the intermembrane space, thereby blocking ATP synthesis and, in turn, leading to Neuropathy, Ataxia, and Retinitis Pigmentosa (NARP) syndrome. This study seeks to explore the possibility of [Ca2+]m overload mediating the pathological roles of high glucose in defective respiratory chain-mediated mitochondrial stress. NARP cybrids are the in vitro experimental models of cells with F1FO-ATPase defects, with these cells harboring 98% of mtDNA T8993G mutations. Their counterparts, 143B osteosarcoma cell lines, are the parental cell lines used for comparison. We observed that NARP cells mediated and enhanced the death of cells (apoptosis) when incubated with hydrogen peroxide (H2O2) and high glucose, as depicted using the MTT assay of cell viability. Furthermore, using fluorescence probe-coupled laser scanning confocal imaging microscopy, NARP cells were found to significantly enable mitochondrial reactive oxygen species (mROS) formation and enhance the depolarization of the mitochondrial membrane potential (ΔΨm). Elucidating the mechanisms of sugar-enhanced toxicity on the mitochondria may, in the future, help to alleviate the symptoms of patients with NARP syndromes and other neurodegenerative diseases.

The agronomic characteristics and combining ability of glutinous Thermo-sensitive Genic Male Sterile rice (Oryza sativa) lines

Glutinous rice (Oryza sativa), despite its unique culinary qualities, typically yields less than other rice varieties, posing agricultural and economic challenges. The construction of a glutinous thermo-sensitive genic male sterile (TGMS) rice variety could indeed be a way to improve the yield and cultivation efficiency of glutinous rice. Among them, the two-line system operates by crossing a TGMS line, which is sterile at high temperatures but fertile at lower temperatures, with a normal fertile line. In this study, we conducted to access morphological characteristics and combining ability of some glutinous TGMS lines. The parental lines were characterized on 13 agronomic traits including growth duration, plant height, flag leaf width, flag leaf length, panicle length, rachis length, number of primary branches, number of secondary branches, number of panicles per plant, percentage of filled grains, P1000 seeds, grain yield, and amylose content. The obtained results revealed significant variations in growth traits, yield, and amylose content of studied rice lines. Hierarchical clustering based on principal component analysis of rice lines on all measured traits showed three major clusters with high genetic diversity. Furthermore, fifteen glutinous TGMS rice lines and three testers were used in a Line x Tester mating system to produce 45 hybrids. The 45 hybrids were evaluated on 6 traits related to grain yield and morphology in a randomized complete block design experiment with three replications. A line x tester analysis was conducted to estimate the combining ability, genetic variance, and the contribution of parental lines to genetic variation in hybrids. These findings were valuable for rice breeders to orient the strategy for breeding of hybrid glutinous rice varieties with high efficiency.

PAI1 Regulates Cell Morphology and Migration Markers in Trastuzumab-Resistant HER2-Positive Breast Cancer Cells.

HER2-positive breast cancer is a significant cause of mortality. Overcoming trastuzumab resistance requires a deeper understanding of its molecular mechanisms to develop effective therapies. This study investigates the role of plasminogen activator inhibitor-1 (PAI1) in migration and drug resistance in trastuzumab-resistant HER2-positive breast cancer. Trastuzumab resistance poses a significant challenge in clinical management due to its association with aggressive disease behaviour and limited treatment options. This study focuses on PAI1, a key player in the TGF-β signalling pathway, which is implicated in cancer progression and metastasis. Trastuzumab-resistant cell lines (SKBR3 and HCC1954) demonstrated markedly elevated PAI1 expression levels, up to 40-fold compared to parental lines. This elevation was accompanied by increased expression of migration markers such as Col4a1, Fibronectin, ICAM1, Timp2, and Vimentin. Through overexpression and silencing experiments, we observed that modulating PAI1 levels significantly impacts cell morphology, transitioning cells from an epithelial to mesenchymal phenotype. Importantly, combining trastuzumab with aleplasinin, a PAI1 inhibitor, synergistically reduced PAI1 expression in both parental and resistant cell lines. This suggests a potential therapeutic strategy to overcome trastuzumab resistance. These findings emphasise PAI1 as a critical mediator of migration and therapeutic response in HER2-positive breast cancer, offering insights into novel treatment approaches targeting PAI1 to improve clinical outcomes in drug resistance.

Evaluation of Genetic Diversity in Sorghum Genotypes for Drought Tolerance Using Mahalanobis’ D2 Analysis

Genetic diversity is vital in plant breeding and crop improvement, enabling the development of varieties resilient to biotic and abiotic stresses. Mahalanobis' D² analysis is a robust multivariate statistical method for assessing genetic diversity, offering precise measures of genetic divergence. This study aimed to evaluate the genetic diversity of twenty sorghum (Sorghum bicolor L. Moench) genotypes under rainfed and irrigated conditions using Mahalanobis’ D² statistics. The experiment was conducted at the Main Agricultural Research Station (MARS), Dharwad, involving twenty genotypes sourced from the AICRP sorghum, Dharwad, India. The genotypes were sown in medium black soil under two moisture levels: rainfed and irrigated. Morphological, physiological, phenological, and yield-related traits were recorded to identify drought-tolerant genotypes. Genetic divergence was estimated using D² statistics, and genotypes were grouped into clusters using Tocher’s method. Under rainfed conditions, the genotypes were classified into six clusters, while under irrigated conditions, they were grouped into three clusters. The intra-cluster distances ranged from 7.79 to 12.14 under rainfed conditions and from 8.14 to 15.74 under irrigated conditions. The inter-cluster distances revealed significant genetic variation, with the highest divergence observed between Clusters III and V under rainfed conditions and Clusters II and III under irrigated conditions. These findings highlight the presence of substantial genetic diversity among sorghum genotypes, facilitating the selection of diverse parental lines for breeding programs aimed at enhancing drought tolerance. The insights gained from this study contribute to the development of resilient sorghum varieties capable of withstanding water scarcity, ensuring sustainable agriculture, and promoting food security in drought-prone regions.

Bread wheat cultivar Popo harbors QTLs for seedling and adult plant resistance to leaf rust in South African and Argentine environments

Leaf rust, caused by the fungus Puccinia triticina Eriks (Pt), is a destructive disease affecting wheat (Triticum aestivum L.) production in many countries, and a serious threat to food security. As a result, several breeding programs have included leaf rust resistance as an important trait. The discovery and identification of new resistance genes that could aid in incorporating durable or long-lasting leaf rust resistance into wheat is fundamental in these breeding programs. The present study aimed to identify quantitative trait loci (QTLs) for leaf rust resistance in 127 recombinant inbred lines (RIL) developed from the cross between the resistant wheat cultivar Popo and the susceptible cultivar Kariega. The RIL population and parental lines were phenotyped for leaf rust infection type and severity at seedling and adult plant stage, respectively. The former in the greenhouse (in Argentina) and the latter in multiple field test environments comprising 3 locations in South Africa (in Tygerhoek in the Western Cape Province during the 2014, 2015, 2017 and 2018 cropping seasons; Clarens during 2014, 2016 and 2017 cropping seasons and in Bethlehem in the Free State Province during 2017 cropping season) and in 1 location in Argentina (during the 2017 and 2018 cropping seasons in Marcos Juárez, Córdoba Province). The population was genotyped using genotyping-by-sequencing. A total of 12,080 silicoDArT and 2,669 SNP markers were used for QTL analysis. In total, 25 putative QTLs for resistance to leaf rust at seedling and adult plant stages were identified, including 5 QTLs for seedling and 20 QTLs for adult plant resistance (APR). Interestingly, both Popo and Kariega contributed with alleles for resistance. Significant loci for reducing leaf rust infection at seedling stage were designated QLr.arc-1A, QLr.arc-2B, QLr.arc-5B, QLr.arc-6A and QLr.arc-6D. Three minor QTLs derived from Popo designated as QLr.arc-1B1, QLr.arc-2D and QLr.arc-3D were also detected from the field tests, explaining 5–10%, 10–16% and 5–7% of the phenotypic variance, respectively. The identified QTLs and their closely linked silicoDArT and SNP-based markers can be used for fine mapping and candidate gene discovery in wheat breeding programs targeting durable leaf rust resistance.

Co-Expression Network Analysis and Introgressive Gene Identification for Fiber Length and Strength Reveal Transcriptional Differences in 15 Cotton Chromosome Substitution Segment Lines and Their Upland and Sea Island Parents.

Fiber length (FL) and strength (FS) are the core indicators for evaluating cotton fiber quality. The corresponding stages of fiber elongation and secondary wall thickening are of great significance in determining FL and FS formation, respectively. QTL mapping and high-throughput sequencing technology have been applied to dissect the molecular mechanism of fiber development. In this study, 15 cotton chromosome segment substitution lines (CSSLs) with significant differences in FL and FS, together with their recurrent parental Gossypium hirsutum line CCRI45 and donor parent G. barbadense line Hai1, were chosen to conduct RNA-seq on developing fiber samples at 10 days post anthesis (DPA) and 20 DPA. Differentially expressed genes (DEGs) were obtained via pairwise comparisons among all 24 samples (each one with three biological repeats). A total of 969 DEGs related to FL-high, 1285 DEGs to FS-high, and 997 DEGs to FQ-high were identified. The functional enrichment analyses of them indicated that the GO terms of cell wall structure and ROS, carbohydrate, and phenylpropanoid metabolism were significantly enriched, while the GO terms of glucose and polysaccharide biosynthesis, and brassinosteroid and glycosylphosphatidylinositol metabolism could make great contributions to FL and FS formation, respectively. Weighted gene co-expressed network analyses (WGCNA) were separately conducted for analyzing FL and FS traits, and their corresponding hub DEGs were screened in significantly correlated expression modules, such as EXPA8, XTH, and HMA in the fiber elongation and WRKY, TDT, and RAC-like 2 during secondary wall thickening. An integrated analysis of these hub DEGs with previous QTL identification results successfully identified a total of 33 candidate introgressive DEGs with non-synonymous mutations between the Gh and Gb species. A common DEG encoding receptor-like protein kinase 1 was reported to likely participate in fiber secondary cell thickening regulation by brassionsteroid signaling. Such valuable information was conducive to enlightening the developing mechanism of cotton fiber and also provided an abundant gene pool for further molecular breeding.

Application of mannitol as pre-treatment and sucrose supports callus induction through anther culture in Gossypium arboreum

Diploid or tree cotton (Gossypium arboreum) species may provide a breeding source for fiber quality and disease resistance. Developing homozygous lines of Gossypium arboreum through anther culture can be a successful strategy for breeding the parental lines needed for cotton hybrids. This study describes callus induction on four G. arboreum genoytypes under various concentrations of sucrose as well as plant growth regulators. Explant materials were derived from immature anther and grew on modified Murashige and Skoog (MS) media. Prior to main treatment, mannitol application were applied to anthers with concentration of 0.7 M and modified MS media consisted of 3%, 5%, and 7% sucrose, as well as an addition of IAA, NAA, BAP, 2,4-D and Kin. Optimum callus formation was observed in Marvi genotype, in a medium containg IAA as well as sucrose between 5-7%. Furthermore, combination of IAA and NAA with sucrose 7% indicates highest number of embryogenic calli. However, Marvi genotype showed decreased rate of callus formation when the amount of sucrose increased on MS supplemented with NAA medium. The results indicated that the best formula on each genotypes were different. However, MS media with IAA (1 mg/l) + kin (0.2 mg/l) + CM (70 ml/l) + sucrose 5% provided the best performance to produce embryogenic calli on each genotypes. Our report can be utilized as an opportunity and strategy to obtain double haploid lines as a parental source in hybrid production.

Insights into heterosis from histone modifications in the flag leaf of inter-subspecific hybrid rice

BackgroundInter-subspecific hybrid rice represents a significant breakthrough in agricultural genetics, offering higher yields and better resilience to various environmental stresses. While the utilization of these hybrids has shed light on the genetic processes underlying hybridization, understanding the molecular mechanisms driving heterosis remains a complex and ongoing challenge. Here, chromatin immunoprecipitation-sequencing (ChIP-seq) was used to analyze genome-wide profiles of H3K4me3 and H3K27me3 modifications in the inter-subspecific hybrid rice ZY19 and its parents, Z04A and ZHF1015, then combined them with the transcriptome and DNA methylation data to uncover the effects of histone modifications on gene expression and the contribution of epigenetic modifications to heterosis.ResultsIn the hybrid, there were 8,126 and 1,610 different peaks for H3K4me3 and H3K27me3 modifications when compared to its parents, respectively, with the majority of them originating from the parental lines. The different modifications between the hybrid and its parents were more frequently observed as higher levels in the hybrid than in the parents. In ZY19, there were 476 and 84 allele-specific genes with H3K4me3 and H3K27me3 modifications identified, representing 7.9% and 12% of the total analyzed genes, respectively. Only a small portion of genes that showed differences in parental H3K4me3 and H3K27me3 modifications which demonstrated allele-specific histone modifications (ASHM) in the hybrid. The H3K4me3 modification level in the hybrid was significantly lower compared to the parents. In the hybrid, DNA methylation occurs more frequently among histone modification target genes. Additionally, over 62.58% of differentially expressed genes (DEGs) were affected by epigenetic variations. Notably, there was a strong correlation observed between variations in H3K4me3 modifications and gene expression levels in the hybrid and its parents.ConclusionThese findings highlight the substantial impact of histone modifications and DNA methylation on gene expression during hybridization. Epigenetic variations play a crucial role in controlling the differential expression of genes, with potential implications for heterosis.

An autonucleolytic suspension HEK293F host cell line for high-titer serum-free AAV5 and AAV9 production with reduced levels of DNA impurity

We sought to engineer mammalian cells to secrete nuclease activity as a step toward removing the need to purchase commercial nucleases as process additions in bioprocessing of AAV5 and AAV9 as gene therapy vectors. Engineering HeLa cells with a serratial nuclease transgene did not bring about nuclease activity in surrounding media whereas engineering serum-free, suspension-adapted HEK293F cells with a staphylococcal nuclease transgene did result in detectable nuclease activity in surrounding media of the resultant stable transfectant cell line, "NuPro-1S." When cultivated in serum-free media, NuPro-1S cells yielded 3.06×1010 AAV5 viral genomes (vg)/mL via transient transfection, compared with 3.85×109 vg/mL from the parental HEK293F cell line. AAV9 production, followed by purification by ultracentrifugation, yielded 1.8×1013 vg/mL from NuPro-1S cells compared with 7.35×1012 vg/mL from HEK293F cells. AAV9 from both HEK293F and NuPro-1S showed almost identical ability to transduce cells embedded in a scaffold tissue mimic or cells of mouse neonate brain tissue invivo. Comparison of agarose gel data indicated that the DNA content of AAV5 and AAV9 process streams from NuPro-1S cells was reduced by approximately 60% compared with HEK293F cells. A similar reduction in HEK293F cells was only achievable with a 50U/mL Benzonase treatment.