Molecular Markers Research Articles

Plastome structure, phylogeny and evolution of plastid genes in Reevesia (Helicteroideae, Malvaceae).

Reevesia is an eastern Asian-eastern North American disjunction genus in the family Malvaceae s.l. and comprises approximately 25 species. The relationships within the genus are not well understood. Here, 15 plastomes representing 12 Reevesia species were compared, with the aim of better understanding the species circumscription and phylogenetic relationships within the genus and among genera in the family Malvaceae s.l. The 11 newly sequenced plastomes range between 161,532 and 161, 945bp in length. The genomes contain 114 unique genes, 18 of which are duplicated in the inverted repeats (IRs). Gene content of these plastomes is nearly identical. All the protein-coding genes are under purifying selection in the Reevesia plastomes compared. The top ten hypervariable regions, SSRs, and the long repeats identified are potential molecular markers for future population genetic and phylogenetic studies. Phylogenetic analysis based on the whole plastomes confirmed the monophyly of Reevesia and a close relationship with Durio (traditional Bombacaceae) in subfamily Helicteroideae, but not with the morphologically similar genera Pterospermum and Sterculia (both of traditional Sterculiaceae). Phylogenetic relationships within Reevesia suggested that two species, R. pubescens and R. thyrsoidea, as newly defined, are not monophyletic. Six taxa, R. membranacea, R. xuefengensis, R. botingensis, R. lofouensis, R. longipetiolata and R. pycnantha, are suggested to be recognized.

Different control of resistance to two Colletotrichum orbiculare pathogenic races 0 and 1 in cucumber (Cucumis sativus L.).

Quantitative trait locus analysis identified independent novel loci in cucumbers responsible for resistance to races 0 and 1 of the anthracnose fungal pathogen Colletotrichum orbiculare. Cucumbers have been reported to be vulnerable to Colletotrichum orbiculare, causing anthracnose disease with significant yield loss under favorable conditions. The deployment of a single recessive Cssgr gene in cucumber breeding for anthracnose resistance was effective until a recent report on high-virulent strains infecting cucumbers in Japan conquering the resistance. QTL mapping was conducted to identify the resistance loci in the cucumber accession Ban Kyuri (G100) against C. orbiculare strains 104-T and CcM-1 of pathogenic races 0 and 1, respectively. A single dominant locus An5 was detected in the disease resistance hotspot on chromosome 5 for resistance to 104-T. Resistance to CcM-1 was governed by three loci with additive effects located on chromosomes 2 (An2) and 1 (An1.1 and An1.2). Molecular markers were developed based on variant calling between the corresponding QTL regions in the de novo assembly of the G100 genome and the publicly available cucumber genomes. Multiple backcrossed populations were deployed to fine-map An5 locus and narrow the region to approximately 222 kbp. Accumulation of An2 and An1.1 alleles displayed an adequate resistance to CcM-1 strain. This study provides functional molecular markers for pyramiding resistance loci that confer sufficient resistance against anthracnose in cucumbers.

Genetic and epigenetic alterations in precursor lesions of endometrial endometrioid carcinoma.

The hyperplasia-carcinoma sequence is a stepwise tumourigenic programme towards endometrial cancer in which normal endometrial epithelium becomes neoplastic through non-atypical endometrial hyperplasia (NAEH) and atypical endometrial hyperplasia (AEH), under the influence of unopposed oestrogen. NAEH and AEH are known to exhibit polyclonal and monoclonal cell growth, respectively; yet, aside from focal PTEN protein loss, the genetic and epigenetic alterations that occur during the cellular transition remain largely unknown. We sought to explore the potential molecular mechanisms that promote the NAEH-AEH transition and identify molecular markers that could help to differentiate between these two states. We conducted target-panel sequencing on the coding exons of 596 genes, including 96 endometrial cancer driver genes, and DNA methylome microarrays for 48 NAEH and 44 AEH lesions that were separately collected via macro- or micro-dissection from the endometrial tissues of 30 cases. Sequencing analyses revealed acquisition of the PTEN mutation and the clonal expansion of tumour cells in AEH samples. Further, across the transition, alterations to the DNA methylome were characterised by hypermethylation of promoter/enhancer regions and CpG islands, as well as hypo- and hyper-methylation of DNA-binding regions for transcription factors relevant to endometrial cell differentiation and/or tumourigenesis, including FOXA2, SOX17, and HAND2. The identified DNA methylation signature distinguishing NAEH and AEH lesions was reproducible in a validation cohort with modest discriminative capability. These findings not only support the concept that the transition from NAEH to AEH is an essential step within neoplastic cell transformation of endometrial epithelium but also provide deep insight into the molecular mechanism of the tumourigenic programme. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Large-scale transcriptomic and genomic analyses reveal a novel functional gene SERPINB6 for chicken carcass traits

BackgroundCarcass traits are crucial indicators of meat production efficiency. However, the molecular regulatory mechanisms associated with these traits remain unclear.ResultsIn this study, we conducted comprehensive transcriptomic and genomic analyses on 399 Tiannong partridge chickens to identify key genes and variants associated with carcass traits and to elucidate the underlying regulatory mechanisms. Based on association analyses with the elastic net (EN) model, we identified 12 candidate genes (AMY1A, AP3B2, CEBPG, EEF2, EIF4EBP1, FGFR1, FOXD3, GOLM1, LOC107052698, PABPC1, SERPINB6 and TBC1D16) for 4 carcass-related traits, namely live weight, dressed weight, eviscerated weight, and breast muscle weight. SERPINB6 was identified as the only overlapping gene by 3 analyses, EN model analysis, weighted gene co-expression network analysis and differential expression analysis. Cell-level experiments confirmed that SERPINB6 promotes the proliferation of chicken DF1 cells and primary myoblasts. Further expression genome-wide association study and association analysis indicated that rs317934171 is the critical site that enhances SERPINB6 expression. Furthermore, a dual-luciferase reporter assay proved that gga-miR-1615 targets the 3′UTR of SERPINB6.ConclusionsCollectively, our findings reveal that SERPINB6 serves as a novel gene for chicken carcass traits by promoting fibroblast and myoblast proliferation. Additionally, the downstream variant rs317934171 regulates SERPINB6 expression. These results identify a new target gene and molecular marker for the molecular mechanisms of chicken carcass traits.

Prognostic significance of Klotho expression in patients with renal cell carcinoma.

Various molecular markers have been investigated in renal cell carcinoma (RCC) without significant reliability. We analyzed Klotho (tumor suppressive protein) expression in RCC to investigate its association with tumor-stage, grade, disease-free-survival (DFS) and overall-survival (OS). Data of histologically confirmed patients of RCC with complete clinical follow-up were retrieved from Medical-Record-Library. Tissue sections of tumor and normal parenchyma were prepared from the blocks. Immunohistochemical studies for Klotho were done with commercially available kit (EPR6856, Ab181373; Abcam, Cambridge MA, USA). Klotho expression was scored between 0-3 and grouped into weak/absent (0, 1) and moderate/strong (2, 3). Tumors stages and grades were grouped into low stage (I and II) and high stage (III and IV) and into low grade (grade 1 and 2) and high grade (grade 3 and 4) according to WHO/ISUP grading. The histopathologists were blinded as to the clinical and follow-up data. Various prognostic factors were analyzed with respect to Klotho expression. Kaplan-Meier curves were created for DFS and OS. Fifty-four patients of mean age 55.15 ± 13.34 years and M:F ratio of 1.8:1 were included. Normal renal tissue had strong expression of Klotho in all. In tumor tissue 20 (37%) had negative, 7 (13%) had weak, 14 (25.9%) had moderate and 13 (24.1%) had strong Klotho expression. Significantly more patients had absent/weak Klotho expression with higher grade (16/24 (66.7%) vs 7/25 (28%); p = 0.007), higher stage (22/33 (66%) vs 5/21 (23.8%); p = 0.002), LVI (12/14 (85.7%) vs 2/14 (14.3%); p = 0.002), sinus-fat-invasion (16/21 (76.2%) vs 5/21 (23.8%); p = 0.002), renal-vein-involvement (14/18 (77.8%) vs 4/18 (22.2%); p = 0.004), necrosis (17/26 (65.3%) vs 9/26 (34.6%); p = 0.029) and metastasis (8/9 (88.9%) vs 1/9 (11.1%); p = 0.01). Median DFS and OS were significantly lower in patients with weak/absent Klotho expression (12 vs 23 months, p = 0.023 and 15 vs 33 months, p = 0.006 respectively). Kaplan-Meier curves showed lower estimated DFS and OS in patients with weak/absent expression. We conclude that Klotho expression in renal tumor could be a good prognostic marker in patients with RCC.

Analysis and validation of biomarkers of immune cell-related genes in postmenopausal osteoporosis: An observational study.

Postmenopausal osteoporosis (PMOP) is a common metabolic inflammatory disease. In conditions of estrogen deficiency, chronic activation of the immune system leads to a hypo-inflammatory phenotype and alterations in its cytokine and immune cell profile, although immune cells play an important role in the pathology of osteoporosis, studies on this have been rare. Therefore, it is important to investigate the role of immune cell-related genes in PMOP. PMOP-related datasets were downloaded from the Gene Expression Omnibus database. Immune cells scores between high bone mineral density (BMD) and low BMD samples were assessed based on the single sample gene set enrichment analysis method. Subsequently, weighted gene co-expression network analysis was performed to identify modules highly associated with immune cells and obtain module genes. Differential analysis between high BMD and low BMD was also performed to obtain differentially expressed genes. Module genes are intersected with differentially expressed genes to obtain candidate genes, and functional enrichment analysis was performed. Machine learning methods were used to filter out the signature genes. The receiver operating characteristic (ROC) curves of the signature genes and the nomogram were plotted to determine whether the signature genes can be used as a molecular marker. Gene set enrichment analysis was also performed to explore the potential mechanism of the signature genes. Finally, RNA expression of signature genes was validated in blood samples from PMOP patients and normal control by real-time quantitative polymerase chain reaction. Our study of PMOP patients identified differences in immune cells (activated dendritic cell, CD56 bright natural killer cell, Central memory CD4 T cell, Effector memory CD4 T cell, Mast cell, Natural killer T cell, T follicular helper cell, Type 1 T-helper cell, and Type 17 T-helper cell) between high and low BMD patients. We obtained a total of 73 candidate genes based on modular genes and differential genes, and obtained 5 signature genes by least absolute shrinkage and selection operator and random forest model screening. ROC, principal component analysis, and t-distributed stochastic neighbor embedding down scaling analysis revealed that the 5 signature genes had good discriminatory ability between high and low BMD samples. A logistic regression model was constructed based on 5 signature genes, and both ROC and column line plots indicated that the model accuracy and applicability were good. Five signature genes were found to be associated with proteasome, mitochondria, and lysosome by gene set enrichment analysis. The real-time quantitative polymerase chain reaction results showed that the expression of the signature genes was significantly different between the 2 groups. HIST1H2AG, PYGM, NCKAP1, POMP, and LYPLA1 might play key roles in PMOP and be served as the biomarkers of PMOP.

Correlation of TRPA1 RNAscope and Agonist Responses.

The TRPA1 ion channel is a sensitive detector of reactive chemicals, found primarily on sensory neurons. The phenotype exhibited by mice lacking TRPA1 suggests its potential as a target for pharmacological intervention. Antibody-based detection for distribution analysis is a standard technique. In the case of TRPA1, however, there is no antibody with a plausible validation in knockout animals or functional studies, but many that have failed in this regard. To this end we employed the single molecule in situ hybridization technique RNAscope on sensory neurons immediately after detection of calcium responses to the TRPA1 agonist allyl isothiocyanate. There is a clearly positive correlation between TRPA1 calcium imaging and RNAscope detection (R = 0.43), although less than what might have been expected. Thus, the technique of choice should be carefully considered to suit the research question. The marginal correlation between TRPV1 RNAscope and the specific agonist capsaicin indicates that such validation is advisable for every RNAscope target. Given the recent description of a long-awaited TRPA1 reporter mouse, TRPA1 RNAscope detection might still have its use cases, for detection of RNA at particular sites, for example, defined structurally or by other molecular markers.

Novel PHOTOPERIOD-1 gene variants associate with yield-related and root-angle traits in European bread wheat

Key messagePHOTOPERIOD-1 homoeologous gene copies play a pivotal role in regulation of flowering time in wheat. Here, we show that their influence also extends to spike and shoot architecture and even impacts root development.The sequence diversity of three homoeologous copies of the PHOTOPERIOD-1 gene in European winter wheat was analyzed by Oxford Nanopore amplicon-based multiplex sequencing and molecular markers in a panel of 194 cultivars representing breeding progress over the past 5 decades. A strong, consistent association with an average 8% increase in grain yield was observed for the PpdA1-Hap1 haplotype across multiple environments. This haplotype was found to be linked in 51% of cultivars to the 2NS/2AS translocation, originally introduced from Aegilops ventricosa, which leads to an overestimation of its effect. However, even in cultivars without the 2NS/2AS translocation, PpdA1-Hap1 was significantly associated with increased grain yield, kernel per spike and kernel per m2 under optimal growth conditions, conferring a 4% yield advantage compared to haplotype PpdA1-Hap4. In contrast to Ppd-B1 and Ppd-D1, the Ppd-A1 gene exhibits novel structural variations and a high number of SNPs, highlighting the evolutionary changes that have occurred in this region over the course of wheat breeding history. Additionally, cultivars carrying the photoperiod-insensitive Ppd-D1a allele not only exhibit earlier heading, but also deeper roots compared to those with photoperiod-sensitive alleles under German conditions. PCR and KASP assays have been developed that can be effectively employed in marker-assisted breeding programs to introduce these favorable haplotypes.

PCDHGA10 as a Potential Biomarker of Lung Squamous Cell Carcinoma Based on Bioinformatics and Experimental Verification.

Procalcitonin gamma subfamily A, 10 (PCDHGA10) is a member of the procalcitonin gamma gene cluster, which is associated with neuronal synapse development. However, there are lack of studies on the role and potential prognostic value of PCDHGA10 in lung squamous cell carcinoma (LUSC). We analyzed the RNAseq data of PCDHGA10 to compare their expression differences. Then survival analysis, tumor microenvironment (TME) analysis, and mutation analysis were carried out. Additionally, we performed gene ontology (GO) and kyoto gene encyclopedia (KEGG) enrichment analyses to explore potential signal pathways. PCDHGA10 protein expression was evaluated using immunohistochemistry (IHC) on tissue microarrays (HLugS180Su02). By microarray analysis and database analysis, we found that PCDHGA10 was significantly highly expressed in LUSC. Sufferers with elevated PCDHGA10 levels exhibited a worse prognosis, according to the survival analysis. The PCDHGA10 mutation was also linked to LUSC patient prognosis. Besides, PCDHGA10 was closely related to tumor immune cell infiltration andimmunecheckpoints. In conclusion, PCDHGA10 is expected to become a new molecular marker for LUSC.

Selection efficiency of molecular markers associated to Yellow Mosaic Virus (YMV) tolerance and identification of donors for earliness, yield and YMV tolerance in blackgram (Vigna mungo (L.) Hepper) using GT Biplot analysis

Selection efficiency of molecular markers associated to Yellow Mosaic Virus (YMV) tolerance and identification of donors for earliness, yield and YMV tolerance in blackgram (Vigna mungo (L.) Hepper) using GT Biplot analysis

Towards a species-level phylogeny for Neotropical Myrtaceae: Notes on topology and resources for future studies.

Increasingly complete phylogenies underpin studies in systematics, ecology, and evolution. Myrteae (Myrtaceae), with ~2700 species, is a key component of the exceptionally diverse Neotropical flora, but given its complicated taxonomy, automated assembling of molecular supermatrices from public databases often lead to unreliable topologies due to poor species identification. Here, we build a taxonomically verified molecular supermatrix of Neotropical Myrteae by assembling 3909 published and 1004 unpublished sequences from two nuclear and seven plastid molecular markers. We infer a time-calibrated phylogenetic tree that covers 712 species of Myrteae (~28% of the total diversity in the clade) and evaluate geographic and taxonomic gaps in sampling. The tree inferred from the fully concatenated matrix mostly reflects the topology of the plastid data set and there is a moderate to strong incongruence between trees inferred from nuclear and plastid partitions. Large, species-rich genera are still the poorest sampled within the group. Eastern South America is the best-represented area in proportion to its species diversity, while Western Amazon, Mesoamerica, and the Caribbean are the least represented. We provide a time-calibrated tree that can be more reliably used to address finer-scale eco-evolutionary questions that involve this group in the Neotropics. Gaps to be filled by future studies include improving representation of taxa and areas that remain poorly sampled, investigating causes of conflict between nuclear and plastid partitions, and the role of hybridization and incomplete lineage sorting in relationships that are poorly supported.

Probing Genetics and Environmental Factors underlying Uterine Fibroid Tumorigenesis in Ghana, West Africa

Uterine fibroid (UF) is the most prevalent benign tumour that affects millions of women globally, with a high incidence of 70% amongst women of reproductive age. UF has been associated with various complications, such as recurrent surgeries, infertility, anemia and pregnancy loss. Notably, women of African descent often experience more severe symptoms and complications. Although hormones, growth factors, and genetic alterations are widely associated with UF, the precise mechanism underlying its pathogenesis is not fully understood. Recent evidence suggests altered microbiota may serve as a potential risk factor for UF development. Altered microbiota can contribute to tumorigenesis via epigenetic changes to host cells or toxic effects from invasion. The lack of curative-drug treatment poses significant challenges to patients with UF. Patients often undergo surgeries that require the removal of the uterus or tumour, which can negatively impact fertility. Furthermore, uterine fibroids’ diagnosis relies on expensive imaging technologies such as ultrasound, which may not be readily available in developing countries. Moreso, diagnosis is often conducted only after patients’ symptoms become severe. Although late presentation may contribute to severe symptoms and complications among women with UF in Africa, other factors that influence severity and increase incidence in this population remain unknown. A comprehensive assessment of UF predisposing factors in high-risk populations such as Ghana could give better insights into disease pathogenesis. Hence, this study aims to assess: UF-associated demographic factors, the role of uterine microbiota dysbiosis on UF tumorigenesis; and molecular markers associated with UF in the Ghanaian population. Epidemiological data and clinical samples (tissues, blood and cervico-vaginal swabs) will be obtained. The characterization of samples will involve metagenomics, whole genome sequencing, functional validation of SNPs and SNP genotyping. The association of risk alleles with disease phenotypes will be assessed via regression analysis using PLINK v.1.9. The findings will provide information on potential disease markers that can be explored for better management strategies for UF in high-risk populations.

Serum AXL is a potential molecular marker for predicting COVID-19 progression

BackgroundThe severity, symptoms, and outcome of COVID-19 is thought to be closely linked to how the virus enters host cells. This process involves the key roles of angiotensin-converting enzyme 2 (ACE2) and the Tyrosine protein kinase receptor UFO (AXL) receptors. However, there is limited research on the circulating levels of ACE2 and AXL and their implications in COVID-19.MethodsA control group of 71 uninfected individuals was also included in the study. According to the Guidance for Corona Virus Disease 2019 (10th edition), a cohort of 358 COVID-19 patients were categorized into non-severe and severe cases. Serum ACE2/AXL levels in COVID-19 patients were detected by enzyme-linked immunosorbent assay (ELISA) at different time points post-COVID-19 infection, including days 0-7, 8-15, 31-179 and >180 days. Serum SARS-CoV-2 IgG/IgM antibodies in COVID-19 patients at the same intervals were assessed by using an iFlash 3000 Chemiluminescence Immunoassay Analyzer. The receiver operating characteristic (ROC) curves were used to assess the diagnostic value of the biological markers, and the association between laboratory parameters and illness progression were explored.ResultsCompared with the uninfected group, the levels of ACE2 and AXL in the COVID-19 group were decreased, and the SARS-COV-2 IgG level was increased. AXL (AUC = 0.774) demonstrated a stronger predictive ability for COVID-19 than ACE2. In the first week after infection, only the level of AXL was statistically different between severe group and non-severe group. After first week, the levels of ACE2 and AXL were different in two groups. Moreover, in severe COVID-19 cases, the serum ACE2, AXL, and SARS-COV-2 IgM levels reached a peak during days 8–15 before declining, whereas serum SARS-COV-2 IgG levels continued to rise, reaching a peak at day 31-180 days before decreasing. In addition, the AXL level continued to decrease and the SARS-COV-2 IgG level continued to increase in the infected group after 180 days compared to the uninfected group.ConclusionsThe levels of serum ACE2 and AXL correlate with COVID-19 severity. However, AXL can also provide early warning of clinical deterioration in the first week after infection. AXL appears to be a superior potential molecular marker for predicting COVID-19 progression.

Development of Novel KASP Markers for Improved Germination in Deep-Sown Direct Seeded Rice

BackgroundThe lack of stable-high yielding and direct-seeded adapted varieties with better germination ability from deeper soil depth and availability of molecular markers are major limitation in achieving the maximum yield potential of rice under water and resource limited conditions. Development of high-throughput and trait-linked markers are of great interest in genomics-assisted breeding. The aim of present study was to develop and validate novel KASP (Kompetitive Allele-Specific PCR) markers associated with traits improving germination and seedling vigor of deep sown direct seeded rice (DSR).ResultsOut of 58 designed KASP assays, four KASP assays did not show any polymorphism in any of the eleven genetic backgrounds considered in the present study. The 54 polymorphic KASP assays were then validated for their robustness and reliability on the F1s plants developed from eight different crosses considered in the present study. The third next validation was carried out on 256 F3:F4 and 713 BC3F2:3 progenies. Finally, the reliability of the KASP assays was accessed on a set of random 50 samples from F3:F4 and 80–100 samples from BC3F2:3 progenies using the 10 random markers. From the 54 polymorphic KASP, based on the false positive rate, false negative rate, KASP utility in different genetic backgrounds and significant differences in the phenotypic values of the positive (desirable) and negative (undesirable) traits, a total of 12 KASP assays have been selected. These 12 KASP include 5 KASP on chromosome 3, 1 on chromosome 4, 3 on chromosome 7 and 3 on chromosome 8. The two SNPs lying in the exon regions of LOC_Os04g34290 and LOC_Os08g32100 led to non-synonymous mutations indicating a possible deleterious effect of the SNP variants on the protein structure.ConclusionThe present research work will provide trait-linked KASP assays, improved breeding material possessing favourable alleles and breeding material in form of expected pre-direct-seeded adapted rice varieties. The marker can be utilized in introgression program during pyramiding of valuable QTLs/genes providing adaptation to rice under DSR. The functional studies of the genes LOC_Os04g34290 and LOC_Os08g32100 possessing two validated SNPs may provide valuable information about these genes.

Genetic Profile of Greek Indigenous Honey Bee Populations, Local Extinctions, Geographical Distinction, and Patterns, by Using mtDNA COI and Nuclear CSD Gene Analysis Markers

Evaluation and conservation of local genetic resources of the domestic honey bee populations is important, especially in regions with high diversity levels as well as high honey bee colony density. Greece is rich in honey bee biodiversity, hosting several subspecies, with the status of them being, though, doubtful. The purpose of the present study was to investigate the genetic relationships of both stationary and movable honey bee populations, originating from many location throughout Greece. Two molecular markers were utilized, namely, the conserved mitochondrial COI gene and the highly variable complementary sex determination (CSD) gene. Samples were collected from nine distant populations: eight populations from colonies that followed the traditional stationary beekeeping model and one following the modern migratory beekeeping model type, where the hives are transferred from place to place according to the season. Regardless the beekeeping model, all populations were characterized by sufficient genetic diversity indicating no signs of inbreeding or any bottleneck effects. Nevertheless, genetic differentiation and phylogenetic analysis in comparison with haplotypes obtained from GenBank revealed a genetic admixture pattern suggesting that movement causes genetic homogeneity, occasionally in the stable reared populations as well. Interestingly, two populations, namely, Kastoria and Protokklisi, belonging to A. m. macedonica population, were significantly differentiated, supporting the maintenance of their genetic integrity. Unfortunately, on the other hand, genetic structure of the populations from Crete (Sasalos population) provided evidence that the indigenous breed from the island, A. m. adami, has probably gone extinct. Future management strategies should focus on the conservation of the local genetic resources in which distinct genetic identity has been sustained.

Extracorporeal photopheresis (ECP) in the treatment of chronic lung allograft dysfunction (CLAD): a prospective, multicentre, open-label, randomised controlled trial studying the addition of ECP to standard care in the treatment of bilateral lung transplant patients with CLAD (E-CLAD UK)

BackgroundLong-term survival after lung transplantation is limited compared with other organ transplants. The main cause is development of progressive immune-mediated damage to the lung allograft. This damage, which can develop...

Advances in Breeding for Oil Quality Enhancement in Indian Mustard (Brassica spp. L.): Achievements, Challenges, and Research Opportunities

Major objectives in oil crop improvement are enhancement of seed and oil yield, quality of oil according to its use, i.e. edible or industrial uses, breeding of varieties that fit in different cropping systems and breeding biotic and abiotic stress resistant/tolerant varieties. Despite traditional breeding approaches, including pure line breeding, yielding only modest gains in productivity, recent advancements in mustard breeding have led to significant breakthroughs in both productivity and oil quality. This review discusses the innovative breeding strategies that have contributed to these advancements, with a focus on hybrid development, oil quality enhancement, and biotechnological approaches. To enhance productivity, researchers at the University of Delhi have developed hybrid seed production techniques using transgenic Barnase-barstar systems and cytoplasmic male sterility (CMS) systems. These systems enable large-scale hybrid seed production, with field trials demonstrating significant yield heterosis ranging from 31% to 55% compared to national check varieties. In addition to productivity, improving oil and meal quality has been a key objective. By integrating genes from canola-quality mustard lines, breeders have achieved reductions in erucic acid and glucosinolates, enhancing the health profile and industrial applicability of mustard oil. A high-density linkage map developed using an F1 double haploid mapping population has facilitated the marker-assisted backcross breeding of desirable traits, enabling precise transfer of key quality traits. Transgenic approaches, such as antisense RNA technology, have led to the development of high-oleic, low-linoleic mustard lines with improved fatty acid profiles. These advancements reflect a strategic combination of conventional and biotechnological methods, demonstrating a clear pathway for boosting mustard yields while enhancing oil quality. Molecular markers reported for genetic diversity assessment, mapping and tagging genes/QTLs for different qualitative and quantitative traits and their use in marker-assisted selection have been presented. This progress not only addresses current challenges but also sets the stage for future research aimed at further optimizing productivity, oil quality, and resistance to pests and diseases in mustard cultivation.

Tracing Geographic and Molecular Footprints of Copepod Crustaceans Causing Multifocal Purple Spots Syndrome in the Caribbean Sea Fan Gorgonia ventalina

The recent rise in ocean temperatures, accompanied by other environmental changes, has notably increased the occurrence and spread of diseases in Octocorallia, many species of which are integral to shallow tropical and subtropical coral reef ecosystems. This study focuses on the understanding of these diseases, which has been largely limited to symptomatic descriptions, with clear etiological factors identified in only a fraction of cases. A key example is the multifocal purple spots syndrome (MFPS) affecting the common Caribbean octocoral sea fan Gorgonia ventalina, linked to the gall-forming copepods of the genus Sphaerippe, a member of the widespread family, Lamippidae. The specialized nature of these copepods as endoparasites in octocorals suggests the potential for the discovery of similar diseases across this host spectrum. Our investigation employed four molecular markers to study disease hotspots in Saint Eustatius, Curaçao, northwest and southwest Cuba, and Bonaire. This led to the discovery of a group of copepod species in these varied Caribbean locations. Importantly, these species are morphologically indistinguishable through traditional methods, challenging established taxonomic approaches. The observed diversity of symbionts, despite the host species’ genetic uniformity, is likely due to variations in larval dispersal mechanisms. Our phylogenetic analyses confirmed that the Lamippidae copepods belong to the order, Poecilostomatoida (Copepoda), and revealed their sister group relationship with the Anchimolgidae, Rhynchomolgidae, and Xarifiidae clades, known for their symbiotic relationships with scleractinian corals. These results add to our understanding of the evolutionary and ecological interactions of copepods and their hosts, and the diseases that they cause, and are important data in a changing climate.

Regulation of cellular and molecular markers of epithelial-mesenchymal transition by Brazilin in breast cancer cells

Breast cancer is the most common invasive neoplasm and the leading cause of cancer death in women worldwide. The main cause of mortality in cancer patients is invasion and metastasis, where the epithelial-mesenchymal transition (EMT) is a crucial player in these processes. Pharmacological therapy has plants as its primary source, including isoflavonoids. Brazilin is an isoflavonoid isolated from Haematoxilum brasiletto that has shown antiproliferative activity in several cancer cell lines. In this study, we evaluated the effect of Brazilin on canonical markers of EMT such as E-cadherin, vimentin, Twist, and matrix metalloproteases (MMPs). By Western blot, we evaluated E-cadherin, vimentin, and Twist expression and the subcellular localization by immunofluorescence. Using gelatin zymography, we determined the levels of secretion of MMPs. We used Transwell chambers coated with matrigel to determine the in vitro invasion of breast cancer cells treated with Brazilin. Interestingly, our results show that Brazilin increases 50% in E-cadherin expression and decreases 50% in vimentin and Twist expression, MMPs, and cell invasion in triple-negative breast cancer (TNBC) MDA-MB-231 and to a lesser extend in MCF7 ER+ breast cancer cells. Together, these findings position Brazilin as a new molecule with great potential for use as complementary or alternative treatment in breast cancer therapy in the future.

Validation of an Organ Mapping Antibody Panel for Cyclical Immunofluorescence Microscopy on Normal Human Kidneys.

The lack of standardization in antibody validation remains a major contributor to irreproducibility of human research. To address this, we have applied a standardized approach to validate a panel antibodies to identify 18 major cell types and 5 extracellular matrix compartments in the human kidney by immunofluorescence (IF) microscopy. We have used these to generate an organ mapping antibody panel for 2-D and 3-D Cyclical Immunofluorescence (CyCIF) to provide a more detailed method to evaluate of tissue segmentation and volumes using a larger panel of markers than would normally be possible using standard fluorescence microscopy. CyCIF also makes it possible to perform multiplexed IF microscopy of whole slide images, which is a distinct advantage over other multiplexed imaging technologies that are applicable to limited fields of view. This enables a broader view of cell distributions across larger anatomical regions, allowing a better chance to capture localized regions of dysfunction in diseased tissues. These methods are broadly accessible to any laboratory with a fluorescence microscope, enabling spatial cellular phenotyping in normal and disease states. We also provide a detailed solution for image alignment between CyCIF cycles that can be used by investigators to perform these studies without programming experience using open-sourced software. This ability to perform multiplexed imaging without specialized instrumentation or computational skills, opens the door to integration with more highly dimensional molecular imaging modalities such as spatial transcriptomics and imaging mass spectrometry, enabling the discovery of molecular markers of specific cell types and how these are altered in disease.