Cancer-associated fibroblasts: a versatile mediator in tumor progression, metastasis, and targeted therapy.

Tumor microenvironment (TME) has been demonstrated to play a significant role in tumor initiation, progression, and metastasis. Cancer-associated fibroblasts (CAFs) are the major component of TME and exhibit heterogeneous properties in their communication with tumor cells. This heterogeneity of CAFs can be attributed to various origins, including quiescent fibroblasts, mesenchymal stem cells (MSCs), adipocytes, pericytes, endothelial cells, and mesothelial cells. Moreover, single-cell RNA sequencing has identified diverse phenotypes of CAFs, with myofibroblastic CAFs (myCAFs) and inflammatory CAFs (iCAFs) being the most acknowledged, alongside newly discovered subtypes like antigen-presenting CAFs (apCAFs). Due to these heterogeneities, CAFs exert multiple functions in tumorigenesis, cancer stemness, angiogenesis, immunosuppression, metabolism, and metastasis. As a result, targeted therapies aimed at the TME, particularly focusing on CAFs, are rapidly developing, fueling the promising future of advanced tumor-targeted therapy.

Gene Expression Profiles of AHNAK2, DCSTAMP, FN1, and TERT Correlate With Mutational Status and Recurrence in Papillary Thyroid Carcinoma.

Papillary thyroid carcinoma (PTC), the most common malignancy of follicular cell derivation, is generally associated with good prognosis. Nevertheless, it is important to identify patients with aggressive PTCs and unfavorable outcome. Molecular markers such as BRAFV600E mutation and TERT promoter mutations have been proposed for risk stratification. While TERT promoter mutations have been frequently associated with aggressive PTCs, the association of BRAFV600E mutation with increased recurrence and mortality is less clear and has been controversially discussed. The aim of the present study was to analyze whether differentially expressed genes can predict BRAFV600E mutations as well as TERT promoter mutations in PTCs. RNA sequencing identified a large number of differentially expressed genes between BRAFV600E and BRAFwildtype PTCs. Of those, AHNAK2, DCSTAMP, and FN1 could be confirmed in a larger cohort (n = 91) to be significantly upregulated in BRAFV600E mutant PTCs using quantitative RT-PCR. Moreover, individual PTC expression values of DCSTAMP and FN1 were able to predict the BRAFV600E mutation status with high sensitivity and specificity. The expression of TERT was detected in all PTCs harboring TERT promoter mutations and in 19% of PTCs without TERT promoter mutations. Tumors with both TERT expression and TERT promoter mutations were particularly associated with aggressive clinicopathological features and a shorter recurrence-free survival. Altogether, it will be interesting to explore the biological function of AHNAK2, DCSTAMP, and FN1 in PTC in more detail. The analysis of their expression patterns could allow the characterization of PTC subtypes and thus enabling a more individualized surgical and medical treatment.

Downregulation of JAM3 occurs in cholangiocarcinoma by hypermethylation: A potential molecular marker for diagnosis and prognosis.

Junctional adhesion molecular 3 (JAM3) is downregulated by hypermethylation in cancers but is unclear in cholangiocarcinoma. The JAM3 expression level was checked in cholangiocarcinoma cell lines and tissues. Methylated JAM3 was detected in cell lines, tissues and plasma cell-free DNAs (cfDNA). The roles of JAM3 in cholangiocarcinoma were studied by transfection of siRNA and pCMV3-JAM3. The survival analysis was based on the Gene Set Cancer Analysis (GSCA) database. JAM3 was downregulated in HCCC-9810 and HuCCT1 cell lines and tissues by hypermethylation. Methylated JAM3 was detected in cfDNAs with 53.3% sensitivity and 96.6% specificity. Transfection of pCMV3-JAM3 into HCCC-9810 and HuCCT1 induced apoptosis and suppressed cell proliferation, migration and invasion. The depletion of JAM3 in RBE cells using siRNA decreased apoptosis and increased cell proliferation, migration and invasion. Hypermethylation of JAM3 was associated with tumour differentiation, metastasis and TNM stage. Downregulation and hypermethylation of JAM3 were related to poor progression-free survival. Junctional adhesion molecular 3 may function as a tumour suppressor in cholangiocarcinoma. Methylated JAM3 DNA may represent a non-invasive molecular marker for the early detection of cholangiocarcinoma and prognosis.

Identification of sex-specific markers using genome re-sequencing in the blunt snout bream (Megalobrama amblycephala).

BACKGROUND: The blunt snout bream (Megalobrama amblycephala) is an important economic freshwater fish in China with tender flesh and high nutritional value. With the cultivation of superior new varieties and the expansion of breeding scale, it becomes imperative to employ sex-control technology to cultivate monosexual populations of M. amblycephala, thereby preventing the deterioration of desirable traits. The development of specific markers capable of accurately identifying the sex of M. amblycephala would facilitate the determination of the genetic sex of the breeding population before gonad maturation, thereby expediting the processes of sex-controlled breeding of M. amblycephala. RESULTS: A whole-genome re-sequencing was performed for 116 females and 141 males M. amblycephala collected from nine populations. Seven candidate male-specific sequences were identified through comparative analysis of male and female genomes, which were further compared with the sequencing data of 257 individuals, and finally three male-specific sequences were generated. These three sequences were further validated by PCR amplification in 32 males and 32 females to confirm their potential as male-specific molecular markers for M. amblycephala. One of these markers showed potential applicability in M. pellegrini as well, enabling males to be identified using this specific molecular marker. CONCLUSIONS: The study provides a high-efficiency and cost-effective approach for the genetic sex identification in two species of Megalobrama. The developed markers in this study have great potential in facilitating sex-controlled breeding of M. amblycephala and M. pellegrini, while also contributing valuable insights into the underlying mechanisms of fish sex determination.

Genome-wide identification of the endonuclease family genes implicates potential roles of TaENDO23 in drought-stressed response and grain development in wheat.

BACKGROUND: Endonucleases play a crucial role in plant growth and stress response by breaking down nuclear DNA. However, the specific members and biological functions of the endonuclease encoding genes in wheat remain to be determined. RESULTS: In this study, we identified a total of 26 TaENDO family genes at the wheat genome-wide level. These genes were located on chromosomes 2 A, 2B, 2D, 3 A, 3B, and 3D and classified into four groups, each sharing similar gene structures and conserved motifs. Furthermore, we identified diverse stress-response and growth-related cis-elements in the promoter of TaENDO genes, which were broadly expressed in different organs, and several TaENDO genes were significantly induced under drought and salt stresses. We further examined the biological function of TaENDO23 gene since it was rapidly induced under drought stress and exhibited high expression in spikes and grains. Subcellular localization analysis revealed that TaENDO23 was localized in the cytoplasm of wheat protoplasts. qRT-PCR results indicated that the expression of TaENDO23 increased under PEG6000 and abscisic acid treatments, but decreased under NaCl treatment. TaENDO23 mainly expressed in leaves and spikes. A kompetitive allele-specific PCR (KASP) marker was developed to identify single nucleotide polymorphisms in TaENDO23 gene in 256 wheat accessions. The alleles with TaENDO23-HapI haplotypes had higher grain weight and size compared to TaENDO23-HapII. The geographical and annual frequency distributions of the two TaENDO23 haplotypes revealed that the elite haplotype TaENDO23-HapI was positively selected in the wheat breeding process. CONCLUSION: We systematically analyzed the evolutionary relationships, gene structure characteristics, and expression patterns of TaENDO genes in wheat. The expression of TaENDO23, in particular, was induced under drought stress, mainly expressed in the leaves and grains. The KASP marker of TaENDO23 gene successfully distinguished between the wheat accessions, revealing TaENDO23-HapI as the elite haplotype associated with improved grain weight and size. These findings provide insights into the evolution and characteristics of TaENDO genes at the genome-wide level in wheat, laying the foundation for further biological analysis of TaENDO23 gene, especially in response to drought stress and grain development.

Rice Big Grain1 improves grain yield in ectopically expressing rice and heterologously expressing tobacco plants.

Functional genomics through transgenesis has provided faster and more reliable methods for identifying, characterizing, and utilizing genes or quantitative trait loci linked to agronomic traits to target yield. The present study explored the role of Big Grain1 (BG1) gene of rice (Oryza sativa L.) in yield improvement of crop plants. We aimed to identify the genetic variation of OsBG1 in various indica rice cultivars by studying the allelic polymorphism of the gene, while also investigating the gene's potential to increase crop yield through the transgenic approach. Our study reports the presence of an extra 393 bp sequence having two 6 bp enhancer elements in the 3' regulatory sequence of OsBG1 in the large-grain cultivar IR64 but not in the small-grain cultivar Badshahbhog. A single copy of the OsBG1 gene in both the cultivars and a 4.1-fold higher expression of OsBG1 in IR64 than in Badshahbhog imply that the grain size is positively correlated with the level of OsBG1 expression in rice. The ectopic expression of OsBG1 under the endosperm-specific glutelin C promoter in Badshahbhog enhanced the flag leaf length, panicle weight, and panicle length by an average of 33.2%, 33.7%, and 30.5%, respectively. The length of anthers, spikelet fertility, and grain yield per plant increased in transgenic rice lines by an average of 27.5%, 8.3%, and 54.4%, respectively. Heterologous expression of OsBG1 under the constitutive 2xCaMV35S promoter improved the number of seed pods per plant and seed yield per plant in transgenic tobacco lines by an average of 2.2-fold and 2.6-fold, respectively. Improving crop yield is crucial to ensure food security and socio-economic stability, and identifying suitable genetic factor is the essential step towards this endeavor. Our findings suggest that the OsBG1 gene is a promising candidate for improving the grain yield of monocot and dicot plant systems by molecular breeding and genetic engineering.

Development of Yein-Early, a Unique Fruit-Color and Leaf-Shape Mutant of Citrus unshiu, and Its Specific Selection Marker.

Citrus unshiu Marc. cv. Miyagawa-wase is one of the most widely cultivated citrus varieties on Jeju Island in Republic of Korea. Mutation breeding is a useful tool for inducing genetic diversity by causing genomic mutations in a short period of time. We previously conducted mutation breeding using gamma irradiation to develop new varieties of C. unshiu. Here, we describe one of these varieties, Yein-early, which has a redder peel, greater hardness, and higher sugar content compared with the wild type (WT). Yein-early leaves also showed a unique phenotype compared with the WT, characterized by longer longitudinal length, shorter transverse length, stronger curling, and longer petiole length. Genome resequencing of Yein-early and the WT uncovered significant single-nucleotide polymorphisms (SNPs) and insertions/deletions (InDels). These variations were crucial in identifying molecular markers unique to Yein-early. In addition, we developed an allele-specific PCR marker specifically targeting a homozygous SNP in Yein-early that distinguishes it from the WT and other citrus varieties. This study contributes to the understanding of pigment synthesis in fruits and provides a valuable tool for selection of the novel Yein-early variety in citrus breeding programs.

Employment of pqqE gene as molecular marker for the traceability of Gram negative phosphate solubilizing bacteria associated to plants.

Insoluble phosphorous compounds solubilization by soil bacteria is of great relevance since it puts available the phosphorus to be used by plants. The production of organic acids is the main microbiological mechanism by which insoluble inorganic phosphorus compounds are solubilized. In Gram negative bacteria, gluconic acid is synthesized by the activity of the holoenzyme glucose dehydrogenase-pyrroloquinoline quinine named GDH-PQQ. The use of marker genes is a very useful tool to evaluate the persistence of the introduced bacteria and allow to follow-up the effect of biotic and abiotic factors on these beneficial microorganisms in the soil. In previous studies we detected the presence of the pqqE gene in a great percentage of both non-culturable and culturable native soil bacteria. The objective of this study was to analyze the phylogeny of the sequence of pqqE gene and its potential for the study of phosphate solubilizing bacteria from pure and mixed bacterial cultures and rhizospheric soil samples. For this, the presence of the pqqE gene in the genome of phosphate solubilizing bacteria that belong to several bacteria was determined by PCR. Also, this gene was analyzed from mixed bacterial cultures and rhizospheric soil associated to peanut plants inoculated or not with phosphate solubilizing bacteria. For this, degenerate primers designed from several bacterial genera and specific primers for the genus Pseudomonas spp., designed in this study, were used. DNA template used from simple or mixed bacterial cultures and from rhizospheric soil samples was obtained using two different DNA extraction techniques. Results indicated that pqqE gene amplification product was found in the genome of all Gram negative phosphate solubilizing bacteria analyzed. It was possible to detect this gene in the DNA obtained from mixed cultures where these bacteria grew in interaction with other microorganisms and in that obtained from rhizospheric soil samples inoculated or not with these bacteria. The phylogenetic analysis indicated that pqqE gene is a conserved gene within related genera. In conclusion, pqqE gene could be a potential marker for the study of phosphate solubilizing bacterial populations.

Comparison of the chloroplast genomics of nine endangered Habenaria species and phylogenetic analysis.

BACKGROUND: Habenaria, a genus in the family Orchidaceae, are the nearly cosmopolitan orchids, and most species have significant medicinal and ornamental values. Despite the morphological and molecular data that have been studied in recent years, the phylogenetic relationship is still unclear. RESULTS: We sequenced, assembled, and annotated the chloroplast (cp) genomes of two species (Habenaria aitchisonii Rchb.f. and Habenaria tibetica Schltr.ex Limpricht) of Habenaria grown on the Qinghai-Tibetan Plateau (QTP), and compared them with seven previously published cp genomes which may aid in the genomic profiling of these species. The two genomes ranged from 155,259-155,269 bp in length and both included 132 genes, encoding 86 proteins, 38 tRNAs and 8 rRNAs. In the cp genomes, the tandem repeats (797), SSRs (2195) and diverse loci (3214) were identified. Comparative analyses of codon usage, amino frequency, microsatellite, oligo repeats and transition and transversion substitutions revealed similarities between the species. Moreover, we identified 16 highly polymorphic regions with a nucleotide diversity above 0.02, which may be suitable for robust authentic barcoding and inferring in the phylogeny of Habenaria species. Among the polymorphic regions, positive selection was significantly exerted on several genes, such as cemA, petA, and ycf1. This finding may suggest an important adaptation strategy for the two Habenaria species on the QTP. The phylogenetic relationship revealed that H. aitchisonii and H. tibetica were more closely related to each other than to the other species, and the other seven species were clustered in three groups. In addition, the estimated divergence time suggested that the two species separated from the others approximately 0.39 Mya in the Neogene period. Our findings also suggest that Habenaria can be divided into different sections. CONCLUSIONS: The results of this study enriched the genomics resources of Habenaria, and SSR marker may aid in the conservation management of two endangered species.

Investigation of genetic diversity using molecular and biochemical markers associated with powdery mildew resistance in different flax (Linum usitatissimum L.) genotypes.

Under greenhouse conditions, the resistance of 18 different genotypes of flax to powdery mildew was evaluated. To investigate genetic diversity and identify the molecular and biochemical markers linked to powdery mildew resistance in the tested genotypes, two molecular marker systems-start codon targeted (SCoT) and inter-simple sequence repeat (ISSR)-as well as a biochemical marker (protein profiles, antioxidant enzyme activity, and secondary metabolites) were used. Based on the results, the genotypes were classified into four categories: highly susceptible, susceptible, moderately susceptible, and moderately resistant. The genotypes differed significantly in powdery mildew severity: Polk had a severity of 92.03% and Leona had a severity of 18.10%. Compared to the other genotypes, the moderately resistant genotypes had higher levels of flavonoids, antioxidant enzymes, phenolics, and straw yield; nevertheless, their hydrogen peroxide and malondialdehyde levels were lower. Protein profiles revealed 93.75% polymorphism, although the ISSR marker displayed more polymorphism (78.4%) than the SCoT marker (59.7%). Specific molecular and biochemical markers associated with powdery mildew resistance were identified. The 18 genotypes of flax were divided into two major clusters by the dendrogram based on the combined data of molecular markers. The first main cluster included Leona (genotype number 7), considered moderate resistance to powdery mildew and a separate phenetic line. The second main cluster included the other 17 genotypes, which are grouped together in a sub-cluster. This means that, besides SCoT, ISSR markers can be a useful supplementary technique for molecular flax characterization and for identifying genetic associations between flax genotypes under powdery mildew infection.

Fine mapping of TFL, a major gene regulating fruit length in snake gourd (Trichosanthes anguina L).

Fruit length is a crucial agronomic trait of snake gourd (Trichosanthes anguina L); however, genes associated with fruit length have not been characterised. In this study, F2 snake gourd populations were generated by crossing the inbred lines, S1 and S2 (fruit lengths: 110 and 20 cm, respectively). Subsequently, bulk segregant analysis, sequencing, and fine-mapping were performed on the F2 population to identify target genes. Our findings suggest that the fruit length of snake gourd is regulated by a major-effect regulatory gene. Mining of genes regulating fruit length in snake gourd to provide a basis for subsequent selection and breeding of new varieties. Genotype-phenotype association analysis was performed on the segregating F2 population comprising 6,000 plants; the results indicate that the target gene is located on Chr4 (61,846,126-61,865,087 bp, 18.9-kb interval), which only carries the annotated candidate gene, Tan0010544 (designated TFL). TFL belongs to the MADS-box family, one of the largest transcription factor families. Sequence analysis revealed a non-synonymous mutation of base C to G at position 202 in the coding sequence of TFL, resulting in the substitution of amino acid Gln to Glu at position 68 in the protein sequence. Subsequently, an InDel marker was developed to aid the marker-assisted selection of TFL. The TFL in the expression parents within the same period was analysed using quantitative real-time PCR; the TFL expression was significantly higher in short fruits than long fruits. Therefore, TFL can be a candidate gene for determining the fruit length in snake gourd. Collectively, these findings improve our understanding of the genetic components associated with fruit length in snake gourds, which could aid the development of enhanced breeding strategies for plant species.

High-yield hybrid breeding of Camellia oleifolia based on ISSR molecular markers.

BACKGROUND: C. Oleifera is among the world's largest four woody plants known for their edible oil production, yet the contribution rate of improved varieties is less than 20%. The species traditional breeding is lengthy cycle (20-30 years), occupation of land resources, high labor cost, and low accuracy and efficiency, which can be enhanced by molecular marker-assisted selection. However, the lack of high-quality molecular markers hinders the species genetic analysis and molecular breeding. RESULTS: Through quantitative traits characterization, genetic diversity assessment, and association studies, we generated a selection population with wide genetic diversity, and identified five excellent high-yield parental combinations associated with four reliable high-yield ISSR markers. Early selection criteria were determined based on kernel fresh weight and cultivated 1-year seedling height, aided by the identification of these 4 ISSR markers. Specific assignment of selected individuals as paternal and maternal parents was made to capitalize on their unique attributes. CONCLUSIONS: Our results indicated that molecular markers-assisted breeding can effectively shorten, enhance selection accuracy and efficiency and facilitate the development of a new breeding system for C. oleifera.

A 5.2-kb insertion in the coding sequence of PavSCPL, a serine carboxypeptidase-like enhances fruit firmness in Prunus avium.

Fruit firmness is an important trait in sweet cherry breeding because it directly positively influences fruit transportability, storage and shelf life. However, the underlying genes responsible and the molecular mechanisms that control fruit firmness remain unknown. In this study, we identified a candidate gene, PavSCPL, encoding a serine carboxypeptidase-like protein with natural allelic variation, that controls fruit firmness in sweet cherry using map-based cloning and functionally characterized PavSCPL during sweet cherry fruit softening. Genetic analysis revealed that fruit firmness in the 'Rainier' × 'Summit' F1 population was controlled by a single dominant gene. Bulked segregant analysis combined with fine mapping narrowed the candidate gene to a 473-kb region (7418778-7 891 914 bp) on chromosome 6 which included 72 genes. The candidate gene PavSCPL, and a null allele harbouring a 5244-bp insertion in the second exon that completely inactivated PavSCPL expression and resulted in the extra-hard-flesh phenotype, were identified by RNA-sequencing analysis and gene cloning. Quantitative RT-PCR analysis revealed that the PavSCPL expression level was increased with fruit softening. Virus-induced gene silencing of PavSCPL enhanced fruit firmness and suppressed the activities of certain pectin-degrading enzymes in the fruit. In addition, we developed functional molecular markers for PavSCPL and the Pavscpl5.2-k allele that co-segregated with the fruit firmness trait. Overall, this research identified a crucial functional gene for fruit firmness. The results provide insights into the genetic control and molecular mechanism of the fruit firmness trait and present useful molecular markers for molecular-assisted breeding for fruit firmness in sweet cherry.

Identification of major QTLs for drought tolerance in soybean, together with a novel candidate gene, GmUAA6.

Drought tolerance is a complex trait in soybean that is controlled by polygenetic quantitative trait loci (QTLs). In this study, wilting score, days-to-wilting, leaf relative water content, and leaf relative conductivity were used to identify QTLs associated with drought tolerance in recombinant inbred lines derived from a cross between a drought-sensitive variety, Lin, and a drought-tolerant variety, Meng. A total of 33 drought-tolerance QTLs were detected. Of these 17 were major QTLs. In addition, 15 were novel drought-tolerance QTLs. The most predominant QTL was on chromosome 11. This was detected in at least three environments. The overlapped mapping interval of the four measured traits was 0.2 cM in genetic distance (about 220 kb in physical length). Glyma.11g143500 (designated as GmUAA6), which encodes a UDP-N-acetylglucosamine transporter, was identified as the most likely candidate gene. The allele of GmUAA6 from Lin (GmUAA6Lin) was associated with improved soybean drought tolerance. Overexpression of GmUAA6Lin in Arabidopsis and soybean hairy roots enhanced drought tolerance. Furthermore, a 3-bp insertion/deletion (InDel) in the coding sequence of GmUAA6 explained up to 49.9% of the phenotypic variation in drought tolerance-related traits, suggesting that this InDel might be used in future marker-assisted selection of drought-tolerant lines in soybean breeding programs.

Molecular characterization of Indian races of Fusarium oxysporum f. sp. lentis (Fol) based on secreted in Xylem (SIX) effector genes and development of a SIX11 gene-based molecular marker for specific detection of Fol.

Fusarium wilt of lentil caused by Fusarium oxysporum f. sp. lentis (Fol) is a destructive pathogen limiting lentil production in India. In the present study, Secreted in Xylem (SIX) effectors genes were explored in Indian races of Fol and also a diagnostic tool for reliable detection of the disease was developed. Four SIX effectors genes, SIX11, SIX13, SIX6 and SIX2 were identified in 12 isolates of Fol belonging to seven races. SIX11 was present in all the races while SIX 13 was absent in race 6 and SIX6 was present only in race 4. The phylogenetic analysis revealed the conserved nature of the SIX genes within the forma specialis and showed sequence homology with F. oxysporum f. sp. pisi. The presence of three effectors, SIX11, SIX13 and SIX6 in race 4 correlates with high disease incidence in lentil germplasms. The in-silico characterization revealed the presence of signal peptide and localization of the effectors. Further SIX11 effector gene present in all the isolates was used to develop Fol-specific molecular marker for accurate detection. The marker developed could differentiate F. oxysporum f. sp. lycopersici, F. solani, F. oxysporum, Rhizoctonia solani and Sclerotium rolfsii and had a detection limit of 0.01ng µL- 1. The effector-based marker detection helps in the unambiguous detection of the pathogen under field conditions.

Rapid breeding of an early maturing, high-quality, and high-y.ielding rice cultivar using marker­assisted selection coupled with optimized anther culture.

With the global shift towards healthier eating habits, the focus of the rice industry has evolved from quantity to quality. In China, the Yangtze River Basin is the main area consuming long-grain and high-quality indica rice. Hubei Province, a significant rice-producing area, currently cultivates a limited range of rice varieties, risking degradation and diminishing economic returns. Therefore, it is imperative to cultivate elite rice varieties tailored to the local production conditions and can significantly enhance the added value. This study bred the novel rice cultivar "Runxiangyu", characterized by early maturity, high quality, and high yield. It is a hybrid of Ezhong 5, known for its moderate height and excellent quality, albeit with a long growth period and lack of fragrance, and Yuzhenxiang, renowned for its high quality, short growth period, and fragrance but limited by its tall stature and poor tillering ability. The breeding process utilized optimized anther culture coupled with molecular marker-assisted selection (MAS) and phenotype analysis. In the field, the developed cultivar was 120.9 cm tall and had an entire growth period of 117.5 days, demonstrating moderate disease resistance and excellent heat tolerance. Its grains are fragrant, meeting the national standard of grade two high-quality rice set by the Food Quality Supervision and Inspection Center of the Ministry of Agriculture and Rural Areas). Exhibiting superior agronomic traits, such as plant type, height, growth period, and stress resistance, along with and quality attributes, including grain shape, chalkiness, fragrance, and taste, "Runxiangyu" was certified by the Agricultural Crop Variety Certification Commission of Hubei in 2022. These findings suggested that molecular MAS coupled with optimized anther culture and multi-site phenotype analysis is an efficient and rapid method for crop breeding. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01495-4.

Cytogenetic and Genomic Characterization of a Novel Wheat-Tetraploid Thinopyrum elongatum 1BS⋠1EL Translocation Line with Stripe Rust Resistance.

Stripe rust, caused by Puccinia striiformis f. sp. tritici, is a destructive wheat disease pathogen. Thinopyrum elongatum is a valuable germplasm including diploid, tetraploid, and decaploid with plenty of biotic and abiotic resistance. In a previous study, we generated a stripe rust-resistant wheat-tetraploid Th. elongatum 1E/1D substitution line, K17-841-1. To further apply the wild germplasm for wheat breeding, we selected and obtained a new homozygous wheat-tetraploid Th. elongatum translocation line, T1BS⋅1EL, using genomic in situ hybridization, fluorescence in situ hybridization (FISH), oligo-FISH painting, and the wheat 55K single nucleotide polymorphism genotyping array. The T1BS⋅1EL is highly resistant to stripe rust at the seedling and adult stages. Pedigree and molecular marker analyses revealed that the resistance gene was located on the chromosome arm 1EL of tetraploid Th. elongatum, tentatively named Yr1EL. In addition, we developed and validated 32 simple sequence repeat markers and two kompetitive allele-specific PCR assays that were specific to the tetraploid Th. elongatum chromosome arm 1EL to facilitate marker-assisted selection for alien 1EL stripe rust resistance breeding. This will help us explore and locate the stripe rust resistance gene mapping on the 1E chromosome and deploy it in the wheat breeding program.

Molecular Markers and Marker-Assisted Selection Provide Genetic Insights for Identifying Key Quantitative Trait Locus for Watermelon Rind Thickness.

Rind thickness (RT) is an important agronomic trait in watermelon [Citrullus lanatus (Thunb.) Mansf.] and affects watermelon storability. However, genetic studies on this trait, as well as gene regulation studies, are scarce and of limited production significance. We constructed a temporary F2 generation using the highly differentiated thick-rind watermelon 'XiaoXiGua-4' and the thin-rind watermelon 'DuanMan' as parents and localized the Cla97C02G044120 gene, which controls the thickness of watermelon rind, to the intervals of chromosome 2, CL2-32303995 and CL2-32316840, through 2 years of genetic analysis. No exonic mutations were found in this gene, but two promoter mutations resulted in changes in the promoter progenitor. Fluorescence quantitative PCR analysis revealed highly significant differences in expression at 1 d and 28 d, and the expression was significantly lower in thick-skinned watermelon varieties. Marker-assisted selection (MAS) for this trait was performed using the Caps marker CL2-32303995 and the InDel marker CL2-32316840, which not only verified the stability of the localization interval but also distinguished thick rind from thin rind. These results can be used for germplasm resource screening and have strong breeding significance.

Comparative Analysis of the Mitochondrial Genome of Eggplant (Solanum melongena L.) to Identify Cytoplasmic Male Sterility Candidate Genes.

Cytoplasmic male sterility (CMS) is important for commercial hybrid seed production. However, it is still not used in eggplant (Solanum melongena L.), and corresponding regulatory genes and mechanisms of action have not been reported. We report CMS line 327A, which was derived from the hybridization between cultivated and wild eggplants. By looking at different stages of anther development under a microscope, we saw that the 327A anther's tapetum layer vacuolized during meiosis, which caused abortion. To investigate the 327A CMS regulatory genes, the mitochondrial genomes of 327A and its maintainer line 327B were assembled de novo. It was found that 15 unique ORFs (Open Reading Frame) were identified in 327A. RT-PCR and RT-QPCAR tests confirmed that orf312a and orf172a, 327A-specific ORFs with a transmembrane domain, were strongly expressed in sterile anthers of 327A. In addition, orf312a has a chimeric structure with the ribosomal protein subunit rpl16. Therefore, orf312a and orf172a can be considered strong candidate genes for CMS. Concurrently, we analyzed the characteristics of CMS to develop a functional molecular marker, CMS312, targeting a future theoretical basis for eggplant CMS three-line molecular breeding.

Candidate Gene Identification and Transcriptome Analysis of Tomato male sterile-30 and Functional Marker Development for ms-30 and Its Alleles, ms-33, 7B-1, and stamenless-2.

Male sterility is a valuable trait for hybrid seed production in tomato (Solanum lycopersicum). The mutants male sterile-30 (ms-30) and ms-33 of tomato exhibit twisted stamens, exposed stigmas, and complete male sterility, thus holding potential for application in hybrid seed production. In this study, the ms-30 and ms-33 loci were fine-mapped to 53.3 kb and 111.2 kb intervals, respectively. Tomato PISTILLATA (TPI, syn. SlGLO2), a B-class MADS-box transcription factor gene, was identified as the most likely candidate gene for both loci. TPI is also the candidate gene of tomato male sterile mutant 7B-1 and sl-2. Allelism tests revealed that ms-30, ms-33, 7B-1, and sl-2 were allelic. Sequencing analysis showed sequence alterations in the TPI gene in all these mutants, with ms-30 exhibiting a transversion (G to T) that resulted in a missense mutation (S to I); ms-33 showing a transition (A to T) that led to alternative splicing, resulting in a loss of 46 amino acids in protein; and 7B-1 and sl-2 mutants showing the insertion of an approximately 4.8 kb retrotransposon. On the basis of these sequence alterations, a Kompetitive Allele Specific PCR marker, a sequencing marker, and an Insertion/Deletion marker were developed. Phenotypic analysis of the TPI gene-edited mutants and allelism tests indicated that the gene TPI is responsible for ms-30 and its alleles. Transcriptome analysis of ms-30 and quantitative RT-PCR revealed some differentially expressed genes associated with stamen and carpel development. These findings will aid in the marker-assisted selection for ms-30 and its alleles in tomato breeding and support the functional analysis of the TPI gene.