Sirtuin 3 relieves inflammatory responses elicited by lipopolysaccharide via the PGC1α-NFκB pathway in bovine mammary epithelial cells.

Excessive inflammation in bovine mammary endothelial cells (BMEC) due to mastitis leads to disease progression and eventual culling of cattle. Sirtuin 3 (SIRT3), a mitochondrial deacetylase, downregulates pro-inflammatory cytokines in BMEC exposed to high concentrations of nonesterified fatty acids by blunting nuclear factor-κB (NFκB) signaling. In nonruminants, SIRT3 is under the control of PGC1α, a transcriptional cofactor. Specific aims were to study (1) the effect of SIRT3 on inflammatory responses of lipopolysaccharide (LPS)-challenged bovine mammary epithelial cells (bovine mammary alveolar cells-T, MAC-T) models, and (2) the role of PGC1α in the attenuation of NFκB signaling via SIRT3. To address these objectives, first, MAC-T cells were incubated in triplicate with 0, 50, 100, 150, or 200 µg/mL LPS (derived from Escherichia coli O55:B5) for 12 h with or without a 2-h incubation of the NFκB inhibitor ammonium pyrrolidine dithiocarbamate (APDC, 10 µM). Second, SIRT3 was overexpressed using adenoviral expression (Ad-SIRT3) at different multiplicity of infection (MOI) for 6 h followed by a 12 h incubation with 150 µg/mL LPS. Third, cells were treated with the PGC1α agonist ZLN005 (10 µg/mL) for 24 h and then challenged with 150 µg/mL LPS for 12 h. Fourth, cells were initially treated with the PGC1α inhibitor SR-18292 (100 µM) for 6 h followed by a 6-h culture with or without 50 MOI Ad-SIRT3 and a challenge with 150 µg/mL LPS for 12 h. Data were analyzed using one-way ANOVA with subsequent Bonferroni correction. Linear and quadratic contrasts were used to determine dose-responses to LPS. There were linear and quadratic effects of LPS dosage on cell viability. Incubation with 150 and 200 µg/mL LPS for 12 h decreased cell viability to 78.6 and 34.9%, respectively. Compared with controls, expression of IL1B, IL6, and TNFA was upregulated by 5.2-, 5.9-, and 2.7-fold with 150 µg/mL LPS; concentrations of IL-1ß, IL-6, and tumor necrosis factor-α (TNF-α) in cell medium also increased. Compared with the LPS group, LPS+APDC increased cell viability and reversed the upregulation of IL1B, IL6, and TNFA expression. However, mRNA and protein abundance of SIRT3 decreased linearly with increasing LPS dose. Ad-SIRT3 infection (50 MOI) reduced IL1B, IL6, and TNFA expression and also their concentrations in cell medium, and decreased pNFκB P65/NFκB P65 ratio and nuclear abundance of NFκB P65. The PGC1α agonist increased SIRT3 expression, whereas it decreased cytokine expression, pNFκB P65/NFκB P65 ratio, and prevented NFκB P65 nuclear translocation. Contrary to the agonist, the PGC1α inhibitor had opposite effects, and elevated the concentrations of IL-1ß, IL-6, and TNF-α in cell medium. Overall, data suggested that SIRT3 activity could attenuate LPS-induced inflammatory responses in mammary cells via alterations in the PGC1α-NFκB pathway. As such, there may be potential benefits for targeting SIRT3 in vivo to help prevent or alleviate negative effects of mastitis.

Long-read genome assembly and genetic architecture of fruit shape in the bottle gourd.

The bottle gourd (Lagenaria siceraria, Cucurbitaceae) is an important horticultural crop exhibiting tremendous diversity in fruit shape. The genetic architecture of fruit shape variation in this species remains unknown. We assembled a long-read-based, high-quality reference genome (ZAAS_Lsic_2.0) with a contig N50 value over 390-fold greater than the existing reference genomes. We then focused on dissection of fruit shape using a one-step geometric morphometrics-based functional mapping approach. We identified 11 quantitative trait loci (QTLs) responsible for fruit shape (fsQTLs), reconstructed their visible effects and revealed syntenic relationships of bottle gourd fsQTLs with 12 fsQTLs previously reported in cucumber, melon or watermelon. Homologs of several well-known and newly identified fruit shape genes, including SUN, OFP, AP2 and auxin transporters, were comapped with bottle gourd QTLs.

Metabolomics analysis reveals metabolic changes associated with trans-resveratrol treatment in experimental cryptorchidism mice.

This study aimed to analyse global metabolomic changes associated with trans-resveratrol (RSV) treatment in mice with cryptorchidism using untargeted metabolomics. Cryptorchidism was established surgically in Kunming mice, which were then treated with 20µg g-1 day-1, s.c., RSV for 35 consecutive days. Typical manifestations of spermatogenesis arrest were seen in mice with cryptorchidism, and RSV treatment for 35 days restored spermatogenesis. Liquid chromatography-tandem mass spectrometry was used to profile the metabolome of testes from mice in the control (non-cryptorchid, untreated), cryptorchid and RSV-treated cryptorchid groups. In all, 1386 and 179 differential metabolites were detected in the positive and negative modes respectively. Seven and six potential biomarkers were screened for spermatogenesis arrest and restoration respectively. Pathway analysis showed changes in 197 metabolic pathways. The hexosamine biosynthesis pathway was inhibited in the cryptorchid group, which probably resulted in a decrease in the end product, uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). Immunoblot analysis showed that total testicular protein O-linked ß-N-acetylglucosamine glycosylation was related to spermatogenesis arrest, further indicating a decrease in UDP-GlcNAc in the cryptorchid group. Thus, untargeted metabolomics revealed the biochemical pathways associated with the restoration of metabolic status in the cryptorchid group following RSV treatment and the findings could be used to monitor the response to RSV treatment. This study provides a meaningful foundation for the future clinical application of RSV in the treatment of spermatogenesis dysfunction.

Orphan genes are involved in drought adaptations and ecoclimatic-oriented selections in domesticated cowpea.

Orphan genes (OGs) are genes that are restricted to a single species or a particular taxonomic group. To date, little is known about the functions of OGs in domesticated crops. Here, we report our findings on the relationships between OGs and environmental adaptation in cowpea (Vigna unguiculata). We identified 578 expressed OGs, of which 73.2% were predicted to be non-coding. Transcriptomic analyses revealed a high rate of OGs that were drought inducible in roots when compared with conserved genes. Co-expression analysis further revealed the possible involvement of OGs in stress response pathways. Overexpression of UP12_8740, a drought-inducible OG, conferred enhanced tolerance to osmotic stresses and soil drought. By combining Capture-Seq and fluorescence-based Kompetitive allele-specific PCR (KASP), we efficiently genotyped single nucleotide polymorphisms (SNPs) on OGs across a 223 accession cowpea germplasm collection. Population genomic parameters, including polymorphism information content (PIC), expected heterozygosity (He), nucleotide diversity (π), and Tajima's D statistics, that were calculated based on these SNPs, showed distinct signatures between the grain- and vegetable-type subpopulations of cowpea. This study reinforces the idea that OGs are a valuable resource for identifying new genes related to species-specific environmental adaptations and fosters new insights that artificial selection on OGs might have contributed to balancing the adaptive and agronomic traits in domesticated crops in various ecoclimatic conditions.

Fine mapping Ruv2, a new rust resistance gene in cowpea (Vigna unguiculata), to a 193-kb region enriched with NBS-type genes.

KEY MESSAGE: A new rust resistance gene Ruv2 was fine-mapped in cowpea to a 193-kb region on chromosome 2, which harboured 23 predicted gene models enriched with NBS-type genes. ZN016 is a landrace vegetable cowpea highly resistant to rust. Two previous studies using mixed-spores inoculation suggested different modes of inheritance of rust resistance in ZN016. In this study, we initially developed a detached leaf assay with a purified single-rust isolate (Auv-LS). Using this approach, we assessed the inheritance of rust resistance in a recombinant inbred line (RIL) population and an F2 population, both derived from the cross of "ZN016" and the susceptible cultivar "Zhijiang282." A single dominant gene mode against Auv-LS was revealed in both populations. QTL mapping showed that this gene was coincident with the Ruv2 locus on LG7, one of the three resistance QTLs previously mapped based on mixed-spores inoculation data. Therefore, Ruv2 was considered as specifically against the rust isolate Auv-LS. Through an analysis of the RIL recombinants at Ruv2, we fine-mapped the gene to an ~ 0.45-cM interval between SNP markers 2_09656 and 2_00973, which corresponded to an ~ 193-kb region on chromosome 2 that harboured 23 predicted gene models enriched with NBS-type genes. Re-sequencing of the two parents revealed polymorphisms in four genes predictively to cause substantial protein structural changes, rendering them valuable candidate genes for future validation. Cross-species syntenic analysis indicated that Ruv2 may represent a novel rust resistance gene in food legumes. A cleaved amplified polymorphic sequences marker tightly linked to Ruv2 was developed to facilitate breeding. This work establishes a basis for map-based cloning of Ruv2 and breeding for rust resistance in cowpea and other legume crops.

Genomic regions, cellular components and gene regulatory basis underlying pod length variations in cowpea (V. unguiculata L. Walp).

Cowpea (V. unguiculata L. Walp) is a climate resilient legume crop important for food security. Cultivated cowpea (V. unguiculata L) generally comprises the bushy, short-podded grain cowpea dominant in Africa and the climbing, long-podded vegetable cowpea popular in Asia. How selection has contributed to the diversification of the two types of cowpea remains largely unknown. In the current study, a novel genotyping assay for over 50 000 SNPs was employed to delineate genomic regions governing pod length. Major, minor and epistatic QTLs were identified through QTL mapping. Seventy-two SNPs associated with pod length were detected by genome-wide association studies (GWAS). Population stratification analysis revealed subdivision among a cowpea germplasm collection consisting of 299 accessions, which is consistent with pod length groups. Genomic scan for selective signals suggested that domestication of vegetable cowpea was accompanied by selection of multiple traits including pod length, while the further improvement process was featured by selection of pod length primarily. Pod growth kinetics assay demonstrated that more durable cell proliferation rather than cell elongation or enlargement was the main reason for longer pods. Transcriptomic analysis suggested the involvement of sugar, gibberellin and nutritional signalling in regulation of pod length. This study establishes the basis for map-based cloning of pod length genes in cowpea and for marker-assisted selection of this trait in breeding programmes.

Population genomic analyses from low-coverage RAD-Seq data: a case study on the non-model cucurbit bottle gourd.

Restriction site-associated DNA sequencing (RAD-Seq), a next-generation sequencing-based genome 'complexity reduction' protocol, has been useful in population genomics in species with a reference genome. However, the application of this protocol to natural populations of genomically underinvestigated species, particularly under low-to-medium sequencing depth, has not been well justified. In this study, a Bayesian method was developed for calling genotypes from an F2 population of bottle gourd [Lagenaria siceraria (Mol.) Standl.] to construct a high-density genetic map. Low-depth genome shotgun sequencing allowed the assembly of scaffolds/contigs comprising approximately 50% of the estimated genome, of which 922 were anchored for identifying syntenic regions between species. RAD-Seq genotyping of a natural population comprising 80 accessions identified 3226 single nuclear polymorphisms (SNPs), based on which two sub-gene pools were suggested for association with fruit shape. The two sub-gene pools were moderately differentiated, as reflected by the Hudson's F(ST) value of 0.14, and they represent regions on LG7 with strikingly elevated F(ST) values. Seven-fold reduction in heterozygosity and two times increase in LD (r²) were observed in the same region for the round-fruited sub-gene pool. Outlier test suggested the locus LX3405 on LG7 to be a candidate site under selection. Comparative genomic analysis revealed that the cucumber genome region syntenic to the high FST island on LG7 harbors an ortholog of the tomato fruit shape gene OVATE. Our results point to a bright future of applying RAD-Seq to population genomic studies for non-model species even under low-to-medium sequencing efforts. The genomic resources provide valuable information for cucurbit genome research.

Differential selection of yield and quality traits has shaped genomic signatures of cowpea domestication and improvement.

Cowpeas (tropical legumes) are important in ensuring food and nutritional security in developing countries, especially in sub-Saharan Africa. Herein, we report two high-quality genome assemblies of grain and vegetable cowpeas and we re-sequenced 344 accessions to characterize the genomic variations landscape. We identified 39 loci for ten important agronomic traits and more than 541 potential loci that underwent selection during cowpea domestication and improvement. In particular, the synchronous selections of the pod-shattering loci and their neighboring stress-relevant loci probably led to the enhancement of pod-shattering resistance and the compromise of stress resistance during the domestication from grain to vegetable cowpeas. Moreover, differential selections on multiple loci associated with pod length, grain number per pod, seed weight, pod and seed soluble sugars, and seed crude proteins shaped the yield and quality diversity in cowpeas. Our findings provide genomic insights into cowpea domestication and improvement footprints, enabling further genome-informed cultivar improvement of cowpeas.

QTL mapping and epistatic interaction analysis in asparagus bean for several characterized and novel horticulturally important traits.

BACKGROUND: Asparagus bean (Vigna. unguiculata. ssp sesquipedalis) is a subspecies and special vegetable type of cowpea (Vigna. unguiculata L. Walp.) important in Asia. Genetic basis of horticulturally important traits of asparagus bean is still poorly understood, hindering the utilization of targeted, DNA marker-assisted breeding in this crop. Here we report the identification of quantitative trait loci (QTLs) and epistatic interactions for four horticultural traits, namely, days to first flowering (FLD), nodes to first flower (NFF), leaf senescence (LS) and pod number per plant (PN) using a recombinant inbred line (RIL) population of asparagus bean. RESULTS: A similar genetic mode of one major QTL plus a few minor QTLs was found to dominate each of the four traits, with the number of QTLs for individual traits ranging from three to four. These QTLs were distributed on 7 of the 11 chromosomes. Major QTLs for FLD, NFF and LS were co-localized on LG 11, indicative of tight linkage. Genome wide epistasis analysis detected two and one interactive locus pairs that significantly affect FLD and LS, respectively, and the epistatic QTLs for FLD appeared to work in different ways. Synteny based comparison of QTL locations revealed conservation of chromosome regions controlling these traits in related legume crops. CONCLUSION: Major, minor, and epistatic QTLs were found to contribute to the inheritance of the FLD, NFF, LS, and PN. Positions of many of these QTLs are conserved among closely related legume species, indicating common mechanisms they share. To our best knowledge, this is the first QTL mapping report using an asparagus bean × asparagus bean intervarietal population and provides marker-trait associations for marker-assisted approaches to selection.

Genome-wide characterization and expression analysis of the MLO gene family sheds light on powdery mildew resistance in Lagenaria siceraria.

MLO (mildew locus O) genes play a vital role in plant disease defense system, especially powdery mildew (PM). Lagenaria siceraria is a distinct Cucurbitaceae crop, and PM is one of the most serious diseases threatening crop production and quality. Although MLOs have been exploited in many Cucurbitaceae species, genome-wide mining of MLO gene family in bottle gourd has not been surveyed yet. Here we identified 16 MLO genes in our recently assembled L. siceraria genome. A total of 343 unique MLO protein sequences from 20 species were characterized and compared to deduce a generally high level of purifying selection and the occurrence of regions related to candidate susceptibility factors in the evolutional divergence. LsMLOs were clustered in six clades containing seven conserved transmembrane domains and 10 clade-specific motifs along with deletion and variation. Three genes (LsMLO3, LsMLO6, and LsMLO13) in clade V showed high sequence identity with orthologues involved in PM susceptibility. The expression pattern of LsMLOs was tissue-specific but not cultivar-specific. Furthermore, it was indicated by qRT-PCR and RNA-seq that LsMLO3 and LsMLO13 were highly upregulated in response to PM stress. Subsequent sequence analysis revealed the structural deletion of LsMLO13 and a single nonsynonymous substitution of LsMLO3 in the PM-resistant genotype. Taken all together, it is speculated that LsMLO13 is likely a major PM susceptibility factor. The results of this study provide new insights into MLO family genes in bottle gourd and find a potential candidate S gene for PM tolerance breeding.

Genome-wide association analysis reveals the optimal genomic regions for pod size in bean.

The snap bean is the most commonly grown vegetable legume worldwide, and its pod size is both an important yield and appearance quality trait. However, the improvement of pod size in snap beans grown in China has been largely hindered by a lack of information on the specific genes that determine pod size. In this study, we identified 88 snap bean accessions and evaluated their pod size traits. Through a genome-wide association study (GWAS), 57 single nucleotide polymorphisms (SNPs) significantly associated with pod size were detected. Candidate gene analysis showed that cytochrome P450 family genes, WRKY, and MYB transcription factors were the predominant candidate genes for pod development, and eight of these 26 candidate genes showed relatively higher expression patterns in flowers and young pods. A significant pod length (PL) SNP and a single pod weight (SPW) SNP were successfully converted into kompetitive allele-specific polymerase chain reaction (KASP) markers and validated in the panel. These results enhance our understanding of the genetic basis of pod size, and also provide genetic resources for the molecular breeding of pod size in snap beans.

Genotypic differences in pod wall and seed growth relate to invertase activities and assimilate transport pathways in asparagus bean.

BACKGROUND AND AIMS: Coordination of sugar transport and metabolism between developing seeds and their enclosing fruit tissues is little understood. In this study the physiological mechanism is examined using two genotypes of asparagus bean (Vigna unguiculata ssp. sesquipedialis) differing in pod wall and seed growth rates. Pod growth dominates over seed growth in genotype 'Zhijiang 121' but not in 'Zhijiang 282' in which a 'bulging pod' phenotype is apparent from 8 d post-anthesis (dpa) onward. METHODS: Seed and pod wall growth rates and degree of pod-bulging were measured in the two genotypes together with assays of activities of sucrose-degrading enzymes and sugar content in pod wall and seed and evaluation of cellular pathways of phloem unloading in seed coat using a symplasmic fluorescent dye, 5(6)-carboxyfluorescein (CF). KEY RESULTS: Activities of cell wall, cytoplasmic and vacuolar invertases (CWIN, CIN and VIN) were significantly smaller in pod walls of '282' than in '121' at 10 dpa onwards. Low INV activities were associated with weak pod wall growth of '282'. In seed coats, CF was confined within the vasculature in '282' but moved beyond the vasculature in '121', indicating apoplasmic and symplasmic phloem unloading, respectively. Higher CWIN activity in '282' seed coats at 6-8 dpa correlated with high hexose concentration in embryos and enhanced early seed growth. However, CWIN activity in '282' decreased significantly compared with '121' from 10 dpa onwards, coinciding with earlier commencement of nuclei endoreduplication in their embryos. CONCLUSIONS: The study shows genotypic differences between 'bulging pod' and 'non-bulging' phenotypes of asparagus bean in sucrose metabolism in relation to the pathway of phloem unloading in developing seed coats, and to pod and seed growth. Low INV activity in pod wall corresponds to its shortened and weak growth period; by contrast, the apoplasmic path in the seed coat is associated with high CWIN activity and strong early seed growth.

Insight into the bZIP gene family in Lagenaria siceraria: Genome and transcriptome analysis to understand gene diversification in Cucurbitaceae and the roles of LsbZIP gene expression and function under cold stress.

The basic leucine zipper (bZIP) as a well-known transcription factor family, figures prominently in diverse biological and developmental processes and response to abiotic/biotic stresses. However, no knowledge of the bZIP family is available for the important edible Cucurbitaceae crop bottle gourd. Herein, we identified 65 putative LsbZIP genes and characterized their gene structure, phylogenetic and orthologous relationships, gene expression profiles in different tissues and cultivars, and responsive genes under cold stress. The phylogenetic tree of 16 released Cucurbitaceae plant genomes revealed the evolutionary convergence and divergence of bZIP family. Based on the specific domains, LsbZIP family were classified into 12 clades (A-K, S) with similar motifs and exon-intron distribution. 65 LsbZIP genes have undergone 19 segmental and two tandem duplication events with purifying selection. The expression profiling of LsbZIP genes showed tissue-specific but no cultivar-specific pattern. The cold stress-responsive candidate LsbZIP genes were analyzed and validated by RNA-Seq and RT-PCR, providing new insights of transcriptional regulation of bZIP family genes in bottle gourd and their potential functions in cold-tolerant variety breeding.

Unravelling the Genetic Architecture of Rust Resistance in the Common Bean (Phaseolus vulgaris L.) by Combining QTL-Seq and GWAS Analysis.

The common bean (Phaseolus vulgaris L.) is the most important legume crop directly used for human consumption worldwide. Bean rust, caused by Uromyces appendiculatus, is a devastating disease and usually causes severe loss of seed yield and pod quality. Deployment of resistant cultivars is the best strategy to combat this disease. However, despite being the largest snap bean-producing country, the genetic basis research of rust resistance has largely lagged in China. In this study, an RIL population and a diversity panel were evaluated for rust resistance against a purified rust isolate Cua-LS using a detached leaf assay. Deploying a QTL-Seq analysis in the RIL population, a 1.81 Mb interval on chromosome 4, a 2.73 Mb interval on chromosome 5 and a 1.26 Mb interval on chromosome 6 were identified as major QTLs for rust resistance, designated as Qur-1, Qur-2 and Qur-3, respectively. Through a GWAS diversity panel, 64 significant SNPs associated with rust resistance were detected, distributed in all 11 chromosomes and explaining 19-49% of the phenotypic variation. Synteny analysis showed that Qur-2 was validated in GWAS, but the rust QTL/SNPs detected in our study were different from the known genes, except Ur-11. A total of 114 candidate genes, including the typical NBS-LRR genes, protein kinase superfamily proteins and ABC transporter family proteins, were identified and proposed as the likely candidates. The identified 17 resistant accessions will enrich the resistant germplasm resources, and the detected QTLs/SNPs will facilitate the molecular breeding of rust resistance in the common bean.

Elucidation of the Flavor Aspects and Flavor-Associated Genomic Regions in Bottle Gourd (Lagenaria siceraria) by Metabolomic Analysis and QTL-seq.

Bottle gourd (Lagenaria siceraria) is a commercially important cucurbitaceous vegetable with health-promoting properties whose collections and cultivars differ considerably in their flavor aspects. However, the metabolomic profile related to flavor has not yet been elucidated. In the present study, a comprehensive metabolite analysis revealed the metabolite profile of the strong-flavor collection "J120" and weak-flavor collection "G32". The major differentially expressed metabolites included carboxylic acids, their derivatives, and organooxygen compounds, which are involved in amino acid biosynthesis and metabolism. QTL-seq was used to identify candidate genomic regions controlling flavor in a MAGIC population comprising 377 elite lines. Three significant genomic regions were identified, and candidate genes likely associated with flavor were screened. Our study provides useful information for understanding the metabolic causes of flavor variation among bottle gourd collections and cultivars. Furthermore, the identified candidate genomic regions may facilitate rational breeding programs to improve bottle gourd quality.

Identification of Bottle Gourd (Lagenaria siceraria) OVATE Family Genes and Functional Characterization of LsOVATE1.

The OVATE gene family is a class of conserved transcription factors that play significant roles in plant growth, development, and abiotic stress, and also affect fruit shape in vegetable crops. Bottle gourd (Lagenaria siceraria), commonly known as calabash or gourd, is an annual climber belonging to the Cucurbitaceae family. Studies on bottle gourd OVATE genes are limited. In this study, we performed genome-wide identification of the OVATE gene family in bottle gourd, and identified a total of 20 OVATE family genes. The identified genes were unevenly distributed across 11 bottle gourd chromosomes. We also analyzed the gene homology, amino acid sequence conservation, and three-dimensional protein structure (via prediction) of the 20 OVATE family genes. We used RNA-seq data to perform expression analysis, which found 20 OVATE family genes to be differentially expressed based on spatial and temporal characteristics, suggesting that they have varying functions in the growth and development of bottle gourd. In situ hybridization and subcellular localization analysis showed that the expression characteristics of the LsOVATE1 gene, located on chromosome 7 homologous to OVATE, is a candidate gene for affecting the fruit shape of bottle gourd. In addition, RT-qPCR data from bottle gourd roots, stems, leaves, and flowers showed different spatial expression of the LsOVATE1 gene. The ectopic expression of LsOVATE1 in tomato generated a phenotype with a distinct fruit shape and development. Transgenic-positive plants that overexpressed LsOVATE1 had cone-shaped fruit, calyx hypertrophy, petal degeneration, and petal retention after flowering. Our results indicate that LsOVATE1 could serve important roles in bottle gourd development and fruit shape determination, and provide a basis for future research into the function of LsOVATE1.

Partial sequencing of the bottle gourd genome reveals markers useful for phylogenetic analysis and breeding.

BACKGROUND: Bottle gourd [Lagenaria siceraria (Mol.) Standl.] is an important cucurbit crop worldwide. Archaeological research indicates that bottle gourd was domesticated more than 10,000 years ago, making it one of the earliest plants cultivated by man. In spite of its widespread importance and long history of cultivation almost nothing has been known about the genome of this species thus far. RESULTS: We report here the partial sequencing of bottle gourd genome using the 454 GS-FLX Titanium sequencing platform. A total of 150,253 sequence reads, which were assembled into 3,994 contigs and 82,522 singletons were generated. The total length of the non-redundant singletons/assemblies is 32 Mb, theoretically covering ~ 10% of the bottle gourd genome. Functional annotation of the sequences revealed a broad range of functional types, covering all the three top-level ontologies. Comparison of the gene sequences between bottle gourd and the model cucurbit cucumber (Cucumis sativus) revealed a 90% sequence similarity on average. Using the sequence information, 4395 microsatellite-containing sequences were identified and 400 SSR markers were developed, of which 94% amplified bands of anticipated sizes. Transferability of these markers to four other cucurbit species showed obvious decline with increasing phylogenetic distance. From analyzing polymorphisms of a subset of 14 SSR markers assayed on 44 representative China bottle gourd varieties/landraces, a principal coordinates (PCo) analysis output and a UPGMA-based dendrogram were constructed. Bottle gourd accessions tended to group by fruit shape rather than geographic origin, although in certain subclades the lines from the same or close origin did tend to cluster. CONCLUSIONS: This work provides an initial basis for genome characterization, gene isolation and comparative genomics analysis in bottle gourd. The SSR markers developed would facilitate marker assisted breeding schemes for efficient introduction of desired traits.

Identification and Validation of a Core Single-Nucleotide Polymorphism Marker Set for Genetic Diversity Assessment, Fingerprinting Identification, and Core Collection Development in Bottle Gourd.

Germplasm collections are indispensable resources for the mining of important genes and variety improvement. To preserve and utilize germplasm collections in bottle gourd, we identified and validated a highly informative core single-nucleotide polymorphism (SNP) marker set from 1,100 SNPs. This marker set consisted of 22 uniformly distributed core SNPs with abundant polymorphisms, which were established to have strong representativeness and discriminatory power based on analyses of 206 bottle gourd germplasm collections and a multiparent advanced generation inter-cross (MAGIC) population. The core SNP markers were used to assess genetic diversity and population structure, and to fingerprint important accessions, which could provide an optimized procedure for seed authentication. Furthermore, using the core SNP marker set, we developed an accessible core population of 150 accessions that represents 100% of the genetic variation in bottle gourds. This core population will make an important contribution to the preservation and utilization of bottle gourd germplasm collections, cultivar identification, and marker-assisted breeding.

Sirtuin 3 Restores Synthesis and Secretion of Very Low-Density Lipoproteins in Cow Hepatocytes Challenged with Nonesterified Fatty Acids In Vitro.

Fatty liver is closely associated with elevated concentrations of nonesterified fatty acids (NEFA) and a low level of very low-density lipoproteins (VLDL) in blood of dairy cows. High NEFA inhibit the VLDL synthesis and assembly, and cause hepatic triacylglycerol (TAG) deposition. Sirtuin 3 (SIRT3), a mitochondrial deacetylase, antagonizes NEFA-induced TAG accumulation through modulating expressions of fatty acid synthesis and oxidation genes in cow hepatocytes. However, the role of SIRT3 in the VLDL synthesis and assembly was largely unknown. Here we aimed to test whether SIRT3 would recover the synthesis and assembly of VLDL in cow hepatocytes induced by high NEFA. Primary cow hepatocytes were isolated from 3 Holstein cows. Hepatocytes were infected with SIRT3 overexpression adenovirus (Ad-SIRT3), SIRT3-short interfering (si) RNA, or first infected with Ad-SIRT3 and then incubated with 1.0 mM NEFA (Ad-SIRT3 + NEFA). Expressions of key genes in VLDL synthesis and the VLDL contents in cell culture supernatants were measured. SIRT3 overexpression significantly increased the mRNA abundance of microsomal triglyceride transfer protein (MTP), apolipoprotein B100 (ApoB100) and ApoE (p < 0.01), and raised VLDL contents in the supernatants (p < 0.01). However, SIRT3 silencing displayed a reverse effect in comparison to SIRT3 overexpression. Compared with NEFA treatment alone, the Ad-SIRT3 + NEFA significantly upregulated the mRNA abundance of MTP, ApoB100 and ApoE (p < 0.01), and increased VLDL contents in the supernatants (p < 0.01). Our data demonstrated that SIRT3 restored the synthesis and assembly of VLDL in cow hepatocytes challenged with NEFA, providing an in vitro basis for further investigations testing its feasibility against hepatic TAG accumulation in dairy cows during the perinatal period.

Novel Genomic Regions of Fusarium Wilt Resistance in Bottle Gourd [Lagenaria siceraria (Mol.) Standl.] Discovered in Genome-Wide Association Study.

Fusarium wilt (FW) is a typical soil-borne disease that seriously affects the yield and fruit quality of bottle gourd. Thus, to improve resistance to FW in bottle gourd, the genetic mechanism underlying FW resistance needs to be explored. In this study, we performed a genome-wide association study (GWAS) based on 5,330 single-nucleotide polymorphisms (SNPs) and 89 bottle gourd accessions. The GWAS results revealed a total of 10 SNPs (P ≤ 0.01, -log10 P ≥ 2.0) significantly associated with FW resistance that were detected in at least two environments (2019DI, 2020DI, and the average across the 2 years); these SNPs were located on chromosomes 1, 2, 3, 4, 8, and 9. Linkage disequilibrium (LD) block structure analysis predicted three potential candidate genes for FW resistance. Genes HG_GLEAN_10001030 and HG_GLEAN_10001042 were within the range of the mean LD block of the marker BGReSe_14202; gene HG_GLEAN_10011803 was 280 kb upstream of the marker BGReSe_00818. Real-time quantitative PCR (qRT-PCR) analysis showed that HG_GLEAN_10011803 was significantly up-regulated in FW-infected plants of YD-4, Yin-10, and Hanbi; HG_GLEAN_10001030 and HG_GLEAN_10001042 were specifically up-regulated in FW-infected plants of YD-4. Therefore, gene HG_GLEAN_10011803 is likely the major effect candidate gene for resistance against FW in bottle gourd. This work provides scientific evidence for the exploration of candidate gene and development of functional markers in FW-resistant bottle gourd breeding programs.