Genetic Links Between Metabolic Syndrome and Osteoarthritis: Insights from Cross-Trait Analysis.

BACKGROUND: Previous observational studies have indicated a bidirectional association between metabolic syndrome (MetS) and osteoarthritis (OA). However, it remains unclear whether these bidirectional associations reflect causal relationships or shared genetic factors, and the underlying biological mechanisms of this association are not fully understood. METHODS: Leveraging summary statistics from genome-wide association studies (GWASs) conducted by the UK Biobank and the Glucose and Insulin-related Traits Consortium (MAGIC), we performed global genetic correlation analyses, genome-wide cross-trait meta-analyses, and a bidirectional two-sample Mendelian randomization analyses using summary statistics from GWASs to comprehensively assess the relationship of MetS and OA. RESULTS: We first detected an extensive genetic correlation between MetS and OA (rg=0.393, P=1.52×10-18), which was consistent in four MetS components, including waist circumference, triglycerides, hypertension and high-density lipoprotein cholesterol and OA with rg ranging from -0.229 to 0.490. We then discovered 32 variants jointly associated with MetS and OA through multi-trait Analysis of GWAS. Co-localization analysis founded 12 genes shared between MetS and OA, with functional implications in several biological pathways. Finally, MR analysis suggested genetic liability to MetS significantly increased the risk of OA, but no reverse causality was found. CONCLUSION: Our results illustrate a common genetic architecture, pleiotropic loci, as well as causality between MetS and OA, potentially enhancing our knowledge of high comorbidity and genetic processes that overlap between the two disorders.

Development and validation of functional kompetitive allele-specific PCR markers for herbicide resistance in Brassica napus.

Effective weed control in the field is essential for maintaining favorable growing conditions and rapeseed yields. Sulfonylurea herbicides are one kind of most widely used herbicides worldwide, which control weeds by inhibiting acetolactate synthase (ALS). Molecular markers have been designed from polymorphic sites within the sequences of ALS genes, aiding marker-assisted selection in breeding herbicide-resistant rapeseed cultivars. However, most of them are not breeder friendly and have relatively limited application due to higher costs and lower throughput in the breeding projects. The aims of this study were to develop high throughput kompetitive allele-specific PCR (KASP) assays for herbicide resistance. We first cloned and sequenced BnALS1 and BnALS3 genes from susceptible cultivars and resistant 5N (als1als1/als3als3 double mutant). Sequence alignments of BnALS1 and BnALS3 genes for cultivars and 5N showed single nucleotide polymorphisms (SNPs) at positions 1676 and 1667 respectively. These two SNPs for BnALS1 and BnALS3 resulted in amino acid substitutions and were used to develop a KASP assay. These functional markers were validated in three distinct BC1F2 populations. The KASP assay developed in this study will be valuable for the high-throughput selection of elite materials with high herbicide resistance in rapeseed breeding programs.

A shared genetic contribution to osteoarthritis and COVID-19 outcomes: a large-scale genome-wide cross-trait analysis.

Background: Patients with osteoarthritis (OA) are exposed to an increased risk of adverse outcomes of COVID-19, and they tend to experience disruption in access to healthcare services and exercise facilities. However, a deep understanding of this comorbidity phenomenon and the underlying genetic architecture of the two diseases is still unclear. In this study, we aimed to untangle the relationship between OA and COVID-19 outcomes by conducting a large-scale genome-wide cross-trait analysis. Methods: Genetic correlation and causal relationships between OA and COVID-19 outcomes (critical COVID-19, COVID-19 hospitalization, and COVID-19 infection) were estimated by linkage disequilibrium score regression and Mendelian Randomization approaches. We further applied Multi-Trait Analysis of GWAS and colocalization analysis to identify putative functional genes associated with both OA and COVID-19 outcomes. Results: Significant positive genetic correlations between OA susceptibility and both critical COVID-19 (rg=0.266, P=0.0097) and COVID-19 hospitalization (rg=0.361, P=0.0006) were detected. However, there was no evidence to support causal genetic relationships between OA and critical COVID-19 (OR=1.17[1.00-1.36], P=0.049) or OA and COVID-19 hospitalization OR=1.08[0.97-1.20], P=0.143). These results were robustly consistent after the removal of obesity-related single nucleotide polymorphisms (SNPs). Moreover, we identified a strong association signal located near the FYCO1 gene (lead SNPs: rs71325101 for critical COVID-19, Pmeta=1.02×10-34; rs13079478 for COVID-19 hospitalization, Pmeta=1.09×10-25). Conclusion: Our findings further confirmed the comorbidity of OA and COVID-19 severity, but indicate a non-causal impact of OA on COVID-19 outcomes. The study offers an instructive perspective that OA patients did not generate negative COVID-19 outcomes during the pandemic in a causal way. Further clinical guidance can be formulated to enhance the quality of self-management in vulnerable OA patients.

Association between air pollution exposure and outpatient visits for dermatomyositis in a humid subtropical region of China: a time-series study.

In recent years, a growing number of studies have found that air pollution plays critical roles in the onset and development of autoimmune diseases, but few studies have shown an association between air pollutants and dermatomyositis (DM). We sought to investigate the relationship between short-term exposure to air pollution and outpatient visits for DM and to quantify the burden of DM due to exposure to air pollutants in Hefei, China. Daily records of hospital outpatient visits for DM, air pollutants and meteorological factors data in Hefei from January 1, 2018 to December 31, 2021 were obtained. We used a distributed lag non-linear model (DLNM) in conjunction with a generalized linear model (GLM) to explore the association between air pollution and outpatient visits for DM, and conducted stratified analyses by gender, age and season. Moreover, we used attributable fraction (AF) and attributable number (AN) to reflect the burden of disease. A total of 4028 DM clinic visits were recorded during this period. High concentration nitrogen dioxide (NO2) exposure was associated with increased risk of DM outpatient visits (relative risk (RR) 1.063, 95% confidence interval (CI) 1.015-1.114, lag 0-5). Intriguingly, exposure to high concentration ozone (O3) was associated with reduced risk of outpatient visits for DM (RR 0.974, 95% CI 0. 0.954-0.993, lag 0-6). The results of stratified analyses showed that the cold season (vs. warm season) were more susceptible to outpatient visits for DM associated with NO2 and O3 exposure. In addition, we observed that an increased risk of DM outpatient visits was attributable to high concentration NO2 exposure, while high concentration O3 exposure was associated with a decreased risk of DM outpatient visits. This study provided a scientific basis for the etiology research and health protection of DM.

Arthritis and incident pulmonary diseases in middle-aged and elderly Chinese: a longitudinal population-based study.

BACKGROUND: The coexistence of arthritis and pulmonary abnormalities has long been observed, but the causal inter-relationships among them are still uncertain especially in elderly adults. METHODS: We extracted data from The China Health and Retirement Longitudinal Study (CHARLS). A total of 7534 participants without chronic lung diseases or/and asthma at the baseline and have complete follow-up information were included. Multivariate Cox proportional hazards models were used to estimate the adjusted hazard ratios (HRs) for developing chronic lung diseases or asthma. We also utilized generalized linear models to examine the association between arthritis and baseline peak expiratory flow (PEF). RESULTS: During 50,615 and 51,975 person-years of follow-up, 629 and 188 participants incident chronic lung diseases and asthma, respectively. Compared to those without arthritis, participants with arthritis had a higher risk of chronic lung diseases (HR = 1.54, 95%CI = 1.31-1.81, P = 1.23 × 10-7) and asthma (HR = 1.70, 95%CI = 1.27-2.28, P = 3.78 × 10-4). Arthritis subjects demonstrated significantly lower PEF than those without arthritis [ß = - 11.85 (95%CI = - 17.56, - 6.14), P = 4.81 × 10-5]. The results were stable after excluding these participates who incident chronic lung diseases or asthma in the first 1 year of follow-up. CONCLUSION: Arthritis increased the risk of pulmonary diseases among middle-aged and elderly Chinese. Early detection and treatment of pulmonary abnormalities among arthritis patients could help decrease the mortality and reduce the global burden of arthritis. Key Points • The coexistence of arthritis and pulmonary abnormalities has long been observed, but whether arthritis status can trigger pulmonary disorders is still uncertain. • Arthritis status are associated with increased risk of pulmonary diseases (chronic lung diseases/asthma) among middle-aged and elderly Chinese. • Early detection and treatment of pulmonary abnormalities among arthritis patients could help decrease the mortality and reduce the global burden of arthritis.

CRISPR/Cas9-Mediated Gene Editing of BnFAD2 and BnFAE1 Modifies Fatty Acid Profiles in Brassica napus.

Fatty acid (FA) composition determines the quality of oil from oilseed crops, and thus is a major target for genetic improvement. FAD2 (Fatty acid dehydrogenase 2) and FAE1 (fatty acid elongase 1) are critical FA synthetic genes, and have been the focus of genetic manipulation to alter fatty acid composition in oilseed plants. In this study, to improve the nutritional quality of rapeseed cultivar CY2 (about 50% oil content; of which 40% erucic acid), we generated novel knockout plants by CRISPR/Cas9 mediated genome editing of BnFAD2 and BnFAE1 genes. Two guide RNAs were designed to target one copy of the BnFAD2 gene and two copies of the BnFAE1 gene, respectively. A number of lines with mutations at three target sites of BnFAD2 and BnFAE1 genes were identified by sequence analysis. Three of these lines showed mutations in all three target sites of the BnFAD2 and BnFAE1 genes. Fatty acid composition analysis of seeds revealed that mutations at all three sites resulted in significantly increased oleic acid (70-80%) content compared with that of CY2 (20%), greatly reduced erucic acid levels and slightly decreased polyunsaturated fatty acids content. Our results confirmed that the CRISPR/Cas9 system is an effective tool for improving this important trait.

The effect of air pollution exposure on risk of outpatient visits for Sjogren's syndrome: A time-series study.

BACKGROUND: Emerging evidence showed that air pollutants are associated with development and recurrence of autoimmune disorders, but there is scarce evidence regarding the relationship between air pollutants and Sjogren's syndrome (SS). We sought to investigate whether air pollutants affect the risk of outpatient visits for SS and to quantify the burden of SS visits attributable to air pollution exposure in Hefei, China. METHODS: Daily data on outpatient visits for SS, air pollutants and meteorological data in Hefei, China, from January 1, 2015 to December 31, 2020 were obtained. A distributed lag non-linear model in conjunction with a generalized linear model were employed to assess the relationship between air pollution and SS outpatient visits. Stratified analyses were further performed by gender, age and season. Attributable fraction (AF) and attributable number (AN) were used to reflect disease burden. RESULTS: There were 4501 records of outpatient visits for SS. Exposure to PM2.5 was associated with increased risk of SS outpatient visits (relative risk (RR) = 1.218, 95% confidence interval (CI): 1.017-1.458, lag 0-14 day). An increase of 24 µg/m3 (interquartile range) in NO2 concentration was associated with 26.3% increase in the risk of SS outpatient visits (RR = 1.263, 95%CI: 1.105-1.445, lag 0-10 day). In contrast, exposure to O3 was associated with decreased risk of SS outpatient visits (RR = 0.692, 95%CI: 0.510-0.939, per 63 µg/m3 in O3 exposure, lag 0-27 day). Stratified analyses showed that females (vs. males) was more vulnerable to SS outpatient visits associated with NO2 and O3 exposure. SS patients aged ≥65 years (vs. aged <65 years) were susceptible to PM2.5 exposure. Exposure to PM2.5 or NO2 in the cold season was associated with higher risk of SS outpatient visits than that in the warm season. In addition, the AN (232, 95%CI: 119, 324) and AF (5.16%, 95%CI: 2.55%, 7.21%) of NO2 exposure were higher than those of PM2.5 exposure. CONCLUSION: PM2.5 and NO2 exposure are associated with increased risk of SS outpatient visits, while O3 exposure appears to be associated with decreased risk of SS outpatient visits. The effect of air pollutants exposure on risk of SS outpatients can be modified by age, gender and season. The burden of SS outpatient visits attributable to NO2 exposure is higher than those attributable to PM2.5 exposure.

Does smoking protect against developing osteoarthritis? Evidence from a genetically informed perspective.

OBJECTIVE: Cumulative evidence from observational studies showed an inverse association between smoking and osteoarthritis (OA), but this association could be confounded by obesity. This study aimed to decipher the causal effect of smoking on osteoarthritis risk from a genetically informed perspective. METHODS: We performed a two-sample univariable and multivariable MR to evaluate the independent effect of smoking on OA risk. Summary-level data were obtained from the largest available genome-wide association studies (GWASs) of smoking initiation, body mass index (BMI) and OA. Genetic variants predicted the exposure were selected as instruments from the respective GWAS. RESULTS: Genetically liability for smoking initiation had an effect estimate consistent with increased risk for overall OA (odds ratio (OR)=1.61, 95% confidence interval (CI)=1.43-1.81, P=7.50 × 10-15), hip OA (OR=1.62, 95%CI=1.29-2.02, P=2.93 × 10-5), knee OA (OR=1.54, 95%CI=1.29-1.84, P =1.80 × 10-6) and hip and/or knee OA (OR=1.56, 95%CI=1.34-1.80, P=3.63 × 10-9), respectively. We also found that genetic liability of BMI was significantly associated with OA risk and the OR per genetically predicted 1 kg/m2 increase of BMI ranged from 2.19 to 2.64. Additionally, multivariate MR analysis revealed a strong evidence for an effect of smoking initiation on the risk of overall OA (OR=1.45, 95%CI=1.31-1.61, P=3.69 × 10-12) and its subtypes after controlling for BMI. CONCLUSION: Our findings support an independent deleterious causal effect for smoking upon OA risk, which further strengthen the importance of smoking cessation interventions and obesity management in the general population, in order to lessen the huge burden of OA in the global aging era.

Environmental factors and risk of gout.

Gout is a chronic disease with inflammatory arthritis caused by monosodium urate (MSU) crystals deposition, an elevated serum urate level (hyperuricaemia) is the critical factor leading to MSU crystals deposition and promoting the progression of gout. The onset and development of gout is generally the result of multiple factors, such as diet, heredity and environmental factors. Although genetics and diet are thought to play as major factors, a growing body of research evidence has highlighted that environmental factors also play a significant role in the onset and exacerbation of gout. Recent studies have shown that air pollutants such as particulate matter, sulfur dioxide (SO2) and carbon monoxide (CO) may increase the risk of hospitalizations for gout, and that the changes in temperature and humidity may affect uric acid (UA) levels. There is also seasonal trend in gout. It has been demonstrated that environmental factors may induce or accelerate the production and release of pro-inflammatory mediators, causing an unbalance oxidative stress and systemic inflammation, and then participating in the overall process or a certain link of gout. Moreover, several environmental factors have shown the ability to induce the production urate and regulate the innate immune pathways, involving in the pathogenesis of gout. Nevertheless, the role of environmental factors in the etiology of gout remains unclear. In this review, we summarized the recent literatures and aimed to discuss the relationship between environmental factors (such as microclimate, season, ambient/indoor air pollution and extreme weather) and gout. We further discussed the inflammatory mechanisms of environmental factors and gout and the comprehensive effects of environmental factors on gout. We also made a prospect of the management and treatment of gout, with special consideration to environmental factors associated with gout.

No Genetic Causal Association Between Periodontitis and Arthritis: A Bidirectional Two-Sample Mendelian Randomization Analysis.

Objectives: Periodontitis (PD) has been linked to arthritis in previous epidemiological observational studies; however, the results are inconclusive. It remains unclear whether the association between PD and arthritis is causal. The purpose of this study was to investigate the causal association of PD with arthritis, including rheumatoid arthritis (RA) and osteoarthritis (OA). Methods: We performed a two-sample bidirectional Mendelian randomization (MR) analysis using publicly released genome-wide association studies (GWAS) statistics. The inverse-variance weighted (IVW) method was used as the primary analysis. We applied four complementary methods, including weighted median, weighted mode, MR-Egger regression and MR pleiotropy residual sum and outlier (MR-PRESSO) to detect and correct for the effect of horizontal pleiotropy. Results: Genetically determined PD did not have a causal effect on OA (OR = 1.06, 95% CI: 0.99-1.15, P = 0.09) and RA (OR = 0.99, 95% CI: 0.87-1.13, P = 0.89). Furthermore, we did not find a significant causal effect of arthritis on PD in the reverse MR analysis. The results of MR-Egger regression, Weighted Median, and Weighted Mode methods were consistent with those of the IVW method. Horizontal pleiotropy was unlikely to distort the causal estimates according to the sensitivity analysis. Conclusions: Our MR analysis reveals non-causal association of PD with arthritis, despite observational studies reporting an association between PD and arthritis.

Therapeutic Potential of Galectin-1 and Galectin-3 in Autoimmune Diseases.

Galectins are a highly conserved protein family that binds to ß-galactosides. Different members of this family play a variety of biological functions in physiological and pathological processes such as angiogenesis, regulation of immune cell activity, and cell adhesion. Galectins are widely distributed and play a vital role both inside and outside cells. They can regulate homeostasis and immune function in vivo through mechanisms such as apoptosis. Recent studies have indicated that galectins exhibit pleiotropic roles in inflammation. Furthermore, emerging studies have found that galectins are involved in the occurrence and development of autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), type 1 diabetes (T1D), and systemic sclerosis (SSc) by regulating cell adhesion, apoptosis, and other mechanisms. This review will briefly discuss the biological characteristics of the two most widely expressed and extensively explored members of the galectin family, galectin-1 and galectin-3, as well as their pathogenetic and therapeutic roles in autoimmune diseases. This information may provide a novel and promising therapeutic target for autoimmune diseases.

Causal Effects of Gut Microbiome on Systemic Lupus Erythematosus: A Two-Sample Mendelian Randomization Study.

The observational association between gut microbiome and systemic lupus erythematosus (SLE) has been well documented. However, whether the association is causal remains unclear. The present study used publicly available genome-wide association study (GWAS) summary data to perform two-sample Mendelian randomization (MR), aiming to examine the causal links between gut microbiome and SLE. Two sets of MR analyses were conducted. A group of single nucleotide polymorphisms (SNPs) that less than the genome-wide statistical significance threshold (5 × 10-8) served as instrumental variables. To obtain a comprehensive conclusion, the other group where SNPs were smaller than the locus-wide significance level (1 × 10-5) were selected as instrumental variables. Based on the locus-wide significance level, the results indicated that there were causal effects of gut microbiome components on SLE risk. The inverse variance weighted (IVW) method suggested that Bacilli and Lactobacillales were positively correlated with the risk of SLE and Bacillales, Coprobacter and Lachnospira were negatively correlated with SLE risk. The results of weighted median method supported that Bacilli, Lactobacillales, and Eggerthella were risk factors for SLE and Bacillales and Coprobacter served as protective factors for SLE. The estimates of MR Egger suggested that genetically predicted Ruminiclostridium6 was negatively associated with SLE. Based on the genome-wide statistical significance threshold, the results showed that Actinobacteria might reduce the SLE risk. However, Mendelian randomization pleiotropy residual sum and outlier (MR-PRESSO) detected significant horizontal pleiotropy between the instrumental variables of Ruminiclostridium6 and outcome. This study support that there are beneficial or detrimental causal effects of gut microbiome components on SLE risk.

Isolation, characterization and cytoprotective effects against UV radiation of exopolysaccharide produced from Paenibacillus polymyxa PYQ1.

A novel exopolysaccharide (EPS) from Paenibacillus polymyxa PYQ1 was extracted, well purified and characterized. This EPS was homogeneous glucomannan-type polysaccharide with the average molecular weight of 4.38 × 106 Da. Structural characterization indicated that the monosaccharides of EPS were pyranoses connected by ß-glycosidic linkages. Furthermore, our results showed the protective benefits of EPS against UVC induced cytotoxicity in HaCaT cells through scavenging excessive reactive oxygen species, mitigating the decrease of mitochondrial membrane potential, improving catalase activity and maintaining membrane integrity. Taken together, this study qualified EPS from P. polymyxa PYQ1 was a promising natural polymer which worth further investigation as a skin-care agent.

LMI1-like genes involved in leaf margin development of Brassica napus.

In rapeseed (Brassica napus L.), leaf margins are variable and can be entire, serrate, or lobed. In our previous study, the lobed-leaf gene (LOBED-LEAF 1, BnLL1) was mapped to a 32.1 kb section of B. napus A10. Two LMI1-like genes, BnaA10g26320D and BnaA10g26330D, were considered the potential genes that controlled the lobed-leaf trait in rapeseed. In the present study, these two genes and another homologous gene (BnaC04g00850D) were transformed into Arabidopsis thaliana (L.) Heynh. plants to identify their functions. All three LMI1-like genes of B. napus produced serrate leaf margins. The expression analysis indicated that the expression level of BnaA10g26320D determined the difference between lobed- and entire-leaved lines in rapeseed. Therefore, it is likely that BnaA10g26320D corresponds to BnLL1.

Transcriptome analysis of Brassica napus pod using RNA-Seq and identification of lipid-related candidate genes.

BACKGROUND: Brassica napus is an important oilseed crop. Dissection of the genetic architecture underlying oil-related biological processes will greatly facilitates the genetic improvement of rapeseed. The differential gene expression during pod development offers a snapshot on the genes responsible for oil accumulation in. To identify candidate genes in the linkage peaks reported previously, we used RNA sequencing (RNA-Seq) technology to analyze the pod transcriptomes of German cultivar Sollux and Chinese inbred line Gaoyou. METHODS: The RNA samples were collected for RNA-Seq at 5-7, 15-17 and 25-27 days after flowering (DAF). Bioinformatics analysis was performed to investigate differentially expressed genes (DEGs). Gene annotation analysis was integrated with QTL mapping and Brassica napus pod transcriptome profiling to detect potential candidate genes in oilseed. RESULTS: Four hundred sixty five and two thousand, one hundred fourteen candidate DEGs were identified, respectively, between two varieties at the same stages and across different periods of each variety. Then, 33 DEGs between Sollux and Gaoyou were identified as the candidate genes affecting seed oil content by combining those DEGs with the quantitative trait locus (QTL) mapping results, of which, one was found to be homologous to Arabidopsis thaliana lipid-related genes. DISCUSSION: Intervarietal DEGs of lipid pathways in QTL regions represent important candidate genes for oil-related traits. Integrated analysis of transcriptome profiling, QTL mapping and comparative genomics with other relative species leads to efficient identification of most plausible functional genes underlying oil-content related characters, offering valuable resources for bettering breeding program of Brassica napus. CONCLUSIONS: This study provided a comprehensive overview on the pod transcriptomes of two varieties with different oil-contents at the three developmental stages.

Characterization of the quantitative trait locus OilA1 for oil content in Brassica napus.

Increasing seed oil content has become one of the most important breeding criteria in rapeseed (Brassica napus). However, oil content is a complex quantitative trait. QTL mapping in a double haploid population (SG population) emerging from a cross between a German (Sollux) and Chinese (Gaoyou) cultivars revealed one QTL for oil content on linkage group A1 (OilA1), which was mapped to a 17 cM genetic interval. To further validate and characterize the OilA1, we constructed a high-resolution map using B. rapa sequence resources and developed a set of near-isogenic lines (NILs) by employing a DH line SG-DH267 as donor and Chinese parent Gaoyou as recurrent background. The results showed highly conserved synteny order between B. rapa and B. napus within the linkage group A1 and revealed a possible centromere region between two markers ZAASA1-38 and NTP3 (2.5 cM). OilA1 was firstly validated by 250 BC5F2 plants and was confirmed in a 10.6 cM interval between the markers ZAASA1-47 and ZAASA1-77. Further substitution mapping was conducted by using two generations of QTL-NILs, 283 lines from eight BC5F3:4 families and 428 plants from six BC5F4 sub-NILs and thus narrowed the OilA1 interval to 6.9 cM and 4.3 cM (1.4 Mb), respectively. Field investigations with two replications using homozygous BC5F3:4 sister sub-NILs indicated that NILs, which carry a Sollux chromosome segment across the target region showed significant higher oil content (1.26 %, p < 0.001) than their sister NILs containing Gaoyou chromosome. The OilA1 locus is of particular interest for breeding purpose in China because 80 % of Chinese cultivars do not carry this desirable allele.

Molecular mapping of Arabidopsis thaliana lipid-related orthologous genes in Brassica napus.

Quantitative Trait Loci (QTL) for oil content has been previously analyzed in a SG-DH population from a cross between a Chinese cultivar and a European cultivar of Brassica napus. Eight QTL with additive and epistatic effects, and with environmental interactions were evaluated. Here we present an integrated linkage map of this population predominantly based on informative markers derived from Brassica sequences, including 249 orthologous A. thaliana genes, where nearly half (112) are acyl lipid metabolism related genes. Comparative genomic analysis between B. napus and A. thaliana revealed 33 colinearity regions. Each of the conserved A. thaliana segments is present two to six times in the B. napus genome. Approximately half of the mapped lipid-related orthologous gene loci (76/137) were assigned in these conserved colinearity regions. QTL analysis for seed oil content was performed using the new map and phenotypic data from 11 different field trials. Nine significant QTL were identified on linkage groups A1, A5, A7, A9, C2, C3, C6 and C8, together explaining 57.79% of the total phenotypic variation. A total of 14 lipid related candidate gene loci were located in the confidence intervals of six of these QTL, of which ten were assigned in the conserved colinearity regions and felled in the most frequently overlapped QTL intervals. The information obtained from this study demonstrates the potential role of the suggested candidate genes in rapeseed kernel oil accumulation.

Autoscreening of restriction endonucleases for PCR-restriction fragment length polymorphism identification of fungal species, with Pleurotus spp. as an example.

A molecular method based on PCR-restriction fragment length polymorphism (RFLP) analysis of internal transcribed spacer (ITS) ribosomal DNA sequences was designed to rapidly identify fungal species, with members of the genus Pleurotus as an example. Based on the results of phylogenetic analysis of ITS sequences from Pleurotus, a PCR-RFLP endonuclease autoscreening (PRE Auto) program was developed to screen restriction endonucleases for discriminating multiple sequences from different species. The PRE Auto program analyzes the endonuclease recognition sites and calculates the sizes of the fragments in the sequences that are imported into the program in groups according to species recognition. Every restriction endonuclease is scored through the calculation of the average coefficient for the sequence groups and the average coefficient for the sequences within a group, and then virtual electrophoresis maps for the selected restriction enzymes, based on the results of the scoring system, are displayed for the rapid determination of the candidate endonucleases. A total of 85 haplotypes representing 151 ITS sequences were used for the analysis, and 2,992 restriction endonucleases were screened to find the candidates for the identification of species. This method was verified by an experiment with 28 samples representing 12 species of Pleurotus. The results of the digestion by the restriction enzymes showed the same patterns of DNA fragments anticipated by the PRE Auto program, apart from those for four misidentified samples. ITS sequences from 14 samples (of which nine sequences were obtained in this study), including four originally misidentified samples, confirmed the species identities revealed by the PCR-RFLP analysis. The method developed here can be used for the identification of species of other living microorganisms.