Identification of QTNs, QTN-by-environment interactions, and their candidate genes for salt tolerance related traits in soybean.

BACKGROUND: Salt stress significantly reduces soybean yield. To improve salt tolerance in soybean, it is important to mine the genes associated with salt tolerance traits. RESULTS: Salt tolerance traits of 286 soybean accessions were measured four times between 2009 and 2015. The results were associated with 740,754 single nucleotide polymorphisms (SNPs) to identify quantitative trait nucleotides (QTNs) and QTN-by-environment interactions (QEIs) using three-variance-component multi-locus random-SNP-effect mixed linear model (3VmrMLM). As a result, eight salt tolerance genes (GmCHX1, GsPRX9, Gm5PTase8, GmWRKY, GmCHX20a, GmNHX1, GmSK1, and GmLEA2-1) near 179 significant and 79 suggested QTNs and two salt tolerance genes (GmWRKY49 and GmSK1) near 45 significant and 14 suggested QEIs were associated with salt tolerance index traits in previous studies. Six candidate genes and three gene-by-environment interactions (GEIs) were predicted to be associated with these index traits. Analysis of four salt tolerance related traits under control and salt treatments revealed six genes associated with salt tolerance (GmHDA13, GmPHO1, GmERF5, GmNAC06, GmbZIP132, and GmHsp90s) around 166 QEIs were verified in previous studies. Five candidate GEIs were confirmed to be associated with salt stress by at least one haplotype analysis. The elite molecular modules of seven candidate genes with selection signs were extracted from wild soybean, and these genes could be applied to soybean molecular breeding. Two of these genes, Glyma06g04840 and Glyma07g18150, were confirmed by qRT-PCR and are expected to be key players in responding to salt stress. CONCLUSIONS: Around the QTNs and QEIs identified in this study, 16 known genes, 6 candidate genes, and 8 candidate GEIs were found to be associated with soybean salt tolerance, of which Glyma07g18150 was further confirmed by qRT-PCR.

An efficient multi-locus mixed model framework for the detection of small and linked QTLs in F2.

In the genetic system that regulates complex traits, metabolites, gene expression levels, RNA editing levels and DNA methylation, a series of small and linked genes exist. To date, however, little is known about how to design an efficient framework for the detection of these kinds of genes. In this article, we propose a genome-wide composite interval mapping (GCIM) in F2. First, controlling polygenic background via selecting markers in the genome scanning of linkage analysis was replaced by estimating polygenic variance in a genome-wide association study. This can control large, middle and minor polygenic backgrounds in genome scanning. Then, additive and dominant effects for each putative quantitative trait locus (QTL) were separately scanned so that a negative logarithm P-value curve against genome position could be separately obtained for each kind of effect. In each curve, all the peaks were identified as potential QTLs. Thus, almost all the small-effect and linked QTLs are included in a multi-locus model. Finally, adaptive least absolute shrinkage and selection operator (adaptive lasso) was used to estimate all the effects in the multi-locus model, and all the nonzero effects were further identified by likelihood ratio test for true QTL identification. This method was used to reanalyze four rice traits. Among 25 known genes detected in this study, 16 small-effect genes were identified only by GCIM. To further demonstrate GCIM, a series of Monte Carlo simulation experiments was performed. As a result, GCIM is demonstrated to be more powerful than the widely used methods for the detection of closely linked and small-effect QTLs.

Methodological implementation of mixed linear models in multi-locus genome-wide association studies.

The mixed linear model has been widely used in genome-wide association studies (GWAS), but its application to multi-locus GWAS analysis has not been explored and assessed. Here, we implemented a fast multi-locus random-SNP-effect EMMA (FASTmrEMMA) model for GWAS. The model is built on random single nucleotide polymorphism (SNP) effects and a new algorithm. This algorithm whitens the covariance matrix of the polygenic matrix K and environmental noise, and specifies the number of nonzero eigenvalues as one. The model first chooses all putative quantitative trait nucleotides (QTNs) with ≤ 0.005 P-values and then includes them in a multi-locus model for true QTN detection. Owing to the multi-locus feature, the Bonferroni correction is replaced by a less stringent selection criterion. Results from analyses of both simulated and real data showed that FASTmrEMMA is more powerful in QTN detection and model fit, has less bias in QTN effect estimation and requires a less running time than existing single- and multi-locus methods, such as empirical Bayes, settlement of mixed linear model under progressively exclusive relationship (SUPER), efficient mixed model association (EMMA), compressed MLM (CMLM) and enriched CMLM (ECMLM). FASTmrEMMA provides an alternative for multi-locus GWAS.

Effect of marker segregation distortion on high density linkage map construction and QTL mapping in Soybean (Glycine max L.).

Marker segregation distortion is a natural phenomenon. Severely distorted markers are usually excluded in the construction of linkage maps. We investigated the effect of marker segregation distortion on linkage map construction and quantitative trait locus (QTL) mapping. A total of 519 recombinant inbred lines of soybean from orthogonal and reciprocal crosses between LSZZH and NN493-1 were genotyped by specific length amplified fragment markers and seed linoleic acid content was measured in three environments. As a result, twenty linkage groups were constructed with 11,846 markers, including 1513 (12.77%) significantly distorted markers, on 20 chromosomes, and the map length was 2475.86 cM with an average marker-interval of 0.21 cM. The inclusion of distorted markers in the analysis was shown to not only improve the grouping of the markers from the same chromosomes, and the consistency of linkage maps with genome, but also increase genome coverage by markers. Combining genotypic data from both orthogonal and reciprocal crosses decreased the proportion of distorted markers and then improved the quality of linkage maps. Validation of the linkage maps was confirmed by the high collinearity between positions of markers in the soybean reference genome and in linkage maps and by the high consistency of 24 QTL regions in this study compared with the previously reported QTLs and lipid metabolism related genes. Additionally, linkage maps that include distorted markers could add more information to the outputs from QTL mapping. These results provide important information for linkage mapping, gene cloning and marker-assisted selection in soybean.

Research advances in cartilage stem cells markers and induced differentiation.

Cartilage stem cells (CSCs) are cells that self-proliferate, have surface antigen expression, and have multidirectional differentiation potential in the articular cartilage. CSCs, as an ideal source of stem cells, has a good application prospect in stem cell therapy. This article reviews the CSCs markers, cartilage differentiation signaling pathway, and clinical treatment of osteoarthritis.

[The effect of low-magnitude high-frequency loading on peri-implant bone healing and implant osseointegration in Beagle dogs].

PURPOSE: Low-magnitude, high-frequency（LMHF） loading has been confirmed to play an important role in bone healing. The present study aimed to evaluate the effect of LMHF loading applied directly to titanium dental implants on peri-implant bone healing and implant. METHODS: The mandibular premolars and molars were extracted from six male Beagle dogs. Three months post-extraction, six titanium implants (Aadva Standard Implant Narrow, 3.3 mm×8 mm) were inserted into the mandibular premolar and molar area (three implants per side) in each of the six dogs. In each animal, one side was randomly selected to undergo daily LMHF loading (experimental group), while the other side had no further intervention (control group). The loading was applied directly to the implant abutment using an individual jig and a custom-made loading device (8 µm,100 Hz). The implant stability quotient (ISQ) was tested every week. Three dogs were euthanized after 2 weeks, and three were euthanized after 8 weeks. Tissue samples were fixed and stained for micro-computed tomography(micro-CT) and histomorphometric analyses. The data were statistically analyzed using SPSS 13.0 software package. RESULTS: The experimental group had significantly increased peri-implant bone volume relative to tissue volume in region of interest 2 (100-500 µm) compared with the control group after 2 weeks of loading (P<0.05); however, there was no significant difference between the two groups after 8 weeks (P>0.05). The ISQ value and the micro-CT results did not differ between the two groups during the study period. CONCLUSIONS: LMHF loading positively influences peri-implant bone healing and osseointegration in the early healing period.

[Role of ATP6V1H gene in bone metabolism].

Osteoporosis is one of the common clinical orthopedic diseases, which can lead to a variety of complications. There are many pathogenic factors in this disease. The latest research found that ATP6V1H is a new gene leading to the occurrence of osteoporosis, and it is likely to become a new target for the future drug treatment of osteoporosis.This paper introduces the biological structure and characteristics of H subunit, summed up the human body caused by loss of ATP6V1H and animal models such as zebrafish, mice bone loss and osteoporosis symptom such as related research reports of the loss, from osteoclast, osteoblast and marrow stromal cell level and the connection between the various subunits further expounds the H subunit regulate bone dynamic balance of mechanism, to explore ATP6V1H in bone developmentand bone related diseases has laid a solid foundation, also provide new ideas for clinical treatment of osteoporosis.

[Expression of BMP/Smads in rabbit condylar cartilage during mandibular forward positioning].

OBJECTIVE: To identify the relationship between the expression of BMP/Smads in condylar cartilage and condylar growth modifications in rabbits during mandibular forward positioning. METHODS: Sixty male rabbits with 8 weeks of age were randomly divided into the experimental group (n=36) and control group (n=24). The mandibles of rabbits in the experimental groups were induced to forward position by a functional appliance. The rabbits in the experimental group and control group were sacrificed after 3 days and 1, 2, 4, 8, 12 weeks, respectively. The expression of BMP-2, Smad1/5, 4 and 6 in condylar cartilage was examined by immunohistochemical staining. RESULT: The expression of BMP-2, Smad1/5, 4 and 6 was mainly found in the chondrocytes from the transitional zone and hypertrophic zone, and was also found in the chondrocytes and osteoblasts of the mineralized zone. Compared with those of the age-matched controls, the positive signals for BMP-2, Smad1/5, 4 and 6 in the experimental animals were stronger at early stage (P<0.05), coinciding with the remodeling in condylar cartilage after functional appliance. CONCLUSION: The expression of BMP-2, Smad1/5, 4 and 6 is associated with the adaptive remodeling of the condylar cartilage after functional appliance.

Mapping small-effect and linked quantitative trait loci for complex traits in backcross or DH populations via a multi-locus GWAS methodology.

Composite interval mapping (CIM) is the most widely-used method in linkage analysis. Its main feature is the ability to control genomic background effects via inclusion of co-factors in its genetic model. However, the result often depends on how the co-factors are selected, especially for small-effect and linked quantitative trait loci (QTL). To address this issue, here we proposed a new method under the framework of genome-wide association studies (GWAS). First, a single-locus random-SNP-effect mixed linear model method for GWAS was used to scan each putative QTL on the genome in backcross or doubled haploid populations. Here, controlling background via selecting markers in the CIM was replaced by estimating polygenic variance. Then, all the peaks in the negative logarithm P-value curve were selected as the positions of multiple putative QTL to be included in a multi-locus genetic model, and true QTL were automatically identified by empirical Bayes. This called genome-wide CIM (GCIM). A series of simulated and real datasets was used to validate the new method. As a result, the new method had higher power in QTL detection, greater accuracy in QTL effect estimation, and stronger robustness under various backgrounds as compared with the CIM and empirical Bayes methods.

Improving power and accuracy of genome-wide association studies via a multi-locus mixed linear model methodology.

Genome-wide association studies (GWAS) have been widely used in genetic dissection of complex traits. However, common methods are all based on a fixed-SNP-effect mixed linear model (MLM) and single marker analysis, such as efficient mixed model analysis (EMMA). These methods require Bonferroni correction for multiple tests, which often is too conservative when the number of markers is extremely large. To address this concern, we proposed a random-SNP-effect MLM (RMLM) and a multi-locus RMLM (MRMLM) for GWAS. The RMLM simply treats the SNP-effect as random, but it allows a modified Bonferroni correction to be used to calculate the threshold p value for significance tests. The MRMLM is a multi-locus model including markers selected from the RMLM method with a less stringent selection criterion. Due to the multi-locus nature, no multiple test correction is needed. Simulation studies show that the MRMLM is more powerful in QTN detection and more accurate in QTN effect estimation than the RMLM, which in turn is more powerful and accurate than the EMMA. To demonstrate the new methods, we analyzed six flowering time related traits in Arabidopsis thaliana and detected more genes than previous reported using the EMMA. Therefore, the MRMLM provides an alternative for multi-locus GWAS.

Identification of domestication-related loci associated with flowering time and seed size in soybean with the RAD-seq genotyping method.

Flowering time and seed size are traits related to domestication. However, identification of domestication-related loci/genes of controlling the traits in soybean is rarely reported. In this study, we identified a total of 48 domestication-related loci based on RAD-seq genotyping of a natural population comprising 286 accessions. Among these, four on chromosome 12 and additional two on chromosomes 11 and 15 were associated with flowering time, and four on chromosomes 11 and 16 were associated with seed size. Of the five genes associated with flowering time and the three genes associated with seed size, three genes Glyma11g18720, Glyma11g15480 and Glyma15g35080 were homologous to Arabidopsis genes, additional five genes were found for the first time to be associated with these two traits. Glyma11g18720 and Glyma05g28130 were co-expressed with five genes homologous to flowering time genes in Arabidopsis, and Glyma11g15480 was co-expressed with 24 genes homologous to seed development genes in Arabidopsis. This study indicates that integration of population divergence analysis, genome-wide association study and expression analysis is an efficient approach to identify candidate domestication-related genes.

Epistatic association mapping for alkaline and salinity tolerance traits in the soybean germination stage.

Soil salinity and alkalinity are important abiotic components that frequently have critical effects on crop growth, productivity and quality. Developing soybean cultivars with high salt tolerance is recognized as an efficient way to maintain sustainable soybean production in a salt stress environment. However, the genetic mechanism of the tolerance must first be elucidated. In this study, 257 soybean cultivars with 135 SSR markers were used to perform epistatic association mapping for salt tolerance. Tolerance was evaluated by assessing the main root length (RL), the fresh and dry weights of roots (FWR and DWR), the biomass of seedlings (BS) and the length of hypocotyls (LH) of healthy seedlings after treatments with control, 100 mM NaCl or 10 mM Na2CO3 solutions for approximately one week under greenhouse conditions. A total of 83 QTL-by-environment (QE) interactions for salt tolerance index were detected: 24 for LR, 12 for FWR, 11 for DWR, 15 for LH and 21 for BS, as well as one epistatic QTL for FWR. Furthermore, 86 QE interactions for alkaline tolerance index were found: 17 for LR, 16 for FWR, 17 for DWR, 18 for LH and 18 for BS. A total of 77 QE interactions for the original trait indicator were detected: 17 for LR, 14 for FWR, 4 for DWR, 21 for LH and 21 for BS, as well as 3 epistatic QTL for BS. Small-effect QTL were frequently observed. Several soybean genes with homology to Arabidopsis thaliana and soybean salt tolerance genes were found in close proximity to the above QTL. Using the novel alleles of the QTL detected above, some elite parental combinations were designed, although these QTL need to be further confirmed. The above results provide a valuable foundation for fine mapping, cloning and molecular breeding by design for soybean alkaline and salt tolerance.

An efficient hierarchical generalized linear mixed model for mapping QTL of ordinal traits in crop cultivars.

Many important phenotypic traits in plants are ordinal. However, relatively little is known about the methodologies for ordinal trait association studies. In this study, we proposed a hierarchical generalized linear mixed model for mapping quantitative trait locus (QTL) of ordinal traits in crop cultivars. In this model, all the main-effect QTL and QTL-by-environment interaction were treated as random, while population mean, environmental effect and population structure were fixed. In the estimation of parameters, the pseudo data normal approximation of likelihood function and empirical Bayes approach were adopted. A series of Monte Carlo simulation experiments were performed to confirm the reliability of new method. The result showed that new method works well with satisfactory statistical power and precision. The new method was also adopted to dissect the genetic basis of soybean alkaline-salt tolerance in 257 soybean cultivars obtained, by stratified random sampling, from 6 geographic ecotypes in China. As a result, 6 main-effect QTL and 3 QTL-by-environment interactions were identified.

Combination of intermittent hydrostatic pressure linking TGF-ß1, TNF-α on modulation of proteoglycan 4 metabolism in rat temporomandibular synovial fibroblasts.

OBJECTIVE: This study was designed to evaluate the combined effects of intermittent hydrostatic pressure (IHP) and TGF-ß1 or TNF-α on proteoglycan4 (PRG4) expression in rat temporomandibular synovial fibroblasts (SFs). STUDY DESIGN: Rat SFs were isolated and expanded in monolayer cultures and subjected to IHP in the presence of TGF-ß1 or TNF-α. Quantitative real-time RT-PCR was applied to analyze the PRG4 expression levels. Enzyme-linked immunosorbent assay was also used for the quantification of PRG4 accumulation in the culture medium while immunofluorescence staining was used to detect intracellular PRG4 protein expression. RESULTS: The combination of IHP and TGF-ß1 induced greater PRG4 expression than either stimulus alone. In contrast, TNF-α inhibited PRG4 expression, and this was partially alleviated by IHP. CONCLUSIONS: Our study demonstrates a beneficial role of IHP, which can be used successfully in combination with TGF-ß1 to enhance PRG4 production, and can partially counteract TNF-α-induced PRG4 inhibition in isolated rat SFs.

Feruloylacetone as the model compound of half-curcumin: synthesis and antioxidant properties.

In order to clarify the contribution of phenolic and enolic hydroxyl group to the antioxidant capacity of feruloylacetone, a model compound of half-curcumin, 6-(p-hydroxy-m-methoxyphenyl)-5-hexene-2,4-dione (FT), 6-(p-benzyloxy-m-methoxyphenyl)-5-hexene-2,4-dione (BMFT), 6-(m,p-dihydroxyphenyl)-5-hexene-2,4-dione (DDFT), 6-(p-hydroxy-m-methoxyphenyl)hexane-2,4-dione (DHFT), 6-(p-hydroxy-m-methoxyphenyl)-5-hexene-2,4-diol (THFT), and ethyl 2-(p-hydroxy-m-methoxybenzylidene)-3-oxobutanoate (EOFT) were synthesized. The radical-scavenging abilities of these compounds were tested by trapping 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) cationic radical (ABTS(+·)), 2,2'-diphenyl-1-picrylhydrazyl (DPPH), and galvinoxyl radicals. The reductive capacities were screened by quenching singlet oxygen and by inhibiting the oxidation of linoleic acid. They were also employed to inhibit the oxidation of DNA mediated by hydroxyl radical and 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH). In addition, they were applied to protect erythrocytes against AAPH- and hemin-induced hemolysis. The obtained results revealed that the antioxidant capacity of half-curcumin was derived from the phenolic-OH and the conjugated linkage between phenolic and enolic-OH. The enolic-OH itself cannot trap radicals.

Phenolic and enolic hydroxyl groups in curcumin: which plays the major role in scavenging radicals?

The aim of this work is to clarify the antioxidant abilities of phenolic and enolic hydroxyl groups in curcumin. 1,7-bis(4-benzyloxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione (BEC), 1,7-bis(4-hydroxy-3-methoxyphenyl)heptane-3,5-diol (OHC), 1,7-bis(4-hydroxy-3-methoxyphenyl)heptane-3,5-dione (THC), and 1,7-bis(3,4-dihydroxyphenyl)-1,6-heptadiene-3,5-dione (BDC) are synthesized to determine the antioxidant activities by using antiradical assays against 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical, galvinoxyl radical, and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) cation radical (ABTS*+) and by protecting DNA and erythrocyte against 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH) induced oxidation. The phenolic hydroxyl is the main group for curcumin to trap DPPH, galvinoxyl, and ABTS*+ radicals. The conjugative system between enolic and phenolic hydroxyl groups is beneficial for curcumin to protect erythrocytes against hemin-induced hemolysis and to protect DNA against AAPH-induced oxidation, but is not beneficial for curcumin to protect erythrocytes against AAPH-induced hemolysis. More hydroxyl groups enhance the antioxidant effectiveness of curcumin in the experimental systems employed herein. Therefore, curcumin acts as an antioxidant through the phenolic hydroxyl group.

[Effect of hydrostatic pressure on ultrastructure of synovial fibroblasts from rat temporomandibular joint].

OBJECTIVE: To investigate the role of apoptosis in the remodeling of temporomandibular joint (TMJ) under pressure. METHODS: Synovial fibroblasts obtained from rat temporomandibular joint were subjected to different hydrostatic pressure for 12 h. Changes of ultrastructure were observed by transmission electron microscope. RESULTS: At 30 kPa, the ultrastructure of synovial fibroblasts showed no obvious changes. At 60 kPa, the chromatin was condensated, the baryon took on crescent and the mitochondria seemed varicose. At 90 kPa, the apoptosis-like body was wrapped by membrane and embedded in the high density chromatin. CONCLUSIONS: Apoptosis-like change took place in ultrastructure of synovial fibroblasts under hydrostatic pressure, and the degree of the change was related to the hydrostatic pressure exerted.

[The temporal and spatial expression of CBFalpha1 in rabbit condylar cartilage during forward mandibular positioning].

PURPOSE: To identify the relationship between the expression of Core binding factor alpha1 (Cbfalpha1) in condylar cartilage and condylar growth modifications in growing rabbits following forward positioning mandibles with functional appliance. METHODS: Sixty rabbits with age of 8 weeks were randomly divided into the experimental group (n=36) and control group (n=24). Rabbits in the experimental groups were induced to forward their mandibles by a functional appliance. The animals in the two groups were killed after 3 days and 1, 2, 4, 8 and 12 weeks, respectively. The temporal and spatial expression of Cbfalpha1 in condylar cartilage was examined by immunohistochemical staining and measured with Image Computer Analysis Software. Date was statistically analyzed by Student's t test and one-way ANOVA. RESULTS: The expression of Cbfalpha1 was mainly found in the chondrocytes from the transitive zone and hypertrophic zone, and was also observed in the chondrocytes and osteoblasts of the mineralized zone. Compared with those of the age-matched controls, the positive signals for Cbfalpha1 of each zone in the experimental animals were significantly stronger (P<0.05), and the highest expression of transitive zone, hypertrophic zone and mineralized zone was detected after 4 weeks of treatment (71.00+/-1.52, 50.00+/-0.75, 82.00+/-0.39). CONCLUSION: The pattern of Cbfalpha1 expression is concerned with the adaptive remodeling of the condylar cartilage after functional appliance.

[The morphology of the cells in the cartilage of rabbit mandibular condyle].

PURPOSE: To study the tissue layers and their function of the cartilage in mandibular condyle in rabbits. METHODS: Six adult Japanese white rabbits were subjected. Their temporomandibular joints were studied by immunohistochemistry for FGFR3 and PCNA, and in situ hybridization for aggrecan and collagen II mRNA expression, as well as ultrastructure. RESULTS: The upper proliferative cells did not express FGFR3, but the lower proliferative cells expressed FGFR3. Only few cells in the upper proliferative layer were PCNA positive, but all cells in the lower proliferative layer were positive for PCNA. No collagen II mRNA expression was found in the upper proliferative cell, but aggrecan and collagen II mRNA coexpressed in the lower proliferative layer. The cells in both layers were different in ultrastructure. CONCLUSION: The cartilage in mandibular condyle should have the 5 following tissue layer: fibrous layer, proliferative layer, transitional layer, cartilaginous layer and calcified cartilaginous layer. The cells in the proliferative layer are undifferentiated and the cells in the differentiated layer are prechondrocytes.