Detection of QTLs controlling fast kernel dehydration in maize (Zea mays L.).

In order to understand the effect of grain moisture of inbred lines at the silking and physiological maturity stages on kernel dehydration rate, 59 maize inbred lines from six subgroups were selected. Grain moisture was measured and QTLs associated with kernel dehydration were mapped. A rapid dehydration evaluation and association analysis revealed eight inbred lines with faster dehydration rate, including Yuanwu 02, K36, Zhonger/O2, Lo1125, Han 49, Qi 319, Hua 160, and PH4CV. A single sequence repeat analysis using 85 pairs detected five QTLs with phenotypic variation contribution ≥10% in the permanent F2 generation populations Zheng 58 x S1776 and Chang 7-2 x K1131, which had LOD threshold values ≥ 3 in both 2013 and 2014. The chromosome region of qFkdr7b had not previously been reported and is preliminarily identified as a new major QTL. A false positive field verification of grain dehydration rate of 53 inbred lines indicated that the screening result of the rapid dehydration inbred lines by specific amplification with marker Phi114 was most similar to the field assessment result, followed by markers Phi127 and Phi029. The rapid dehydration lines selected based on primer Phi114 amplification were also similar to the field dehydration rate and can thus be used for molecular marker-assisted selection. A significant effort is needed to improve stress resistance and shorten the growth period via fast kernel dehydration in intermediate materials of the inbred lines K36, Zhonger/ O2, Lo1125, Han 49, Hua 160, and PH4CV, and further using the selected lines for new combinations.

Genetic analyses of the major and minor locus groups of bacterial wilt resistance in tobacco using a diallel cross design.

Tobacco germplasm samples with various levels of resistance to bacterial wilt were selected to construct F1 combinations of parental inbred lines and orthogonal diallel crosses using samples collected in 2009 (15 germplasms), 2010 (15 germplasms), and 2011 (16 germplasms). A total of 1/2P (P + 1) experimental materials were used for analysis. Based on the analyses of major and minor locus groups, genetic effects on the incidence rate and index of bacterial wilt in tobacco were investigated on the 15th and 25th day during the early stage. Significant effects were observed in major locus groups, but not in minor locus groups. Specifically, adjacent major locus groups (J1 = 13,056 and J1 = 13,055; J1 = 14,080 and J1 = 14,079) were detected in both the first and second analyses with considerable effects. Based on the additive effects of minor locus groups on the rate and index of bacterial wilt, the effects on the incidence rates of Yunyan 85, DB101, and RG11 as well as the effects on the disease index of the latter two germplasms reached the maximum. This was consistent with the disease resistance indicators of these tobacco varieties in the field (corresponding broad heritability >20%). Genetic homozygous dominant loci (+ +) increased the rate of bacterial wilt (susceptible), whereas homozygous recessive loci (- -) reduced the index of bacterial wilt (resistant) with considerable additive effects and low dominant effects, suggesting that the inheritance of the bacterial wilt rate and index in tobacco mainly relies on additive inheritance.

Association between polymorphisms in exons 4 and 10 of the BPI gene and immune indices in Sutai pigs.

The bactericidal/permeability-increasing protein (BPI) gene has been identified as a candidate gene for disease-resistance breeding. We evaluated whether polymorphisms in exons 4 and 10 of the BPI gene are associated with immune indices [interleukin-2 (IL-2), IL-4, IL-6, interferon-b (IFN-b), IL-10, and IL-12]. In this study, we identified one mutation (C522T) in the BPI exon 4 site and two mutations (A1060G and T1151G) in the BPI exon 10 site. Correlation analysis revealed that in the Sutai pig population, the effect of genotypes at the BPI exon 4 site on the level of IL-6 was significant (P < 0.05), with an effective genotype of CD; moreover, the effect of genotypes at the BPI exon 10 site on the level of IL-12 was significant (P < 0.05), and the effective genotype was AB. The optimal combined genotype was CD-AB, which was more effective regarding the IL-6 and IL-12 levels compared to the other combined genotypes (P < 0.05). These results indicate that single nucleotide polymorphisms and the combined genotypes of BPI exons 4 and 10 affect immune indices in Sutai pigs. Therefore, these genotypes should be further examined as effective markers for disease-resistant breeding of pigs.

Identification of BPI protein produced in different expression system and its association with Escherichia coli F18 susceptibility.

The super antibiotic bactericidal/permeability-increasing (BPI) protein is a member of a new generation of proteins that have been implicated as endotoxin-neutralizing agents. In this study, recombinant porcine BPI protein was obtained by generating porcine BPI encoding prokaryotic, eukaryotic, and yeast expression vectors. Recombinant protein expression was detected in yeast GS115, Escherichia coli, and 293-6E cells by gel electrophoresis and Western blotting. Escherichia coli F18 is the primary Gram-negative bacteria in the gut and the main pathogen leading to diarrhea and edema dis-ease in weaning piglets. Therefore, E. coli F18-resistant and -sensitive Sutai piglets were used to test differential expression of BPI protein by Western blotting and to investigate the potential correlation between BPI protein expression and E. coli F18-susceptibility. Recombinant porcine BPI protein expression was not detected in the prokaryotic and yeast expression systems; however, soluble protein was detected in the eukaryotic expression system. These data indicate the strong bacterio-static action of the BPI protein and confirm the feasibility of obtaining large amounts of recombinant porcine BPI recombinant protein using this eukaryotic expression system. In addition, the BPI protein expres-sion levels in the E. coli F18-resistant group were significantly higher than those in the sensitive group, indicating that high BPI protein ex-pression is associated with resistance to E. coli F18. Our findings pro-vide a basis for further investigations into the development of a drug designed to confer resistance to E. coli F18 in weaning piglets.

Influence of sirolimus-induced TGF-ß secretion on mouse Treg cell proliferation.

We examined the effects of co-culturing CD4+ CD25+ Treg cells with sirolimus or cyclosporin A on Treg cell proliferation and differentiation and on transforming growth factor-ß (TGF-ß) and Foxp3 expression. CD4+ CD25+ Treg cells were harvested from mononuclear cells of spleens of C57BL/6 mice using immunomagnetic beads and divided into control, sirolimus, and cyclosporine groups. Following a 96-h co-culture, Treg cells were assayed by flow cytometry. FoxP3 and TGF-ß mRNA levels and secretion were assayed by reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay. Smad protein of the TGF-ß signaling pathway was assayed by western blot and its effect on CD4+ CD25+ FoxP3+ Treg cell proliferation was determined. Sirolimus-promoted differentiation and proliferation was examined using a TGF-ß neutralizing antibody. Sirolimus-treated CD4+ T cell TGF-ß secretion increased 2.5X over control levels (P < 0.01), but that of the cyclosporine group decreased marginally (P > 0.05). The CD4+ cell proportion decreased significantly (41.25 vs 69.22%, P < 0.01) and slightly (65.21 vs 69.22, P > 0.05) in the cyclosporine and sirolimus groups, respectively. T cell Foxp3 mRNA expression was significantly higher in the sirolimus-treated than in the cyclosporine (53.7 vs 40.2%, P < 0.05) and control groups (P < 0.01), but was significantly lower in the cyclosporine group than in controls (23.6 vs 40.2%, P < 0.01). Overall, sirolimus promoted CD4+ CD25+ Treg cell proliferation and growth in vitro, whereas cyclosporin A inhibited proliferation. Sirolimus might promote CD4+ CD25+ FoxP3+ regulatory T cell proliferation by inducing TGF-ß secretion in vivo.

TLR4 gene expression in pig populations and its association with resistance to Escherichia coli F18.

TLR4 is the main recognition receptor of bacterial lipopolysaccharides, which play an important role in innate and adaptive immunity. We used real-time PCR to analyze the tissue expression profile and differential expression of TLR4 in 4 pig populations (Escherichia coli F18-resistant Sutai, E. coli F18-sensitive Sutai, Large White, Meishan), in order to determine the role that the TLR4 gene plays in resistance to E. coli F18. We found that TLR4 expressed consistently in the 4 populations, with relatively high levels in immune tissues and the highest level in the lung. Generally, the expression of TLR4 in E. coli F18-sensitive individuals was the highest, followed by that in E. coli F18-resistant, Large White and Meishan. In the spleen, lung, kidney, lymph nodes, and thymus gland, TLR4 expression is significantly higher in the E. coli F18-sensitive than in the other 3 populations; there were no significant differences among E. coli F18-resistant Sutai, Large White, and Meishan. In addition, Gene Ontology and pathway analysis showed that TLR4 takes part in the inflammatory response. We found that porcine TLR4 has consistent tissue specificity in each breed, and downregulation of expression of the TLR4 gene is related to resistance to E. coli F18 in weaning piglets.

Age-dependent expression of the BPI gene in Sutai piglets.

We compared and analyzed the expression of the BPI gene of Sutai piglets ranging from newborn to post-weaning days 8, 18, 30, and 35 by the real-time PCR method, in order to determine if it is involved in protection against disease caused by ETEC F18. There was a significant difference between 18 and 35-day expression in the jejunum. There were also significant differences between 35-day expression and expression at the other development stages in the duodenum. There were no significant differences in expression at 8, 18, and 30 days in the jejunum. We conclude that the porcine BPI gene may be the direct factor that resisted the ETEC F18 in weaning piglets, and that the resistance to ETEC F18 in weaning piglets is related to up-regulation of mRNA expression of BPI gene to a certain extent.