Cost-effective duplex Kompetitive Allele Specific PCR markers for homologous genes facilitating wheat breeding.

BACKGROUND: Owing to successful cloning of wheat functional genes in recent years, more traits can be selected by diagnostic markers, and consequently, effective molecular markers will be powerful tools in wheat breeding programs. RESULTS: The present study proposed a cost-effective duplex Kompetitive Allele Specific PCR (dKASP) marker system that combined multiplex PCR and KASP™ technology to yield twice the efficiency at half the cost compared with the common KASP™ markers and provide great assistance in breeding selection. Three dKASP markers for the major genes controlling plant height (Rht-B1/Rht-D1), grain hardness (Pina-D1/Pinb-D1), and high-molecular-weight glutenin subunits (Glu-A1/Glu-D1) were successfully developed and applied in approved wheat varieties growing in the middle and lower reaches of the Yangtze River and advanced lines from our breeding program. Three markers were used to test six loci with high efficiency. In the approved wheat varieties, Rht-B1b was the most important dwarfing allele, and the number of accessions carrying Pinb-D1b was much greater than that of the accessions carrying Pina-D1b. Moreover, the number of accessions carrying favorable alleles for weak-gluten wheat (Null/Dx2) was much greater than that of the accessions carrying favorable alleles for strong-gluten wheat (Ax1 or Ax2*/Dx5). In the advanced lines, Rht-B1b and Pinb-D1b showed a significant increase compared with the approved varieties, and the strong-gluten (Ax1 or Ax2*/Dx5) and weak-gluten (Null/Dx2) types also increased. CONCLUSION: A cost-effective dKASP marker system that combined multiplex PCR and KASP™ technology was proposed to achieve double the efficiency at half the cost compared with the common KASP™ markers. Three dKASP markers for the major genes controlling PH (Rht-B1/Rht-D1), GH (Pina-D1/Pinb-D1), and HMW-GS (Glu-A1/Glu-D1) were successfully developed, which would greatly improve the efficiency of marker-assisted selection of wheat.

Genome-wide association studies and transcriptome sequencing analysis reveal novel genes associated with Al tolerance in wheat.

Aluminum (Al) toxicity is a major threat to the productivity and quality of wheat on acid soil. Identifying novel Al tolerance genes is crucial for breeders to pyramid different tolerance mechanisms thus leading to greater Al tolerance. We aim to identify novel quantitative trait loci (QTL) and key candidate genes associated with Al tolerance in wheat. Herein, we investigated the genotypic variation in Al tolerance among 334 wheat varieties using an acid soil assay. Genome-wide association study (GWAS) and transcriptome were carried out to identify key genes for Al tolerance. GWAS identified several QTL associated with acid soil tolerance including one major QTL on chromosome 1A, in addition to the QTL on 4D where TaALMT1 is located. The four significant markers around the newly identified QTL explained 27.2% of the phenotypic variation. With the existence of reported markers for TaALMT1, more than 97% of the genotypes showed tolerance to Al. For those genotypes with the existence of the novel QTL on 1A but without TaALMT1, more than 90% of genotypes showed medium or high tolerance to Al, confirming the existence of the Al tolerance gene(s) on chromosome 1A. By combining GWAS and RNA-seq analysis, we identified 11 candidate genes associated with Al tolerance. The results provide new insights into the genetic basis of Al tolerance in wheat. The identified genes can be used for the breeding of Al tolerant accessions.

Characterization of the baking-induced changes in starch molecular and crystalline structures in sugar-snap cookies.

This study aims to understand the starch molecular structural changes from baking sugar-snap cookies. Changes in the whole-molecule size distribution and chain-length distribution of the parent wheat flour and from final cookie products were measured by size-exclusion chromatography with and without enzymatic debranching, and the results fitted by two biosynthesis-based models. Fraction crystallinity was also analyzed. After cooking, there was a significant decrease in average molecular sizes of amylopectin and in the average lengths of amylose chains, and some starch granules lost birefringence. However, the chain-length distributions of amylopectin showed no noticeable difference, resulting in little change in relative crystallinity and gelatinization temperatures. Both the short-range ordered structure and the periodic lamellar structure were disrupted. This study provides new insight into starch structural changes in sugar-snap cookies after baking, which play an important role in determining final cookie quality. For example, a decrease in size of amylose chains influences cookie sensory properties, and thus can be used as an additional tool for choice of grains.

miR­183­5p attenuates cerebral ischemia injury by negatively regulating PTEN.

Cerebral ischemia is a common cerebrovascular disease caused by the occlusion of a cerebral blood vessel. MicroRNAs (miRNAs/miRs) are emerging regulators of various human diseases, including cerebral ischemia. Upregulation of miR­183­5p has been reported to alleviate liver injury induced by ischemia­reperfusion (I/R). However, the effect of miR­183­5p on cerebral ischemia injury remains unknown. The present study evaluated the effects of miR­183­5p on ischemia injury using ischemic models of mouse brains exposed to transient middle cerebral artery occlusion and Neuro­2A (N2A) neuroblastoma cells exposed to oxygen­glucose­deprivation (OGD) and subsequently reoxygenated. Ischemia was evaluated in mice using neurological function scores, cerebral edema, 2,3,5­triphenyltetrazoliumchloride, Nissl and Fluoro­Jade B staining assays. In addition, miR­183­5p expression, N2A cell viability and the expression levels of apoptosis­associated proteins were detected by quantitative PCR, Cell Counting Kit­8 assay, flow cytometry and western blotting. The association between miR­183­5p and phosphatase and tensin homolog (PTEN) was also confirmed by a luciferase reporter assay. The results revealed that miR­183­5p expression was decreased and brain damage was increased in ischemic mice compared with the sham group. Additionally, miR­183­5p levels were reduced, and apoptosis was increased in N2A cells exposed to ischemia compared with the control group. Following transfection with agomiR­183­5p, cerebral ischemic injury and apoptosis levels were reduced in the in vivo I/R stroke model and OGD­induced N2A cells. In addition, PTEN was determined to be a target of miR­183­5p following elucidation of a direct binding site. Overexpression of PTEN reversed the miR­183­5p­induced N2A cell apoptosis inhibition and survival after OGD. The results of the present study suggested that miR­183­5p reduced ischemic injury by negatively regulating PTEN, which may aid the development of a novel therapeutic strategy for cerebral ischemia.

Effects of amylose and amylopectin fine structure on sugar-snap cookie dough rheology and cookie quality.

Starch, the most abundant component of wheat flour, has a significant influence on sugar-snap cookie quality, but the underlying mechanisms are not well understood. In this study, the solvent retention capacity and rheological properties of sugar-snap cookie dough, and cookie physical characteristics and textural properties, were analyzed. Starch molecular structures were measured by size-exclusion chromatography (SEC) and the results parameterized using two biosynthesis-based mathematical models to obtain structure-property correlations. The results show that the viscoelasticity of sugar-snap cookie dough is positively correlated with the length of amylopectin short chains. In addition, the length of amylose short chains is positively correlated with cookie thickness and negatively correlated with its spread ratio. Starch molecular structure shows no influence on cookie hardness and fracturability. Mechanisms are proposed for these results, which could lead to new ways to improve sugar-snap cookie quality by choosing starches with appropriate structural features.

Long Non-coding RNA MINCR Regulates miR-876-5p/GSPT1 Axis to Aggravate Glioma Progression.

Emerging evidence underlined the crucial roles played by long non-coding RNAs (lncRNAs) in glioma. MINCR has been reported in multiple malignancies. Here, we studied its function and potential mechanism in glioma, which remain unclear. Gene expressions were analyzed by qRT-PCR assay. Both in vitro and in vivo assays were conducted to evaluate the cellular function of MINCR in glioma. The subcellular situation of MINCR was detected by subcellular fractionation and FISH assays. Luciferase reporter, RNA pull-down and RNA immunoprecipitation (RIP) assays were combined to investigate potential mechanisms of relevant genes. MINCR was up-regulated in glioma. MINCR depletion markedly refrained glioma cell proliferation, migration and invasion via sponging miR-876-5p. MiR-876-5p suppressed the malignant behaviors of glioma via binding to GSPT1. MINCR shared the binding sites with the 3'-untranslated region of GSPT1 and prevented the binding of miR-876-5p to GSPT1 mRNA, thus up-regulating the level of GSPT1. Moreover, miR-876 inhibition and GSPT1 up-regulation counteracted the functional effect induced by silencing MINCR on glioma progression. Our findings uncovered that MINCR might aggravated glioma cell proliferation and migration via acting as competing endogenous RNA (ceRNA), indicating prospective novel therapeutic target for glioma.

Comparison of pasting properties measured from the whole grain flour and extracted starch in barley (Hordeum vulgare L.).

Pasting properties of barley starch are important characteristics from a processing standpoint. The isolation of starch form barley grains is time consuming thus the whole grain flour is always used. To compare pasting properties of starch with those of the whole grain flour, we used a Rapid Visco Analyser (RVA) to measure pasting properties of three types of samples: grain flour and starches isolated using two different extraction methods. We also investigated compositional, morphological and structural properties of the two starch samples. Significant differences in pasting properties were found among the three sample types, but most of the parameters of pasting properties displayed significant correlations between flour and starch. No significant differences were found in amylose/amylopectin ratio, granule morphology, granule size distribution and crystal structure between starches extracted using two different methods. However, the starch isolated from water homogenization had a higher protein content and lower total starch, amylose and amylopectin contents than the starch extracted with homogenized extraction under alkaline conditions. We concluded that the whole grain flour can be used to predict the pasting properties in breeding programs.

Comparative Mapping and Candidate Gene Analysis of SSIIa Associated with Grain Amylopectin Content in Barley (Hordeum vulgare L.).

Amylopectin concentration in barley endosperm has important effects on grain quality and end-use. In this study, quantitative trait locus (QTL) analysis together with genome-wide association studies (GWAS) were performed to identify markers linked to grain amylopectin content respectively using a doubled haploid (DH) population of 178 lines and a collection of 185 diverse barley germplasms both genotyped by genotyping-by-sequencing (GBS). A stable QTL on chromosome 7H and 11 associated single nucleotide polymorphisms (SNPs) were detected. In the co-localized region, the SSIIa (SSII-3) gene was predicted as the candidate gene. Then we isolated and characterized biparental SSIIa alleles of the DH population, investigated the expression pattern by quantitative real-time PCR (qRT-PCR), and revealed that a 33-bp deletion in exon 2 is responsible for reducing SSIIa transcript, thus resulting in a reduced amylopectin content. A sequence-based molecular marker was developed for the SSIIa allele and validated the effectivity, which would provide help for barley breeding.

Identification of C-type lectin-domain proteins (CTLDPs) in silkworm Bombyx mori.

C-type lectins (CTLs) represent a large family of proteins that can bind carbohydrate moieties normally in a calcium-dependent manner. CTLs play important roles in mediating cell adhesion and the recognition of pathogens in the immune system. In the present study, we have identified 23 CTL genes in domestic silkworm Bombyx mori. CTL-domain proteins (CTLDPs) are classified into three groups based on the number of carbohydrate-recognition domains (CRDs) and the domain architectures. These include twelve CTL-S (Single-CRD), six immulectins (Dual-CRD) and five CTL-X (CRD with other domains). We studied their phylogenetic features, analyzed the conserved residues, predicted tertiary structures, and examined the tissue expression profile and immune inducibility. Through bioinformatics analysis, we have putatively identified ten secretory and two cytoplasmic CTL-S; four secretory and two cytoplasmic immulectins; one secretory, one cytoplasmic and three transmembrane forms of CTL-X. Most B. mori CTLDPs form monophyletic groups with orthologs from Lepidoptera, Diptera, Coleoptera and Hymenoptera species. Immulectins of B. mori and Manduca sexta evolved from common ancestor genes perhaps due to gene duplication events of CTL-S ancestor genes. Homology modeling revealed that the overall structures of B. mori CTL domains are analogous to those of humans with a variable loop region. We examined the expression profile of CTLDP genes in naïve and immune-stimulated tissues. The expression and induction of CTLDP genes were related to the tissues and microorganisms. Together, our gene identification, sequence comparison, phylogenetic analysis, homology modeling and expression analysis laid a good foundation for the further studies of B. mori CTLDPs and comparative genomics.