Wheat Grain Yield and Protein Content as Influenced by Sodium Bisulfite

Two wheat varieties with similar yield and significantly different protein content were chosen to study the effect of foliage spraying sodium bisulfite (NaHSO3) at low concentration to wheat plants receiving different levels of nitrogen on nitrate reduction along with yield and protein content of grain, so as to discuss the relationship between carbon metabolism and nitrogen metabolism, and find out the intrinsic mechanism of grain yield and protein content formation. The results showed that spraying NaHSO3 at grain-filing stage increased the yield of grain, but declined the capacity of nitrate reduction, which might result in decrease of protein content in the end. Whether receiving NaHSO, or not, the variety with high protein content had higher final protein level and nitrate reduction capacity than that with low protein content.

ADDITION OF SODIUM BISULFITE TO BILIVERDIN

Summary:Sodium bisulfite could add to the central methine bridge of biliver- din in methanol,and give sodium bilirubin-10-sulfonate,however no bilirubin was formed.

The bisulfite genomic sequencing protocol

The bisulfite genomic sequencing (BGS) protocol has gained worldwide popularity as the method of choice for analyzing DNA methylation. It is this popular because it is a powerful protocol and it may be coupled with many other applications. However, users often run into a slew ofproblems, including incomplete conversion,overly degraded DNA, sub-optimal PCR amplifications, false positives, uninformative results, or altogether failed experiments. We pinpoint the reasons why these problems arise and carefully explain the critical steps toward accomplishing a successful experiment step-by-step. This protocol has worked successfully (>99.9% conversion) on as little as 100 ng of DNA derived from nearly 10-year-old DNA samples extracted from whole blood stored at -80°C and resulted in enough converted DNA for more than 50 PCRreactions. The aim of this article is to makelearning and usage of BGS easier, more efficient and standardized for all users.

The effectiveness of bisulfite-activated permanganate technology to enhance the coagulation efficiency of Microcystis aeruginosa

The effects of bisulfite-activated permanganate technology(PM/BS)as a pre-oxidation process on enhancing Microcystis aeruginosa(M.aeruginosa)removal by post coagulation were investigated.The results demonstrated that pretreatment with PM/BS process effectively promoted the algae removal by coagulation with Al2(SO4)3 as the coagulant and this phenomenon was more obvious with the increase of water hardness.Compared to the sole coagulation,PM/BS pre-oxidation combing with coagulation could neutralize the zeta potential of algal cells effectively,decrease the algal cell size,and lead to the formation of more compact flocs due to the in-situ generated Mn02.The effect of oxidant dosages on algal organic matter(AOM)was also studied and no obvious release of macromolecular substances was observed with the dosage of KMn04 increasing from 3.0 mg/L to 7.0 mg/L,suggesting the integrity of algal cells at a high KMn04 dosage.Moreover,PM/BS pre-oxidation could lead to the decrease of most analyzed disinfection by-products(DBPs)at a Al2(SO4)3 dosage of 40.0 mg/L.The algae removal efficiency was also significantly enhanced by PM/BS pre-oxidation in the test using real algae-laden water.This study indicated that PM/BS process might be a potential assistant technology for algae removal by subsequent coagulation.

Differential methylation analysis for bisulfite sequencing using DSS

Bisulfite sequencing(BS-seq)technology measures DNA methylation at single nucleotide resolution.A key task in BSseq data analysis is to identify differentially methylation(DM)under different conditions.Here we provide a tutorial for BS-seq DM analysis using Bioconductor package DSS.DSS uses a beta-binomial model to characterize the sequence counts from BS-seq,and implements rigorous statistical method for hypothesis testing.It provides flexible functionalities for a variety of DM analyses.

A carbazole-hemicyanine dye based ratiometric fluorescent probe for selective detection of bisulfite(HSO_3^-)in cells and C. elegans

Bisulfite(HSO_3) is an important sulfur dioxide(SO_2) derivative, which plays a major role in many physiological processes and is also closely associated with a variety of diseases. Thus the development of highly selective and sensitive fluorescent probes is essential to detect HSO_3 in living cells. In this work,we report the synthesis and analysis of a ratiometric fluorescent probe for selective detection of HSO_3 based on the 1,4-nucleophilic addition reaction with the carbazole as an electron donor(D) and aldehyde group as an electron acceptor(A). The addition of HSO_3 and other ions to our probe can be observed by UV vis and fluorescence spectrometry. Our investigation proved that the probe is highly selective and sensitive for HSO_3 and ratiometric changes. Moreover, the probe has good cell permeability and was successfully applied to the detection of exogenous HSO_3 in Hela cells and C. elegans.

Sodium Bisulfite： An Efficient Catalyst for Ether Formation via Dehydration of Aromatic/Aliphatic Alcohol

Straightforward etherification of benzyl alcohols （1） via intermolecular dehydration can be efficiently catalyzed by sodium bisulfite under solvent-free conditions. In the presence of 0.3 mol% or 0.6 mol% amount of sodium bisulfite, symmetric and unsymmetric ethers are prepared from the corresponding alcohols in high yields （up to 95%）. Etherification of benzhydryl alcohols is also discussed.

Des=gn of efficient simplified genomic DNA and bisulfite sequencing in large plant populations

The next generation sequencing enables generation of high resolution and high throughput data for structure sequence of any genome at a fast declining cost. This opens opportunity for population based genetic and genomic analyses. In many applications, whole genome sequencing or re-sequencing is unnecessary or prohibited by budget limits. The Reduced Representation Genome Sequencing （RRGS）, which sequences only a small proportion of the genome of interest, has been proposed to deal with the situations. Several forms of RRGS are proposed and implemented in the literature. When applied to plant or crop species, the current RRGS protocols shared a key drawback that a significantly high proportion （up to 60%） of sequence reads to be generated may be of non-genomic origin but attributed to chloroplast DNA or rRNA genes, leaving an exceptional low efficiency of the sequencing experiment. We recommended and discussed here the design of optimized simplified genomic DNA and bisulfite sequencing strategies, which may greatly improves efficiency of the sequencing experiments by bringing down the presentation of the undesirable sequencing reads to less than 10% in the whole sequence reads. The optimized RAD- seq and RRBS-seq methods are potentially useful for sequence variant screening and genotyping in large plant/crop populations.

Genome‑wide DNA methylation and transcriptome analyses reveal the key gene for wool type variation in sheep

Background Wool fibers are valuable materials for textile industry.Typical wool fibers are divided into medullated and non-medullated types,with the former generated from primary wool follicles and the latter by either primary or secondary wool follicles.The medullated wool is a common wool type in the ancestors of fine wool sheep before breeding.The fine wool sheep have a non-medullated coat.However,the critical period determining the type of wool follicles is the embryonic stage,which limits the phenotypic observation and variant contrast,making both selection and studies of wool type variation fairly difficult.Results During the breeding of a modern fine(MF)wool sheep population with multiple-ovulation and embryo transfer technique,we serendipitously discovered lambs with ancestral-like coarse(ALC)wool.Whole-genome rese-quencing confirmed ALC wool lambs as a variant type from the MF wool population.We mapped the significantly associated methylation locus on chromosome 4 by using whole genome bisulfite sequencing signals,and in turn identified the SOSTDC1 gene as exons hypermethylated in ALC wool lambs compare to their half/full sibling MF wool lambs.Transcriptome sequencing found that SOSTDC1 was expressed dozens of times more in ALC wool lamb skin than that of MF and was at the top of all differentially expressed genes.An analogy with the transcriptome of coarse/fine wool breeds revealed that differentially expressed genes and enriched pathways at postnatal lamb stage in ALC/MF were highly similar to those at the embryonic stage in the former.Further experiments validated that the SOSTDC1 gene was specifically highly expressed in the nucleus of the dermal papilla of primary wool follicles.Conclusion In this study,we conducted genome-wide differential methylation site association analysis on differen-tial wool type trait,and located the only CpG locus that strongly associated with primary wool follicle development.Combined with transcriptome analysis,SOSTDC1 was identified as the only gene at this locus that was specifically overexpressed in the primary wool follicle stem cells of ALC wool lamb skin.The discovery of this key gene and its epigenetic regulation contributes to understanding the domestication and breeding of fine wool sheep.

Promoter hypermethylation and loss of CD133 gene expression in colorectal cancers

AIM: To understand CD133 promoter hypermethyl-ation and expression in 32 colorectal cancer cell lines. METHODS: Nucleic acid was isolated from 32 colorectal cancer cell lines and CD133 expression levels were measured by reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR. Promoter methylation status of the CD133 gene was analyzed with a methylation-specific PCR after sodium-bisulfi te modification and by clonal sequencing analysis. The correlation between expression and promoter methylation of CD133 gene was confirmed with treatment of 5-aza-2’-deoxycytidine. RESULTS: We measured CD133 expression levels in 32 colorectal cancer cell lines. RT-PCR analysis showed undetectable or low levels of CD133 expression in 34.4%of cell lines. To verify the relation between CD133 expression and methylation status of the CD133 gene promoter in colorectal carcinogenesis, CD133 gene promoter hypermethylation was analyzed in 32 cancer cell lines. Promoter hypermethylation was detected in 13 (40.6%) of the cell lines using methylation specificPCR and confirmed by bisulfite sequencing analysis. Treatment of 11 of the cell lines with the demethylation agent 5-aza-2’-deoxycytidine recovered CD133 expression in most of them. CONCLUSION: Transcriptional repression of CD133 is caused by promoter hypermethylation of the CD133 CpG islands in some of colorectal cancer cell lines. The study may contribute to the understanding of the role of CD133 inactivation in the progression of colorectal cancers.

MGMT is down-regulated independently of promoter DNA methylation in rats with all-trans retinoic acidinduced spina bifida aperta

O6-methylguanine DNA methyltransferase(MGMT), a DNA repair enzyme, has been reported in some congenital malformations, but it is less frequently reported in neural tube defects. This study investigated MGMT mRNA expression and methylation levels in the early embryo and in different embryonic stages, as well as the relationship between MGMT and neural tube defects. Spina bifida aperta was induced in rats by a single intragastric administration of all-trans retinoic acid on embryonic day(E) 10, whereas normal control rats received the same amount of olive oil on the same embryonic day. DNA damage was assessed by detecting γ-H2 A.X in spina bifida aperta rats. Real time-polymerase chain reaction was used to examine mRNA expression of MGMT in normal control and spina bifida aperta rats. In normal controls, the MGMT mRNA expression decreased with increasing embryonic days, and was remarkably reduced from E11 to E14, reaching a minimum at E18. In the spina bifida aperta model, γ-H2 A.X protein expression was increased, and mRNA expression of MGMT was markedly decreased on E14, E16, and E18. Bisulfite sequencing polymerase chain reaction for MGMT promoter methylation demonstrated that almost all CpG sites in the MGMT promoter remained unmethylated in both spina bifida aperta rats and normal controls, and there was no significant difference in methylation level between the two groups on either E14 or E18. Our results show that DNA damage occurs in spina bifida aperta rats. The mRNA expression of MGMT is downregulated, and this downregulation is independent of promoter DNA methylation.

A stage-scanning laser confocal microscope and protocol for DNA methylation sequencing

Recent understanding of the role of epigenetic regulation in health and disease has necessitated the development of newer and efficient methods to map the methylation pattern of target gene. In this article we report construction of a stage-scanning laser confocal microscope (SLCM) and associated protocol that determines the methylation status of target gene. We have adapted restricted Sanger’s sequencing where fluorescine labeled primers and dideoxy guanine fraction alone are used for target amplification and termination at cytosine positions. Amplified ssDNA bands are separated in 6% denaturing PAGE and scanned using SLCM to sequence the positions of methylated cytosines. We demonstrate that our me- thodology can detect < 100 femtomoles of DNA, and resolve the position of cytosine within ± 2 nucleotide. In a calibration run using a designer DNA of 99 bases, our methodology had resolved all the 11 cytosine positions of the DNA. We have further demonstrated the utility of apparatus by mapping methylation status in the Exon-1 region of a gene, E-Cadherin, in the plasma DNA sample of a healthy subject. We believe our approach constitute a low cost alternative to conventional DNA sequencers and can help develop methylation based DNA biomarkers for the diagnosis of disease and in therapeutics.

Epigenome-wide DNA methylation analysis reveals differentially methylation patterns in skeletal muscle between Chinese Chenghua and Qingyu pigs

Chenghua(CH)pig and Qingyu(QY)pig are typical Chinese native fatty breeds.CH pig is mainly distributed in Chengdu Plain,while QY pig is widely distributed throughout the mountain areas around the Sichuan Basin.There are significant differences in their phenotypic traits,including body image,growth performance,and meat quality.This study compared several meat quality traits of CH and QY pigs and conducted a genome-wide DNA methylation analysis using reduced representation bisulfite sequencing(RRBS).It was observed that the pH at 45 min(pH_(45min),P=5.22e-13),lightness at 45 min(L^(*)_(45min),P=4.85e-5),and lightness at 24 h(L^(*)_(24h),P=3.57e-5)of CH pigs were higher than those of QY pigs.We detected 10699 differentially methylated cytosines(DMCs)and 2760 differentially methylated genes(DMGs)associated with these DMCs.Functional analysis showed that these DMGs were mainly enriched in the AMPK signaling pathway,Type II diabetes mellitus,Insulin signaling pathway,mTOR signaling pathway,and Insulin resistance.Furthermore,15 DMGs were associated with fat metabolism(ACACA,CAB39,CRADD,CRTC2,FASN,and GCK),muscle development(HK2,IKBKB,MTOR,PIK3CD,PPARGC1A,and RPTOR),or meat quality traits(PCK1,PRKAG2,and SLC2A4).The findings may help to understand further the epigenetic regulation mechanisms of meat quality traits in pigs and provide new basic data for the study of local pigs.

Eagles report:Developing cancer biomarkers from genomewide DNA methylation analyses

Analyses of DNA methylation in human cancers have identified hypermethylation of individual genes and diminished methylation at repeat elements as common alterations,and have thereby provided important mechanistic insights into cancer biology as well as biomarkers for cancer detection,prognosis and prediction of therapy responses.The techniques available in the past were best suited for investigations of individual candidate genes and sequences,whereas recently developed high-throughput techniques promise to generate unbiased and comprehensive surveys of DNA methylation states across entire genomes.In this minireview we give a short overview of established and novel techniques and outline some major questions that can now be addressed to develop further cancer biomarkers and therapies based on DNA methylation.

Dynamic changes of genomic methylation profiles at different growth stages in Chinese Tan sheep

Background:Tan sheep,an important local sheep breed in China,is famous for their fur quality.One-month-old Tan sheep have white,curly hair with beautiful flower spikes,commonly known as“nine bends”,which has high economic value.However,the“nine bends”characteristic gradually disappears with age;consequently,the economic value of the Tan sheep decreases.Age-related changes in DNA methylation have been reported and may be responsible for age-induced changes in gene expression.Until now,no genome-wide surveys have been conducted to identify potential DNA methylation sites involved in different sheep growth stages.In this study we investigated the dynamic changes of genome-wide DNA methylation profiles in Tan sheep using DNA from skin and deep whole-genome bisulfite sequencing,and compared the DNA methylation levels at three different growth stages:1,24,and 48 months old(mon1,mon24,and mon48,respectively).Results:In this study,11 skin samples from three growth stages(four for mon1,four for mon24,and three for mon48)were used for DNA methylation analysis and gene expression profiling.There were 52,288 and 236 differentially methylated genes(DMGs)identified between mon1 and mon24,mon1 and mon48,and mon24 and mon48,respectively.Of the differentially methylated regions,1.11%,7.61%,and 7.65% were in the promoter in mon1 vs.mon24,mon24 vs.mon48,and mon1 vs.mon48,respectively.DMGs were enriched in the MAPK and WNT signaling pathways,which are related to age growth and hair follicle morphogenesis processes.There were 51 DMGs associated with age growth and curly fleece formation.Four DMGs between mon1 and mon48(KRT71,CD44,ROR2 and ZDHHC13)were further validated by bisulfite sequencing.Conclusions:This study revealed dynamic changes in the genomic methylation profiles of mon1,mon24,and mon48 sheep,and the percentages of methylated cytosines were 3.38%,2.85% and 4.17%,respectively.Of the DMGs,KRT71 and CD44 were highly methylated in mon1,and ROR2 and ZDHHC13 were highly methylated in mon48.These findings provide foundational information that may be used to develop strategies for potentially retaining the lamb fur and thus improving the economic value of Tan sheep.

Genome-wide DNA methylation landscape of four Chinese populations and epigenetic variation linked to Tibetan high-altitude adaptation

DNA methylation(DNAm)is one of the major epigenetic mechanisms in humans and is important in diverse cellular processes.The variation of DNAm in the human population is related to both genetic and environmental factors.However,the DNAm profiles have not been investigated in the Chinese population of diverse ethnicities.Here,we performed double-strand bisulfite sequencing(DSBS)for 32 Chinese individuals representing four major ethnic groups including Han Chinese,Tibetan,Zhuang,and Mongolian.We identified a total of 604,649 SNPs and quantified DNAm at more than 14 million Cp Gs in the population.We found global DNAm-based epigenetic structure is different from the genetic structure of the population,and ethnic difference only partially explains the variation of DNAm.Surprisingly,non-ethnic-specific DNAm variations showed stronger correlation with the global genetic divergence than these ethnic-specific DNAm.Differentially methylated regions(DMRs)among these ethnic groups were found around genes in diverse biological processes.Especially,these DMR-genes between Tibetan and nonTibetans were enriched around high-altitude genes including EPAS1 and EGLN1,suggesting DNAm alteration plays an important role in high-altitude adaptation.Our results provide the first batch of epigenetic maps for Chinese populations and the first evidence of the association of epigenetic changes with Tibetans'high-altitude adaptation.

Regulation of Leaf Longevity by DML3-Mediated DNA Demethylation

Leaf senescence is driven by the expression of senescence-associated genes(SAGs).Development-specific genes often undergo DNA demethylation in their promoter and other regions,which regulates gene expression.Whether and how DNA demethylation regulates the expression of SAGs and thus leaf senescence remain elusive.Whole-genome bisulfite sequencing(WGBS)analyses of wild-type(WT)and demeter-like 3(dml3)Arabidopsis leaves at three developmental stages revealed hypermethylation during leaf senescence in dml3 compared with WT,and 20556 differentially methylated regions(DMRs)were identified by comparing the methylomes of dml3 and WT in the CG,CHG,and CHH contexts.Furthermore,we identified that 335 DMR-associated genes(DMGs),such as NAC016 and SEN1,are upregulated during leaf senescence,and found an inverse correlation between the DNA methylation levels(especially in the promoter regions)and the transcript abundances of the related SAGs in WT.In contrast,in dml3 the promoters of SAGs were hypermethylated and their transcript levels were remarkably reduced,and leaf senescence was significantly delayed.Collectively,our study unraveled a novel epigenetic regulatory mechanism underlying leaf senescence in which DML3 is expressed at the onset of and during senescence to demethylate promoter,gene body or 3'UTR regions to activate a set of SAGs.

Covering Your Bases： Inheritance of DNA Methylation in Plant Genomes

Cytosine methylation is an important base modification that is inherited across mitotic and meiotic cell divisions in plant genomes. Heritable methylation variants can contribute to within-species phenotypic variation. Few methylation variants were known until recently, making it possible to begin to address major unanswered questions： the extent of natural methylation variation within plant genomes, its effects on phenotypic variation, its degree of depend- ence on genotype, and how it fits into an evolutionary context. Techniques like whole-genome bisulfite sequencing （WGBS） make it possible to determine cytosine methylation states at single-base resolution across entire genomes and populations. Application of this method to natural and novel experimental populations is revealing answers to these long-standing questions about the role of DNA methylation in plant genomes.

Crop Epigenomics： Identifying, Unlocking, and Harnessing Cryptic Variation in Crop Genomes

DNA methylation is a key chromatin modification in plant genomes that is meiotically and mitotically her- itable, and at times is associated with gene expression and morphological variation. Benefiting from the increased availability of high-quality reference genome assemblies and methods to profile single-base res- olution DNA methylation states, DNA methylomes for many crop species are available. These efforts are making it possible to begin answering crucial questions, including understanding the role of DNA methyl- ation in developmental processes, its role in crop species evolution, and whether DNA methylation is dynamically altered and heritable in response to changes in the environment. These genome-wide maps provide evidence for the existence of silent epialleles in plant genomes which, once identified, can be tar- geted for reactivation leading to phenotypic variation.