Identification and Bioinformatics Analysis of SnRK2 and CIPK Family Genes in Sorghum

Through bioinformatic data mining, 10 SnRK2 and 31 CIPK genes were identified from sorghum genome. They are unevenly distributed in the sorghum chromosomes. Most SnRK2 genes have 8 introns, while the CIPK genes have a few （no intron or less than 3 introns） or more than I0 introns. Phylogenetic analysis revealed that SnRK2 genes belong to one cluster and CIPK genes form the other independent cluster. The sorghum SnRK2s are subgrouped into three parts, and CIPK into five parts. More than half SnRK2 and CIPK genes present in homologous pairs, suggesting gene duplication may be due to the amplification of SnRK family genes. The kinase domains of SnRK2 family are highly conserved with 88.40% identity, but those of the CIPK family are less conserved with 63.72% identity. And the identity of sorghum CBLinteracting NAF domains of CIPKs is 61.66%. What＇s more, regarding to the sorghum SnRK2 and CIPK kinases, they are characterized with distinct motifs and their subcellular localization is not necessarily the same, which suggests they may be divergent in functions. Due to less conserved sequences, complex subcellular localization, and more family members, sorghum CIPK genes may play more flexible and multiple biological functions. According to the phylogenetic analysis of SnRK genes and SnRK functional studies in other plants, it is speculated that sorghum SnRK2 and CIPK genes may play important roles in stress response, growth and development.

Identification of a major QTL for Hessian fly resistance in wheat cultivar‘Chokwang’

The Hessian fly(HF,Mayetiola destructor)is one of the destructive pests of wheat(Triticum aestivum L.)worldwide.Resistant cultivars can effectively minimize wheat damage due to this insect pest.To identify new quantitative trait loci(QTL)for HF resistance,a population of recombinant inbred lines(RILs)was developed from a cross between the HF-resistant wheat cultivar‘Chokwang’and susceptible wheat‘Ning 7840’,and phenotyped for responses to HF attack.A linkage map was constructed using 1147 single nucleotide polymorphism(SNP)markers generated from genotyping-by-sequencing(GBS).One major QTL,QHf.hwwg-6 BS,for HF-resistance was identified on chromosome arm 6 BS,which explained up to84.0%of the phenotypic variation and was contributed by Chokwang.Two RILs showed recombination in the candidate interval of QHf.hwwg-6 BS,which delimited QHf.hwwg-6 BS to a 4.75 Mb physical interval between 6,028,601 bp and 10,779,424 bp on chromosome arm 6 BS of IWGSC Chinese Spring reference genome Ref Seq v2.0.Seven GBS-SNPs in the candidate interval were converted into Kompetitive allele specific polymerase chain reaction(KASP)markers.Two of them,KASP-6 B112698 and KASP-6 B7901215,were validated in a U.S.winter wheat panel.KASP-6 B112698 was nearly diagnostic,thus can be used to screen QHf.hwwg-6 BS and pyramid it with other resistance genes in breeding programs.

Metabolism of minor isoforms of prion proteins Cytosolic prion protein and transmembrane prion protein

Transmissible spongiform encephalopathy or prion disease is triggered by the conversion from cellular prion protein to pathogenic prion protein. Growing evidence has concentrated on prion protein configuration changes and their correlation with prion disease transmissibility and patho- genicity. In vivo and in vitro studies have shown that several cytosolic forms of prion protein with specific topological structure can destroy intracellular stability and contribute to prion protein pathogenicity. In this study, the latest molecular chaperone system associated with endoplasmic re- ticulum-associated protein degradation, the endoplasmic reticulum resident protein quality-control system and the ubiquitination proteasome system, is outlined. The molecular chaperone system directly correlates with the prion protein degradation pathway. Understanding the molecular mechanisms will help provide a fascinating avenue for further investigations on prion disease treatment and prion protein-induced neurodegenerative diseases.

Genome-wide linkage mapping of QTL for root hair length in a Chinese common wheat population

Root hairs are fast growing,ephemeral tubular extensions of the root epidermis that aid nutrient and water uptake.The aim of the present study was to identify QTL for root hair length(RHL)using 227 F8 recombinant inbred lines(RILs)derived from a cross of Zhou 8425 B(Z8425 B)and Chinese Spring(CS),and to develop convenient molecular markers for markerassisted breeding in wheat.Analysis of variance of root hair length showed significant differences(P<0.01)among RILs.The genetic map for QTL analysis consisted of 3389 unique SNP markers.Using composite interval mapping,four major QTL(LOD>2.5)for RHL were identified on chromosomes 1 B(2),2 D and 6 D and four putative QTL(2≤LOD≤2.5)were detected on chromosomes 1 A,3 A,6 B,and 7 B,explaining 3.32%–6.52%of the phenotypic variance.The positive alleles for increased RHL of QTL on chromosomes 2 D,6 B and 6 D(QRhl.cau-2 D,q Rhl.cau-6 B,and QRhl.cau-6 D)were contributed by Z8425 B,and CS contributed positive QTL alleles on chromosomes 1 A(q Rhl.cau-1 A),1 B(QRhl.cau-1 B.1 and QRhl.cau-1 B.2),3 A(q Rhl.cau-3 A)and 7 B(q Rhl.cau-7 B).STARP markers were developed for QRhl.cau-1 B.1,QRhl.cau-2 D,QRhl.cau-6 D,and q Rhl.cau-7 B.Haplotype and association analysis indicated that the positive allele of QRhl.cau-6 D had been strongly selected in Chinese wheat breeding programs.Collectively,the identified QTL for root hair length are likely to be useful for marker-assisted selection.

Screening of Microalgae for Biodiesel Feedstock

Three heterotrophic microalgae identified as Scenedesmus sp. Y5, Scenedesmus sp. Y7 and Chorellasp. Y9 were isolated and screened from natural water based on biomass yield and lipid productivity. Fatty acids’ composition analysis showed that both Y5 and Y7 mainly contained C16:0, C18:1 (n - 9), C18:2 (n - 6) and C18:3 (n - 3) and Y9 mainly contained C16:0, C18:0 and C18:2 (n - 6), suggesting that these microalgae can be ideal feedstock for biodiesel. Considering the specific growth rate and lipid productivity, the culture conditions were optimized for Scenedesmus sp. Y5, Scenedesmus sp. Y7 and Chorellasp. Y9. Based on the optimization of cultural conditions, all of these three microalgae were tested in fed-batch fermentation, and their biomass productivities were 4.960 g·L-1·d-1, 5.907 g·L-1·d-1 and 4.038 g·L-1·d-1;lipid productivities reached 1.5120 g·L-1·d-1, 1.233 g·L-1·d-1 and 0.8112 g·L-1·d-1, respectively.

An efficient method for the sanitary vitrification of bovine oocytes in straws

Background：At present,vitrification has been widely applied to humans,mice and farm animals.To improve the efficiency of vitrification in straw,bovine oocytes were used to test a new two-step vitrification method in this study.Results：When in vitro matured oocytes were exposed to 20%ethylene glycol（EG20） for 5 min and 40%ethylene glycol（EG40） for 30 s,followed by treatment with 30%glycerol（Gly30）,Gly40 or Gly50,a volume expansion was observed in Gly30 and Gly40 but not Gly50.This indicates that the intracellular osmotic pressure after a 30 s differs between EG40 and ranged between Gly40（approximately 5.6 mol/L） and Gly50（approximately 7.0 mol/L）.Since oocytes are in EG40 just for only a short period of time（30 s） and at a lower temperature（4℃）,we hypothesize that the main function of this step in to induce dehydration.Based on these results,we omitted the EG40 step,before oocytes were pretreated in EG20 for 5 min,exposed to pre-cooled（4℃） Gly50,for 30 s,and then dipped into liquid nitrogen.After warming,81.1%of the oocytes survived,and the surviving oocytes developed into cleavage stage embryos（63.5%） or blastocysts（20.0%） after parthenogenetic activation.Conclusions：These results demonstrate that in a two-step vitrification procedure,the permeability effect in the second step is not necessary.It is possible that the second step is only required to provide adequate osmotic pressure to condense the intracellular concentration of CPAs to a level required for successful vitrification.

A MITE insertion into the 3′-UTR regulates the transcription of TaHSP16.9 in common wheat

Miniature inverted-repeat transposable elements(MITEs) are a type of DNA transposon frequently inserted into promoters, untranslated regions(UTR), introns, or coding sequences of genes. We found a 276-bp tourist-like MITE insertion in the 3′-UTR of a 16.9 k Da small heat shock protein gene(TaH SP16.9-3A) on chromosome 3A of common wheat. Haplotype analysis revealed two haplotypes, s HSP-W(wild type without MITE insertion) and s HSP-M(mutant with MITE insertion), present in wheat germplasm. Both semiquantitative PCR and quantitative real-time PCR analyses showed increased transcription levels of TaH SP16.9-3A in s HSP-M compared with those of s HSP-W after heat treatment at 42 °C. It appeared that the MITE insertion into the 3′-UTR enhances the transcription of TaH SP16.9-3A.

Genetic improvement of heat tolerance in wheat:Recent progress in understanding the underlying molecular mechanisms

As a cool season crop, wheat(Triticum aestivum L.) has an optimal daytime growing temperature of 15 ℃ during the reproductive stage. With global climate change, heat stress is becoming an increasingly severe constraint on wheat production. In this review, we summarize recent progress in understanding the molecular mechanisms of heat tolerance in wheat. We firstly describe the impact of heat tolerance on morphology and physiology and its potential effect on agronomic traits. We then review recent discoveries in determining the genetic and molecular factors affecting heat tolerance, including the effects of phytohormone signaling and epigenetic regulation. Finally, we discuss integrative strategies to improve heat tolerance by utilization of existing germplasm including modern cultivars, landraces and related species.

A genetic linkage map with 178 SSR and 1901 SNP markers constructed using a RIL population in wheat (Triticum aestivum L.)

The construction of high density genetic linkage map provides a powerful tool to detect and map quantitative trait loci(QTLs) controlling agronomically important traits. In this study, simple sequence repeat(SSR) markers and Illumina 9K i Select single nucleotide polymorphism(SNP) genechip were employed to construct one genetic linkage map of common wheat(Triticum aestivum L.) using 191 recombinant inbred lines(RILs) derived from cross Yu 8679×Jing 411. This map included 1 901 SNP loci and 178 SSR loci, covering 1 659.9 c M and 1 000 marker bins, with an average interval distance of 1.66 c M. A, B and D genomes covered 719.1, 703.5 and 237.3 c M, with an average interval distance of 1.66, 1.45 and 2.9 c M, respectively. Notably, the genetic linkage map covered 20 chromosomes, with the exception of chromosome 5D. Bioinformatics analysis revealed that 1 754(92.27%) of 1 901 mapped SNP loci could be aligned to 1 215 distinct wheat unigenes, among which 1 184(97.4%) were located on o ne single chromosome, and the rest 31(2.6%) were located on 2 to 3 chromosomes. By performing in silico comparison, 214 chromosome deletion bin-mapped expressed sequence tags(ESTs), 1 043 Brachypodium genes and 1 033 rice genes were further added onto the genetic linkage map. This map not only integrated genetic and physical maps, SSR and SNP loci, respectively, but also provided the information of Brachypodium and rice genes corresponding to 1 754 SNP loci. Therefore, it will be a useful tool for comparative genomics analysis, fine mapping of QTL/gene controlling agronomically important traits and marker-assisted selection breeding in wheat.

Incidence and prevalence levels of three aphid-transmitted viruses in crucifer crops in China

Poleroviruses,which are distributed worldwide,infect many crops of economic importance and cause severe plant diseases.Brassica yellows virus(Br YV),which has three genotypes,A,B,and C,is a newly identified polerovirus infecting crucifer crops in China,but its distribution is still unclear.Here,we report the distribution and prevalence levels of the three Br YV genotypes in crucifer crops in China.A total of 570 crucifer leaf samples randomly collected from 22 provinces,four ethnic minority autonomous regions,and three municipalities in China were tested for Br YV.RT-PCR detection showed that 97 of the field samples were positive for Br YV,and the average incidence of Br YV was 17.0%.The virus was detected in 22 provinces,with high incidences in north,northwest,and northeast China.The multiplex RT-PCR amplification of the three Br YV genotypes revealed that both single and mixed infections occurred.Among the Br YV infections,38.1% were mixinfected by more than two viral genotypes,and 8.2% samples were mix-infected by three viral genotypes.Our findings indicated a widespread prevalence of Br YV in China,and Br YV mixed infections with Turnip mosaic virus and Cucumber mosaic virus in crucifer crops are common.This study is the first large-scale survey of Br YV in crucifer crops in China.The information generated in this investigation will contribute to the national prevention and control of viral diseases.

Simulation of gene pyramiding in Drosophila melanogaster

Gene pyramiding has been successfully practiced in plant breeding for developing new breeds or lines in which favorable genes from several different lines were integrated. But it has not been used in animal breeding, and some theoretical investigation and simulation analysis with respect to its strategies, feasibility and efficiency are needed before it can be implemented in animals. In this study, we used four different pure lines of Drosophila melanogaster, each of which is homozygous at a specific mutant gene with a visible effect on phenotype, to simulate the gene pyramiding process and analyze the duration and population size required in different pyramiding strategies. We finally got the ideal individuals, which are homozygous at the four target genes simultaneously. This study demonstrates that gene pyramiding is feasible in animal breeding and the interaction between genes may affect the final results.

Generation of pigs with a Belgian Blue mutation in MSTN using CRISPR/Cpf1-assisted ssODN-mediated homologous recombination

CRISPR/Cpf1 has emerged recently as an effective tool for genome editing in many organisms,but its use in pigs to generate precise genetic modifications has seldom been described.Myostatin(MSTN)is a well-characterized negative regulator of muscle development,and natural mutations in this gene cause a double-muscled phenotype in many species.However,to the best of our knowledge,no naturally occurring mutation in MSTN has been found in pigs.In addition,no living pig models with sophisticated modifications orthologous to natural mutations in MSTN have yet been reported.In this study,we exploited the CRISPR/Cpf1 system to introduce a predefined modification orthologous to the natural MSTN mutation found in Belgian Blue cattle(thus known as the Belgian Blue mutation).Our research demonstrated that the cutting efficiency of CRISPR/Cpf1 was 12.3%in mixed porcine fetal fibroblasts in drug free medium,and 41.7%in clonal colonies obtained using G418 selection.Then,the Cpf1-sgRNA vector,ssODN template,and a self-excision cassette were co-transfected into porcine fetal fibroblasts.After G418 selection,8 clonal colonies were examined and 5 with genetic modification were found.Of these 5,2 harbored the precise 11-bp deletion.Using 1 heterozygous clonal colony,2 cloned Duroc piglets were successfully generated,which was heterozygous for the Belgian Blue mutation.In summary,our results demonstrate that CRISPR/Cpf1 system can be used efficiently to generate double-stranded breaks,and also to mediate homologous recombination to introduce precise genomic modifications in pigs.

Dietary Daidzein Enhances Antiapoptotic Effect of 17β-Estradiol (E_2) on Breast Cancer MCF-7 Cells

Objective： To investigate whether dietary daidzein interact with endogenous 17β-Estradiol （E2） to give rise to additive or inhibitory effects on proliferation and apoptosis in breast cancer cells. Methods： Cell cycle distribution and apoptosis induction were analyzed by using flow cytometry when breast cancer cell lines MCF-7 were cotreated with daidzein （1, 5 μmol/L） and E2 （0.1-10 nmol/L） for 5 days. Whether daidzein could alter E2-modulated mRNA expression of estrogen receptor alpha （ERα）, estrogen receptor beta （ERI3） and ERβ-estrogen response element （ERE） dependent transcription was investigated by RT-PCR and luciferase induction assays. The effects of daidzein on E2-modulated expression of proapoptotic p53, bax and antiapoptotic bcl-2 at both mRNA and protein levels were also investigated by RT-PCR and Western blot. Results： Daidzein enhanced the antiapoptotic effect in an Ea dose-dependent manner, but had no effect on E2-induced proliferation. Daidzein antagonized E2-induced ERβ mRNA expression and ERβ-ERE dependent transcription. In addition, daidzein only antagonized E2-upregulated expression of p53 and bax, but had no effect on E2-upregulated expression of bcl-2. Conclusion： Daidzein enhances the antiapoptotic effect of E2 on breast cancer cells by inhibiting E2-mediated p53-bax proapoptotic pathway. These results suggest that dietary daidzein may enhance deleterious effect of endogenous E2 in hormone-dependent breast cancer.

Revolutionize livestock breeding in the future: an animal embryo-stem cell breeding system in a dish

Meat and milk production needs to increase ～ 70–80% relative to its current levels for satisfying the human needs in 2050.However,it is impossible to achieve such genetic gain by conventional animal breeding systems.Based on recent advances with regard to in vitro induction of germ cell from pluripotent stem cells,herein we propose a novel embryo-stem cell breeding system.Distinct from the conventional breeding system in farm animals that involves selecting and mating individuals,the novel breeding system completes breeding cycles from parental to offspring embryos directly by selecting and mating embryos in a dish.In comparison to the conventional dairy breeding scheme,this system can rapidly achieve 30–40 times more genetic gain by significantly shortening generation interval and enhancing selection intensity.However,several major obstacles must be overcome before we can fully use this system in livestock breeding,which include derivation and mantaince of pluripotent stem cells in domestic animals,as well as in vitro induction of primordial germ cells,and subsequent haploid gametes.Thus,we also discuss the potential efforts needed in solving the obstacles for application this novel system,and elaborate on their groundbreaking potential in livestock breeding.This novel system would provide a revolutionary animal breeding system by offering an unprecedented opportunity for meeting the fast-growing meat and milk demand of humans.

Proteomic Identification of Rhythmic Proteins in Maize Seedling Leaves

Plant leaves respond to day/night cycling in a number of physiological ways. At the mRNA level, the expression of some genes changes during the 24 h period. To determine which proteins exhibited a rhythmic pattern of expression, proteomic profiles in maize seedling leaves were analyzed by high-throughput two-dimensional gel electrophoresis, combined with MALDI-TOF MS technology. Of the 464 proteins that were detected with silver staining in a pH range of 4-7, 17 （3.66%） showed clock rhythmicity in their abundance. These proteins belonged to diverse functional groups and proteins involved in photosynthesis and carbon metabolism were over-represented. These findings provide a new perspective on the relationship between the physiological functions of leaves and the clock rhythmic system.

Comparative genetic mapping revealed powdery mildew resistance gene MlWE4 derived from wild emmer is located in same genomic region of Pm36 and Ml3D232 on chromosome 5BL

Powdery mildew, caused by Blumeria graminis f. sp. tritici, is one of the most devastating wheat diseases. Wild emmer wheat（Triticum turgidum ssp. dicoccoides） is a promising source of disease resistance for wheat. A powdery mildew resistance gene conferring resistance to B. graminis f. sp. tritici isolate E09, originating from wild emmer wheat, has been transferred into the hexaploid wheat line WE4 through crossing and backcrossing. Genetic analyses indicated that the powdery mildew resistance was controlled by a single dominant gene, temporarily designated Ml WE4. By mean of comparative genomics and bulked segregant analysis, a genetic linkage map of Ml WE4 was constructed, and Ml WE4 was mapped on the distal region of chromosome arm 5BL. Comparative genetic linkage maps showed that genes Ml WE4, Pm36 and Ml3D232 were co-segregated with markers XBD37670 and XBD37680, indicating they are likely the same gene or alleles in the same locus. The co-segregated markers provide a starting point for chromosome landing and map-based cloning of Ml WE4, Pm36 and Ml3D232.

Development of a Porcine cDNA Microarray:Analysis of Clenbuterol Responding Genes in Pig(Sus scrofa) Internal Organs

Pig （Sus scrofa） fat accumulation can be reduced by feeding with high dosages of clenbuterol, but the molecular mechanism has not yet been explained. In our study, a porcine cDNA microarray representing 3 358 pig genes was successfully developed. This microarray is the first porcine DNA microarray in China and its false positive rate is 0.98%, which means the microarray platform is reliable. The microarray can be used to study gene expression profiles in multiple pig tissues because the present genes percentage of adipose, skeletal muscle, heart, liver, lung, kidney, and spleen were all more than 60%. This microarray was used to identify the genes responding to clenbuterol stimulation in pig internal organs, including heart, liver, lung, spleen, and kidney. Many genes were identified including enzymes involved in lipids metabolism （lipoprotein lipase up-regulated in liver, heart and lung, ATP-citrate lyase and carnitine palmitoyltransferase II precursor up-regulated in liver, succinyl-CoA up-regulated in lung, mitochondrial malate dehydrogenase down-regulated in spleen）, and signaling pathway genes （cAMP-protein kinase A signaling pathway was found up-regulated in liver, heart, lung, and kidney as reported previously, while transforming growth factor was found down-regulated in heart and lung）. However, no common gene responding to clenbuterol administration was found in all tissues. The expression levels of 14 genes were analyzed using real-time PCR with 82.1% of them induced to express similar magnitudes as in the microarray analyses. This work offers some understanding of how clenbuterol so effectively reduces pig adipose accumulation on the molecular level.

Wheat 14-3-3 Protein Conferring Growth Retardation in Arabidopsis

14-3-3 proteins belong to a family of phosphoserine/threonine-binding modules and participate in a wide array of signal transduction and regulatory events. Our previous study demonstrated that Ta14-3-3 was significantly down-regulated in leaf and root tissues of hybrid wheat at the tillering stage. In this paper, three homoeologous Ta14-3-3 genes were cloned from common wheat （Triticum aestivum L., 2n=6x=42, AABBDD） and mapped on chromosomes 2A, 2B, and 2D, respectively. Transgenic Arabidopsis plants ectopically overexpressing Ta14-3-3 displayed shorter primary roots, delayed flowering and retarded growth rates, indicating that Ta14-3-3 acted as a growth inhibitor in Arabidopsis. In wheat, Ta14-3-3 was down-regulated in roots and leaves of hybrids as compared to their parental lines. We proposed that Ta14-3-3 proteins might regulate growth vigor in hybrid wheat.

QTL mapping revealed TaVp-1A conferred pre-harvest sprouting resistance in wheat population Yanda 1817×Beinong 6

Pre-harvest sprouting （PHS） occurs frequently in most of the wheat cultivation area worldwide, which severely reduces yield and end-use quality, resulting in substantial economic loss. In this study, quantitative trait loci （QTL） for PHS resistance were mapped using an available high-density single nucleotide polymorphism （SNP） and simple sequence repeat （SSR） genetic linkage map developed from a 269 recombinant inbred lines （RILs） population of Yanda 1817xBeinong 6. Using phenotypic data on two locations （Beijing and Shijiazhuang, China） in two years （2012 and 2013 harvesting seasons）, five QTLs, designated as QPhs.cau-3A. 1, QPhs.cau-3A.2, QPhs.cau-5B, QPhs.cau-4A, and QPhs.cau-6A, for PHS （GP） were detected by inclusive composite interval mapping （ICIM） （LOD≥2.5）. Two major QTLs, QPhs.cau-3A.2 and QPhs.cau-5B, were mapped on 3AL and 5BS chromosome arms, explaining 6.29-21.65% and 4.36-5.94% of the phenotypic variance, respectively. Precise mapping and comparative genomic analysis revealed that the TaVp-1A flanking region on 3AL is responsible for QPhs.cau-3A.2. SNP markers flanking QPhs.cau-3A.2 genomic region were developed and could be used for introgression of PHS tolerance into high yielding wheat varieties through marker-assisted selection （MAS）.

Fine mapping of powdery mildew resistance gene PmTm4 in wheat using comparative genomics

Powdery mildew,caused by Blumeria graminis f.sp.tritici,is one of the most severe wheat diseases.Mining powdery mildew resistance genes in wheat cultivars and their appliance in breeding program is a promising way to control this disease.Genetic analysis revealed that a single dominant resistance gene named PmTm4 originated from Chinese wheat line Tangmai 4 confers resistance to prevailing isolates of B.graminis f.sp.tritici isolate E09.Detailed comparative genomics analyses helped to develop closely linked markers to PmTm4 and a fine genetic map was constructed using large F2population,in which PmTm4 was located into a 0.66-c M genetic interval.The orthologous subgenome region of PmTm4in Aegilops tauschii was identified,and two resistance gene analogs（RGA）were characterized from the corresponding sequence scaffolds of Ae.tauschii draft assembly.The closely linked markers and identified Ae.tauschii orthologs in the mapping interval provide an entry point for chromosome landing and map-based cloning of PmTm4.