Genetic Diversity and Population Structure Analysis of Barley Landraces from Shanghai Region Using Genotyping-by-Sequencing

Barley(Hordeum vulgare L.)is an important economic crop for food,feed and industrial raw materials.In the present research,112 barley landraces from the Shanghai region were genotyped using genotyping-by-sequencing(GBS),and the genetic diversity and population structure were analyzed.The results showed that 210,268 Single Nucleotide Polymorphisms(SNPs)were present in total,and the average poly-morphism information content(PIC)was 0.1642.Genetic diversity and population structure analyses suggested that these barley landraces were differentiated and could be divided into three sub-groups,with morphological traits of row-type and adherence of the hulls the main distinguishing factors between groups.Genotypes with similar or duplicated names were also investigated according to their genetic backgrounds and seed appearances.This study provided valuable information on barley landraces from the Shanghai region,and showed that all these barley landraces should be protected and used for future breeding programs.

Synthesis of betanin by expression of the core betalain biosynthetic pathway in carrot

Betalain has received increased attention because of its high nutritional value and crucial physiological functions.Based on the elucidation of its core biosynthetic pathway,betalain can be produced in additional plants by metabolic engineering.Synthesis of betalain in carrot(Daucus carota L.)can improve its nutritional quality and economic value by extracting betalain from the fleshy root,non-edible part,and processing residue of carrot.In this study,two different constructs,namely,pYB:mCD(AomelOS,BvCYP76AD1S,and BvDODA1S)and p YB:CDD(BvCYP76AD1S,BvDODA1S,and MjcDOPA5GTS),were introduced into carrot for betanin synthesis by Agrobacterium-mediated transformation.Betanin can be synthetized in both transgenic calli,and p YB:m CD-transgenic callus can be used to produce betacyanin by suspension culture.However,pYB:mCD-transgenic seedlings can synthetize betanin only by tyrosine feeding.The p YB:CDD-transgenic lines can synthetize betanin in whole plants.The betanin content in fleshy root of pYB:CDD-transgenic carrot was(63.4±9)μg·g^(-1)fresh weight according to quantitative analysis.These betanin-producing carrot plant materials can be used to synthesize betanin for industrial application or consumption as dietary sources.

Genome-Wide Identification of Candidate Genes Associated with β-glucan Traits in a Hulled and Hulless Barley (Hordeum vulgare L.) Population

Barley grain is a valuable source ofβ-glucan,which is an important component of dietary fiber with significant human health benefits.Although the genetic basis ofβ-glucan biosynthesis has been widely studied,a genome-wide association study(GWAS)is still required for a scan of the candidate genes related to the complex quantitative trait based on the high-quality barley reference genome.In this study,a GWAS was conducted using a population composed of 87 barley landraces(39 hulled and 48 hulless,β-glucan from 2.07%to 6.56%)with 191,098 nucleotide polymorphisms(SNPs)markers to cover the chromosomes with the highest density.The population was divided into four sub-populations(POP1~POP4),and theβ-glucan content in POP2 was significantly higher than that in other groups,in which most of the hulless barley landraces are from Qinghai-Tibet Plateau in China.Among seven SNP markers identified by GWAS,two(SNP2 and SNP3)of them showed positive correlation toβ-glucan trait and the remaining five(SNP1,SNP4,SNP5,SNP6 and SNP7)showed the negative relationship.Two candidate genes linked to SNP7,HORVU7Hr1G000320 and HORVU7Hr1G000040,belong to the nucleotide triphosphate hydrolase super-family which is probable to affect the activities ofβ-glucan synthase.Another candidate gene associated with SNP1,HORVU1Hr1G000010,is possibly involved in sugar response.In conclusion,our results provide new insights into the genetic basis ofβ-glucan accumulation in barley grains,and the discovery of new SNP markers distributed in each chromosome and the associated candidate genes will be valuable for the breeding of functional barley varieties with high β-glucan.

Phytohormones Accumulation and Distribution in Shoots and Roots of Haploid,Diploid and Tetraploid Barley Seedlings Derived from Microspore Culture

Phytohormones play important roles in plant growth and development,and polyploids are thought to be an important method for plant breeding.However,the relationship between ploidy and phytohormone is still unclear.In this study,barley at three ploidy levels were produced by microspore culture.Therefore,we further analyzed the phytohormone content in the shoots and roots of the three kinds of barley materials to study the effect of ploidy on phytohormones accumulation and distribution.The results showed that Abscisic acid(ABA),gibberellin(GA),jasmonic acid(JA),auxin(IAA),salicylic acid(SA)and cytokinin(CTK)were successfully determined in shoots and roots using LC-MS(liquid chromatography mass spectrometry).By comparing the shoots of the haploid and diploid plants,it was found that the distribution trend of the six phytohormones was consistent,and another consistent trend was found in the roots of the diploid and tetraploid plants.In addition,we further analyzed the shoot/root ratio of the different phytohormones to identify the potential differences for haploid,diploid and tetraploid.Here,the relationship between ploidy and phytohormone we provided would provide new insights into understanding the new phenotypes that occur in polyploid species.

Mycorrhizal Synthesis Involving Boletus edulis and Pinus massoniana

Mycorrhizae involving Boletus edulis and Pinus massoniana have been successfully established and their morphological characteristics described.Optimal conditions for mycorrhizal synthesis were determined using orthogonal testing of three parameters:substrate,age of host plant and inoculation procedure.Following integration analysis of plant survival rates,diameter of seedling rootstock,height of seedling shoot,number of mycorrhizae,ratio of shoot/root biomass,number of mycorrhizae,and the ratio of infected/non-infected root tips,we concluded that a double-layered growth substrate that simulated natural soil structure was superior for mycorrhizal synthesis compared with the more commonly used single-layered peat moss and vermiculite substrate,especially when sepiolite powder was also added.

Transcriptome Profiling of Barley Cultivar Hua 30 MDEC in Response to Agrobacterium Infection

Agrobacterium-mediated transformation has been widely used in plants.However,the mechanism in plant cells’response to Agrobacterium infection was very complex.The mechanism of the determinants in host cell remains obscure,especially in barley,which is recalcitrant for Agrobacterium-mediated transformation.In the present study,microspore-derived embryogenic calli(MDEC)from barley elite cultivar were employed as unique subjects to characterize the mechanisms during the Agrobacterium infection process.Hua 30 MDEC can be successfully infected by Agrobacterium.RNA-sequencing at different infection points(0,2,6,12,24 hpi)was performed.The average expressional intensity of the whole genomics increased from 0 to 2 hpi,and then decreased subsequently.More upregulated than downregulated differentially expressed genes(DEGs)were counted at the same time.GO enrichment analysis showed that protein modification was significantly overrepresented in upregulated DEGs.Chromosome-related biological processes,gene expression and cellular metabolic processes were significantly overrepresented in downregulated DEGs.KEGG analysis showed that plant defense responses,phenylpropanoid biosynthesis and biosynthesis of amino acids were significantly enriched across the infection time course.Nine DEGs related to defense responses were identified.All DEGs were upregulated from 2 to 24 hpi.We speculate that these genes are possibly related to Agrobacterium infection.These findings will provide deep insights into the molecular events occurring during the process of Agrobacterium-mediated transformation.

Analysis of Bacterial Communities Present in Agaricus bisporus Phase Ⅱ Compost Using Amplified Ribosomal DNA Restriction Analysis(ARDRA)(Ⅱ)——Analysis of Bacterial Communities

Changes in the bacterial communities inhabiting Agaricus bisporus compost during Phase Ⅱ treatment were monitored using amplified rDNA restriction analysis(ARDRA).Sequence analysis data indicated that the bacterial communities associated with the compost samples were far richer in composition when determined by ARDRA compared with traditional methods based on bacterial isolation.Furthermore,the composition of the communities,especially in terms of the dominant bacteria during each of the four treatment stages(beginning of Phase Ⅱ,after pasteurization,5 days aeration,end of Phase Ⅱ,respectively),were very different.Restriction endonuclease digestion of mainly bacterial clones from four 16S rDNA libraries,C1,C3,C6 and C7,previously constructed on the basis of 16S rDNA fragments amplified from the four different stages of the Phase Ⅱ composting process,revealed the presence of unique microbial types.Thermophilic bacteria belonging to the Bacilli,and previously unreported strains of Trichococcus,Planococcus,Caryophanon,and subclass γ-Proteobacteria,were identified among the clones from the compost sampled during the aeration period(C6).Bacteria belonging to Thermus thermophilus and subclass α-Proteobacteria were detected in C1 and C7 composts sampled at the beginning and the end of Phase Ⅱ,respectively.Clones of some uncultured bacteria were also scored.

Generating homozygous mutant populations of barley microspores by ethyl methanesulfonate treatment

Induced mutations are important for genetic research and breeding.Mutations induced by physical or chemical mutagenesis are usually heterozygous during the early generations.However,mutations must be fixed prior to phenotyping or field trials,which requires additional rounds of self-pollination.Microspore culture is an effective method to produce double-haploid(DH)plants that are fixed homozygotes.In this study,we conducted ethyl methanesulfonate(EMS)-induced mutagenesis of microspore cultures of barley(Hordeum vulgare)cultivar‘Hua30’and landrace‘HTX’.The EMS concentrations were negatively correlated with the efficiency of callus induction and the frequency of mutant plant regeneration.The two genotypes showed different regeneration efficiencies.The phenotypic variation of the regenerated M1 plants and the presence of genome-wide nucleotide mutations,revealed by whole-genome sequencing,highlight the utility of EMS-induced mutagenesis of isolated microspore cultures for developing DH mutants.Genome-wide analysis of the mutation frequency in the regenerated plants revealed that a considerable proportion of mutations resulted from microspore culture(somaclonal variation)rather than EMS-induced mutagenesis.In addition to producing a population of 1972 homozygous mutant lines that are available for future field trials,this study lays the foundation for optimizing the regeneration efficiency of DH plants and the richness of mutations(mainly by fine-tuning the mutagen dosage).

Assessing the product quality and biological activities of barley(Hordeum vulgare L.)grasses at different harvest times

Barley(Hordeum vulgare L.)grasses(BG)have attracted considerable interest due to their multiple physiological activities and health benefits.In this paper,eight BG at four different growth stages were collected:seedling,tillering,stem elongation,and heading,and their product qualities and biological activities were examined and studied.Results demonstrated that harvest time obviously influenced proximate composition,surface color,and amino acid profile.Z21 and Z23 harvested at the seedling and early tillering stages,respectively,had higher total phenolic content(TPC)and total flavonoid content(TFC)than the other BG.Moreover,Z21 and Z23 possessed better DPPH radical-scavenging ability,antioxidant capacity,andα-glucosidase inhibitory activity,which were positively correlated with TPC,TFC,and protein and total amino acid contents.Our findings suggested that the early tillering stages were the preferable harvest times for BG with good product quality and biological properties.

Auxin biosynthesis by the YUCCA6 flavin monooxygenase gene in woodland strawberry

Auxin has been regarded as the main signal molecule coordinating the growth and ripening of fruits in strawberry, the reference genomic system for Rosaceae. The mechanisms regulating auxin biosynthesis in strawberry are largely elusive. Recently, we demonstrated that two YUCCA genes are involved in flower and fruit development in cultivated strawberry. Here, we show that the woodland strawberry （Fragaria vesca L.） genome harbors nine loci for YUCCA genes and eight of them encode functional proteins. Transcription pattern in different plant organs was different for all eight FvYUCs. Functionality of the FvYUC6 gene was studied in transgenic strawberry over- expressing FvYUC6, which showed typical high-auxin pheno- types. Overexpression of FvYUC6 also delayed flowering and led to complete male sterility in F. vesta. Additionally, specific repression of FvYUC6 expression by RNA interference signifi- cantly inhibited vegetative growth and reduced plant fertility. The development of leaves, roots, flowers, and fruits was greatly affected in FvYUC6-repressed plants. Expression of a subset of auxin-responsive genes was well correlated with the changes of FvYUC6 transcript levels and free indole-3-acetic acid levels in transgenic strawberry. These observations are consistent with an important role of FvYUC6 in auxin synthesis, and support a main role of the gene product in vegetative and reproductive development in woodland strawberry.

Efficient genome editing in Claviceps purpurea using a CRISPR/Cas9 ribonucleoprotein method

Claviceps purpurea produces many pharmacologically important ergot alkaloids(EAS),which are widely used to treat migraine and hypertension and to aid childbirth.Although an EAS biosynthetic cluster of C.purpurea has been discovered more than 20 years ago,the complete biosynthetic pathway of EAS has not been fully characterized until now.The main obstacle to elucidating this pathway and strain modification is the lack of efficient genome-editing tools for C.purpurea.The conventional gene manipulation method for C.purpurea relies on homologous recombination(HR),although the efficiency of HR in C.purpurea is very low(~1-5%).Consequently,the disruption of target genes is laborious and time-consuming.Although CRISPR/Cas9 genome-editing methods based on in vivo Cas9 expression and gRNA transcription have been reported recently,their gene-disruption efficiency is still very low.Here,we developed an efficient genome-editing system in C.purpurea based on in vitro assembled CRISPR/Cas9 gRNA ribonucleoprotein complexes.As proof of principle,three target genes were efficiently knocked out using this CRISPR/Cas9 ribonucleoprotein complex-mediated HR system,with editing efficiencies ranging from 50%to 100%.Inactivation of the three genes,which are closely related to uridine biosynthesis(ura5),hypha morphology(rac),and EAS production(easA),resulted in a uridine auxotrophic mutant,a mutant with a drastically different phenotype in axenic culture,and a mutant that did not produce EAS,respectively.Our ribonucleoprotein-based genome-editing system has a great advantage over conventional and in vivo CRISPR/Cas9 methods for genome editing in C.purpurea,which will greatly facilitate elucidation of the EAS biosynthetic pathway and other future basic and applied research on C.purpurea.