Tissue differential expression of lycopene β-cyclase gene in papaya

Carotene pigments in flowers and fruits are distinct features related to fitness advantages such as attracting insects forpollination and birds for seed dispersal.In papaya,the flesh color of the fruit is considered a quality trait that correlateswith nutritional value and is linked to shelf-life of the fruit.To elucidate the carotenoid biosynthesis pathway in papaya,we took a candidate gene approach to clone the lycopene β-cyclase gene,LCY-B.A papaya LCY-B ortholog,cpLCY-B,was successfully identified from both cDNA and bacterial artificial chromosome(BAC)libraries and complete genomicsequence was obtained from the positive BAC including the promoter region.This cpLCY-B shared 80% amino acididentity with citrus LCY-B.However,full genomic sequences from both yellow- and red-fleshed papaya were identical.Quantitative real-time PCR(qPCR)revealed similar levels of expression at six different maturing stages of fruits forboth yellow-and red-fleshed genotypes.Further expression analyses of cpLCY-B showed that its expression levels wereseven- and three-fold higher in leaves and,respectively,flowers than in fruits,suggesting that cpLCY-B is down-regulatedduring the fruit ripening process.

Cloning and characterization of a FLORICAULA/LEAFY ortholog, PFL, in polygamous papaya

The homologous genes FLORICAULA (FLO) in Antirrhinum and LEAFY (LFY) in Arabidopsis are known to regu- late the initiation of flowering in these two distantly related plant species. These genes are necessary also for the expression of downstream genes that control floral organ identity. We used Arabidopsis LFY cDNA as a probe to clone and sequence a papaya ortholog of LFY, PFL. It encodes a protein that shares 61% identity with the Arabidopsis LFY gene and 71% identity with the LFY homologs of the two woody tree species: California sycamore (Platanus racemosa) and black cottonwood (Populus trichocarpa). Despite the high sequence similarity within two conserved regions, the N-terminal proline-rich motif in papaya PFL differs from other members in the family. This difference may not affect the gene function of papaya PFL, since an equally divergent but a functional LFY ortholog NEEDLY of Pinus radiata has been reported. Genomic and BAC Southern analyses indicated that there is only one copy of PFL in the papaya genome. In situ hybridization experiments demonstrated that PFL is expressed at a relatively low level in leaf primordia, but it is expressed at a high level in the floral meristem. Quantitative PCR analyses revealed that PFL was expressed in flower buds of all three sex types - male, female, and hermaphrodite with marginal difference between hermaphrodite and unisexual flowers. These data suggest that PFL may play a similar role as LFY in flower development and has limited effect on sex differentiation in papaya.

Diversity of metabolite accumulation patterns in inner and outer seed coats of pomegranate:exploring their relationship with genetic mechanisms of seed coat development

The expanded outer seed coat and the rigid inner seed coat of pomegranate seeds,both affect the sensory qualities of the fruit and its acceptability to consumers.Pomegranate seeds are also an appealing model for the study of seed coat differentiation and development.We conducted nontarget metabolic profiling to detect metabolites that contribute to the morphological differentiation of the seed coats along with transcriptomic profiling to unravel the genetic mechanisms underlying this process.Comparisons of metabolites in the lignin biosynthetic pathway accumulating in seed coat layers at different developmental stages revealed that monolignols,including coniferyl alcohol and sinapyl alcohol,greatly accumulated in inner seed coats and monolignol glucosides greatly accumulated in outer seed coats.Strong expression of genes involved in monolignol biosynthesis and transport might explain the spatial patterns of biosynthesis and accumulation of these metabolites.Hemicellulose constituents and flavonoids in particular accumulated in the inner seed coat,and candidate genes that might be involved in their accumulation were also identified.Genes encoding transcription factors regulating monolignol,cellulose,and hemicellulose metabolism were chosen by coexpression analysis.These results provide insights into metabolic factors influencing seed coat differentiation and a reference for studying seed coat developmental biology and pomegranate genetic improvement.

Papaya CpbHLH1/2 regulate carotenoid biosynthesis-related genes during papaya fruit ripening

The ripening of papaya is a physiological and metabolic process associated with accumulation of carotenoids,alternation of flesh color and flavor,which depending on genotype and external factors such as light and hormone.Transcription factors regulating carotenoid biosynthesis have not been analyzed during papaya fruit ripening.RNA-Seq experiments were implemented using different ripening stages of papaya fruit from two papaya varieties.Cis-elements in lycopeneβ-cyclase genes(CpCYC-B and CpLCY-B)were identified,and followed by genome-wide analysis to identify transcription factors binding to these cis-elements,resulting in the identification of CpbHLH1 and CpbHLH2,two bHLH genes.The expressions of CpbHLH1/2 were changed during fruit development,coupled with transcript increase of carotenoid biosynthesis-related genes including CpCYC-B,CpLCY-B,CpPDS2,CpZDS,CpLCY-E,and CpCHY-B.Yeast onehybrid(Y1H)and transient expression assay revealed that CpbHLH1/2 could bind to the promoters of CpCYC-B and CpLCY-B,and regulate their transcriptions.In response to strong light,the results of elevated expression of carotenoid biosynthesis-related genes and the changed expression of CpbHLH1/2 indicated that CpbHLH1/2 were involved in light-mediated mechanisms of regulating critical genes in the carotenoid biosynthesis pathway.Collectively,our findings demonstrated several TF family members participating in the regulation of carotenoid genes and proved that CpbHLH1 and CpbHLH2 individually regulated the transcription of lycopeneβ-cyclase genes(CpCYC-B and CpLCY-B).This study yielded novel findings on regulatory mechanism of carotenoid biosynthesis during papaya fruit ripening.

DNA methylome and transcriptome landscapes revealed differential characteristics of dioecious flowers in papaya

Separate sexes in dioecious plants display different morphology and physiological characteristics.The differences between the two sexes lie in their highly differentiated floral characteristics and in sex-related phenotype,which is genetically determined and epigenetically modified.In dioecious papaya(Carica papaya L.),global comparisons of epigenetic DNA methylation and gene expressions were still limited.We conducted bisulfite sequencing of early-stage flowers grown in three seasons(spring,summer and winter)and compared their methylome and transcriptome profiles to investigate the differential characteristics of male and female in papaya.Methylation variances between female and male papaya were conserved among three different seasons.However,combined genome-scale transcriptomic evidence revealed that most methylation variances did not have influence on the expression profiles of neighboring genes,and the differentially expressed genes were most overrepresented in phytohormone signal transduction pathways.Further analyses showed diverse stress-responsive methylation alteration in male and female flowers.Male flower methylation was more responsive to stress whereas female flower methylation varied less under stress.Early flowering of male papaya in spring might be associated with the variation in the transcription of CpSVP and CpAP1 coinciding with their gene-specific hypomethylation.These findings provide insights into the sex-specific DNA methylation and gene expression landscapes of dioecious papaya and a foundation to investigate the correlation between differentiated floral characteristics and their candidate genes.

Auxin regulation involved in gynoecium morphogenesis of papaya flowers

The morphogenesis of gynoecium is crucial for propagation and productivity of fruit crops.For trioecious papaya(Carica papaya),highly differentiated morphology of gynoecium in flowers of different sex types is controlled by gene networks and influenced by environmental factors,but the regulatory mechanism in gynoecium morphogenesis is unclear.Gynodioecious and dioecious papaya varieties were used for analysis of differentially expressed genes followed by experiments using auxin and an auxin transporter inhibitor.We first compared differential gene expression in functional and rudimentary gynoecium at early stage of their development and detected significant difference in phytohormone modulating and transduction processes,particularly auxin.Enhanced auxin signal transduction in rudimentary gynoecium was observed.To determine the role auxin plays in the papaya gynoecium,auxin transport inhibitor(N-1-Naphthylphthalamic acid,NPA)and synthetic auxin analogs with different concentrations gradient were sprayed to the trunk apex of male and female plants of dioecious papaya.Weakening of auxin transport by 10 mg/L NPA treatment resulted in female fertility restoration in male flowers,while female flowers did not show changes.NPA treatment with higher concentration(30 and 50 mg/L)caused deformed flowers in both male and female plants.We hypothesize that the occurrence of rudimentary gynoecium patterning might associate with auxin homeostasis alteration.Proper auxin concentration and auxin homeostasis might be crucial for functional gynoecium morphogenesis in papaya flowers.These results will lead to further investigation on the auxin homeostasis and gynoecium morphogenesis in papaya.

Sex biased expression of hormone related genes at early stage of sex differentiation in papaya flowers

Sex types of papaya are controlled by a pair of nascent sex chromosomes,but molecular genetic mechanisms of sex determination and sex differentiation in papaya are still unclear.We performed comparative analysis of transcriptomic profiles of male and female floral buds at the early development stage before the initiation of reproductive organ primordia at which there is no morphological difference between male and female flowers.A total of 1734 differentially expressed genes(DEGs)were identified,of which 923 showed female-biased expression and 811 showed male-biased expression.Functional annotation revealed that genes related to plant hormone biosynthesis and signaling pathways,especially in abscisic acid and auxin pathways,were overrepresented in the DEGs.Transcription factor binding motifs,such as MYB2,GAMYB,and AP2/EREBP,were enriched in the promoters of the hormone-related DEGs,and transcription factors with those motifs also exhibited differential expression between sex types.Among these DEGs,we also identified 11 genes in the non-recombining region of the papaya sex chromosomes and 9 genes involved in stamen and carpel development.Our results suggested that sex differentiation in papaya may be regulated by multiple layers of regulation and coordination and involved transcriptional,epigenetic,and phytohormone regulation.Hormones,especially ABA and auxin,transcription factors,and genes in the non-recombination region of the sex chromosome could be involved in this process.Our findings may facilitate the elucidation of signal transduction and gene interaction in sex differentiation of unisexual flowers in papaya.

The role of cis-elements in the evolution of crassulacean acid metabolism photosynthesis

Crassulacean acid metabolism(CAM)photosynthesis is an innovation of carbon concentrating mechanism that is characterized by nocturnal CO2 fixation.Recent progresses in genomics,transcriptomics,proteomics,and metabolomics of CAM species yielded new knowledge and abundant genomic resources.In this review,we will discuss the pattern of cis-elements in stomata movement-related genes and CAM CO2 fixation genes,and analyze the expression dynamic of CAM related genes in green leaf tissues.We propose that CAM photosynthesis evolved through the re-organization of existing enzymes and associated membrane transporters in central metabolism and stomatal movement-related genes,at least in part by selection of existing circadian clock cis-regulatory elements in their promoter regions.Better understanding of CAM evolution will help us to design crops that can thrive in arid or semiarid regions,which are likely to expand due to global climate change.

PGD: Pineapple Genomics Database

Pineapple occupies an important phylogenetic position as its reference genome is a model for studying the evolution the Bromeliaceae family and the crassulacean acid metabolism(CAM)photosynthesis.Here,we developed a pineapple genomics database(PGD,http://pineapple.angiosperms.org/pineapple/html/index.html)as a central online platform for storing and integrating genomic,transcriptomic,function annotation and genetic marker data for pineapple(Ananas comosus(L.)Merr.).The PGD currently hosts significant search tools and available datasets for researchers to study comparative genomics,gene expression,gene co-expression molecular marker,and gene annotation of A.comosus(L).PGD also performed a series of additional pages for a genomic browser that visualizes genomic data interactively,bulk data download,a detailed user manual,and data integration information.PGD was developed with the capacity to integrate future data resources,and will be used as a long-term and open access database to facilitate the study of the biology,distribution,and the evolution of pineapple and the relative plant species.An email-based helpdesk is also available to offer support with the website and requests of specific datasets from the research community.

A near-complete genome assembly of the allotetrapolyploid Cenchrus fungigraminus(JUJUNCAO)provides insights into its evolution and C4 photosynthesis

JUJUNCAO(Cenchrus fungigraminus;2n=4x=28)is a Cenchrus grass with the highest biomass production among cultivated plants,and it can be used for mushroom cultivation,animal feed,and biofuel production.Here,we report a nearly complete genome assembly of JUJUNCAO and reveal that JUJUNCAO is an allopolyploid that originated2.7 million years ago(mya).Its genome consists of two subgenomes,and subgenome A shares high collinear synteny with pearl millet.We also investigated the genome evolution of JUJUNCAO and suggest that the ancestral karyotype of Cenchrus split into the A and B ancestral karyotypes of JUJUNCAO.Comparative transcriptome and DNA methylome analyses revealed functional divergence of homeologous gene pairs between the two subgenomes,which was a further indication of asymmetric DNA methylation.The three types of centromeric repeat in the JUJUNCAO genome(CEN137,CEN148,and CEN156)may have evolved independently within each subgenome,with some introgressions of CEN156 from the B to the A subgenome.We investigated the photosynthetic characteristics of JUJUNCAO,revealing its typical C4 Kranz anatomy and high photosynthetic efficiency.NADP-ME and PEPCK appear to cooperate in the major C4 decarboxylation reaction of JUJUNCAO,which is different from other C4 photosynthetic subtypes and may contribute to its high photosynthetic efficiency and biomass yield.Taken together,our results provide insights into the highly efficient photosynthetic mechanism of JUJUNCAO and provide a valuable reference genome for future genetic and evolutionary studies,as well as genetic improvement of Cenchrus grasses.

Pistachio genomes provide insights into nut tree domestication and ZW sex chromosome evolution

Pistachio is a nut crop domesticated in the Fertile Crescent and a dioecious species with ZW sex chromosomes.We sequenced the genomes of Pistacia vera cultivar(cv.)Siirt,the female parent,and P.vera cv.Bagyolu,the male parent.Two chromosome-level reference genomes of pistachio were generated,and Z and W chromosomes were assembled.The ZW chromosomes originated from an autosome following the first inversion,which occurred approximately 8.18 Mya.Three inversion events in the W chromosome led to the formation of a 12.7-Mb(22.8%of the W chromosome)non-recombining region.These W-specific sequences contain several genes of interest that may have played a pivotal role in sex determination and contributed to the initiation and evolution of a ZW sex chromosome system in pistachio.The W-specific genes,including defA,defA-like,DYT1,two PTEN1,and two tandem duplications of six VPS13A paralogs,are strong candidates for sex determination or differentiation.Demographic history analysis of resequenced genomes suggest that cultivated pistachio underwent severe domestication bottlenecks approximately 7640 years ago,dating the domestication event close to the archeological record of pistachio domestication in Iran.We identified 390,211,and 290 potential selective sweeps in 3 cultivar subgroups that underlie agronomic traits such as nut development and quality,grafting success,flowering time shift,and drought tolerance.These findings have improved our understanding of the genomic basis of sex determination/differentiation and horticulturally important traits and will accelerate the improvement of pistachio cultivars and rootstocks.

Allele-defined genome reveals biallelic differentiation during cassava evolution

Dear Editor,Generation of heterozygous genomes by hybridization between or within species can help maintain plant diversity and serve as a potential source of new species(Baek et al.,2018).Moreover,genomic heterozygosity is associated with genomic coadaptation,developmental stability,and heterosis.Accurate definition of alleles in haplotypes is necessary to precisely characterize allelic variation controlling agriculturally important traits(Shi et al.,2019).Currently,most released genomes have mosaic assembly of haplotypes due to random selection or collapse of alleles during genome assembly(Shi et al.,2019),which masked allelic variation and functional differentiation of divergent alleles in heterozygous species.

Unraveling a genetic roadmap for improved taste in the domesticated apple

Although taste is an important aspect of fruit quality, an understanding of its genetic control remains elusive in apple and other fruit crops. In this study, we conducted genomic sequence analysis of 497 Malus accessions and revealed erosion of genetic diversity caused by apple breeding and possible independent domestication events of dessert and cider apples. Signatures of selection for fruit acidity and size, but not for fruit sugar content, were detected during the processes of both domestication and improvement. Furthermore, we found that single mutations in major genes affecting fruit taste, including Ma1, MdTDT, and MdSOT2, dramatically decrease malate, citrate, and sorbitol accumulation, respectively, and correspond to important domestication events. Interestingly, Ma1 was identified to have pleiotropic effects on both organic acid content and sugar:acid ratio, suggesting that it plays a vital role in determining fruit taste. Fruit taste is unlikely to have been negatively affected by linkage drag associated with selection for larger fruit that resulted from the pyramiding of multiple genes with minor effects on fruit size. Collectively, our study provides new insights into the genetic basis of fruit quality and its evolutionary roadmap during apple domestication, pinpointing several candidate genes for genetic manipulation of fruit taste in apple.

PacBio Sequencing Reveals Transposable Elements as a Key Contributor to Genomic Plasticity and Virulence Variation in Magnaporthe oryzae

Dear Editor ：The sustainable cultivation of rice, which serves as staple food crop for more than half of the world＇s population, is under serious threat due to the huge yield losses inflicted by rice blast disease caused by the globally destructive fungus Magnaporthe oryzae （Pyricularia oryzae） （Dean et al., 2012; Nalley et al., 2016; Deng et al., 2017）. This filamentous ascomycete fungus is also capable of causing blast infection on other economically important cereal crops, including wheat, millet, and barley, making it the world＇s most important plant pathogenic fungus （Zhong et al., 2016）.

Identifying a melanogenesis-related candidate gene by a high-quality genome assembly and population diversity analysis in Hypsizygus marmoreus

Hypsizygus marmoreus is one of the most important edible fungi in Basidiomycete division and includes white and gray strains.However,very limited knowledge is known about the genomic structures and the genetic basis for the white/gray diversity of this mushroom.Here,we report the near-complete high-quality H.marmoreus genome at the chromosomal level.Comparative genomics analysis indicates that chromosome structures were relatively conserved,and variations in collinearity and chromosome number were mainly attributed by chromosome split/fusion events in Aragicales,whereas the fungi genome experienced many genomic chromosome fracture,fusion,and genomic replication events after the split of Aragicales from Basidiomycetes.Resequencing of 57 strains allows us to classify the population into four major groups and associate genetic variations with morphological features,indicating that white strains were not originated independently.We further generated genetic populations and identified a cytochrome P450 as the candidate causal gene for the melanogenesis in H.marmoreus based on bulked segregant analysis (BSA)and comparative transcriptome analysis.The high-quality H.marmoreus genome and diversity data compiled in this study provide new knowledge and resources for the molecular breeding of H.marmoreus as well as the evolution of Basidiomycete.