| Black pepper(Piper nigrum),a perennial vine,is known as the "King of Spices" and one of the most important spice crops in the world.It has a wide range of medicinal and industrial value.Piper is also important for evolutionary studies due to its included among the strikingly diverse and long-isolated lineages of basal angiosperms.Piperales belongs to magnoliids,which represent a primitive group of angiosperms,and have similar morphological characteristics of monocots and eudicots.Based on this,it makes the phylogenetic position of the whole magnoliids with monocots and eudicots controversial.Piperine,as the most important functional substance of black pepper,in addition to its widespread use in cooking,also has a wide range of roles in chemoprevention,immunomodulation,anti-oxidation,anti-cancer,anti-inflammatory,detoxification and promotion of the absorption of Chinese herbal medicines and traditional medicines as well as bioavailability.At present,the basic research on black pepper is relatively weak due to the lack of a high quality reference genome and the complex metabolism of piperine and other key functional substances.It seriously hinders the molecular breeding of black pepper for further development and utilization of its resources,and restricts its potential application on the field of piperine.Therefore,this study carried out genome sequencing and chromosome-scale high-quality assembly for the black pepper.Based on this genome,we performed a comprehensive analysis of the genome characteristics and phylogenetic position.Further studies in the metabolic network of the core genes and gene families of piperine biosynthesis has been done.According to these studies,we have a preliminary insight of the piperine biosynthesis and get the molecular foundation for genetic improvement.The main results are as follows:1.A high-quality chromosome-scale reference genome of black pepperAs the first sequenced species of Piperales in the whole basal angiosperm group,the genome size of black pepper is 761.74 Mb with heterozygosity of 1.33%2 and repetitive sequence content of 59.54%.So,it can be defined as a complex genome with high heterozygosity and repetitive sequences.Based on this,this study combines PacBio sequencing,10x Genomics,BioNano Direct Label and Stain(DLS)optical mapping and Hi-C chromatin interaction mapping to assemble and anchor the chromosome of black pepper.What' s more,we also use the Illumina data for postprocessing the potential InDel and incorrecting assembly with the small fragment.The preliminarily assembly of black pepper genome("Piper_nigrum_v1")from PacBio and 10x Genomics contains 1,277 scaffolds with an N50 of 2.3 Mb and assembled genome size of 791.0 Mb.After further assistance assembly using BioNano and Hi-C data,the assembled genome size was 761.2 Mb,which contained 45 scaffolds sequences,and the N50 reached 29.8 Mb.The final chromosome-scale genome assembly of black pepper was named"Piper_nigrum_v3".Furthermore,there is high completeness and accuracy of the assembled genome when evaluated using Illumina reads,RNA-seq reads,CEGMA and BUSCO analysis2.Complete annotation of gene structure and function of black pepper genomeFirstly,the repetitive sequences of the black pepper genome were annotated and masked by ab initio prediction and homologous sequence alignment.The results showed that the repetitive sequences accounted for 54.85%of the total genome size,54.01%of which belonged to interspersed repeats sequences and 40.55%of which belonged to LTR-retrotransposons(LTR/Gypsy and LTR/Copia accounted for 27.63%and 9.95%respectively).Then,the BRAKER2 was used to predict the gene structure of black pepper by combining ab initio prediction,UniProt protein database,RNA-seq and Iso-seq transcriptome data.Based on this,a total of 63,466 protein-coding genes were annotated In addition,1,514 transpfer RNA(tRNA),1,206 ribosomal RNA(rRNA),1,533 small nuclear RNA(snRNA),256 microRNAs,6,509 long-chain non-coding RNA(lncRNAs),59 transcription factor(TFs)families,157 transcription regulators(TRs)and 646 chromatin regulatory factors(CRs)were also annotated.For gene function annotation,48,277 and 46,256 genes were aligned to NR and UniProt database,further 3,652 protein families and 2,071 GO classifications were identified by InterProScan analysis.KEGG annotation showed that 11,362 protein-coding genes were annotated to KO function and 57,700 genes were annotated to 330 metabolic pathways.Furthermore,a total of 10 gene clusters related to alkaloid metabolism pathway were identified by secondary metabolites annotation analysis3.Identification of the genome-wide replication events in black pepperSynteny analysis showed that there were 1,295 syntenic blocks in the black pepper genome,accounting for 45.10%of the total number of genes annotated in the genome.Among 66.0%of the homologous genes were located in different inter-chromosomally and 34.0%were located within chromosomes.Secondly,synteny dot plot analysis showed that there was a large number of replication regions in and between chromosomes of black pepper.In addition,the distribution of synonymous substitution rates(Ks)of the reciprocal best hit gene pairs and syntenic block gene pairs in the black pepper genome both showed that there was a single peak at about 0.1.The whole genome replication events of black pepper were identified,and the occurrence time was calculated to be 17.2-17.9 MYA4.Determined the phylogenetic position of black pepper and magnoliidsBased on the comparative genome and phylogenetic analysis of 21 typical species,the phylogenetic location of black pepper and its magnoliids was determined.In this study,82 single-copy orthologous genes families were identified from 21 species of black pepper,9 eudicots,3 monocots,3 magnoliids,one amborella,2 gymnosperms and 2 lower plants as outgroups.Subsequently,82 single-copy orthologous genes were used to construct species phylogenetic tree.The results showed that magnoliids was placed sisterly to the whole monocots-eudicots.Our results further suggest that Piperales,represented by Piper nigrum,first diverged from the Magnoliales(Liriodendron chinense)plus Laurales(Cinnamomum kanehirae and Persea americana)approximately 175-187 MYA(95%highest posterior density(HPD)interval)5.Revealed the molecular characteristics of piperine biosynthesis in black pepperBy analyzing the gene family expansions and RNA-seq transcriptome data of different tissues in black pepper,the core metabolic pathway of piperine biosynthesis that represents as the phenylpropane metabolic pathway,lysine metabolic pathway,and acyl transfer metabolic process were revealed.The analysis of gene family expansion showed that 91 gene families were expanded in black pepper,of which 35 gene families were significantly expansion(family-wide P-value ?0.01).Black pepper-specific gene family expansion-related genes were significantly enriched in secondary metabolism-related functions and disease resistance-related genes.RNA-seq transcriptomic data from different tissues showed that genes involved in phenylpropane and lysine metabolism pathways were specifically over-expressed in berry,and the expansion gene family of BAHD-AT and SCPL-AT were accompanied by high expression in berry tissue,which linked phenylpropane and lysine metabolism.Furthermore,the evolution analysis at sequence level of expanding gene family of the phenylpropane metabolic pathway,lysine metabolic pathway,BAHD-AT and SCPL-AT showed purifying selection and positive selection in different degrees. |
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