The chromosome-scale genome of black wolfberry (Lycium ruthenicum) provides useful genomic resources for identifying genes related to anthocyanin biosynthesis and disease resistance

Abstract

The black wolfberry (L ycium ruthenicum; 2n = 2x = 24) is an important medicinal plant with ecological and economic value. Its fruits have numerous beneficial pharmacological activities, especially those of anthocyanins, polysaccharides, and alkaloids, and have high nutritional value. However, the lack of available genomic resources for this species has hindered research on its medicinal and evolutionary mechanisms. In this study, we developed the telomere-to-telomere (T2T) nearly gapless genome of L. ruthenicum (2.26 Gb) by integrating PacBio HiFi, Nanopore Ultra-Long, and Hi-C technologies. The assembled genome comprised 12 chromosomes with 37,149 protein-coding genes functionally annotated. Approximately 80% of the repetitive sequences were identified, of which long terminal repeats (LTRs) were the most abundant, accounting for 73.01%. The abundance of LTRs might be the main reason for the larger genome of this species compared to that of other Lycium species. The species-specific genes of L. ruthenicum were related to defense mechanisms, salt tolerance, drought resistance, and oxidative stress, further demonstrating their superior adaptability to arid environments. Based on the assembled genome and fruit transcriptome data, we further constructed an anthocyanin biosynthesis pathway and identified 19 candidate structural genes and seven transcription factors that regulate anthocyanin biosynthesis in the fruit developmental stage of L. ruthenicum, most of which were highly expressed at a later stage in fruit development. Furthermore, 154 potential disease resistance-related nucleotide-binding genes have been identified in the L. ruthenicum genome. The whole-genome and proximal, dispersed, and tandem duplication genes in the L. ruthenicum genome enriched the number of genes involved in anthocyanin synthesis and resistance-related pathways. These results provide an important genetic basis for understanding genome evolution and biosynthesis of pharmacologically active components in the Lycium genus.

Keywords: Anthocyanin biosynthesis; Comparative genomics; Gene duplication; Genome; Lycium ruthenicum.

Graphical abstract

Fig. 1

Overview of the Lyciumruthenicum genome. A. Image of L. ruthenicum. B. Hi-C interactions heat map of 12 chromosomes from L. ruthenicum, and individual chromosomes were represented by blue boxes. C. Circular plot showing basic genomic information of L. ruthenicum genome. (a): chromosome lengths, (b): gene density, (c): repeat sequence density, (d): GC content, and (e): the interior relationship between different chromosomes.

Fig. 2

Phylogeny and gene family analyses of Lyciumruthenicum. A. Phylogenetic tree of L. ruthenicum and other ten plant species. The divergence time (million years ago, Mya) of each node was represented by a black number with the confidence range in brackets. The numbers of expanded, contracted gene families are shown in red and orange, while no changed gene families are shown in blue. Distribution of gene numbers and family sizes of 11 species (left). B. Gene family clustering diagram of five Solanoideae species (Solanum tuberosum, S. lycopersicum, L. ruthenicum, L. barbarum, L. ferocissimum). The letters in parentheses represent different species, and the numbers represent the number of common and unique gene families. C and D. KEGG enrichment for expanded (left) and contracted (right) gene families in L. ruthenicum.

Fig. 3

Genome evolution of Lyciumruthenicum. A. Ks distributions of paralogs and orthologous genes in the genomes of Ipomoea triloba, Vitis vinifera, Nicotiana tabacum, Solanumlycopersicum, L. barbarum, L. ferocissimum and L. ruthenicum (Lru). B. Syntenic depth analysis between L. ruthenicum and V. vinifera. C. Insertion time of LTRs in L. ruthenicum, L. barbarum, and L. ferocissimum; Mya: Million years ago. D. Homologous dot plot between L. ruthenicum and V. vinifera. Collinear blocks between the L. ruthenicum and V. vinifera chromosomes are highlighted by the red solid box.

Fig. 4

Duplicated gene in Lyciumruthenicum genome. A and B, Ka/Ks ratio and value of five types duplicated gene. C, KEGG and D, GO enrichment for five different types duplicated genes (P < 0.05). DSD: dispersed duplication, PD: proximal duplication, TD: tandem duplication, TRD: transposed duplication, and WGD: whole-genome duplication.

Fig. 5

Anthocyanin biosynthesis in Lyciumruthenicum fruit. A and B. KEGG enrichment in upregulated and downregulated genes in L. ruthenicum fruit. C. Heatmap showing the differential expression of anthocyanin biosynthesis genes according to the transcriptome data of L. ruthenicum fruit. Different colors are used to represent different types of duplication genes. DSD: dispersed duplication, PD: proximal duplication, TD: tandem duplication, and WGD: whole-genome duplication. D. Heatmap showing the expression of transcription factor according to the transcriptome data of L. ruthenicum fruit.

Fig. 6

Phylogenetic and transcriptome analyses of NBS (nucleotide-binding site) genes in the Lyciumruthenicum fruit. A. Clustered distribution of 58 NBS genes on chromosomes, with gene locations marked in black. B. Phylogenetic tree based on 58 NBS disease-resistant protein amino acid sequence in L. ruthenicum fruit. CC-NBS-LRR: Coiled-Coil (CC)-NBS-leucine-rich repeat (LRR); Tir-NBS-LRR: Toll interleukin-1 receptor (TIR)-NBS-LRR. C. The expression of 58 NBS disease-resistant genes in the L. ruthenicum fruit.