Unraveling the Molecular Basis of Color Variation in Dioscorea alata Tubers: Integrated Transcriptome and Metabolomics Analysis.

Dioscorea alata L. (Dioscoreaceae) is a widely cultivated tuber crop with variations in tuber color, offering potential value as health-promoting foods. This study focused on the comparison of D. alata tubers possessing two distinct colors, white and purple, to explore the underlying mechanisms of color variation. Flavonoids, a group of polyphenols known to influence plant color and exhibit antioxidant properties, were of particular interest. The total phenol and total flavonoid analyses revealed that purple tubers (PTs) have a significantly higher content of these metabolites than white tubers (WTs) and a higher antioxidant activity than WTs, suggesting potential health benefits of PT D. alata. The transcriptome analysis identified 108 differentially expressed genes associated with the flavonoid synthesis pathway, with 57 genes up-regulated in PTs, including CHS, CHI, DFR, FLS, F3H, F3'5'H, LAR, ANS, and ANR. The metabolomics analysis demonstrated that 424 metabolites, including 104 flavonoids and 8 tannins, accumulated differentially in PTs and WTs. Notably, five of the top ten up-regulated metabolites were flavonoids, including 6-hydroxykaempferol-7-O-glucoside, pinocembrin-7-O-(6″-O-malonyl)glucoside, 6-hydroxykaempferol-3,7,6-O-triglycoside, 6-hydroxykaempferol-7-O-triglycoside, and cyanidin-3-O-(6″-O-feruloyl)sophoroside-5-O-glucoside, with the latter being a precursor to anthocyanin synthesis. Integrating transcriptome and metabolomics data revealed that the 57 genes regulated 20 metabolites within the flavonoid synthesis pathway, potentially influencing the tubers' color variation. The high polyphenol content and antioxidant activity of PTs indicate their suitability as nutritious and health-promoting food sources. Taken together, the findings of this study provide insights into the molecular basis of tuber color variation in D. alata and underscore the potential applications of purple tubers in the food industry and human health promotion. The findings contribute to the understanding of flavonoid biosynthesis and pigment accumulation in D. alata tubers, opening avenues for future research on enhancing the nutritional quality of D. alata cultivars.

Low-coverage whole genome sequencing of eleven species/subspecies in Dioscorea sect. Stenophora (Dioscoreaceae): comparative plastome analyses, molecular markers development and phylogenetic inference.

Dioscorea sect. Stenophora (Dioscoreaceae) comprises about 30 species that are distributed in the temperate and subtropical regions of the Northern Hemisphere. Despite being evolutionarily "primitive" and medically valuable, genomic resources and molecular studies of this section are still scarce. Here, we conducted low-coverage whole genome sequencing of 11 Stenophora species/subspecies to retrieve their plastome information (whole plastome characteristics, plastome-divergent hotspots, plastome-derived SSRs, etc.) and polymorphic nuclear SSRs, as well as performed comparative plastome and phylogenetic analyses within this section. The plastomes of Stenophora species/subspecies ranged from 153,691 bp (D. zingiberensis) to 154,149 bp (D. biformifolia) in length, and they all contained the same 114 unique genes. All these plastomes were highly conserved in gene structure, gene order and GC content, although variations at the IR/SC borders contributed to the whole length differences among them. The number of plastome-derived SSRs among Stenophora species/subspecies varied from 74 (D. futschauensis) to 93 (D. zingiberensis), with A/T found to be the most frequent one. Seven highly variable regions and 12 polymorphic nuclear SSRs were identified in this section, thereby providing important information for further taxonomical, phylogenetic and population genetic studies. Phylogenomic analyses based on whole plastome sequences and 80 common protein coding genes strongly supported D. biformifolia and D. banzhuana constituted the successive sister species to the remaining sampled species, which could be furtherly divided into three clades. Overall, this study provided a new perspective for plastome evolution of Stenophora, and proved the role of plastome phylogenomic in improving the phylogenetic resolution in this section. These results also provided an important reference for the protection and utilization of this economically important section.

Pan-Plastome of Greater Yam (Dioscorea alata) in China: Intraspecific Genetic Variation, Comparative Genomics, and Phylogenetic Analyses.

Dioscorea alata L. (Dioscoreaceae), commonly known as greater yam, water yam, or winged yam, is a popular tuber vegetable/food crop worldwide, with nutritional, health, and economical importance. China is an important domestication center of D. alata, and hundreds of cultivars (accessions) have been established. However, genetic variations among Chinese accessions remain ambiguous, and genomic resources currently available for the molecular breeding of this species in China are very scarce. In this study, we generated the first pan-plastome of D. alata, based on 44 Chinese accessions and 8 African accessions, and investigated the genetic variations, plastome evolution, and phylogenetic relationships within D. alata and among members of the section Enantiophyllum. The D. alata pan-plastome encoded 113 unique genes and ranged in size from 153,114 to 153,161 bp. A total of four whole-plastome haplotypes (Haps I-IV) were identified in the Chinese accessions, showing no geographical differentiation, while all eight African accessions shared the same whole-plastome haplotype (Hap I). Comparative genomic analyses revealed that all four whole plastome haplotypes harbored identical GC content, gene content, gene order, and IR/SC boundary structures, which were also highly congruent with other species of Enantiophyllum. In addition, four highly divergent regions, i.e., trnC-petN, trnL-rpl32, ndhD-ccsA, and exon 3 of clpP, were identified as potential DNA barcodes. Phylogenetic analyses clearly separated all the D. alata accessions into four distinct clades corresponding to the four haplotypes, and strongly supported that D. alata was more closely related to D. brevipetiolata and D. glabra than D. cirrhosa, D. japonica, and D. polystachya. Overall, these results not only revealed the genetic variations among Chinese D. alata accessions, but also provided the necessary groundwork for molecular-assisted breeding and industrial utilization of this species.

Genome Sequence Resource for Colletotrichum gloeosporioides, an Important Pathogenic Fungus Causing Anthracnose of Dioscorea alata.

Anthracnose disease is one of the most important diseases of Dioscorea alata and many other food yams, which is caused by Colletotrichum gloeosporioides fungus from the Glomerellaceae family of the Sordariomycetes class. In the present study, a C. gloeosporioides starin named CgDa01 was isolated from D. alata, and its genome was sequenced based on Oxford Nanopore technology (ONT) and the Illumina sequencing platform. The high-quality genome of CgDa01 was assembled with a 62.78 Mb genome size and 15,845 predicted protein-coding genes. The proteins of predicted genes were annotated using multiple public databases, including the nonredundant protein database, the InterProScan databases, and Kyoto Encyclopedia of Genes and Genomes. Among the annotated protein-coding genes, 55 were predicted as potential virulence genes by the fungal virulence factor database. The C. gloeosporioides CgDa01 genome assembly described in this study can serve as a resource for better understanding the pathogenic mechanism of C. gloeosporioides on yam hosts.

Morphological Characteristics and Comparative Chloroplast Genome Analyses between Red and White Flower Phenotypes of Pyracantha fortuneana (Maxim.) Li (Rosaceae), with Implications for Taxonomy and Phylogeny.

Pyracantha fortuneana (Maxim.) Li (Rosaceae), commonly known as Chinese firethorn, is an evergreen shrub with high nutritional, medicinal, and horticultural importance. This species typically has white flowers, but a rare red flower phenotype has been found in very few wild populations in western Hubei, China, showing great ornamental potential. In this study, the complete chloroplast genome of the red flower phenotype of P. fortuneana was reported for the first time, using high-throughput sequencing technology. The complete chloroplast genome was 160,361 bp in length and showed a typical quadripartite structure with a pair of inverted repeat (IR) regions (26,350 bp) separated by a large single-copy (LSC) region (88,316 bp) and a small single-copy (SSC) region (19,345 bp). A total of 131 functional genes were annotated in this chloroplast genome, including 86 protein-coding genes (PCGs), eight rRNA genes, and 37 tRNA genes. Comparative chloroplast genome analyses revealed that high genome similarity existed not only between red and white flower phenotypes of P. fortuneana, but also among Pyracantha species. No evidence for positive selection was found in any PCG, suggesting the evolutionary conservation of Pyracantha chloroplast genomes. Furthermore, four mutational hotspots (trnG-trnR-atpA, psbZ-trnG-trnfM-rps14, ycf3-trnS-rps4, and ndhF-rpl32) with π > 0.004 were identified as potential molecular markers for Pyracantha species. Phylogenomic analysis strongly supported that the red flower phenotype of P. fortuneana was nested within the common white flower phenotype. Based on both morphological and molecular evidence, we suggest that the red flower phenotype of P. fortuneana could be considered as a new forma. Overall, the availability of these genetic resources will not only offer valuable information for further studies on molecular taxonomy, phylogeny, and population genetics of Pyracantha species but also could be used as potential genetic resources for Chinese firethorn breeding.

Genome-wide analyses of introgression between two sympatric Asian oak species.

Introgression can be an important source of new alleles for adaption under rapidly changing environments, perhaps even more important than standing variation. Though introgression has been extensively studied in many plants and animals, key questions on the underlying mechanisms of introgression still remain unanswered. In particular, we are yet to determine the genomic distribution of introgressed regions along the genome; whether the extent and patterns of introgression are influenced by ecological factors; and when and how introgression contributes to adaptation. Here, we generated high-quality genomic resources for two sympatric widespread Asian oak species, Quercus acutissima and Q. variabilis, sampled in multiple forests to study introgression between them. We show that introgressed regions are broadly distributed across the genome. Introgression was affected by genetic divergence between pairs of populations and by the similarity of the environments in which they live-populations occupying similar ecological sites tended to share the same introgressed regions. Introgressed genomic footprints of adaptation were preferentially located in regions with suppressed recombination rate. Introgression probably confers adaptation in these oak populations by introducing allelic variation in cis-regulatory elements, in particular through transposable element insertions, thereby altering the regulation of genes related to stress. Our results provide new avenues of research for uncovering mechanisms of adaptation due to hybridization in sympatric species.

Genome sequencing and transcriptome analyses provide insights into the origin and domestication of water caltrop (Trapa spp., Lythraceae).

Humans have domesticated diverse species from across the plant kingdom; however, our current understanding of plant domestication is largely founded on major cereal crops. Here, we examine the evolutionary processes and genetic basis underlying the domestication of water caltrop (Trapa spp., Lythraceae), a traditional, yet presently underutilized non-cereal crop that sustained early Chinese agriculturalists. We generated a chromosome-level genome assembly of tetraploid T. natans, and then divided the allotetraploid genome into two subgenomes. Based on resequencing data from 57 accessions, representing cultivated diploid T. natans, wild T. natans (2x and 4x) and diploid T. incisa, we showed that water caltrop was likely first domesticated in the Yangtze River Valley as early as 6300 yr BP, and experienced a second improvement c. 800 years ago. We also provided strong support for an allotetraploid origin of T. natans within the past 230 000-310 000 years. By integrating selective sweep and transcriptome profiling analyses, we identified a number of genes potentially selected and/or differentially expressed during domestication, some of which likely contributed not only to larger fruit sizes but also to a more vigorous root system, facilitating nutrient uptake, environmental stress response and underwater photosynthesis. Our results shed light on the evolutionary and domestication history of water caltrop, one of the earliest domesticated crops in China. This study has implications for genomic-assisted breeding of this presently underutilized aquatic plant, and improves our general understanding of plant domestication.

Genome-wide Identification and Evolutionary Analysis of NBS-LRR Genes From Secale cereale.

Secale cereale is an important crop in the Triticeae tribe of the Poaceae family, and it has unique agronomic characteristics and genome properties. It possesses resistance to many diseases and serves as an important resource for the breeding of other Triticeae crops. We performed a genome-wide study on S. cereale to identify the largest group of plant disease resistance genes (R genes), the nucleotide-binding site-leucine-rich repeat receptor (NBS-LRR) genes. In its genome, 582 NBS-LRR genes were identified, including one from the RNL subclass and 581 from the CNL subclass. The NBS-LRR gene number in the S. cereale genome is greater than that in barley and the diploid wheat genomes. S. cereale chromosome 4 contains the largest number of NBS-LRR genes among the seven chromosomes, which is different from the pattern in barley and the genomes B and D of wheat but similar to that in the genome A of wheat. Further synteny analysis suggests that more NBS-LRR genes on chromosome 4 have been inherited from a common ancestor by S. cereale and the wheat genome A than the wheat genomes B and D. Phylogenetic analysis revealed that at least 740 NBS-LRR lineages are present in the common ancestor of S. cereale, Hordeum vulgare and Triticum urartu. However, most of them have only been inherited by one or two species, with only 65 of them preserved in all three species. The S. cereale genome inherited 382 of these ancestral NBS-LRR lineages, but 120 of them have been lost in both H. vulgare and T. urartu. This study provides the full NBS-LRR profile of the S. cereale genome, which is a resource for S. cereale breeding and indicates that S. cereale can be an important material for the molecular breeding of other Triticeae crops.

Pre-quaternary diversification and glacial demographic expansions of Cardiocrinum (Liliaceae) in temperate forest biomes of Sino-Japanese Floristic Region.

The Sino-Japanese Floristic Region (SJFR) in East Asia is one of the most diverse temperate floras in the world. However, the relative influence of Neogene palaeogeographical changes and Quaternary climatic fluctuations as causal mechanisms on species diversification remains largely controversial, because most divergence time estimates were inferred from single-locus data and have limited geographic or taxonomic sampling. To evaluate these influences, we use SNP markers from restriction site-associated DNA sequencing (RAD-Seq) loci and expressed sequence tags-simple sequence repeat (EST-SSR) markers to investigate the levels of genetic variation, speciation and demographic history of the temperate-deciduous forest (TDF) endemic Cardiocrinum (Endlicher) Lindley (Liliaceae), a genus comprising three species in China (C. giganteum, C. cathayanum) and Japan (C. cordatum). Phylogenomic and population genomic coalescent-based analyses demonstrated that Late Neogene tectonic/climatic events triggered speciation of Cardiocrinum, and Pleistocene climatic fluctuations had limited influence on its divergence history. Population demographic inference using Approximate Bayesian Computation from EST-SSRs and palaeoclimatic niche models both indicated that all three Cardiocrinum species experienced population expansions during the transition from the LIG to the LGM. We also discussed the implications of these results on the conservation of montane TDF species in the SJFR under ongoing environmental change. Our results improve our understanding of how the constituents of montane TDF across the SJFR responded to previous periods of rapid climate and environmental change in terms of speciation and population demographic processes.

Comparative Genomics and Phylogenomics of East Asian Tulips (Amana, Liliaceae).

The genus Amana Honda (Liliaceae), when it is treated as separate from Tulipa, comprises six perennial herbaceous species that are restricted to China, Japan and the Korean Peninsula. Although all six Amana species have important medicinal and horticultural uses, studies focused on species identification and molecular phylogenetics are few. Here we report the nucleotide sequences of six complete Amana chloroplast (cp) genomes. The cp genomes of Amana range from 150,613 bp to 151,136 bp in length, all including a pair of inverted repeats (25,629-25,859 bp) separated by the large single-copy (81,482-82,218 bp) and small single-copy (17,366-17,465 bp) regions. Each cp genome equivalently contains 112 unique genes consisting of 30 transfer RNA genes, four ribosomal RNA genes, and 78 protein coding genes. Gene content, gene order, AT content, and IR/SC boundary structure are nearly identical among all Amana cp genomes. However, the relative contraction and expansion of the IR/SC borders among the six Amana cp genomes results in length variation among them. Simple sequence repeat (SSR) analyses of these Amana cp genomes indicate that the richest SSRs are A/T mononucleotides. The number of repeats among the six Amana species varies from 54 (A. anhuiensis) to 69 (Amana kuocangshanica) with palindromic (28-35) and forward repeats (23-30) as the most common types. Phylogenomic analyses based on these complete cp genomes and 74 common protein-coding genes strongly support the monophyly of the genus, and a sister relationship between Amana and Erythronium, rather than a shared common ancestor with Tulipa. Nine DNA markers (rps15-ycf1, accD-psaI, petA-psbJ, rpl32-trnL, atpH-atpI, petD-rpoA, trnS-trnG, psbM-trnD, and ycf4-cemA) with number of variable sites greater than 0.9% were identified, and these may be useful for future population genetic and phylogeographic studies of Amana species.

The Complete Chloroplast Genomes of Three Cardiocrinum (Liliaceae) Species: Comparative Genomic and Phylogenetic Analyses.

The genus Cardiocrinum (Endlicher) Lindley (Liliaceae) comprises three herbaceous perennial species that are distributed in East Asian temperate-deciduous forests. Although all three Cardiocrinum species have horticultural and medical uses, studies related to species identification and molecular phylogenetic analysis of this genus have not been reported. Here, we report the complete chloroplast (cp) sequences of each Cardiocrinum species using Illumina paired-end sequencing technology. The cp genomes of C. giganteum, C. cathayanum, and C. cordatum were found to be 152,653, 152,415, and 152,410 bp in length, respectively, including a pair of inverted repeat (IR) regions (26,364-26,500 bp) separated by a large single-copy (LSC) region (82,186-82,368 bp) and a small single-copy (SSC) region (17,309-17,344 bp). Each cp genome contained the same 112 unique genes consisting of 30 transfer RNA genes, 4 ribosomal RNA genes, and 78 protein-coding genes. Gene content, gene order, AT content, and IR/SC boundary structures were almost the same among the three Cardiocrinum cp genomes, yet their lengths varied due to contraction/expansion of the IR/SC borders. Simple sequence repeat (SSR) analysis further indicated the richest SSRs in these cp genomes to be A/T mononucleotides. A total of 45, 57, and 45 repeats were identified in C. giganteum, C. cathayanum, and C. cordatum, respectively. Six cpDNA markers (rps19, rpoC2-rpoC1, trnS-psbZ, trnM-atpE, psaC-ndhE, ycf15-ycf1) with the percentage of variable sites higher than 0.95% were identified. Phylogenomic analyses of the complete cp genomes and 74 protein-coding genes strongly supported the monophyly of Cardiocrinum and a sister relationship between C. cathayanum and C. cordatum. The availability of these cp genomes provides valuable genetic information for further population genetics and phylogeography studies on Cardiocrinum.