Mapping and Identifying a Candidate Gene Plr4, a Recessive Gene Regulating Purple Leaf in Rice, by Using Bulked Segregant and Transcriptome Analysis with Next-Generation Sequencing

The anthocyanin biosynthesis of rice is a major concern due to the potential nutritional value. Purple appears in various organs and tissues of rice such as pericarp, flower organs, leaves, leaf sheaths, internodes, ligules, apex, and stigma. At present, there are many studies on the color of rice pericarp, but the gene and mechanism of other organs such as leaves are still unclear, and the gene regulatory network of specific organ coloring has not been systematically understood. In this study, genetic analysis demonstrated that the purple leaf traits of rice were regulated by a recessive gene. The green leaf cultivar Y58S and purple leaf cultivar XianHongB were used to construct the mapping population. A set of near isogenicline (NIL) (BC3F1) was bred via crossing and back-crossing. The generations of BC3F2 appeared to separate four phenotypes, pl1, pl2, pl3, and pl4, due to the occurrence of a purple color in different organs. We constructed three bulked segregant analysis (BSA) pools (pl1–pl2, pl1–pl3, and pl1–pl4) by using the separated generations of BC3F5 and mapped the purple leaf gene plr4 to the vicinity of 27.9–31.1 Mb on chromosome 4. Subsequently, transcriptome sequencing (RNA-Seq) for pl3 and pl2 was used to analyze the differentially expressed genes in the localization interval, where 12 unigenes exhibited differential expression in which two genes (Os04g0577800, Os04g0616400) were downregulated. The two downregulated genes (Os04g0577800 and Os04g0616400) are possible candidate genes because of the recessive genetic characteristics of the purple leaf genes. These results will facilitate the cloning of plr4 and illustrate the molecular mechanisms of the anthocyanin synthesis pathway.

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PbCOP1.1 Contributes to the Negative Regulation of Anthocyanin Biosynthesis in Pear

Plants ◽

10.3390/plants8020039 ◽

2019 ◽

Vol 8 (2) ◽

pp. 39 ◽

Cited By ~ 4

Author(s):

Meng Wu ◽

Min Si ◽

Xieyu Li ◽

Linyan Song ◽

Jianlong Liu ◽

...

Keyword(s):

Protein Sequence ◽

Molecular Mechanisms ◽

Anthocyanin Biosynthesis ◽

Pcr Analysis ◽

Anthocyanin Synthesis ◽

Light Signaling ◽

Pear Fruit ◽

Phylogenetic Tree Analysis ◽

Expression Levels ◽

Protein Sequence Alignment

The synthesis of anthocyanin in pear (Pyrus bretschneideri) fruit is regulated by light. However, little is known about the molecular mechanisms of pear fruit coloring mediated by upstream light-signaling regulators. Here, the photoresponse factors CONSTITUTIVE PHOTOMORPHOGENIC (COP) 1.1 and 1.2 were cloned from ‘Red Zaosu’ peel to study their functions in pear fruit coloring. The overexpression vectors pBI121-PbCOP1.1 and pBI121-PbCOP1.2 were constructed to analyze their effects on anthocyanin synthesis in pear fruit. A protein sequence alignment and phylogenetic tree analysis revealed that PbCOP1 proteins are highly homologous with those of other species. An analysis of tissue differential expression showed that the greatest expression levels of PbCOP1s occurred in the leaves. Their expression levels increased in the leaves during development, when the leaves changed from red to green. The overexpression of PbCOP1s in the peel resulted in reduced anthocyanin synthesis at the injection sites. A quantitative PCR analysis of the injection sites showed that PbCOP1.1 significantly inhibited the expression of the anthocyanin synthesis-related genes CHI, DFR, UFGT2, bHLH3, HY5 and GST. Based on the above results, we hypothesize that PbCOP1.1 is an anthocyanin synthetic inhibitory factor of pear coloration.

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RNA-Seq-Based Profiling of pl Mutant Reveals Transcriptional Regulation of Anthocyanin Biosynthesis in Rice (Oryza sativa L.)

International Journal of Molecular Sciences ◽

10.3390/ijms22189787 ◽

2021 ◽

Vol 22 (18) ◽

pp. 9787

Author(s):

Ruonan Xu ◽

Ronghui Pan ◽

Yuchan Zhang ◽

Yanlei Feng ◽

Ujjal Kumar Nath ◽

...

Keyword(s):

Molecular Mechanisms ◽

Anthocyanin Biosynthesis ◽

Oryza Sativa L ◽

Transcriptional Profiling ◽

Expression Patterns ◽

Regulatory Gene ◽

Leaf Color ◽

Phenotypic Marker ◽

Bhlh Genes ◽

Purple Leaf

Purple-colored leaves in plants attain much interest for their important biological functions and could be a potential source of phenotypic marker in selecting individuals in breeding. The transcriptional profiling helps to precisely identify mechanisms of leaf pigmentation in crop plants. In this study, two genetically unlike rice genotypes, the mutant purple leaf (pl) and wild (WT) were selected for RNA-sequencing and identifying the differentially expressed genes (DEGs) that are regulating purple leaf color. In total, 609 DEGs were identified, of which 513 and 96 genes were up- and down-regulated, respectively. The identified DEGs are categorized into metabolic process, carboxylic acid biosynthesis, phenylpropanoids, and phenylpropanoid biosynthesis process enrichment by GO analysis. Kyoto Encyclopedia of Genes and Genomes (KEGG) confirmed their association with phenylpropanoid synthesis, flavonoid synthesis, and phenylalanine metabolism. To explore molecular mechanism of purple leaf color, a set of anthocyanin biosynthetic and regulatory gene expression patterns were checked by qPCR. We found that OsPAL (Os02g0626100, Os02g0626400, Os04g0518400, Os05g0427400 and Os02g0627100), OsF3H (Os03g0122300), OsC4HL (Os05g0320700), and Os4CL5 (Os08g0448000) are associated with anthocyanin biosynthesis, and they were up-regulated in pl leaves. Two members of regulatory MYB genes (OsMYB55; Os05g0553400 and Os08g0428200), two bHLH genes (Os01g0196300 and Os04g0300600), and two WD40 genes (Os11g0132700 and Os11g0610700) also showed up-regulation in pl mutant. These genes might have significant and vital roles in pl leaf coloration and could provide reference materials for further experimentation to confirm the molecular mechanisms of anthocyanin biosynthesis in rice.

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The Light-Induced WD40-Repeat Transcription Factor DcTTG1 Regulates Anthocyanin Biosynthesis in Dendrobium candidum

Frontiers in Plant Science ◽

10.3389/fpls.2021.633333 ◽

2021 ◽

Vol 12 ◽

Author(s):

Ning Jia ◽

Jingjing Wang ◽

Yajuan Wang ◽

Wei Ye ◽

Jiameng Liu ◽

...

Keyword(s):

Transcription Factor ◽

Molecular Mechanisms ◽

Anthocyanin Biosynthesis ◽

Raw Material ◽

Anthocyanin Synthesis ◽

Anthocyanin Content ◽

Anthocyanin Accumulation ◽

Wd40 Repeat ◽

Strong Light ◽

Dendrobium Candidum

Dendrobium candidum is used as a traditional Chinese medicine and as a raw material in functional foods. D. candidum stems are green or red, and red stems are richer in anthocyanins. Light is an important environmental factor that induces anthocyanin accumulation in D. candidum. However, the underlying molecular mechanisms have not been fully unraveled. In this study, we exposed D. candidum seedlings to two different light intensities and found that strong light increased the anthocyanin content and the expression of genes involved in anthocyanin biosynthesis. Through transcriptome profiling and expression analysis, we identified a WD40-repeat transcription factor, DcTTG1, whose expression is induced by light. Yeast one-hybrid assays showed that DcTTG1 binds to the promoters of DcCHS2, DcCHI, DcF3H, and DcF3′H, and a transient GUS activity assay indicated that DcTTG1 can induce their expression. In addition, DcTTG1 complemented the anthocyanin deficiency phenotype of the Arabidopsis thaliana ttg1-13 mutant. Collectively, our results suggest that light promotes anthocyanin accumulation in D. candidum seedlings via the upregulation of DcTTG1, which induces anthocyanin synthesis-related gene expression.

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Study on cyanidin metabolism in petals of pink-flowered strawberry based on transcriptome sequencing and metabolite analysis

BMC Plant Biology ◽

10.1186/s12870-019-2048-8 ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 1

Author(s):

Li Xue ◽

Jian Wang ◽

Jun Zhao ◽

Yang Zheng ◽

Hai-Feng Wang ◽

...

Keyword(s):

Regulatory Networks ◽

Molecular Mechanisms ◽

Target Genes ◽

Anthocyanin Biosynthesis ◽

Expression Profiles ◽

Expression Patterns ◽

Anthocyanin Synthesis ◽

Integrated Analysis ◽

Intergeneric Hybridization ◽

Significant Differential Expression

Abstract Background Pink-flowered strawberry is a promising new ornamental flower derived from intergeneric hybridization (Fragaria × Potentilla) with bright color, a prolonged flowering period and edible fruits. Its flower color ranges from light pink to red. Pigment compounds accumulated in its fruits were the same as in cultivated strawberry fruits, but different from that in its flowers. However, the transcriptional events underlying the anthocyanin biosynthetic pathway have not been fully characterized in petal coloration. To gain insights into the regulatory networks related to anthocyanin biosynthesis and identify the key genes, we performed an integrated analysis of the transcriptome and metabolome in petals of pink-flowered strawberry. Results The main pigments of red and dark pink petals were anthocyanins, among which cyanidins were the main compound. There were no anthocyanins detected in the white-flowered hybrids. A total of 50,285 non-redundant unigenes were obtained from the transcriptome databases involved in red petals of pink-flowered strawberry cultivar Sijihong at three development stages. Amongst the unigenes found to show significant differential expression, 57 were associated with anthocyanin or other flavonoid biosynthesis, in which they were regulated by 241 differentially expressed members of transcription factor families, such as 40 MYBs, 47 bHLHs, and 41 NACs. Based on a comprehensive analysis relating pigment compounds to gene expression profiles, the mechanism of flower coloration was examined in pink-flowered strawberry. A new hypothesis was proposed to explain the lack of color phenotype of the white-flowered strawberry hybrids based on the transcriptome analysis. The expression patterns of FpDFR and FpANS genes corresponded to the accumulation patterns of cyanidin contents in pink-flowered strawberry hybrids with different shades of pink. Moreover, FpANS, FpBZ1 and FpUGT75C1 genes were the major factors that led to the absence of anthocyanins in the white petals of pink-flowered strawberry hybrids. Meanwhile, the competitive effect of FpFLS and FpDFR genes might further inhibit anthocyanin synthesis. Conclusions The data presented herein are important for understanding the molecular mechanisms underlying the petal pigmentation and will be powerful for integrating novel potential target genes to breed valuable pink-flowered strawberry cultivars.

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Metabolome and Transcriptome Sequencing Analysis Reveals Anthocyanin Metabolism in Pink Flowers of Anthocyanin-Rich Tea (Camellia sinensis)

Molecules ◽

10.3390/molecules24061064 ◽

2019 ◽

Vol 24 (6) ◽

pp. 1064 ◽

Cited By ~ 15

Author(s):

Dylan Rothenberg ◽

Haijun Yang ◽

Meiban Chen ◽

Wenting Zhang ◽

Lingyun Zhang

Keyword(s):

Camellia Sinensis ◽

Flower Development ◽

Molecular Mechanisms ◽

Anthocyanin Biosynthesis ◽

Functional Enrichment ◽

Anthocyanin Synthesis ◽

Sequencing Analysis ◽

Anthocyanin Accumulation ◽

Structural Genes ◽

Anthocyanin Pathway

Almost all flowers of the tea plant (Camellia sinensis) are white, which has caused few researchers to pay attention to anthocyanin accumulation and color changing in tea flowers. A new purple-leaf cultivar, Baitang purple tea (BTP) was discovered in the Baitang Mountains of Guangdong, whose flowers are naturally pink, and can provide an opportunity to understand anthocyanin metabolic networks and flower color development in tea flowers. In the present study, twelve anthocyanin components were identified in the pink tea flowers, namely cyanidin O-syringic acid, petunidin 3-O-glucoside, pelargonidin 3-O-beta-d-glucoside, which marks the first time these compounds have been found in the tea flowers. The presence of these anthocyanins seem most likely to be the reason for the pink coloration of the flowers. Twenty-one differentially expressed genes (DEGs) involved in anthocyanin pathway were identified using KEGG pathway functional enrichment, and ten of these DEG’s screened using venn and KEGG functional enrichment analysis during five subsequent stages of flower development. By comparing DEGs and their expression levels across multiple flower development stages, we found that anthocyanin biosynthesis and accumulation in BTP flowers mainly occurred between the third and fourth stages (BTP3 to BTP4). Particularly, during the period of peak anthocyanin synthesis 17 structural genes were upregulated, and four structural genes were downregulated only. Ultimately, eight critical genes were identified using weighted gene co-expression network analysis (WGCNA), which were found to have direct impact on biosynthesis and accumulation of three flavonoid compounds, namely cyanidin 3-O-glucoside, petunidin 3-O-glucoside and epicatechin gallate. These results provide useful information about the molecular mechanisms of coloration in rare pink tea flower of anthocyanin-rich tea, enriching the gene resource and guiding further research on anthocyanin accumulation in purple tea.

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De novo transcriptome sequencing of radish (Raphanus sativus L.) fleshy roots: analysis of major genes involved in the anthocyanin synthesis pathway

BMC Molecular and Cell Biology ◽

10.1186/s12860-019-0228-x ◽

2019 ◽

Vol 20 (1) ◽

Cited By ~ 3

Author(s):

Jian Gao ◽

Wen-Bo Li ◽

Hong-Fang Liu ◽

Fa-Bo Chen

Keyword(s):

Raphanus Sativus ◽

Molecular Mechanisms ◽

De Novo ◽

Anthocyanin Biosynthesis ◽

Expression Patterns ◽

Anthocyanin Synthesis ◽

Biosynthesis Pathway ◽

Red Pigment ◽

De Novo Transcriptome ◽

Qrt Pcr

Abstract Background The HongXin radish (Raphanus sativus L.), which contains the natural red pigment (red radish pigment), is grown in the Fuling district of Chongqing City. However, the molecular mechanisms underlying anthocyanin synthesis for the formation of natural red pigment in the fleshy roots of HongXin radish are not well studied. Results De novo transcriptome of HX-1 radish, as well as that of the advanced inbred lines HX-2 and HX-3 were characterized using next generation sequencing (NGS) technology. In total, approximately 66.22 million paired-end reads comprising 34, 927 unigenes (N50 = 1, 621 bp) were obtained. Based on sequence similarity search with known proteins, total of 30, 127 (about 86.26%) unigenes were identified. Additionally, functional annotation and classification of these unigenes indicated that most of the unigenes were predominantly enriched in the metabolic process-related terms, especially for the biosynthetic pathways of secondary metabolites. Moreover, majority of the anthocyanin biosynthesis-related genes (ABRGs) involved in the regulation of anthocyanin biosynthesis were identified by targeted search for their annotation. Subsequently, the expression of 15 putative ABRGs involved in the anthocyanin synthesis-related pathways were validated using quantitative real-time polymerase chain reaction (qRT-PCR). Of those, RsPAL2, RsCHS-B2, RsDFR1, RsDFR2, RsFLS, RsMT3 and RsUFGT73B2-like were identified significantly associated with anthocyanin biosynthesis. Especially for RsDFR1, RsDFR2 and RsFLS, of those, RsDFR1 and RsDFR2 were highest enriched in the HX-3 and WG-3, but RsFLS were down-regulated in HX-3 and WG-3. We proposed that the transcripts of RsDFR1, RsDFR2 and RsFLS might be act as key regulators in anthocyanin biosynthesis pathway. Conclusions The assembled radish transcript sequences were analysed to identify the key ABRGs involved in the regulation of anthocyanin biosynthesis. Additionally, the expression patterns of candidate ABRGs involved in the anthocyanin biosynthetic pathway were validated by qRT-PCR. We proposed that the transcripts of RsDFR1, RsDFR2 and RsFLS might be acted as key regulators in anthocyanin biosynthesis pathway. This study will enhance our understanding of the biosynthesis and metabolism of anthocyanin in radish.

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Higher anthocyanin accumulation associated with higher transcription levels of anthocyanin biosynthesis genes in spinach

Genome ◽

10.1139/gen-2017-0261 ◽

2018 ◽

Vol 61 (7) ◽

pp. 487-496 ◽

Cited By ~ 4

Author(s):

Xiaofeng Cai ◽

Lihao Lin ◽

Xiaoli Wang ◽

Chenxi Xu ◽

Quanhua Wang

Keyword(s):

Molecular Mechanisms ◽

Spinacia Oleracea ◽

Anthocyanin Biosynthesis ◽

Expression Patterns ◽

Regulatory Genes ◽

Anthocyanin Synthesis ◽

Anthocyanin Accumulation ◽

Pigment Formation ◽

Leaf Petiole ◽

Genes Encoding

Spinach (Spinacia oleracea L.) is widely cultivated as an economically important green leafy vegetable crop for fresh and processing consumption. The red–purple spinach shows abundant anthocyanin accumulation in the leaf and leaf petiole. However, the molecular mechanisms of anthocyanin synthesis in this species are still undetermined. In the present study, we investigated pigment formation and identified anthocyanin biosynthetic genes in spinach. We also analyzed the expression of these genes in purple and green cultivars by quantitative PCR. The accumulation of anthocyanin showed that it was the dominant pigment resulting in the red coloration in spinach. In total, 22 biosynthesis genes and 25 regulatory genes were identified in spinach, based on the spinach genomic and transcriptomic database. Furthermore, the expression patterns of genes encoding enzymes indicated that SoPAL, SoUFGT3, and SoUFGT4 were possible candidate genes for anthocyanin biosynthesis in red–purple spinach. The expression patterns of transcription factors indicated that two SoMYB genes, three SobHLH genes, and one SoWD40 gene were drastically up-regulated and co-expression in red–purple spinach, suggesting an essential role of regulatory genes in the anthocyanin biosynthesis of spinach. These results will enhance our understanding of the molecular mechanisms of anthocyanin biosynthesis in purple spinach.

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Transcriptomic dynamics changes related to anthocyanin accumulation in the fleshy roots of carmine radish (Raphanus sativus L.) characterized using RNA-Seq

PeerJ ◽

10.7717/peerj.10978 ◽

2021 ◽

Vol 9 ◽

pp. e10978

Author(s):

Xia Song ◽

Jian Gao ◽

Hua Peng

Keyword(s):

Molecular Mechanisms ◽

Anthocyanin Biosynthesis ◽

Pearson Correlation ◽

Anthocyanin Synthesis ◽

Growth Stages ◽

Rna Seq ◽

Development Stages ◽

Qrt Pcr ◽

Dynamic Development ◽

Dynamic Growth

Carmine radish is famous for containing a natural red pigment (red radish pigment). However, the expression of anthocyanin biosynthesis-related genes during the dynamic development stages of the fleshy roots in carmine radish has not been fully investigated. Here, based on HPLC quantification of anthocyanin levels from our previous study, young fleshy roots of the carmine radish “Hongxin 1” obtained at the dynamic development stages of fleshy roots (seedling stage (SS), initial expansion (IE), full expansion (FE), bolting stage (BS), initial flowering stage (IFS), full bloom stage (FBS) and podding stage (PS)) were used for RNA-Seq. Approximately 126 comodulated DEGs related to anthocyanin biosynthesis (common DEGs in the dynamic growth stages of fleshy roots in carmine radish) were identified, from which most DEGs appeared to be likely to participate in anthocyanin biosynthesis, including two transcription factors, RsMYB and RsRZFP. In addition, some related proteins, e.g., RsCHS, RsDFR, RsANS, RsF′3H, RsF3GGT1, Rs3AT1, RsGSTF12, RsUFGT78D2 and RsUDGT-75C1, were found as candidate contributors to the regulatory mechanism of anthocyanin synthesis in the fleshy roots of carmine radish. In addition, 11 putative DEGs related to anthocyanin synthesis were evaluated by qRT-PCR via the (2-ΔΔCT) method; the Pearson correlation analysis indicated excellent concordance between the RNA-Seq and qRT-PCR results. Furthermore, GO enrichment analysis showed that “anthocyanin-containing compound biosynthetic process” and “anthocyanin-containing compound metabolic process” were commonly overrepresented in the dynamic growth stages of fleshy roots after the initial expansion stage. Moreover, five significantly enriched pathways were identified among the DEGs in the dynamic growth stages of fleshy roots in carmine radish, namely, flavonoid biosynthesis, flavone and flavonol biosynthesis, diterpenoid biosynthesis, anthocyanin biosynthesis, and benzoxazinoid biosynthesis. In conclusion, these results will expand our understanding of the complex molecular mechanisms of anthocyanin biosynthesis in the fleshy roots of carmine radish and the putative candidate genes involved in this process.

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Morphological, cytological, and genetic analyses of the “sango” mutant with the defects in basidiocarp development in edible mushroom Pleurotus pulmonarius

FEMS Microbiology Letters ◽

10.1093/femsle/fnz227 ◽

2019 ◽

Author(s):

Yasuhito Okuda ◽

Mikinari Ito ◽

Yu Shimada ◽

Masato Ishigami ◽

Teruyuki Matsumoto

Keyword(s):

Molecular Mechanisms ◽

Fragment Length Polymorphism ◽

Bulked Segregant Analysis ◽

Recessive Gene ◽

Microscopic Analysis ◽

Edible Mushroom ◽

Wild Type ◽

Pleurotus Pulmonarius ◽

Phenotypic Data ◽

Group Iii

ABSTRACT A spontaneous, morphological variation ‘sango’ was observed in the progeny of a Pleurotus pulmonarius (Fr.) Quél. wild-type basidiocarp (also known as fruiting body) collected from the field. This variant developed wart- and coral-like structures instead of normal basidiocarps. Microscopic analysis showed that the sango phenotype had defects in the differentiation of the pileus and hymenium. Basidiocarp phenotypic data analysis in the progenies revealed that the sango trait is a heritable mutation character controlled by a single recessive gene. This mutation locus was mapped on linkage group III of a previously constructed genetic linkage map by amplified fragment length polymorphism (AFLP) technique in P. pulmonarius. Four AFLP markers identified by bulked segregant analysis showed linkage to the sango mutation locus, with the genetic distance ranging from 0 to 2.1 cM. Of these markers, one marker was co-segregated with the sango mutation locus. This knowledge will be a useful foundation for practical breeding as well as for elucidating molecular mechanisms in basidiocarp development of main edible mushrooms.

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A Comparative Transcriptome Analysis of Purple and Yellow Fleshed Potato Tubers Reveals Long Non-coding RNAs and their Targets Functioned in Anthocyanin Biosynthesis

10.21203/rs.3.rs-515121/v1 ◽

2021 ◽

Author(s):

Ruimin Tang ◽

Haitao Dong ◽

Wanyi Wu ◽

Cailiang Zhao ◽

Xiaoyun Jia ◽

...

Keyword(s):

Molecular Mechanisms ◽

Target Genes ◽

Anthocyanin Biosynthesis ◽

Interaction Network ◽

Anthocyanin Synthesis ◽

Anthocyanin Content ◽

Potato Tubers ◽

Regulatory Relationship ◽

Key Enzyme ◽

Non Coding Rnas

Abstract Background: Purple fleshed potato tubers accumulate large amounts of anthocyanin content, servicing as functional foods and high-value feedstock. Long non-coding RNAs (lncRNAs) have been reported to play an important role in anthocyanin synthesis by regulating gene expression in various action modes. However, the mechanism underlying anthocyanin accumulation mediated by lncRNAs in underground organs remains unclear.Results: To excavate the differentially expressed lncRNAs (DE lncRNAs) between purple and yellow fleshed potato tubers, the transcriptome sequencing was performed and a total of 1421 DE lncRNAs were identified. Gene Ontology and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analyses showed that the target genes of these DE lncRNAs were involved in diverse biological processes and pathways for anthocyanin biosynthesis, reflecting the functional diversity of the corresponding lncRNAs. A lncRNA-mRNA interaction network was created based on their correlation to investigate the regulatory relationship among them. Notably, lncRNAs like XLOC_060098 and XLOC_017372 might contribute to anthocyanin synthesis by targeting the key enzyme genes and transcription factor genes in the pathway. Conclusions: The construction of expression profiling of DE lncRNAs and lncRNA-mRNA relationship network is helpful for further unraveling the molecular mechanisms of lncRNAs in anthocyanin synthesis in potato tubers, and provides theory basis for the cultivation of functional potato varieties and the improvement of nutritional quality of other underground crops.

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