Genetic Analyses of Flower Main Traits from Two Pitayas and Their Progenies: A Cactus Plant.

Elucidation of the genetic foundation governing crucial traits in pitaya flowers is imperative for enhancing both the ornamental and economic values. In this study, the dynamic variation in flower genetics, segregation variation patterns, and a mixed inheritance model of the major and multigene flower traits of 'Dahong' and 'Honghuaqinglong' pitayas and their progenies were explored. The results showed that the main traits of flowers exhibited varying degrees of variation among the reciprocal F1 hybrids, with the data exhibiting the characteristics of quantitative traits. The betalain content, petal number, and stigma number exhibited values below the median values of the parents, suggesting a genetic inclination towards lower values. Perianth width, calyx tube width, petal number, and stigma number had the same genetic effects and significant correlation. Stigma-related traits had a clear maternal inheritance tendency. The heritability of flower length, stigma relative to anther distance, and petal betalain content was governed by two pairs of additive-dominant major genes. Perianth width, calyx tube width, petal number, and stigma number all conformed to the model of two pairs of equal-additive-dominant major genes. This study provides valuable information for parental selection, cross-breeding, and the enhancement of pitaya varieties to meet market preferences and environmental conditions.

Identification of HuSWEET Family in Pitaya (Hylocereus undatus) and Key Roles of HuSWEET12a and HuSWEET13d in Sugar Accumulation.

The sugar composition and content of fruit have a significant impact on their flavor and taste. In pitaya, or dragon fruit, sweetness is a crucial determinant of fruit taste and consumer preference. The sugars will eventually be exported transporters (SWEETs), a novel group of sugar transporters that have various physiological functions, including phloem loading, seed filling, nectar secretion, and fruit development. However, the role of SWEETs in sugar accumulation in pitaya fruit is not yet clear. Here, we identified 19 potential members (HuSWEET genes) of the SWEET family in pitaya and analyzed their conserved motifs, physiochemical characteristics, chromosomal distribution, gene structure, and phylogenetic relationship. Seven highly conserved α-helical transmembrane domains (7-TMs) were found, and the HuSWEET proteins can be divided into three clades based on the phylogenetic analysis. Interestingly, we found two HuSWEET genes, HuSWEET12a and HuSWEET13d, that showed strong preferential expressions in fruits and an upward trend during fruit maturation, suggesting they have key roles in sugar accumulation in pitaya. This can be further roughly demonstrated by the fact that transgenic tomato plants overexpressing HuSWEET12a/13d accumulated high levels of sugar in the mature fruit. Together, our result provides new insights into the regulation of sugar accumulation by SWEET family genes in pitaya fruit, which also set a crucial basis for the further functional study of the HuSWEETs.

Identification of HuSPL family and key role of HuSPL12 in regulation of betalain biosynthesis in pitaya.

The SQUAMOSA promoter binding protein-like (SPL) gene family is a unique family of plant-specific transcription factors (TFs), which plays vital roles in a variety of plant biological processes. Its role in betalain biosynthesis in Hylocereus undantus; however, is still unclear. Here, we report a total of 16 HuSPL genes from the pitaya genome, which were unevenly distributed among nine chromosomes. The HuSPL genes were clustered into seven groups, and most HuSPLs within the same group shared similar exon-intron structures and conserved motifs. Eight segment replication events in the HuSPL gene family were the main driving force behind the gene family expansion. Nine of the HuSPL genes had potential target sites for Hmo-miR156/157b. Hmo-miR156/157b-targeted HuSPLs exhibited differential expression patterns compared with constitutive expression patterns of most Hmo-miR156/157b-nontargeted HuSPLs. The expression of Hmo-miR156/157b gradually increased during fruit maturation, while the expression of Hmo-miR156/157b-targeted HuSPL5/11/14 gradually decreased. In addition, the lowest expression level of Hmo-miR156/157b-targeted HuSPL12 was detected 23rd day after flowering, when the middle pulps started to turn red. HuSPL5, HuSPL11, HuSPL12, and HuSPL14 were nucleus-localized proteins. HuSPL12 could inhibit the expression of HuWRKY40 by binding to its promoter. Results from yeast two-hybrid and bimolecular fluorescence complementation assays showed that HuSPL12 could interact with HuMYB1, HuMYB132, or HuWRKY42 TFs responsible for betalain biosynthesis. The results of the present study provide an essential basis for future regulation of betalain accumulation in pitaya.

Metabolome and transcriptome analyses reveal the molecular mechanisms of LcMYB1 regulating anthocyanin accumulation in litchi hairy roots.

Agrobacterium rhizogenes-mediated hairy root culture offer a promising approach for gene function analysis and production of plant secondary metabolites. Here, we obtained red litchi hairy roots using A. rhizogenes-mediated LcMYB1 transformation. Using high performance liquid chromatography, the main anthocyanins in the red hairy roots were determined to be cyanidin 3-rutinoside and cyanidin 3-glucoside. A total of 164 metabolites were significantly upregulated or downregulated in the red hairy roots, which were mostly involved in flavone and flavonol pathway, and flavonoid pathway. The transcriptome analysis revealed 472 differentially expressed genes (DEGs). Up-regulated genes were considerably enriched in anthocyanin, flavone and flavonol biosynthesis. Integrative metabolite profiling and transcriptome analyses showed that LcF3'H, LcUFGT1, and LcGST4 were key structural genes in anthocyanin biosynthesis. However, the expression of Cinnamyl-alcohol dehydrogenase (CAD) and Peroxidase (POD) leading to the production of lignin were significantly down-regulated, suggesting flavonoids and lignin compete with each other in the phenylpropanoid pathway. A total of 52 DEGs were identified as transcription factors. Correlation analysis showed that 8 transcription factors were positively correlated with LcUFGT1, and LcGST4, involving in anthocyanin biosynthesis. These findings clarify the molecular mechanisms of LcMYB1 regulating anthocyanin accumulation in litchi hairy roots.

Single-nucleus RNA sequencing and mRNA hybridization indicate key bud events and LcFT1 and LcTFL1-2 mRNA transportability during floral transition in litchi.

In flowering plants, floral induction signals intersect at the shoot apex to modulate meristem determinacy and growth form. Here, we report a single-nucleus RNA sequence analysis of litchi apical buds at different developmental stages. A total of 41 641 nuclei expressing 21 402 genes were analyzed, revealing 35 cell clusters corresponding to 12 broad populations. We identify genes associated with floral transition and propose a model that profiles the key events associated with litchi floral meristem identity by analyzing 567 identified floral meristem cells at single cell resolution. Interestingly, single-nucleus RNA-sequencing data indicated that all putative FT and TFL1 genes were not expressed in bud nuclei, but significant expression was detected in bud samples by RT-PCR. Based on the expression patterns and gene silencing results, we highlight the critical role of LcTFL1-2 in inhibiting flowering and propose that the LcFT1/LcTFL1-2 expression ratio may determine the success of floral transition. In addition, the transport of LcFT1 and LcTFL1-2 mRNA from the leaf to the shoot apical meristem is proposed based on in situ and dot-blot hybridization results. These findings allow a more comprehensive understanding of the molecular events during the litchi floral transition, as well as the identification of new regulators.

Transcription factor LcNAC002 coregulates chlorophyll degradation and anthocyanin biosynthesis in litchi.

Chlorophyll degradation and anthocyanin biosynthesis, which often occur almost synchronously during fruit ripening, are crucial for vibrant coloration of fruits. However, the interlink point between their regulatory pathways remains largely unknown. Here, 2 litchi (Litchi chinensis Sonn.) cultivars with distinctively different coloration patterns during ripening, i.e. slow-reddening/stay-green "Feizixiao" (FZX) vs rapid-reddening/degreening "Nuomici" (NMC), were selected as the materials to study the key factors determining coloration. Litchi chinensis STAY-GREEN (LcSGR) was confirmed as the critical gene in pericarp chlorophyll loss and chloroplast breakdown during fruit ripening, as LcSGR directly interacted with pheophorbide a oxygenase (PAO), a key enzyme in chlorophyll degradation via the PAO pathway. Litchi chinensis no apical meristem (NAM), Arabidopsis transcription activation factor 1/2, and cup-shaped cotyledon 2 (LcNAC002) was identified as a positive regulator in the coloration of litchi pericarp. The expression of LcNAC002 was significantly higher in NMC than in FZX. Virus-induced gene silencing of LcNAC002 significantly decreased the expression of LcSGR as well as L. chinensis MYELOBLASTOSIS1 (LcMYB1), and inhibited chlorophyll loss and anthocyanin accumulation. A dual-luciferase reporter assay revealed that LcNAC002 significantly activates the expression of both LcSGR and LcMYB1. Furthermore, yeast-one-hybrid and electrophoretic mobility shift assay results showed that LcNAC002 directly binds to the promoters of LcSGR and LcMYB1. These findings suggest that LcNAC002 is an important ripening-related transcription factor that interlinks chlorophyll degradation and anthocyanin biosynthesis by coactivating the expression of both LcSGR and LcMYB1.

Identification of HubHLH family and key role of HubHLH159 in betalain biosynthesis by activating the transcription of HuADH1, HuCYP76AD1-1, and HuDODA1 in pitaya.

Basic helix-loop-helix (bHLH) proteins are dimeric transcription factors (TFs) involved in various plant physiological and biological processes. Despite this, little is known about the molecular properties and roles of bHLH TFs in pitaya betalain biosynthesis. Here we report the identification of 165 HubHLH genes in H. undantus genome, their chromosomal distribution, physiochemical characteristics, conserved motifs, gene structure, phylogeny and synteny of HubHLH genes. Based on phylogenetic relationship analysis, the 165 HubHLHs were divided into 26 subfamilies and unequally distributed on the 11 chromosomes of pitaya. Based on the pitaya transcriptome data, a candidate gene HubHLH159 was obtained, and the real-time quantitative PCR analysis confirmed that HubHLH159 showed a high expression level in 'Guanhuahong' pitaya (red-pulp) at mature stage, indicating its role in betalain biosynthesis. HubHLH159 is a Group II protein and contains a bHLH domain. It is a nuclear protein with transcriptional activation activity. Dual luciferase reporter assays and virus-induced gene silencing (VIGS) experiments showed that HubHLH159 promotes betalain biosynthesis by activating the expression of HuADH1, HuCYP76AD1-1, and HuDODA1. The results of the present study lay a new theoretical reference for the regulation of pitaya betalain biosynthesis and also provides as essential basis for the future analysis of the functions of HubHLH gene family.

Betalain biosynthesis in red pulp pitaya is regulated via HuMYB132: a R-R type MYB transcription factor.

BACKGROUND: Multiple MYB transcription factors (TFs) are involved in the regulation of plant coloring. Betalain is a kind of natural plant pigment and its biosynthesis is regulated by a number of enzymes. Despite this, little is known about the molecular properties and roles of MYB TFs in pitaya betalain biosynthesis. RESULTS: In the present study, we identified a 1R-MYB gene, HuMYB132, which is preferentially expressed in red-pulp pitaya at the mature stage. It was clustered with Arabidopsis R-R-type genes and had two DNA-binding domains and a histidine-rich region. The expression assays in N. benthamiana and yeast indicated that HuMYB132 is a nucleus-localized protein with transcriptional activation activity. Dual luciferase reporter assay and electrophoretic mobility shift assays (EMSA) demonstrated that HuMYB132 could promote the transcriptional activities of HuADH1, HuCYP76AD1-1, and HuDODA1 by binding to their promoters. Silencing HuMYB132 reduced betalain accumulation and the expression levels of betalain biosynthetic genes in pitaya pulps. CONCLUSIONS: According to our findings, HuMYB132, a R-R type member of 1R-MYB TF subfamily, positively regulates pitaya betalain biosynthesis by regulating the expression of HuADH1, HuCYP76AD1-1, and HuDODA1. The present study provides a new theoretical reference for the management of pitaya betalain biosynthesis and also provides an essential basis for future regulation of betalain biosynthesis in Hylocereus.

Integrated metabolome and transcriptome analysis reveals the cause of anthocyanin biosynthesis deficiency in litchi aril.

Anthocyanins are health-promoting compounds with strong antioxidant properties that play important roles in disease prevention. Litchi chinensis Sonn. is a well-known and economically significant fruit due to its appealing appearance and nutritional value. The mature pericarp of litchi is rich in anthocyanins, whereas the aril (flesh) has an extremely low anthocyanin content. However, the mechanism of anthocyanin differential accumulation in litchi pericarp and aril remained unknown. Here, metabolome and transcriptome analysis were performed to unveil the cause of the deficiency of anthocyanin biosynthesis in litchi aril. Numerous anthocyanin biosynthesis-related metabolites and their derivatives were found in the aril, and the levels of rutin and (-)-epicatechin in the aril were comparable to those found in the pericarp, while anthocyanin levels were negligible. This suggests that the biosynthetic pathway from phenylalanine to cyanidin was present but that a block in cyanidin glycosylation could result in extremely low anthocyanin accumulation in the aril. Furthermore, 54 candidate genes were screened using weighted gene co-expression network analysis (WGCNA), and 9 genes (LcUFGT1, LcGST1, LcMYB1, LcSGR, LcCYP75B1, LcMATE, LcTPP, LcSWEET10, and LcERF61) might play a significant role in regulating anthocyanin biosynthesis. The dual-luciferase reporter (DLR) assay revealed that LcMYB1 strongly activated the promoters of LcUFGT1, LcGST4, and LcSWEET10. The results imply that LcMYB1 is the primary qualitative gene responsible for the deficiency of anthocyanin biosynthesis in litchi aril, which was confirmed by a transient transformation assay. Our findings shed light on the molecular mechanisms underlying tissue-specific anthocyanin accumulation and will help developing new red-fleshed litchi germplasm.

Function of a non-enzymatic hexokinase LcHXK1 as glucose sensor in regulating litchi fruit abscission.

Fruit abscission is a severe hindrance to commercial crop production, and a lack of carbohydrates causes fruit abscission to intensify in a variety of plant species. However, the precise mechanism by which carbohydrates affect fruit setting potential has yet to be determined. In the current study, we noticed negative correlation between hexose level and fruit setting by comparing different cultivars, bearing shoots of varying diameters, and girdling and defoliation treatments. The cumulative fruit-dropping rate was significantly reduced in response to exogenous glucose dipping. These results suggested that hexose, especially glucose, is the key player in lowering litchi fruit abscission. Moreover, five putative litchi hexokinase genes (LcHXKs) were isolated and the subcellular localization as well as activity of their expressed proteins in catalyzing hexose phosphorylation were investigated. LcHXK2 was only found in mitochondria and expressed catalytic protein, whereas the other four HXKs were found in both mitochondria and nuclei and had no activity in catalyzing hexose phosphorylation. LcHXK1 and LcHXK4 were found in the same cluster as previously reported hexose sensors AtHXK1 and MdHXK1. Furthermore, VIGS-mediated silencing assay confirms that LcHXK1 suppression increases fruit abscission. These findings revealed that LcHXK1 functions as hexose sensor, negatively regulating litchi fruit abscission.

Genome-Wide Identification of WRKY Gene Family in Pitaya Reveals the Involvement of HmoWRKY42 in Betalain Biosynthesis.

The WRKY gene family is a plant-specific transcription factor (TF) that regulates many physiological processes and (a) biotic stress responses. Despite this, little is known about the molecular properties and roles of WRKY TFs in pitaya betalain biosynthesis. Here we report the identification of 70 WRKY in Hylocereus undatus, their gene structure, locations on each chromosome, systematic phylogenetic analysis, conserved motif analysis, and synteny of HuWRKY genes. HmoWRKY42 is a Group IIb WRKY protein and contains a coiled-coil motif, a WRKY domain and a C2H2 zinc-finger motif (CX5CX23HXH). Results from yeast one-hybrid and transient dual-luciferase assays showed that HmoWRKY42 was a transcriptional repressor and could repress HmocDOPA5GT1 expression by binding to its promoter. Yeast two-hybrid assays showed that HmoWRKY42 could interact with itself to form homodimers. Knocking out the coiled-coil motif of HmoWRKY42 prevented its self-interaction and prevented it from binding to the HmocDOPA5GT1 promoter. Knocking out the WRKY domain and C2H2 zinc-finger motif sequence of HmoWRKY42 also prevented it from binding to the HmocDOPA5GT1 promoter. The coiled-coil motif, the WRKY domain and the C2H2 zinc finger motif are key motifs for the binding of HmoWRKY42 to the HmocDOPA5GT1 promoter. HmoWRKY42 is localized in the nucleus and possesses trans-activation ability responsible for pitaya betalain biosynthesis by repressing the transcription of HmocDOPA5GT1. As far as we know, no reports are available on the role of HmoWRKY42 in pitaya betalain biosynthesis. The results provide an important foundation for future analyses of the regulation and functions of the HuWRKY gene family.

Proteomic analysis of ubiquitinated proteins in 'Xiangshui' lemon [Citrus limon (L.)] pistils after self- and cross-pollination.

'Xiangshui' lemon exhibits gametophyte self-incompatibility: after self-pollination, the growth of a pollen tube in the style is inhibited. The aim of this work was to study the effect of pollination on the ubiquitylome and proteome in 'Xiangshui' lemon pistil. Here, by using label-free quantification, enrichment by anti-diglycine lysine antibody conjugated agarose beads, high-resolution liquid chromatography tandem mass spectrometry (LC-MS/MS) and the method of shotgun proteomics, we investigated the quantitative proteome, ubiquitylome, and crosstalk between the two datasets in the 'Xiangshui' lemon pistil after pollination. In total, 4682 protein species, 1956 ubiquitinated protein species and 6922 ubiquitination sites were quantified. A principal component analysis revealed that there were some differences in the ubiquitylome and proteome of CK, self-pollination and cross-pollination. The results of the crosstalk showed that the proteome and ubiquitylome were negatively correlated. After self-pollination, the abundance of the ubiquitinated protein species related to programmed cell death, cell aging and sphingolipid metabolic pathways was changed. Furthermore, we analyzed the proteasome pathway in the pistil after self-pollination and further discussed the mechanism by which ubiquitination events affect S-RNase in the style. Overall, pollination greatly changed the ubiquitinated protein species abundance in the pistils of 'Xiangshui' lemon. Accordingly, our work provides a systematic analysis of the proteome and ubiquitylome in 'Xiangshui' lemon pistils under self-incompatibility and sheds light for future studies on the function and mechanism of ubiquitination during self-incompatibility in 'Xiangshui' lemon. SIGNIFICANCE: As a mechanism to prevent self-pollination, self-incompatibility (SI) exists widely in plant species. Although a large number of SI-related genes have been found in fruit trees, there are few studies on the PTMs that affect and are involved in fruit tree responses to SI. This study highlights the fact that the effects of pollination on proteome and ubiquitylome in the 'Xiangshui' lemon pistil, we discussed the correlation between transcriptome and proteome, ubiquitylome and proteome, and we analyzed the expression and the changes of ubiquitination levels of SI related proteins and pathway after self- and cross pollination, and the changes of ubiquitination level of 26S proteasome pathway after cross-pollination. This study provides new insights into the ubiquitination pathway of SI in 'Xiangshui' lemon.

Pitaya Genome and Multiomics Database (PGMD): A Comprehensive and Integrative Resource of Selenicereus undatus.

Pitaya (Selenicereus) is a kind of novel fruit with a delicious taste and superior horticulture ornamental value. The potential economic impact of the pitaya lies in its diverse uses not only as agricultural produce and processed foods but also in industrial and medicinal products. It is also an excellent plant material for basic and applied biological research. A comprehensive database of pitaya would facilitate studies of pitaya and the other Cactaceae plant species. Here, we constructed pitaya genome and multiomics database, which is a collection of the most updated and high-quality pitaya genomic assemblies. The database contains various information such as genomic variation, gene expression, miRNA profiles, metabolite and proteomic data from various tissues and fruit developmental stages of different pitaya cultivars. In PGMD, we also uploaded videos on the flowering process and planting tutorials for practical usage of pitaya. Overall, these valuable data provided in the PGMD will significantly facilitate future studies on population genetics, molecular breeding and function research of pitaya.

Metabolic Profiling of Sugars and Organic Acids, and Expression Analyses of Metabolism-Associated Genes in Two Yellow-Peel Pitaya Species.

Sugar and organic acids are important factors determining pitaya fruit quality. However, changes in sugars and acids, and expressions of metabolism-associated genes during fruit maturation of yellow-peel pitayas are not well-documented. In this study, metabolic and expression analyses in pulps of different fruit developmental stages of 'Wucihuanglong' ('WCHL', Hylocereus undatus) and 'Youcihuanglong' pitaya ('YCHL', Hylocereus megalanthus) were used to explore the sugar and organic acid metabolic process. Total phenols and flavonoids were mainly accumulated at S1 in pitaya pulps. Ascorbic acid contents of 'WCHL' pitaya were higher than that of 'YCHL' pitaya during fruit maturation. Starch was mainly accumulated at early fruit development stages while soluble sugars were rich in late stages. Sucrose, fructose, and glucose were the main sugar components of 'YCHL' pitaya while glucose was dominant in 'WCHL' pitaya. Malic and citric acids were the main organic acids in 'WCHL' and 'YCHL' pitayas, respectively. Based on the transcriptome analyses, 118 genes involved in pitaya sugar and organic acid metabolism were obtained. Results from the correlation analyses between the expression profiling of candidate genes and the contents of sugar and organic acid showed that 51 genes had a significant correlation relationship and probably perform key role in pitaya sugar and organic acid metabolism processes. The finding of the present study provides new information for quality regulation of pitayas.

Integration of genomics, transcriptomics and metabolomics identifies candidate loci underlying fruit weight in loquat.

Fruit weight is an integral part of fruit-quality traits and directly influences commodity values and economic returns of fruit crops. Despite its importance, the molecular mechanisms underlying fruit weight remain understudied, especially for perennial fruit tree crops such as cultivated loquat (Eriobotrya japonica Lindl.). Auxin is known to regulate fruit development, whereas its role and metabolism in fruit development remain obscure in loquat. In this study, we applied a multi-omics approach, integrating whole-genome resequencing-based quantitative trait locus (QTL) mapping with an F1 population, population genomics analysis using germplasm accessions, transcriptome analysis, and metabolic profiling to identify the genomic regions potentially associated with fruit weight in loquat. We identified three major loci associated with fruit weight, supported by both QTL mapping and comparative genomic analysis between small- and big-fruited loquat cultivars. Comparison between two genotypes with contrasting fruit weight performance through transcriptomic and metabolic profiling revealed an important role of auxin in regulating fruit development, especially at the fruit enlarging stage. The multi-omics approach identified two homologs of ETHYLENE INSENSITIVE 4 (EjEIN4) and TORNADO 1 (EjTRN1) as promising candidates controlling fruit weight. Moreover, three single nucleotide polymorphism (SNP) markers were closely associated with fruit weight. Results from this study provided insights from multiple perspectives into the genetic and metabolic controls of fruit weight in loquat. The candidate genomic regions, genes, and sequence variants will facilitate understanding the molecular basis of fruit weight and lay a foundation for future breeding and manipulation of fruit weight in loquat.

HuNAC20 and HuNAC25, Two Novel NAC Genes from Pitaya, Confer Cold Tolerance in Transgenic Arabidopsis.

NAC transcription factors are one of the largest families of transcriptional regulators in plants, and members of the gene family play vital roles in regulating plant growth and development processes including biotic/abiotic stress responses. However, little information is available about the NAC family in pitaya. In this study, we conducted a genome-wide analysis and a total of 64 NACs (named HuNAC1-HuNAC64) were identified in pitaya (Hylocereus). These genes were grouped into fifteen subgroups with diversities in gene proportions, exon-intron structures, and conserved motifs. Genome mapping analysis revealed that HuNAC genes were unevenly scattered on all eleven chromosomes. Synteny analysis indicated that the segmental duplication events played key roles in the expansion of the pitaya NAC gene family. Expression levels of these HuNAC genes were analyzed under cold treatments using qRT-PCR. Four HuNAC genes, i.e., HuNAC7, HuNAC20, HuNAC25, and HuNAC30, were highly induced by cold stress. HuNAC7, HuNAC20, HuNAC25, and HuNAC30 were localized exclusively in the nucleus. HuNAC20, HuNAC25, and HuNAC30 were transcriptional activators while HuNAC7 was a transcriptional repressor. Overexpression of HuNAC20 and HuNAC25 in Arabidopsis thaliana significantly enhanced tolerance to cold stress through decreasing ion leakage, malondialdehyde (MDA), and H2O2 and O2- accumulation, accompanied by upregulating the expression of cold-responsive genes (AtRD29A, AtCOR15A, AtCOR47, and AtKIN1). This study presents comprehensive information on the understanding of the NAC gene family and provides candidate genes to breed new pitaya cultivars with tolerance to cold conditions through genetic transformation.

Two divergent haplotypes from a highly heterozygous lychee genome suggest independent domestication events for early and late-maturing cultivars.

Lychee is an exotic tropical fruit with a distinct flavor. The genome of cultivar 'Feizixiao' was assembled into 15 pseudochromosomes, totaling ~470 Mb. High heterozygosity (2.27%) resulted in two complete haplotypic assemblies. A total of 13,517 allelic genes (42.4%) were differentially expressed in diverse tissues. Analyses of 72 resequenced lychee accessions revealed two independent domestication events. The extremely early maturing cultivars preferentially aligned to one haplotype were domesticated from a wild population in Yunnan, whereas the late-maturing cultivars that mapped mostly to the second haplotype were domesticated independently from a wild population in Hainan. Early maturing cultivars were probably developed in Guangdong via hybridization between extremely early maturing cultivar and late-maturing cultivar individuals. Variable deletions of a 3.7 kb region encompassed by a pair of CONSTANS-like genes probably regulate fruit maturation differences among lychee cultivars. These genomic resources provide insights into the natural history of lychee domestication and will accelerate the improvement of lychee and related crops.

R3-MYB transcription factor LcMYBx from Litchi chinensis negatively regulates anthocyanin biosynthesis by ectopic expression in tobacco.

Anthocyanin accumulation is one of the remarkable physiological changes during fruit ripening. In plants, anthocyanin synthesis is regulated by MYB activators, but the MYB repressors has been recognized recently. Here, we isolated a repressor of anthocyanin synthesis, LcMYBx, from Litchi chinensis Sonn. LcMYBx encoded a typical R3-MYB protein and contained a conserved [D/E]Lx2[R/K]x3Lx6Lx3R motif for interacting with bHLH proteins. Overexpression of LcMYBx in tobacco suppressed anthocyanin accumulation resulting in faded petals from pale-pink to almost white. Gene expression analysis showed the strong down-regulation of endogenous anthocyanin structural and regulatory genes by LcMYBx overexpression. Yeast two-hybrid and bimolecular fluorescence complementation assays indicated that LcMYBx could interact with the transcription factors LcbHLH1 and LcbHLH3. Transient promoter activation assays showed that LcMYBx could inhibit the activation capacity of LcMYB1-LcbHLH3 complex for LcDFR gene. These results suggest that LcMYBx competed with LcMYB1 to LcbHLHs, thus preventing the activation of LcDFR by LcMYB1-LcbHLHs complex and negatively controlling anthocyanin biosynthesis.