Genome-Wide Development of Polymorphic SNP Markers and Evaluation of Genetic Diversity of Litchi (Litchi chinensis Sonn.).

Litchi (Litchi chinensis Sonn.) is a highly valuable fruit crop that is widely grown in tropical and subtropical areas of the world. Studying its genetic diversity and population structure is critical for effective conservation and breeding programs. In this study, we developed 150 single-nucleotide polymorphism (SNP) markers that were evenly spaced across litchi genome and applied them to the evaluation of the genetic diversity of 84 litchi accessions, including old cultivars, modern cultivars, hybrids from known parents and wild accessions. Ninety-one SNP markers, showing high levels of polymorphism and high genotyping success rates, were used for further analysis. The newly developed SNP markers captured a relatively higher level of genetic diversity (He = 0.364) in litchi cultivars and could be successfully applied for the identification of synonymous cultivars and hybrids with close genetic backgrounds. Cluster analysis grouped all genotypes into three clusters that showed perfect association with their fruit maturation period, among which wild accessions clustered with their corresponding domesticated cultivars, and hybrids from different parent combinations showed different inheritance tendencies. Our study not only provided a set of efficient SNP markers for future genetic research, but also laid an important foundation for the conservation and genetic breeding of litchi.

An LcMYB111-LcHY5 Module Differentially Activates an LcFLS Promoter in Different Litchi Cultivars.

Flavonol synthase (FLS) is the crucial enzyme of the flavonol biosynthetic pathways, and its expression is tightly regulated in plants. In our previous study, two alleles of LcFLS,LcFLS-A and LcFLS-B, have been identified in litchi, with extremely early-maturing (EEM) cultivars only harboring LcFLS-A, while middle-to-late-maturing (MLM) cultivars only harbor LcFLS-B. Here, we overexpressed both LcFLS alleles in tobacco, and transgenic tobacco produced lighter-pink flowers and showed increased flavonol levels while it decreased anthocyanin levels compared to WT. Two allelic promoters of LcFLS were identified, with EEM cultivars only harboring proLcFLS-A, while MLM cultivars only harbor proLcFLS-B. One positive and three negative R2R3-MYB transcription regulators of LcFLS expression were identified, among which only positive regulator LcMYB111 showed a consistent expression pattern with LcFLS, which both have higher expression in EEM than that of MLM cultivars. LcMYB111 were further confirmed to specifically activate proLcFLS-A with MYB-binding element (MBE) while being unable to activate proLcFLS-B with mutated MBE (MBEm). LcHY5 were also identified and can interact with LcMYB111 to promote LcFLS expression. Our study elucidates the function of LcFLS and its differential regulation in different litchi cultivars for the first time.

The Upregulated Expression of the Citrus RIN4 Gene in HLB Diseased Citrus Aids Candidatus Liberibacter Asiaticus Infection.

The citrus industry has been threatened by Huanglongbing (HLB) for over a century. Here, an HLB-induced Arabidopsis RPM1-interacting protein 4 (RIN4) homologous gene was cloned from Citrus clementina, and its characteristics and function were analyzed to determine its role during citrus-Candidatus Liberibacter asiaticus (CLas) interactions. Quantitative real-time PCR showed that RIN4 was expressed in roots, stems, leaves and flowers, with the greatest expression level in leaves. Its expression was suppressed by gibberellic acid, indole-3-acetic acid, salicylic acid and jasmonic acid treatments, but was induced by abscisic acid and salt treatments, as well as wounding. The transient expression of a RIN4-GFP showed that RIN4 was localized in the cell membrane. RIN4-overexpressing transgenic C. maxima cv. 'Shatianyou' plants were obtained, and some transgenic plants showed greater sensitivity to CLas infection and earlier HLB symptoms appearance than non-transgenic controls. Results obtained in this study indicated that the upregulated expression of RIN4 in HLB diseased citrus may aid CLas infection.

Metabolite Differences of Polyphenols in Different Litchi Cultivars (Litchi chinensis Sonn.) Based on Extensive Targeted Metabonomics.

Litchi is an important fruit cultivated in tropical and subtropical areas with high nutritious and delicious flavor and the pulp is the main part of the fruit consumed. Previous studies found that litchi had high total phenol content and antioxidant activity, but most of them focused on the identification of single or a few phenolic components with a low throughput test, and the metabolic differences of cultivars are still unknown to a some extent. In this study we used widely targeted metabolome based on ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS/MS) to analyze the polyphenol metabolites of five different genotypes of mature litchi fruit. A total of 126 polyphenol metabolites in eight categories were identified to reveal the composition and differences of polyphenol; 15 common differential metabolites and 20 specific differential metabolites to each cultivar were found for the first time. The results infer that flavonoids, flavonols, hydroxycinnamoyls and catechins are the main polyphenol metabolites of litchi pulp. Cluster analysis showed that there were three groups of polyphenols from high to low; early maturing Feizhixiao is a kind of high polyphenol content cultivars, especially in catechins, anthocyanins, flavonols, quinic acids and hydroxycinnamoyls. The polyphenols in the flesh of mature litchi are rich, and there are significant differences among cultivars; there was a level of correlation between the contents of phenolics and the maturity of litchi cultivars; the content of phenolics in early maturing litchi cultivars appeared higher than those of mid- to late-maturing cultivars. This experiment will provide significant reference information for cultivation, breeding, processing and consumption.

Cloning and Characterization of a Flavonol Synthase Gene From Litchi chinensis and Its Variation Among Litchi Cultivars With Different Fruit Maturation Periods.

Litchi (Litchi chinensis) is an important subtropical fruit tree with high commercial value. However, the short and centralized fruit maturation period of litchi cultivars represents a bottleneck for litchi production. Therefore, the development of novel cultivars with extremely early fruit maturation period is critical. Previously, we showed that the genotypes of extremely early-maturing (EEM), early-maturing (EM), and middle-to-late-maturing (MLM) cultivars at a specific locus SNP51 (substitution type C/T) were consistent with their respective genetic background at the whole-genome level; a homozygous C/C genotype at SNP51 systematically differentiated EEM cultivars from others. The litchi gene on which SNP51 was located was annotated as flavonol synthase (FLS), which catalyzes the formation of flavonols. Here, we further elucidate the variation of the FLS gene from L. chinensis (LcFLS) among EEM, EM, and MLM cultivars. EEM cultivars with a homozygous C/C genotype at SNP51 all contained the same 2,199-bp sequence of the LcFLS gene. For MLM cultivars with a homozygous T/T genotype at SNP51, the sequence lengths of the LcFLS gene were 2,202-2,222 bp. EM cultivars with heterozygous C/T genotypes at SNP51 contained two different alleles of the LcFLS gene: a 2,199-bp sequence identical to that in EEM cultivars and a 2,205-bp sequence identical to that in MLM cultivar 'Heiye.' Moreover, the coding regions of LcFLS genes of other MLM cultivars were almost identical to that of 'Heiye.' Therefore, the LcFLS gene coding region may be used as a source of diagnostic SNP markers to discriminate or identify genotypes with the EEM trait. The expression pattern of the LcFLS gene and accumulation pattern of flavonol from EEM, EM, and MLM cultivars were analyzed and compared using quantitative real-time PCR (qRT-PCR) and high-performance liquid chromatography (HPLC) for mature leaves, flower buds, and fruits, 15, 30, 45, and 60 days after anthesis. Flavonol content and LcFLS gene expression levels were positively correlated in all three cultivars: both decreased from the EEM to MLM cultivars, with moderate levels in the EM cultivars. LcFLS gene function could be further analyzed to elucidate its correlation with phenotype variation among litchi cultivars with different fruit maturation periods.

Comprehensive transcriptomics and proteomics analyses of pollinated and parthenocarpic litchi (Litchi chinensis Sonn.) fruits during early development.

Litchi (Litchi chinensis Sonn.) is an important fruit that is widely cultivated in tropical and subtropical areas. In this study, we used RNA-Seq and iTRAQ technologies to compare the transcriptomes and proteomes of pollinated (polLFs) and parthenocarpic (parLFs) litchi fruits during early development (1 day, 2 days, 4 days and 6 days). We identified 4,864 DEGs in polLFs and 3,672 in parLFs, of which 2,835 were shared and 1,051 were specifically identified in parLFs. Compared to po1LFs, 768 DEGs were identified in parLFs. iTRAQ analysis identified 551 DEPs in polLFs and 1,021 in parLFs, of which 305 were shared and 526 were exclusively identified in parLFs. We found 1,127 DEPs in parLFs compared to polLFs at different stages. Further analysis revealed some DEGs/DEPs associated with abscisic acid, auxin, ethylene, gibberellin, heat shock protein (HSP), histone, ribosomal protein, transcription factor and zinc finger protein (ZFP). WGCNA identified a large set of co-expressed genes/proteins in polLFs and parLFs. In addition, a cross-comparison of transcriptomic and proteomic data identified 357 consistent DEGs/DEPs in polLFs and parLFs. This is the first time that protein/gene changes have been studied in polLFs and parLFs, and the findings improve our understanding of litchi parthenocarpy.

Digital Gene Expression Analysis of Ponkan Mandarin (Citrus reticulata Blanco) in Response to Asia Citrus Psyllid-Vectored Huanglongbing Infection.

Citrus Huanglongbing (HLB), the most destructive citrus disease, can be transmitted by psyllids and diseased budwoods. Although the final symptoms of the two main HLB transmission ways were similar and hard to distinguish, the host responses might be different. In this study, the global gene changes in leaves of ponkan (Citrus reticulata) mandarin trees following psyllid-transmission of HLB were analyzed at the early symptomatic stage (13 weeks post inoculation, wpi) and late symptomatic stage (26 wpi) using digital gene expression (DGE) profiling. At 13 wpi, 2452 genes were downregulated while only 604 genes were upregulated in HLB infected ponkan leaves but no pathway enrichment was identified. Gene function analysis showed impairment in defense at the early stage of infection. At late stage of 26 wpi, however, differentially expressed genes (DEGs) involved in carbohydrate metabolism, plant defense, hormone signaling, secondary metabolism, transcription regulation were overwhelmingly upregulated, indicating that the defense reactions were eventually activated. The results indicated that HLB bacterial infection significantly influenced ponkan gene expression, and a delayed response of the host to the fast growing bacteria might be responsible for its failure in fighting against the bacteria.

Comparative Transcriptome and iTRAQ Proteome Analyses of Citrus Root Responses to Candidatus Liberibacter asiaticus Infection.

Root samples of 'Sanhu' red tangerine trees infected with and without Candidatus Liberibacter asiaticus (CLas) were collected at 50 days post inoculation and subjected to RNA-sequencing and isobaric tags for relative and absolute quantification (iTRAQ) to profile the differentially expressed genes (DEGs) and proteins (DEPs), respectively. Quantitative real-time PCR was subsequently used to confirm the expression of 16 selected DEGs. Results showed that a total of 3956 genes and 78 proteins were differentially regulated by HLB-infection. Among the most highly up-regulated DEPs were sperm specific protein 411, copper ion binding protein, germin-like proteins, subtilisin-like proteins and serine carboxypeptidase-like 40 proteins whose transcript levels were concomitantly up-regulated as shown by RNA-seq data. Comparison between our results and those of the previously reported showed that known HLB-modulated biological pathways including cell-wall modification, protease-involved protein degradation, carbohydrate metabolism, hormone synthesis and signaling, transcription activities, and stress responses were similarly regulated by HLB infection but different or root-specific changes did exist. The root unique changes included the down-regulation in genes of ubiquitin-dependent protein degradation pathway, secondary metabolism, cytochrome P450s, UDP-glucosyl transferases and pentatricopeptide repeat containing proteins. Notably, nutrient absorption was impaired by HLB-infection as the expression of the genes involved in Fe, Zn, N and P adsorption and transportation were significantly changed. HLB-infection induced some cellular defense responses but simultaneously reduced the biosynthesis of the three major classes of secondary metabolites, many of which are known to have anti-pathogen activities. Genes involved in callose deposition were up-regulated whereas those involved in callose degradation were also up-regulated, indicating that the sieve tube elements in roots were hanging on the balance of life and death at this stage. In addition, signs of carbohydrate starvation were already eminent in roots at this stage. Other interesting genes and pathways that were changed by HLB-infection were also discussed based on our findings.