Pecan (Carya illinoinensis) is an important tree nut throughout the world. The high concentration of flavonoid in its kernels makes it an excellent food with health benefits. However, the molecular basis of flavonoid biosynthesis in pecan remains unclear, which hinders quality breeding in this plant. Therefore, in order to find the crucial genes involved in flavonoid biosynthesis, the changes in flavonoid profiles and the transcriptomes of pecan kernels at four developmental stages (late water, gel, dough, and mature stages) were analyzed. As a result, the highest levels of total phenolic, condensed tannin, and flavan-3-ols were observed at the "late water stage". Catechin was the most abundant flavan-3-ol at different development stages. In total, 64 773 unigenes were obtained, and 46 924 (72.44%) unigenes were annotated. After differentially expressed gene (DEG) analysis, 12 750 unique DEGs were identified. Flavonoid-related DEGs of 36 structural genes and eight MYBs were obtained. The structural gene set contained three PALs, three CHSs, two CHIs, one F3H, two F3′Hs, two F3′5′Hs, one DFR, one ANS, two LARs, and two ANRs. The expression patterns of most of the structural genes were consistent with the changes in flavonoid profiles during kernel development. We believe that this RNA-Seq data set will provide valuable resources for unraveling the molecular mechanism of flavonoid metabolism in pecan and will significantly promote genetic studies and quality breeding in this plant.

Phenolics are a group of important plant secondary metabolites that have been proven to possess remarkable antioxidant activity and to be beneficial for human health. Pecan nuts are an excellent source of dietary phenolics. In recent years, many studies have focused on the separation and biochemical analysis of pecan phenolics, but the molecular mechanisms of phenolic metabolism in pecans have not been fully elucidated, which significantly hinders quality breeding research for this plant. Chalcone synthase (CHS) plays crucial roles in phenolic biosynthesis. In this study, three Carya illinoinensis CHSs (CiCHS1, CiCHS2, and CiCHS3), were isolated and analyzed. CiCHS2 and CiCHS3 present high expression levels in different tissues, and they are also highly expressed at the initial developmental stages of kernels in three pecan genotypes. A correlation analysis was performed between the phenolic content and CHSs expression values during kernel development. The results indicated that the expression variations of CiCHS2 and CiCHS3 are significantly related to changes in total phenolic content. Therefore, CiCHSs play crucial roles in phenolic components synthesis in pecan. We believe that the isolation of CiCHSs is helpful for understanding phenolic metabolism in C. illinoinensis, which will improve quality breeding and resistance breeding studies in this plant.

ABSTRACT. Tea is the second most popular non-alcoholic beverage in the world. In recent years, several molecular markers have been used in genetic studies of the tea plant. Yet, only a few single nucleotide polymorphisms (SNPs) have been reported. Here, we identified 818 putative SNPs from expressed sequence tag (EST) databases for the tea plant, which produced a frequency of 1 SNP/170 bp. A direct sequencing method was then used to verify 253 putative SNPs in genome DNA of 17 tea varieties. Fifty (20%) candidate and 299 new SNPs were identified. The haplotype polymorphism and nucleotide diversity of these markers ranged from 0 to 0.960 and 0 to 1.797%, respectively. Using these SNPs, the 17 varieties were classified into 2 groups by cluster analysis. The results indicate that Camellia sinensis-derived ESTs provide a valuable resource for SNP discovery. Furthermore, the abundance of SNPs in tea varieties is anticipated to generate the development of associated genetic studies, in addition to enhancing tea plant-breeding programs.

The ethylene-responsive element (AP2/ERF) is one of the keys and conserved transcription factors (TFs) in plants that play a vital role in regulating plant growth, development, and stress response. A total of 202 AP2/ERF genes were identified from the pecan genome and renamed according to the chromosomal distribution of the CiAP2/ERF genes. They were divided into four subfamilies according to the domain and phylogenetic analysis, including 26 AP2, 168 ERF, six RAV, and two Soloist gene family members. These genes were distributed randomly across the 16 chromosomes, and we found 19 tandem and 146 segmental duplications which arose from ancient duplication events. The gene structure and conserved motif analysis demonstrated the conserved nature of intron/exon organization and motifs among the AP2/ERF genes. Several cis-regulatory elements, which were related to light responsiveness, stress, and defense responses, were identified in the promoter regions of AP2/ERFs. The expression profiling of 202 CiAP2/ERF genes was assessed by using RNA-Seq data and qRT-PCR during development (pistillate flowering development, graft union development, and kernel development) and under abiotic stresses (waterlogging, drought). Moreover, the results suggested that the ERF-VII members may play a critical role in waterlogging stress. These findings provided new insights into AP2/ERF gene evolution and divergence in pecan and can be considered a valuable resource for further functional validation, as well as for utilization in a stress-resistance-variety development program.

Research Highlights: The distribution of simple sequence repeat (SSR) motifs in two draft genomes of pecan was evaluated. Sixty-six SSR loci were validated by PCR amplification in pecan. Twenty-two new development markers can be used for genetic study in genus Carya. Background and Objectives: Pecan has good nutritional and health benefits and is an important crop worldwide. However, the genetic research in this species is insufficient. One of the main reasons for this is the lack of enough accurate, convenient, and economical molecular markers. Among different marker types, SSR loci are enormously useful in genetic studies. However, the number of SSRs in C. illinoinensis (Wangenh.) K. Koch is limited. Materials and Methods: The distribution of SSR motifs in the pecan genome was analyzed. Then, the primers for each SSR were designed. To evaluate their availability, 74 SSR loci were randomly selected and amplified in pecan. Finally, 22 new SSRs and eight former ones were picked to evaluate the genetic diversity in 60 pecan genotypes and to determine their transferability in other Carya species. Results: 145,714 and 143,041 SSR motifs were obtained from two draft genomes of ‘87MX3-2’ and ‘Pawnee’, respectively. In total, 9145 candidate primers were obtained. Sixty-six (89.19%) primers amplified the target products. Among the 30 SSRs, 29 loci showed polymorphism in 60 pecan genotypes. The polymorphic information content (PIC) values ranged from 0.012 to 0.906. In total, 26, 25, and 22 SSRs can be used in C. cathayensis Sarg., C. dabieshanensis W. C. Cheng & R. H. Chang, and C. hunanensis W.C. Liu, respectively. Finally, the dendrogram of all individuals was constructed. The results agree with the geographic origin of the four species and the pedigree relationships between different pecan cultivars. Conclusions: The characterization of SSRs in the pecan genome and the new SSRs will promote the progress of genetic study and breeding in pecan, as well as other species of genus Carya.

Flavonoids influence the flavor and nutritional value of pecan nuts. However, limited information is available regarding the molecular mechanisms underlying pecan flavonoid biosynthesis. Here, we used a high (“YLC28”) and a low (“Oconee”) flavonoid content cultivar as the research objects. The changes in flavonoid content and the gene transcription patterns during kernel development were identified. Different accumulation patterns of total flavonoids (TF) and condensed tannins (CT) were observed between the two cultivars. The contents of TF and CT in “YLC28” were 1.76- and 2.67-fold higher levels than that of “Oconee” on 150 days after full bloom of female flowers, respectively. In total, 30 RNA-Seq libraries were constructed and sequenced. The upregulated genes in “YLC28” were highly enriched in flavonoid-related pathways. Thirty-three structural genes were identified, and the expression of two phenylalanine ammonia lyases, one chalcone synthase, one flavonoid 3’,5’-hydroxylase, and one flavonol synthase exhibited high correlation (r ≥ 0.7, p < 0.01) with the condensed tannin content in “YLC28.” A putative MYB transcription factor, CIL1093S0100, might act as a flavonoid biosynthesis repressor during kernel development. Altogether, these results will be useful for uncovering the molecular mechanisms of flavonoid biosynthesis and subsequently accelerating quality pecan breeding.

Pecan is rich in bioactive components such as fatty acids and flavonoids and is an important nut type worldwide. Therefore, the molecular mechanisms of phytochemical biosynthesis in pecan are a focus of research. Recently, a draft genome and several transcriptomes have been published. However, the full-length mRNA transcripts remain unclear, and the regulatory mechanisms behind the quality components biosynthesis and accumulation have not been fully investigated. In this study, single-molecule long read sequencing technology was used to obtain full-length transcripts of pecan kernels. In total, 37 504 isoforms of 16 702 genes were mapped to the reference genome. The numbers of known isoforms, new isoforms, and novel isoforms were 9013 (24.03%), 26 080 (69.54%), and 2411 (6.51%), respectively. Over 80% of the transcripts (30 751, 81.99%) had functional annotations. A total of 15 465 alternative splicing (AS) events and 65 761 alternative polyadenylation events were detected; wherein, the retained intron was the predominant type (5652, 36.55%) of AS. Furthermore, 1894 long non-coding RNAs and 1643 transcription factors were predicted using bioinformatics methods. Finally, the structural genes associated with fatty acid (FA) and flavonoid biosynthesis were characterized. A high frequency of AS accuracy (70.31%) was observed in FA synthesis-associated genes. The present study provides a full-length transcriptome dataset of pecan kernels, which will significantly enhance the understanding of the regulatory basis of phytochemical biosynthesis during pecan kernel maturation.

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