重测序和转录组分析揭示了核桃（ Juglans regia L.）花青素生物合成相关葡萄糖基转移酶基因的差异表达模式和序列变异

Re-sequencing and transcriptomic analysis reveal differential expression patterns and sequence variation in glucosyltransferase gene related to anthocyanin biosynthesis in walnut ( Juglans regia L.)

Abstract  抽象的

The color of walnut peel, usually green during the development stages, is one of the important appearance qualities of walnut fruit. Red walnut ‘RW-1’ with red peel is a specific germplasm resources due to anthocyanin accumulation, but the mechanism remains unclear. Here, re-sequencing and transcriptome were used to analyze the differences of anthocyanin accumulation in walnut peel with different colors. There were 2,696,620 SNPs and 495,238 InDels between ‘Zhonglin 1’ and ‘RW-1’, and the genetic relationship between them was closer than that of either and walnut reference genome ‘Chandler’. 1,156 differentially expressed genes (DEGs) with structural polymorphisms were screened by combining re-sequencing and transcriptome analysis. LOC108991235 (JrUFGT5), encoding anthocyanidin 3-O-glucosyltransferase, was identified by KEGG function enrichment. The JrUFGT5 gene base substitutions identified in the coding sequence (CDS) region resulted in amino acid variation, but did not occur in the protein domain region. The expression pattern of JrUFGT5 gene was positively correlated with the anthocyanin accumulation in different walnut peels, and the subcellular localization analysis showed JrUFGT5 was located in the cytoplasm and nucleus. Collectively, these findings revealed the differential expression patterns and sequence variation of a crucial anthocyanidin 3-O-glucosyltransferase gene in walnuts with different colors, which is of great significance to the exploration of the mechanism of anthocyanin biosynthesis in red walnut.

核桃青皮的颜色通常在发育阶段为绿色，是核桃果实外观质量的重要特征之一。红色核桃‘RW-1’因其青皮中花青素的积累而呈现红色，是一种特异的种质资源，但其机制尚不清楚。本研究通过重测序和转录组分析，研究了不同颜色核桃青皮中花青素积累的差异。在‘中林1号’和‘RW-1’之间共检测到2,696,620个SNP和495,238个InDel，其遗传关系比它们与核桃参考基因组‘Chandler’的关系更为密切。通过结合重测序和转录组分析，共筛选出具有结构多态性的1,156个差异表达基因（DEGs）。通过KEGG功能富集分析，鉴定出了编码花青素-3-O-葡糖基转移酶的基因LOC108991235（JrUFGT5）。JrUFGT5基因在编码区（CDS）中的碱基替换导致了氨基酸的变化，但未发生在蛋白质结构域区域。JrUFGT5基因的表达模式与不同核桃青皮中花青素的积累呈正相关，并且亚细胞定位分析显示JrUFGT5定位于细胞质和细胞核中。总的来说，这些研究结果揭示了不同颜色核桃中关键花青素-3-O-葡糖基转移酶基因的差异表达模式和序列变异，这对于探索红色核桃中花青素生物合成机制具有重要意义。

Introduction  介绍

Walnut ( Juglans regia L.) is one of the four largest nuts in the world and an indispensable woody oil plant (Zheng et al., 2020). Most walnuts show the characteristics of green peel, branch, leaf and yellow seed coat. With the investigation and collection of walnut germplasm resources, a type of red walnut ‘RW-1’ was picked out as an important research material because of its red peel, branch, leaf and seed coat characteristics, and it has been confirmed that its pigmentation is mainly caused by anthocyanin accumulation (Li et al., 2018a, 2018b; Wang et al., 2022).
核桃（  Juglans regia L.）是世界四大坚果之一，也是不可或缺的木本油料植物（Zheng等，2020）。大多数核桃呈现果皮、枝、叶绿色、种皮黄色的特征。通过对核桃种质资源的调查和收集，红核桃‘RW-1’因其果皮、枝条、叶和种皮的红色特征而被选为重要的研究材料，并已证实其色素沉着主要是由于花青素积累引起的(Li et al., 2018a, 2018b; Wang et al., 2022)。

Anthocyanin is one of the most important chromogenic substances in plants (Zhao et al., 2021), and have many common basic aglycones in the current research. The anthocyanin biosynthesis are catalysed by a series of enzymes (PAL, CHS, CHI, F3H, F3’H, F3’5’H, DFR, and ANS), and transferred to vacuole and other parts for storage after glycosylation, methylation and acylation (Misyura et al., 2013). Glycosylation is a key step in determining the structure of anthocyanin, which can improve the stability and change the colors of anthocyanins (Gachon et al., 2005). This step is catalyzed by UDP glucose: flavonoid (anthocyanin)−3- O-glucosyltransferase (UFGT), which is widely studied in anthocyanin biosynthesis (Sui et al., 2011). Furthermore,  UFGT genes have been isolated from many fruit trees, including kiwifruit (Montefiori et al., 2011), litchi (Zhao et al., 2012), pear (Liu et al., 2019), and so on. The functional verification of these genes is of great significance for further study of the molecular mechanism of pigmentation in walnut.
花青素是植物中最重要的显色物质之一(Zhao et al., 2021)，目前研究中有许多常见的基本苷元。花青素生物合成由一系列酶（PAL、CHS、CHI、F3H、F3'H、F3'5'H、DFR、ANS）催化，经糖基化、甲基化、酰化后转移至液泡等部位储存（Misyura 等人，2013）。糖基化是确定花青素结构的关键步骤，可以提高花青素的稳定性并改变花青素的颜色(Gachon et al., 2005)。该步骤由 UDP 葡萄糖：类黄酮（花青素）−3-  O-葡萄糖基转移酶（UFGT）催化，该酶在花青素生物合成中得到广泛研究（Sui et al., 2011）。此外，  UFGT基因已从许多果树中分离出来，包括猕猴桃(Montefiori et al., 2011)、荔枝(Zhao et al., 2012)、梨(Liu et al., 2019)等。这些基因的功能验证对于进一步研究核桃色素沉着的分子机制具有重要意义。

Genetic polymorphisms are the main determinants of phenotypic variations and affect the expression of individual traits (Wu et al., 2022). As a next-generation sequencing technology, genome re-sequencing identified the single nucleotide polymorphisms (SNPs), insertion/deletion variations (InDel) and other mutations with the reference genome through sequence alignment (Lin et al., 2015). Recently, a highly continuous walnut genome ‘Chandler’ v 2.0 at the chromosome level has been released (Marrano et al., 2020), which provided an important basis for promoting the genomics research of walnut, mining important functional genes and analyzing the growth and development mechanism. Genomics has been used to study the color of walnut tissues; Sideli et al. (2020) conducted GWAS analysis on 168 offspring of ‘Chandler’ × ‘Ldaho’ parents and 528 local walnuts, and found that the color phenotype of walnut pellicle had overlapping regions on ‘Chandler’ genetic map; QTL analysis detected that 12 genes, including the transcription factor MYB113, could be related to the pigmentation of walnut pellicle. The above results provided a reference for our research, and had great significance for the regulatory factors identification of key quality characteristics.
遗传多态性是表型变异的主要决定因素，影响个体性状的表达（Wu et al., 2022）。基因组重测序作为新一代测序技术，通过序列比对，识别出与参考基因组的单核苷酸多态性（SNP）、插入/缺失变异（InDel）等突变（Lin et al., 2015）。近期，发布了染色体水平高度连续的核桃基因组“Chandler”v 2.0（Marrano et al., 2020），为推进核桃基因组学研究、挖掘重要功能基因、分析生长发育等提供了重要基础。发展机制。基因组学已被用来研究核桃组织的颜色；西德利等人。 (2020)对‘Chandler’בLdaho’亲本的168个后代和528个当地核桃进行了GWAS分析，发现核桃皮膜颜色表型在‘Chandler’遗传图谱上存在重叠区域； QTL分析发现转录因子MYB113等12个基因可能与核桃皮膜色素沉着有关。上述结果为我们的研究提供了参考，对于关键质量特征的调控因素识别具有重要意义。

Multi-omics analysis has been widely used to elucidate the mechanism of anthocyanins biosynthesis and accumulation in fruit plants (Shi et al., 2020; Yi et al., 2020). In this study, integrated whole genome re-sequencing and transcriptomic analysis were used to reveal the differences in the anthocyanin accumulation mechanism between green walnut ‘Zhonglin 1’ and red walnut ‘RW-1’. Through the analysis of SNPs, InDels as well as the gene expression level at the rapid accumulation stage of anthocyanins in peels, DEGs with structural differences between ‘Zhonglin 1’ and ‘RW-1’ were identified. Further, the key anthocyanin biosynthesis gene JrUFGT5 was screened, and the expression and structural difference analysis showed that it played an important role in the anthocyanin biosynthesis in red walnut. These results will be helpful to breed cultivars with higher content of red pigment in walnut.
多组学分析已被广泛用于阐明果类植物花青素生物合成和积累的机制(Shi et al., 2020; Yi et al., 2020)。本研究通过整合全基因组重测序和转录组分析，揭示了绿核桃‘中林1号’和红核桃‘RW-1’花青素积累机制的差异。通过对SNP、InDels以及果皮花青素快速积累阶段基因表达水平的分析，鉴定出‘中林1号’和‘RW-1’之间存在结构差异的DEG。进一步筛选了花青素生物合成关键基因JrUFGT5 ，表达量和结构差异分析表明其在红核桃花青素生物合成中发挥重要作用。这些结果有助于选育红色素含量较高的核桃品种。

2. Materials and methods  2 材料与方法

2.1. Plant materials  2.1.植物材料

The green walnut ( J. regia L. ‘Zhonglin 1’) and red walnut ( J. regia L. accession ‘RW-1’) were used as experimental materials ( Zhao et al., 2023). The tender leaves of two walnuts used for re-sequencing were collected at the new  shoot growth stage, and  fruit peels were collected at 30 days after female  flowering (DAF), 60 DAF, and 90 DAF for  gene expression analysis.
以绿核桃（  J. regia L. '中林1号'）和红核桃（  J. regia L. accession 'RW-1'）为实验材料（  Zhao et al., 2023 ）。用于重测序的两个核桃在新梢 生长阶段收集嫩叶，并在雌花后30天（DAF）、60DAF和90DAF收集 果皮 进行 基因表达分析。

2.2. Determination of total anthocyanin content
2.2.总花色苷含量的测定

The total anthocyanins were extracted according the method of  Li et al. (2020). In brief, 0.1 g peels were ground to powder in liquid nitrogen and then homogenized with 1 mL of cold methanol containing 0.1% HCl in the dark at 4 °C. The supernatant was collected by high-speed  centrifugation. The absorbance was measured with a UV spectrophotometer, and each sample had three biological repeats. Anthocyanins (mg g-1)=[(A530 -A620) −0.25 × (A650-A620)]/0.1.
总花青素的提取参照 Li等的方法。 （2020） 。简而言之，将 0.1 g 果皮在液氮中研磨成粉末，然后在 4 °C 的黑暗条件下用 1 mL 含有 0.1% HCl 的冷甲醇均质化。通过高速 离心收集上清液。用紫外分光光度计测量吸光度，每个样品有3个生物重复。花青素(mg g -1 )=[(A 530 -A 620 ) -0.25 × (A 650 -A 620 )]/0.1。

2.3. Genome re-sequencing
2.3.基因组重测序

Genomic DNA from tender leaves of ‘Zhonglin 1’ and ‘RW-1’ were extracted using a CTAB method ( Fallah et al., 2017).  DNA libraries (30×) were constructed and sequenced by Biotechnology Corp. (Beijing, China) on an Illumina HiSeq XTen/NovaSeq/BGI platform, with 150-bp read lengths. The clean reads were mapped to walnut reference genome ‘Chandler’ v2.0 ( Marrano et al., 2020). The call and filter parameters of  SNPs and  InDels refer to the description of  Li et al. (2009).
采用 CTAB 法提取‘中林 1 号’和‘RW-1’嫩叶 基因组 DNA (  Fallah et al., 2017 )。  DNA文库（30×）由中国北京生物技术公司在Illumina HiSeq XTen/NovaSeq/BGI平台上构建和测序，读长为150 bp。干净的读数被映射到核桃参考基因组“Chandler”v2.0（