Re-Sequencing and Transcriptomic Analysis Reveal Differences in Nitrite Reductase in Jujube Fruit (Ziziphus Jujube)

Background Jujube is one of the characteristic fruit tree species in China. ‘Linhuang No. 1’, a cracking-resistant cultivar, and ‘Muzao’, a cracking-susceptible cultivar, were selected as materials by previous study. Whole-genome re-sequencing and transcriptome of ‘Linhuang No. 1’ and ‘Muzao’ allow the screening out of differently expressed genes with different gene structures between them. It could be helpful in explaining divergence/similarity between the two cultivars. Results There are 664,129 mutation sites between ‘Linhuang No. 1’ and ‘Muzao’ by re-sequencing. To determine the genetic relationship of ‘Linhuang 1’, ‘Muzao’ and reference genome ‘Dongzao’, the characteristic mutation sites were analyzed by principal component analysis. The genetic relationship between ‘Linhuang No. 1’ and ‘Muzao’ was closer than that with ‘Dongzao’. 19 differentially expressed genes were screened by combining the transcriptomics with re-sequencing analysis. LOC107427052 (encoding nitrite reductase) was determined by KEGG enrichment analysis for further study. The large base insertion was not in the domain region of the LOC107427052 gene CDS region. As veried by the nding that the base insertion did not affect protein translation. LOC107427052 gene expression levels, the nitrite reductase activities and the nitrite content of ‘Muzao’ were signicantly higher than those of ‘Linhuang No. 1’ at young fruit stage. There was no signicant difference in the product ammonia of nitrite reductase between the two varieties. Our study has laid a foundation for the analysis of genetic information and the comparative nitrite metabolism of ‘Linhuang No. 1’ and ‘Muzao’.


Introduction
Jujube is one of the characteristic fruit tree species in China [1]. Jujube is valued as a traditional herbal medicine, and is cultivated on 2 million hectares in China alone, with an annual production of approximately 7.36 million tons in 2018 (https://data.stats.gov.cn/easyquery.htm?cn=E0103). Jujube cv. Muzao is one of the most widely cultivated jujube varieties in China, combining the characteristics of high yield and high quality [2]. Due to the wide cultivation area of the Muzao tree crop (Ziziphus jujube Mill.), there are many varieties and geographical types of jujube, varying in terms of fruit shape, single fruit weight, drying rate, nutrient composition and fruit characteristics [3]. 'Linhuang No. 1' is a dry-jujube cultivar selected from 'Muzao', which has fruit characteristics similar to those of 'Muzao'. Compared with 'Muzao', 'Linhuang No. 1' shows greater resistance to fruit cracking [4]. 'Linhuang No. 1' has become a valuable genotype for scienti c research.
Whole-genome resequencing is the process of sequencing the genomes of different individuals of a species where the genome has already been sequenced [5]. Through sequence alignment, this methodology is able to identify large numbers of single nucleotide polymorphisms (SNPs), insertion/deletion (indel) variants, structural gene variants (SV), copy number variants (CNV) and other mutations in the wholegenome re-sequenced individuals. This information on genetic variation is an important way to understand the genetic background of a species and to study its evolution. In order to explore the wide phenotypic diversity of sweet cherry varieties with respect to important agronomic traits, such as owering time and defense response to pathogens, Aliki et al. (2020) re-sequenced 21 cherry accessions and found that the majority of high-impact SNPs (e.g., addition of stop codon, frameshifts) were identi ed in genes involved in owering time, dormancy and defense reactions against pathogens [6]. Yu et al. (2018) re-sequenced the genomes of 58 peach cultivars and closely related species to explore the origin and evolutionary history of peaches [7]. The results showed that peaches originated on the Qinghai-Tibet Plateau in Southwest China and had been subjected to frugivore-mediated selection to drive the evolution of fruit traits. The publication of the 'Dongzao' genome sequence provided valuable help for genomic and transcriptomic studies of other jujube cultivars [8]. The genome sequence of jujube cv. Dongzao represents a valuable resource not only for biological discovery and crop improvement but also for evolutionary and comparative genomic analysis. Huang et al. (2016) re-sequenced the genomes of 31 cultivated and wild jujubes to reveal the domestication process of jujube and screened key genes for metabolic effects associated with sweet or sour fruit [9].
In this article, we present analyses of whole-genome re-sequencing and transcriptome of 'Linhuang No. 1' and 'Muzao'. The sequence analysis focused on genomic regions associated with propitious variation, such as single nucleotide polymorphisms (SNPs) and insertions/deletions (InDels). The analysis focused on genes differentially expressed during the full-red period of fruit development. The combination of whole-genome re-sequencing and transcriptome analysis would allow the screening out of differently expressed genes with different gene structures between 'Linhuang No. 1' and 'Muzao', which could be helpful in explaining divergence/similarity of cracking resistance between the two cultivars.

Results
Discovery of SNPs and InDels in 'Linhuang No.1' and 'Muzao' A total of 110 million raw reads were generated for each of 'Linhuang No. 1' and 'Muzao'. After removal of low-quality reads, about 95.93% ('Linhuang No. 1') and 94.74% ('Muzao') of the reads were retained as clean data and used for further investigation. The Q20 percentage (proportion of nucleotides with a quality value greater than 20 in the reads) was greater than 95% and the Q30 percentage was greater than 89%. The mean GC percentage of the clean reads was 33%. The high-quality reads were further mapped onto the reference genome (Ziziphus jujube 1.1), using BWA software. Overall, almost 94% of these reads were uniquely mapped, and covered about 46.5% of the reference genome, with at least 30 × coverage depth (Supplementary materials 1).
Additional le 1: . For all chromosomes, indel polymorphisms were less frequent than SNPs. From the comparison between 'Muzao' and the reference genome, the highest and lowest number of SNPs and indels were also detected on Chromosomes 1 and 12, respectively. From the comparison between 'Linhuang No. 1' and 'Muzao', the highest and lowest numbers of SNP and indel polymorphisms were also found on Chromosomes 1 and 12, respectively. were mutated by SNP or indel, and 19 genes were differentially expressed between the two cultivars ( Fig. 3.A).
We performed cluster analysis on the 19 DEGs ( Fig. 3 Fig. 1). Domain prediction was performed on the protein sequence, and it was found that 'Linhuang No. 1' and 'Muzao' base insertion did not occur in the domain region ( Supplementary Fig. 2). Real-time PCR was used to verify the expression level of the LOC107427052 gene (Fig. 5). The results showed that the LOC107427052 gene expression levels of 'Muzao' were signi cantly higher than

Discussion
The Chinese jujube tree was domesticated from the wild jujube (Z. jujuba Mill. var. spinosa Hu.) [10]. Cultivated jujube trees and wild jujube showed different characteristics, such as tree vs. shrub habit, sparsely thorned vs. heavily thorned, and large vs. small fruits, respectively, through arti cial selection for important agronomic characters[8, 11,12]. Most cultivated jujube varieties produce relatively fewer seeds due to self-incompatibility or cross-incompatibility. Jujube trees can only be bred through seed progeny selection, selection by bud mutation or molecular breeding [13]. These genetic methods resulted in the high heterozygosity, high repeat sequence density and low GC content of the jujube genome [13]. Therefore, sequencing of the jujube genome was very di cult. analyses were combined to screen for differential expression of 'Linhuang No. 1' and 'Muzao' genes with respect to gene structural mutations.
A total of 19 mutant genes were screened for differential expression. There were three signi cantly enriched pathways in 'Linhuang No. In the process of nitrate assimilation in plants, nitrite reductase (NiR) is coupled with nitrate reductase (NR) to complete the inorganic assimilation of nitrate. Nitrite reductase can catalytically reduce nitrate to ammonium [23]. In fact, the reduction reaction rate of nitrate must be strictly regulated. It is necessary to ensure that nitrite and ammonium will not be excessive to avoid plant poisoning, and the supply of ammonium must be ensured. In this process, NiR plays a role in connecting the past and the future. Sivasankar et al. found that the nitrate inducibility is a gene located between the upstream 230 and 180 positions, and the downstream 1 to 67 positions are very important for the minimum induction of nitrate [24]. Ozawa and Kawahigashi (2005) cloned the NiR gene of rice 'Konansou' and overexpressed the gene in a commercial rice variety, Koshihikari [25]. The results showed that, compared with the wild type 'Koshihikari', the introduced exogenous NiR gene made the plant grow better, while callus regeneration ability was also signi cantly improved. Genome re-sequencing Gene cloning and protein sequence alignment The target gene which encoding nitrite reductase (NiR) was ampli ed by cDNA from the pericarp of 'Linhuang No. 1' and 'Muzao' fruits. The primers used were forward primer ATGTCATCGTTCTCTGTTCGGTTT and reverse primer TCAAAACGGGTGTTTCCCTCGA. The ampli ed products were electrophoresed on 1% agarose gel and photographed with a gel imaging analysis system. The PCR products were recovered and puri ed according to the operation steps described in the Gel Recovery Kit (Takara, Japan     LOC107427052 mutation