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 artificial 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 difficult. Liu et al. (2014) published the genome of the first cultivar of the genus Ziziphus, ‘Dongzao’ (437.65 Mbp)[8]. The release of this genome sequence provided a rich resource of genetic information for the breeding of improved jujube accessions, as well as for the molecular improvement of other plants and fruit trees of the family Rhamnaceae.
Huang et al. (2016) reported the genome sequence of another jujube variety, ‘Junzao’, and re-sequenced the genomes of 31 wild jujube and jujube accessions with different geographical distributions[9]. The research of Huang and colleagues revealed the genomic mechanism underlying improvement of fruit sweetness and acidity during domestication and identified four genes related to acidic metabolism pathways, encoding NADP-dependent malic enzyme, pyruvate kinase, isocitrate dehydrogenase and aconitate hydratase, which play key roles in organic acid metabolism. This research provides valuable genomic information and material for jujube breeding. Guo et al. (2020) reported the re-sequencing of 350 jujube accessions[13]. Through a genome-wide association study (GWAS) and selective sweep analysis, variation in the genes involved in the regulation of seven domestication traits were screened for, including fruit shape, kernel size and fruiting branch length. This study provided rich genomic resources for revealing the genetic basis of the domestication and evolution of jujube.
In the current study, the varieties ‘Linhuang No. 1’ (resistant to cracking) and ‘Muzao’ (susceptible to cracking) were used as the research materials, with ‘Dongzao’ used as the reference genome for resequencing analysis. It was found that 4,404,985 and 4,401,491 polymorphic sites were obtained in ‘Linhuang No. 1’ and ‘Muzao’, respectively, whereas 664,129 variant sites were found between ‘Linhuang No. 1’ and ‘Muzao’. Principal component analysis on the characteristic variant sites between ‘Linhuang No. 1’, ‘Muzao’ and ‘Dongzao’ were performed and the results showed that both PC1 and PC2 could distinguish ‘Linhuang No. 1’ and ‘Muzao’ from ‘Dongzao’, indicating that ‘Linhuang No. 1’ and ‘Muzao’ were closer in relation to one another than to ‘Dongzao’.
Mutation is a key element in species evolution. The widespread base substitution and insertion/deletion mutations are important driving forces for genome evolution[14, 15]. Insertion and deletion mutations are more likely to trigger species evolution than are base mutations. The greater the evolutionary distance between species, the more bases are inserted or deleted, and the greater the length of the insertions or deletions[16–18]. Insertions and deletions are the main reasons for the divergence of closely related species. The insertion and deletion of bases can cause DNA sequence changes and cause DNA fragment length polymorphisms. They can even change the structure of genes through insertions or deletions in the exons and introns of the original gene, leading to the generation of new genes in the genome[19]. It was found, through re-sequencing, that there were 781,858 and 775,072 base insertions or deletions in ‘Linhuang No. 1’ and ‘Muzao’, respectively. Compared with ‘Linhuang No. 1’, there were 169,267 base insertions and deletions in ‘Muzao’. To a large extent, this led to the different characteristics between ‘Linhuang 1’and ‘Muzao’.
The transcriptome combines the genetic information of the genome with that of the proteome, with biological functions based on RNA, uses high-throughput sequencing technology (RNA-Seq) to sequence all cDNA libraries in tissues or cells and calculates the gene expression under different processing conditions (by counting the number of relevant reads). The jujube transcriptome has been widely used in research into response to heat stress, cold stress, salt stress and coloring of jujube fruits[20–22]. In the current study, 431 differentially expressed genes were identified by transcriptome analysis of ‘Linhuang No. 1’ and ‘Muzao’ pericarps. At the same time, the transcriptome and resequencing 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 significantly enriched pathways in ‘Linhuang No. 1’ and ‘Muzao’: stilbenoid, diarylheptanoid and gingerol biosynthesis (Rich factor: 14.98), flavonoid biosynthesis (Rich Factor: 8.85) and nitrogen metabolism (Rich factor: 19.48). The gene LOC107427052, encoding nitrite reductase, was screened for further study.
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 significantly improved.
Compared with the reference genome ‘Dongzao’, ‘Linhuang No. 1’ had an insert of 106 bases at the position 12517784 (CDS region) on chromosome NC_029681.1, whereas ‘Muzao’ had an insert of 104 bases at the same position; fragment insertion was verified by cloning. Insertion/deletion mutations are rarer than base substitutions in the evolution of organisms. If the sequence of the coding region has an indel mutation involving a base sequence which is not a multiple of three bases, it will cause serious consequences as a result of a frameshift mutation, which places the mutant under greater pressure in natural selection. In the present study, the domain of the protein encoded by the cloned NiR sequence was predicted, and it was found that the base insertion did not occur in the domain region. The expression levels of the LOC107427052 genes of ‘Linhuang No. 1’ and ‘Muzao’ were consistent with the changes measured in nitrite reductase activity during fruit development. However, the LOC107427052 gene expression level of ‘Muzao’ was significantly higher than that of ‘Linhuang No. 1’ during fruit development. The nitrite reductase activities and the nitrite content of ‘Muzao’ were significantly higher than those of ‘Linhuang No. 1’ at young fruit stage. But there was no significant difference in the product ammonia of nitrite reductase between the two varieties.