Molecular Mechanisms Of Adaptive Differentiation And Ascorbic Acid Synthesis Of Hippophae Rhamnoides Subsp.sinensis And H. Rhamnoides Subsp. Mongolica

Hippophae Linn.is mainly distributed in Eurasia,and the Qinghai-Tibet Plateau and the Hengduan Mountains are the centers of origin and differentiation.H.rhamnoides subsp.sinensis and H.rhamnoides subsp.mongolica are the two most closely related subspecies of Hippophae Linn.H.rhamnoides subsp.sinensis is characterized by strong resistance,smaller fruit,more spines,and abundant secondary fruit metabolites such as ascorbic acid,flavonoids,and carotenoids,etc.It is mainly distributed in the Hengduan Mountains to the three northern regions of China.H.rhamnoides subsp.mongolica has weak resistance,larger fruit,less spines,and relatively low content of secondary metabolites in fruit,which are mainly distributed in the regions east of Altay in Xinjiang and west of Lake Baikal in Russia.Although there is a certain geographical isolation between the two,but no reproductive isolation between them.In the cultivation of new varieties,they are often used as hybrid parents to cultivate good varieties with large fruit,high yield,no thorns,and high content of secondary metabolites.So,what roles do natural selection and artificial domestication play in the adaptive differentiation of H.rhamnoides subsp.sinensis and H.rhamnoides subsp.mongolica?How is the content of ascorbic acid and other substances and the difference in fruit size formed?Based on these,this study used wild H.rhamnoides subsp.sinensis,wild H.rhamnoides subsp.mongolica,and cultivated H.rhamnoides subsp.mongolica populations as materials,based on the completed genomic data of seabuckthorn,adopted whole-genome resequencing,transcriptome sequencing and metabolome detection methods,and adopted relevant software for bioinformatics analysis,and the results were as follows:（1）A total of 797.39 Gbp of Clean Data were obtained from the whole genome resequencing of the three populations,in which the average proportion of Q20 and Q30reached more than 92.38%and 86.68%,respectively.The average comparison rate of each individual plant to the reference genome reached 95.37%,and the genome coverage was86.01%.The average sequencing depth was 8×,which indicated that the sequencing quality was high and could fully meet the needs of this study.（2）The wild H.rhamnoides subsp.sinensis population had the highest level of genetic diversity(π=1.45×10^-3),followed by wild H.rhamnoides subsp.mongolica population(π=1.34×10^-3),and the cultivated H.rhamnoides subsp.mongolica population had the lowest level of genetic diversity(π=1.02×10^-3).The detection of linkage disequilibrium showed that there was a strong linkage disequilibrium among the population loci of cultivated H.rhamnoides subsp.mongolica,and the wild H.rhamnoides subsp.mongolica was more strongly affected by natural selection than the wild H.rhamnoides subsp.sinensis.Between wild H.rhamnoides subsp.sinensis population and wild H.rhamnoides subsp.mongolica population,Chr2 was the chromosome with the most selected loci screened by F_STand XP＿CLR,indicating that the chromosome was the most strongly selected by natural selection.In wild H.rhamnoides subsp.mongolica and cultivated H.rhamnoides subsp.mongolica,Chr3was the chromosome with the most selected loci screened by F_STand XP＿CLR,indicating that the chromosome was the most strongly domesticated.Tajima’s D test showed that the recombination rate of cultivated H.rhamnoides subsp.mongolica population was the highest recombination rate.Under the influence of the Mid-Pleistocene glaciation climatic events and the Kunlun Mountains-Yellow River geological events,H.rhamnoides subsp.sinensis and H.rhamnoides subsp.mongolica began to differentiate about 0.8 million years ago,and formed two separate subspecies about 0.4 million years ago.Compared with the initial differentiation,the effective population size of H.rhamnoides subsp.sinensis increased to 13.44 times of H.rhamnoides subsp.mongolica,and the gene flow decreased to 2.06%.With time,the two species could be divided into two independent species.（3）Natural selection and artificial domestication in three populations have a different degree of adaptability.GO and KEGG analysis results show that the differences genes and metabolic pathways between wild H.rhamnoides subsp.sinensis population and wild H.rhamnoides subsp.mongolica population are not only in terms of regulating the economic characters,also widely occur light use and the ecological environment of cooperation,such as temperature and humidity,and so on.The differential genes and metabolic pathways between the populations of wild H.rhamnoides subsp.mongolica and cultivated H.rhamnoides subsp.mongolica were mainly related to the regulation of physiological activities and the synthesis of metabolites such as sugars,acids,vitamins,and carotenoids,which might be related to the selection of target traits in the early artificial breeding of sea buckthorn.（4）Both natural selection and artificial acclimation can lead to different degrees of differentiation of genes regulating economic traits such as ascorbic acid,flavonoids,carotenoids,fatty acids and fruit size,and artificial acclimation has a rapid and efficient effect.APX1 is an important gene that regulates ascorbic acid synthesis in fruit of sea buckthorn by adaptive selection.The F_STof APX1 is 2.85 between wild H.rhamnoides subsp.sinensis and wild H.rhamnoides subsp.mongolica,and 4.91 between wild H.rhamnoides subsp.mongolica and cultivated H.rhamnoides subsp.mongolica populations,indicating that the gene selected by the artificial selection is more intense.PER45 and AMO2 are important genes that regulate flavonoid biosynthesis in fruit of sea buckthorn by natural selection and artificial domestication selection,respectively.XTH23 and PSY are the key genes that regulate the content of carotenoids in highly differentiated sea-buckthorn populations in China,and PSY can regulate the synthesis of Phytoene in the carotenoid pathway of sea buckthorn fruits.BBE14 and PPCT were highly differentiated genes in wild H.rhamnoides subsp.sinensis population,while4CLL5 and LCAT2 were highly differentiated genes in cultivated H.rhamnoides subsp.mongolica population,indicating that the former was subject to natural selection while the latter was subject to artificial domestication.FW2.2 is a gene that is selected by both natural selection and artificial domestication and has negative regulation function on fruit size.（5）L-galactose pathway,recycling pathway,and D-galacturonic acid pathway are the main pathways of ascorbic acid synthesis in sea buckthorn fruit.Among H.rhamnoides subsp.sinensis and H.rhamnoides subsp.mongolica,the expression levels of GME3 and Gal LDH1 in L-galactose pathway that positively regulate ascorbic acid synthesis in H.rhamnoides subsp.sinensis fruits were significantly higher than those in H.rhamnoides subsp.mongolica fruits at three developmental stages,suggesting that these two genes could promote the accumulation of ascorbic acid in H.rhamnoides subsp.Sinensis fruits.In recycling pathway of promoting ascorbic acid degradation,APX1 genes expressed in H.rhamnoides subsp.mongolica fruits among three developmental stages were significantly higher than that of H.rhamnoides subsp.sinensis,and promote the synthesis of ascorbic acid again gene MDHAR4 fruit expression quantity at the last stage of development in H.rhamnoides subsp.sinensis continue to increase,but in H.rhamnoides subsp.mongolica fruits express continued to reduce the amount.The differential expression of several key genes in the above two cycles may be an important reason for the higher ascorbic acid content in the fruits of H.rhamnoides subsp.sinensis than from H.rhamnoides subsp.mongolica.（6）WGCNA analysis showed that Gal LDH,P4H9 and VTC2 were not only the key core genes in ascorbic acid synthesis,but also significantly correlated with the core genes regulating fruit cell size and number,suggesting that these genes may be closely related to the formation of fruit size of sea buckthorn.In addition,the core gene of positive regulation of ascorbic acid synthesis may be the gene of negative regulation of fruit size,that is,ascorbic acid content and fruit size may be a pair of antagonistic traits.In summary,this paper systematically studied the mechanism of adaptive differentiation three populations caused by natural selection and artificial domestication,and revealed that H.rhamnoides subsp.sinensis and H.rhamnoides subsp.mongolica differentiated into two subspecies about 0.4 million years ago due to climatic and geological influences.The key genes,such as APX1,PER45,XTH23,BBE14 and FW2.2,which regulate ascorbic acid content,flavonoids,carotenoids,fatty acids,and fruit size,were screened out.The different transcription levels of GME3,Gal LDH1,APX1 and MDHAR4 genes during the fruit development stage of H.rhamnoides subsp.sinensis and H.rhamnoides subsp.mongolica are the important reasons for the difference in ascorbic acid content between them.It is premised that ascorbic acid content and fruit size of sea buckthorn may be a pair of antagonistic traits,which provides theoretical support for guiding the molecular breeding of sea buckthorn.