Genetic identification of Changshan Huyou based on whole genome information

Abstract: 【Objective】Changshan Huyou is an excellent local citrus resource in China, but its genetic background is always controversial. The study aimed to explore the genetic background of Changshan Huyou.【Methods】Changshan Huyou was used as the experimental material. Other tested materials included typical citrus species resources and local citrus resources in Zhejiang, such as mandarin orange, pummelo and sour orange. Firstly, the morphological characteristics of leaves and fruits of Changshan Huyou were compared with mandarin orange, pummelo, sour orange and grapefruit. Secondly, the fruit quality of different citrus resources was determined, the transverse and longitudinal diameter of fruit was measured by vernier caliper, and the fruit shape index was calculated, the weights of single fruit, peel, residue and seed were measured by electronic balance, and the edible rate was calculated, the soluble solids and titratable acids were measured by hand-held sugar acid instrument, and the solid acid ratio of fruit was calculated, the content of ascorbic acid was determined by 2,6-dichlorophenol indophenol reagent. The differences in appearance quality and internal quality of the various resources were analyzed. The genome of Changshan Huyou was resequenced, and the sweet orange was used as the reference genome. Changshan Huyou was compared with the sweet orange reference genome through gatk4.0 for SNP-calling, and finally the SNP data of Changshan Huyou, mandarin, sour orange, pummelo and grapefruit were obtained. The SNP information in each window was analyzed, so as to judge the genetic source of each window and obtain the genetic component map of their whole genome. Based on the genomic difference information of mandarin orange, pummelo, Changshan Huyou, sour orange and grapefruit, specific InDel molecular markers were developed, and Changshan Huyou was further identified by the molecular markers.【Results】The morphological analysis of leaves and fruits showed that there were great differences in leaf types of different citrus resources. The leaf morphology of Changshan Huyou was similar to that of sour orange, close to oval or ovate, and its wing leaves were smaller, while the wing leaves of pummelo were larger. Changshan Huyou belongs to small and medium-sized fruit type, which is similar in size to sour orange. The peel is golden yellow, the flesh is light yellow, and the peel thickness is similar to that of sour orange. The leaf type index of Changshan Huyou was not significantly different from that of Zigui sour orange, but significantly different from those of Bendizao mandarin orange, StarRuby grapefruit and Siji pummelo. The fruit quality data showed that Changshan Huyou had no significant difference from sour orange in appearance quality indexes such as single fruit weight, peel thickness, Seed weight, and peel weight, but had significant differences from mandarin orange, grapefruit and pummelo. There were significant differences between Changshan Huyou and sour orange in internal quality indexes such as titratable acid and solid acid ratio. The average content of titratable acid was 0.98%, and the average solid acid ratio was 12.30. The resequencing data showed that more than 87.91% of the genetic regions of Changshan Huyou were hybrid origin between pummelo and mandarin orange, and 9.77% of the genetic regions were homozygous to pummelo. By comparing with the whole genome structure of other tested citrus materials, it was found that the genome structure of Changshan Huyou was mostly similar to that of sour orange, and the sequence similarity with Jiangjin sour orange was 88.34%. By clustering Changshan Huyou with other citrus resources, it was also found that Changshan Huyou was clustered with sour orange, indicating that Changshan huyou would a kind of sour orange. In addition, four pairs of InDel molecular markers were developed according to the different variant ectopic sites of the whole genome, and they could be used to identify the genetic differences among mandarin orange, pummelo, Changshan Huyou, sour orange and grapefruit. Through the verification of molecular markers, it was found that mandarin orange had a long band, pummelo and grapefruit had a short band, sour orange had two bands of pummelo and mandarin, the amplified band size of Changshan Huyou was consistent with the sour orange band, and they were the two bands of mandarin orange and pummelo, which was inconsistent with pummelo and grapefruit, it was further confirmed that Changshan Huyou had a close genetic relationship with sour orange.【Conclusion】Based on the whole genome resequencing data, this study speculated the genetic background of Changshan Huyou, and systematically compared the molecular markers, morphological characteristics and fruit quality data of Changshan Huyou with the citrus resources related to Changshan Huyou. We conclude that 87.91% of the genetic components of Changshan Huyou are heterozygous, the whole genome sequence of Changshan Huyou is highly similar to that of sour orange, and it is also clustered with sour orange, which indicates that Changshan Huyou has the genetic background of sour orange. This is also confirmed by InDel molecular markers. The leaf and fruit morphology of Changshan Huyou is similar to that of sour orange, but the fruit quality has been significantly improved, indicating that Changshan Huyou is a kind of sour orange with improved fruit quality obtained through long-term cultivation and domestication.