Efficient exploration and SSR identification of autotetraploids from the seedlings of thirteen apomictic Citrus genotypes

Abstract：【Objective】Citrus is a crucial part of Chinese fruit crops. There are abundant citrus germplasm resources in China, but many excellent local varieties are gradually eliminated by the market due to the problem of numerous seeds within the fruit. The fruits of triploid plants are generally seedless because of their sterile male and female gametes. Therefore, triploid production is a promising strategy to breed seedless cultivars in citrus. Triploids can be obtained by interploidy crossing between diploids and tetraploids. However, the tetraploid germplasm is rare, which limits the application of this strategy. Exploration of tetraploids is an important prerequisite for triploid production with the aim to improve the seedy local cultivars in our country. For the rootstock improvement, tetraploid plants are also valuable resources because of their higher metabolite content, and better resistance than their diploid par-ents. In this study, we planned to explore tetraploid plants from 13 local cultivars in our country by using the traits of spontaneous doubling of the nucellar cells in polyembryonic citrus varieties. The exploration of tetraploids from the above 13 local cultivars will not only provide excellent tetraploid parents for the production of triploid plants, but also lay the foundation for the basic research about the effect of genome duplication on some important trait change, such as dwarfing, extensive adaptivity and higher medicinal value in tetraploids.【Methods】After the mature fruits were harvested, the seeds were extracted and the seed coats were peeled off, and then they were placed in a thermostat to accelerate germination. When the seeds germinated, they were sown in pots and cultivated in a plant growth chamber. After the seedlings grew up with three or more leaves, putative polyploids were screened according to the morphological feature showing lower height, shorter taproots, less lateral roots, thicker and rounder leaves and declined oil gland density. The ploidy levels of these putative polyploids were further confirmed by flow cytometric analysis and the observation on root tip chromosome numbers. After determination of the ploidy level, some morphological traits, including plant height, root length and diameter, lateral root number, stem diameter, leaf thickness and shape index of the tetraploids and their corresponding diploid parents were measured at the same developmental stage. SSR analysis was used to identify the genetic origin of the explored tetraploids with at least three pairs of SSR primers selected for each cultivar.【Results】The polyembryonic degree of seeds from each cultivar was firstly determined and it showed that the seeds of all 13 cultivars were polyembryonic. Among them, Qu tangerine had the highest number of embryos with an average of 9.4 embryos per seed and Bingtang sweet orange had the lowest number of embryos with an average of 2.2 embryos per seed. Based on the morphological trait screening, we identified 2, 1, 3, 2, 7, 3, 1, 3, 1, 3, 17, 1 and 2 putative polyploids respectively from 343, 499, 892, 385, 519, 290, 457, 241, 119, 690, 828, 114 and 129 seedlings of Qianshanhong tangerine, Bayue tangerine, Qu tangerine, Zao tangerine, Bianping tangerine, Ougan tangerine, Shitougan, Bingtang sweet orange, Jinmi sweet orange, Moping Xiangcheng, Japanese Xiangcheng, Zhique and Youpi kumquat. After further confirmation of ploidy levels concerning above putative tetraploids, we obtained 45 tetraploids and one hexaploid plant from Qu tangerine, with an average occurrence rate of 0.85%, among which the rate of Japanese xiangcheng was the highest with 2.05% and the rate of Bayue tangerine was the lowest with 0.20%. The exploration time from seed germination to obtaining tetraploid seedlings varied among cultivars, with the longest time (42 days) used in Youpi kumquat and the shortest time (23 days) in Shitougan. The morphological traits of tetraploids and their corresponding diploid seedlings from nine cultivars of Qianshanhong tangerine, Qu tangerine, Zao tangerine, Bianping tangerine, Ougan tangerine, Bingtang sweet orange, Moping Xiangcheng, Japanese Xiangcheng and Youpi kumquat were measured. For plant height, tap root length, lateral root numbers and leaf thickness, the tetraploid seedlings of seven cultivars showed significant differences with their diploid parents. For taproot and stem diameter, only the tetraploid seedlings explored from Bingtang sweet orange and Japanese Xiangcheng had significant difference with their diploid parents. For leaf shape index, the tetraploid seedings from Bianping tangerine and Moping Xiangcheng exhibited significant differences with their diploid seedlings. In conclusion, most tetraploid seedlings of all nine cultivars showed lower plant height, shorter and thicker taproot, less lateral root number, thicker and rounder leaves than those of their diploid parents. These results provide supports for the screening of putative tetraploids based on morphological trait observation. For analyzing the genetic origin of the tetraploids obtained in this study, at least three SSR markers were used in each genotype. The results showed that the bands of all 45 tetraploids were identical with those of their corresponding diploids, indicating that all the 45 tetra-ploids might originate from the spontaneous chromosome doubling of nucellar cells of their corresponding diploids. In addition, the bands of the hexaploid from Qu tangerine were also identical with their diploid parent. We speculated that it might derive from chromosome doubling of a triploid zygotic cell, which formed by selfing of a FDR-type 2n gamete with a normal n gamete, and both gametes were produced by Qu tangerine.【Conclusion】This study verified that morphological screening combined with flow cytometry ploidy determination and SSR analysis is an efficient approach to exploring polyploid seedlings from apomictic citrus. Using this method, 45 autotetraploid and one hexaploid plants were obtained from 13 apomictic citrus genotypes. These newly discovered tetraploids are potentially valuable for not only genetic improvement of some elite local citrus cultivars with seeds produced by triploids using interploidy hybridization, but also selection of the promising rootstocks with dwarf, multi-resistance and broad adaptability characteristics to improve the ability to resist various abiotic and biotic stresses.