Efficient exploration and SSR identification of 53 doubled diploid seedlings from six local Citrus cultivars and germplasm resources

Abstract: 【Objective】To improve the quality of citrus seedy scion cultivars and the stress tolerance of citrus rootstocks, we explored and identified citrus doubled diploid seedlings from six local citrus culti-vars and germplasm resources based on the presence of spontaneous doubling of nucellar cells in poly-embryonic seeds. Citrus triploids are usually seedless and can be produced from the hybridization be-tween diploid and tetraploid parents. However, the lack of citrus tetraploid germplasm resources has greatly limited the application of ploidy hybridization in citrus triploid improvement. And it has been extensively reported that tetraploids are more resistant and adaptable than diploids in higher plants. Therefore, it is urgent to explore tetraploid germplasm for the genetic improvement of local citrus culti-vars.【Methods】Following the harvest of the citrus mature fruits, the seeds were extracted with seed coat stripped and placed in a thermostat to accelerate germination. When the seeds germinated, they were sown in pots and cultured in a controlled growth chamber. After the seedlings grew with 2- 3leaves, putative tetraploids were visually screened based on the morphology of their root, leaf and oil gland. The putative tetraploids usually have thicker and shorter taproots, fewer lateral root, smaller leaf index and decreased oil gland density. Their ploidy level was determined using flow cytometry and shoot tip chromosome counting. In addition, the morphological indicators of tetraploids and corresponding diploid seedlings were measured at the same developmental period (about 30-50 d after sowing), in-cluding root length, root diameter, number of lateral roots, stem diameter, leaf length, leaf width and leaf thickness. The genetic origin of tetraploids obtained in this study were further analyzed using 11 pairs of simple sequence repeat (SSR) primers.【Results】Changshan Huyou pummelo, Wenling Gao- cheng, Xinhui sweet orange, a hybrid of blood orange with Clementine, Quzhou Xiangcheng and Sour orange respectively contains 1.4, 3.4, 8.0, 7.1, 5.6 and 5.8 embryos per seed, which indicated that these six genotypes were polyembryonic and different genotypes had variable number of embryos. Based on the morphological observations of root, leaf and oil gland of seedlings, we screened 24, 4, 7, 11, 10 and 88 putative tetraploids respectively from the 890, 72, 373, 709, 303 and 1992 seedlings of Changshan Huyou pummelo, Wenling Gaocheng, Xinhui sweet orange, the hybrid of blood orange with Clemen- tine, Quzhou Xiangcheng and Sour orange. A peak at 50 of elative content of DNA was observed in the plants of the diploid, and a peak at 100 was observed in the plants of the tetraploid by flow cytometry. The number of diploid and tetraploid by shoot tip chromosome counting was 18 and 36 respectively. By analyzing the ploidy level of putative tetraploids, a total of 53 tetraploids were obtained,14 from Chang-shan Huyou pummelo, 2 from Wenling Gaocheng, 4 from Xinhui sweet orange, 3 from a hybrid of blood orange with Clementine, 6 from Quzhou Xiangcheng and 24 from Sour orange, with the tetra- ploid pre-screening accuracy rate of 58.3%, 50.0%, 57.1%, 27.3%, 60.0% and 27.3% respectively. The natural occurrence rate of the tetraploid was 1.57%, 2.78%, 1.07%, 0.42%, 1.98% and 1.20% respective-ly in the above six genotypes. The morphological indicators of tetraploid and corresponding diploid seedlings were measured. The results showed that the taproot length and lateral root number of tetra- ploid seedlings from all genotypes were significantly lower than those of the corresponding diploid plants; root diameter and leaf thickness were significantly higher. For stem dimeter, the tetraploid seed- lings of Wenling Gaocheng and Xinhui sweet orange were significantly smaller than that of diploid plants, the remaining four cultivars had no significant difference. For the leaf shape index, the tetraploid seedlings of Wenling Gaocheng were significantly larger than that of diploid plants, while the tetraploid seedlings of the hybrid of blood orange with Clementine were significantly smaller than that of diploid plants, the remaining four cultivars had no significant difference. These results provided theoretical sup-port for improving the tetraploid pre-screening accuracy rate. Among the 11 polymorphic SSR markers,at least three SSR markers were successfully used for analyzing the genetic origins of tetraploid seed-lings for each genotype. The results showed that the bands of 51 tetraploid seedlings were identical with that of their corresponding diploid genotypes, indicating that all of these 51 seedlings were originated from the chromosome doubling of nucellar cells of their corresponding diploids. And the remaining two tetraploids from Changshan Huyou pummelo showed some bands different from their corresponding diploid, indicating they might be originated from sexual hybridization with unknown pollen parent. 【Conclusion】This study indicates that it is a simple and efficient approach to explore tetraploids from seedling populations of polyembryonic citrus genotypes based on preliminary morphological screening combined with flow cytometry and SSR analysis. These tetraploid germplasm resources explored from the study would be potentially valuable for scion and rootstock improvement and related fundamental researches in citrus crops.