Establishment of Shatian pomelo×P. trifoliata hybrid population and InDel marker identification

Abstract: 【Objective】Hybrid breeding is the main way to breed new citrus varieties, and excellent rootstock breeding is also an important measure to improve the yield and quality of citrus. Pomelo has been developed steadily in China, and the main cultivars are Shatian pomelo and Guanxi pomelo. Due to the nonembryonic characteristics of pomelo, natural interspecific hybridization and seedling variation are easy to occur under seedling raising conditions. After thousands of years of genetic evolution, China’s pomelo germplasm resources have formed a large population, and the population distribution is rich and colorful. The main cultivars of pomelo in China mainly use Poncirus trifoliata and sour pomelo as rootstocks. However, P. trifoliata as a rootstock is not suitable for all pomelo varieties, and the cold resistance of sour pomelo as a rootstock is not as good as P. trifoliata. At the same time, because the tree grafted on sour pomelo is too tall, it is not convenient to manage and harvest. Breeding rootstock with advantages of both rootstocks has important practical value. Shatian pomelo has many seeds with single embryo. Therefore, it is easy to obtain hybrid offspring by conventional sexual hybridization. In this study, a hybridization was made between Shatian pomelo and P. trifoliata for screening rootstock with excellent characteristics of P. trifoliata, such as dwarfing, strong stress resistance, and strong affinity with pomelo in China.【Methods】Five 15- year- old adult Shatian pomelo trees in Yangshuo, Guilin, Guangxi were used as female parent, and Dongkou P. trifoliata in Shaoyang, Hunan was used asmale parent (the P. trifoliata superior line screened in previous study). The hybridization was carried out in Yangshuo, Guangxi from 2018 to 2021. The seeds were compared with the seeds of P. trifoliata and Shatian pomelo (Shatian pomelo × sour pomelo offspring). The seeds were divided into two types: fertile and abortive. The leaves of the offspring were also compared with those of P. trifoliata and Shatian pomelo (Shatian pomelo × sour pomelo offspring), and the leaves were classified to triple compound leaves and single compound leaves. Finally, the genomic DNA of progeny leaves was extracted by modified CTAB method, and the F1 hybrids were identified by InDel molecular markers.【Results】 A total of 274 fruits were collected from the 5 Shatian pomelo trees, and the fruit setting rate was 3.08%. The seed germination rate of hybrid offspring was not significantly different from that of parents. The number of single artificially pollinated fruit seeds was 123 on average, larger and rounder than those of female parent without artificial pollination. The ratio of the plump seeds and abortive seeds of the artificially pollinated fruit was 1∶2.64; the leaves of the hybrid progeny were significantly separated at the seedling stage, and the leaves of the 551 hybrid populations bred from 28 artificially pollinated fruits were all triple compound. The perspective of triple compound leaf morphology was similar to that of the male parent P. trifoliata, but the leaves were larger and slightly longer than those of P. trifoliata. The leaves of 1279 hybrid individuals generated from 42 artificially pollinated fruits were single compound. The leaf phenotypes of the two lines of the triple compound leaves and single compound leaves were stable during the growth and development process. Only a small number of leaves of the triple compound leaves showed occasional variation (four compound leaves and five compound leaves) at the seedling stage. Four pairs of InDel primers from P. trifoliata were screened with different germplasms such as mandarin orange, ponkan, sweet orange, kumquat, citron, P. trifoliata and pomelo. Among them, PT-P13 could not distinguish P. trifoliata and pomelo, PT-P38 could not amplify specific bands of pomelo, PT- P51 could distinguish P. trifoliata and pomelo, but could not distinguish P. trifoliata and mandarin orange, PT-P54 could distinguish P. trifoliata and pomelo, as well as P. trifoliata and citrus germplasm. Therefore, PT- P54 was used to identify the hybrid progeny population. Among them, all plants with triple compound leaves amplified significant differential bands, and some plants with single compound leaves amplified significant differential bands. A total of 698 hybrid seedlings were identified, and the hybrid rate was 38.14%.【Conclusion】Although the hybrid offspring of Shatian pomelo × P. trifoliata could be distinguished by the dominant genetic trait of the triple compound leaves of P. trifoliata, morphological identification has certain limitations. Some trees with single compound leaves might be possible to be sexual hybrid offspring, so it is necessary to combine InDel molecular markers for accurate identification of the true hybrids. In this experiment, a pair of InDel marker primers suitable for detecting the hybrids of Shatian pomelo × P. trifoliata were screened out, which could distinguish other citrus germplasm as well.