Creation of triploid seedling plants of Citrus by crossing Dongshizao pummelo female with tetraploid male parents

Abstract: 【Objective】Citrus is mainly cultivated for fresh consumption in China, so seedless citrus has become the dominant position in market consumption and in breeding programs. In order to enhance the competitiveness of the citrus industry and meet the market demands, it has been the goal of breeders to cultivate seedless varieties of citrus. Triploids are naturally seedless material whose chromosomes are disrupted during meiosis, making it difficult to form normal fertile gametes, resulting in seedless fruits. Triploid citrus typically has larger fruits and possesses stronger resistance and adaptation to the environmental conditions due to chromosome doubling. Ploidy crosses is the most effective strategy to obtain seedless citrus varieties, using monoembryonic diploid and tetraploid ploidy as parents. Therefore, we performed several 2x×4x crosses to create triploid citrus.【Methods】In this study, we selected the Dongshizao pummelo [C. grandis (L.) Osbeck Dongshizao pummelo] as the female parent, which is a local specialty cultivar from Yunnan province, characterized by early-maturing, seedlessness, high sugar and low acidity. Then, we used a late-maturing and productive autotetraploid ZP [C. sinensis (L.) Osbeck Paperrind orange], PT [C. paradisi (L.) Osbeck grapefruit] and an allotetraploid somatic hybrids NH [(C. reticulata Blanco×C. paradisi Macf.) + C. grandis (L.) Osbeck Hirado Buntan pummelo], asthe male parent for the artificial crosses. In the earlier step, the pollinated styles were stained with aniline blue to observe the cross-compatibility between the parents, and in the subsequent steps, the immature seeds obtained from young fruits at 85 and 100 days after pollination (DAP) were cultured in vitro. When seeds grew into seedlings, the ploidy levels were analyzed by flow cytometry. In addition, we also resequenced the maternal Dongshizao pummelo, then the data was mapped to the HWB [Citrus grandis (L.) Osbeck Wanbai pummelo] reference genome to obtain InDel (insertion/deletion) variant loci, which contained 50-200 bp differences. According to these loci, primers were designed upstream and downstream of them, and PCR amplification was performed using the DNA of Dongshizao pummelo, ZP, PT and NH as templates. The amplified products were detected using 2.5% agarose gel, and clear and stable InDel-specific band patterns were selected for the identification of the genetic origin of the polyploid regenerated plants.【Results】The aniline blue staining results of the pollinated styles showed that a large number of pollen tubes could grow down to the bottom of the styles, and showed crosscomptibility in crosses of Dongshizao pummelo × ZP, Dongshizao pummelo × PT and Dongshizao pummelo × NH. Due to the 1∶2 (2x×4x) ratio of maternal and paternal genomes in the endosperm of the progeny, the seeds would be completely sterile in mature fruit. Therefore, the juvenile embryos were cultured in vitro before the seeds were sterilized. At 85 DAP, 771 immature seeds obtained from the young fruits of Dongshizao pummelo × NH cross were cultured in vitro. After shooting and rooting induction, 15 plants were regenerated. Similarily, when 570 and 482 immature seeds of Dongshizao pummelo × PT and Dongshizao pummelo × ZP crosses were rescued at 100 DAP, we obtained 96 and 117 regenerated plants respectively. The regeneration rates of 3 crosses were different, of which the two autotetraploids about 20% were 10 times higher than the allotetraploid (1.9%). We also checked the ploidy levels of the hybrid progenies, 168 out of 228 progeny obtained from the crosses between Dongshizao pummelo and three tetraploid citrus cultivars. In the Dongshizao pummelo × ZP cross, 94 plants were regenerated, in which 60 plants proved to be triploids by flow cytometry analysis, accounting for 63.8%. In the Dongshizao pummelo × PT cross, 1 tetraploid and 60 triploids were detected in 63 regenerated plants, accounting for 95.2% and 1.67% respectively. Similarily, eight plants were detected as triploids in 11 seedlings from the Dongshizao pummelo × NH cross, accounting for 72.7% . In this study, we also developed a group of specific InDel markers for the genetic identification of the polyploid seedlings. Using HWB genome as the reference genome, we developed 10 pairs of InDel primers for hybrid progeny screening using the resequence deta of Dongshizao pummelo, grapefruit and sweet orange, which were called after InDel01-InDel10 respectively. InDel05 and InDel10 could distinguish Dongshizao pummelo from ZP and PT but not NH; InDel07 and InDel08 could distinguish all the parental species, but some of their band patterns were not obvious. Accordingly, the InDel05 primers were selected for the identification of Dongshizao pummelo × PT and Dongshizao pummelo × ZP hybrid progeny, and the InDel07 primers were selected for the identification of Dongshizao pummelo × NH hybrid progeny. The results showed that all the 129 polyploids derived form the crosses were hybrids of both parents.【Conclusion】Using the local specialty cultivar Dongshizao pummelo of Yunnan as the female parent and two autotetraploids (ZP, PT) and one allotetraploid somatic hybrids (NH) as the male parents, three crosses of 128 citrus triploids and one tetraploid were created in a relatively short time after cross-pollination. Our work would lay a foundation for the selection and breeding of new seedless varieties and related molecular research in citrus.