Production of potential seedless pummelo sexual progenies via sexual hybridization using male-sterile cybrid Huayou No. 2 as the female parent

Abstract:【Objective】Seedlessness is an important trait in citrus. Numerous pummelo cultivars in China contain seeds, with certain fruits containing over 150 seeds, reducing the edible portion of the fruit and consumer acceptance. Cytoplasmic male sterility (CMS), controlled by maternally inherited mitochondrial genome and nuclear genome, is one of the main reasons for seedlessness in citrus fruits, and it has been utilized to obtain seedless germplasm in citrus. For instance, the Satsuma mandarin is a typical example of the CMS type. In Japan, it was chosen as maternal parent in cross breeding to develop the superior breeding parent Kiyomi tangor, known for its monoembryony and male sterility. Followingthat, Kiyomi tangor was utilized as the female parent to produce various seedless citrus varieties, such as Shiranui, Harumi, Setoka and Harehime through cross breeding. Therefore, the strategy of utilizing citrus varieties with CMS as the maternal parent to cross with seedy varieties can effectively transfer the trait of CMS to the offspring. This approach enables the production of seedless citrus at the diploid level, greatly improving the efficiency of seedless citrus breeding. Huayou No. 2 pummelo is a new seedless citrus variety produced via protoplast fusion with Satsuma mandarin as callus parent and Hirado Buntan (HB) pummelo as leaf parent, with its mitochondrial genome originated from Satsuma mandarin while the nuclear genome and chloroplast genome inherited from the HB pummelo. Compared to its mesophyll parent HB pummelo, Huayou No. 2 pummelo displayed male sterility and seedlessness (under isolated cultivation), with all other traits staying consistent with HB pummelo, making it ideal for direct cultivation as a new variety. In addition, Huayou No. 2 pummelo carries the CMS cytoplasm from Satsuma mandarin and is a monoembryonic variety. Utilizing it as a female parent for hybridization with other seedy pummelo types holds significant practical value in generating seedless germplasm at the diploid level, as the CMS cytoplasm from Huayou No. 2 pummelo can be transferred to the offspring. With this strategy, in our previous breeding program, Huayou No. 2 pummelo was used as the female parent to cross with Shatian pummelo and Cocktail grapefruit, from which more than 1500 hybrids were obtained. Over 500 hybrids from the cross of Huayou No. 2 pummelo × Shatian pummelo have exhibited male sterility, providing evidence of the success achieved through this strategy.【Methods】Five local pummelo varieties including Gongshuibai, Juhuaxin, Leigong, Majia and Tianyou No. 1 were selected as materials in this study due to their seedy fruits, peel thickness and low edible rate. Five sexual crosses were conducted with Huayou No. 2 pummelo as the female parent and the above five seedy pummelos as the male parents during 2022 to 2023, aiming to produce hybrids that could yield seedless and high- quality fruits with a high edible rate. Controlled pollination was conducted in Huazhong Agricultural University. Following the mature fruits collected, the seeds were extracted and sowed in a growth chamber. After germination, putative polyploids were screened according to the morphological feature and then determined by flow cytometry. By DNA re-sequencing of the parents, InDel (insertion- deletion) markers were mined and used to identify the genetic origin of the plants derived from these crosses.【Results】From the five crosses with Huayou No. 2 pummelo as the female parent, and Gongshuibai, Leigong, Juhuaxin, Majia, and Tianyou No. 1 pummelos as the male parents, 65 mature fruits were obtained, from which 1163 mature seeds were extracted and sowed in the chamber. After germination, a total of 781 seedlings were obtained, including 221 seedlings from the cross of Huayou No. 2 × Gongshuibai pummelo, 238 seedlings from Huayou No. 2 × Leigong pummelo, 104 seedlings from Huayou No. 2 × Majia pummelo, 108 seedlings from Huayou No. 2 × Juhuaxin pummelo and 110 seedlings from Huayou No. 2 × Tianyou No. 1 pummelo, respectively. Based on the morphological trait screening, we identified two putative polyploids from the seedlings derived from the cross of Huayou No. 2 × Tianyou No. 1 pummelo and one of them was verified as tetraploid using flow cytometry. Based on DNA re-sequencing of Huayou No. 2, Gongshuibai pummelo and Leigong pummelo, six pairs of polymorphic InDel primers were mined and used for identifying the genetic origin of the offspring derived from Huayou No. 2 × Gongshuibai pummelo and Huayou No. 2 pummelo × Leigong pummelo. For each cross, 44 plants were randomly selected and the results showed that all the detected offspring displayed the bands only in their male relative parent, suggesting that all the offsprings of both crosses were the hybrids of their parents.【Conclusion】Using a high efficient cell engineering breeding technique in which Huayou No. 2 pummelo was used as the female parent to cross with seedypummelos to produce seedless germplasm at the diploid level, hundreds of diploid hybrid and one tetraploid seedlings for potential seedlessness were obtained, providing abundant materials for pummelo seedless breeding at the diploid level. This study also laid a foundation for providing materials for the researches related to male sterility.