InDel marker- assisted selection of nucellar seedlings in polyembryonic Fortunella hindsii

【Objective】Citrus is a highly heterozygous genome apomixis species, and the polyembryonic seeds frequently contain a certain proportion of sexual offspring(also called zygotic embryos) that exhibit genetic background differences compared to their parents due to genetic recombination. Screening of asexual offspring (nucellar embryos) from the polyembryonic seedlings for genetic transformation experiments could effectively ensure the consistency of the genetic background of explant materials and the scientific reliability of follow-up studies. Based on this, we have developed Insertion-Deletion (InDel) molecular markers for rapid and accurate screening of asexual clonal plants in the offspring of Fortunella hindsii, a short juvenility germplasm of citrus, in order to provide a basis for optimizing and improving the genetic transformation system of F. hindsii.【Methods】The polyembryonic wild kumquat DB02 was used as the maternal parent and the progeny plants produced from DB02 seeds were used as identification materials. The seeds were sown under two conditions, one was in substrate soil and the other was in sterile test tube. Genomic DNA was extracted from the mature leaves of kumquat DB02 and its progeny, and the DNA extraction solution of qualified quality was diluted to the working concentration (about 100 ng · μL-1 ). The re-sequencing data of the maternal DB02 was aligned to the kumquatreference genome, the InDel variant sites were extracted and the (0/1) heterozygous type sites with a difference about 50bp were screened out. The primers were designed within 600 bp around the upstream and downstream of the InDel variant site. PCR amplification products were detected by 3% agarose gel, the markers with clear and stable hybrid bands were selected. All the InDel marker primers were used to amplify the progeny of kumquat DB02 line, and the progeny with only one band was sexual seedlings. The amplification products of the InDel marker primers of the nucellar embryos were all consistent with the maternal parent.【Results】A total of seven pairs of selected InDel markers were used to amplify the maternal parent DB02 kumquat. After detection by agarose gel electrophoresis, all markers contained clearly heterozygous bands in the maternal parent DNA, denoted as“Aa”, which can be used to screen out zygotic embryos and nucellar embryos in subsequent progeny materials. On the one hand, the DB02 progeny seeds were sown in the soil, and a total of 56 seedlings were counted after germination. These seedlings were then numbered. According to the identification results of the amplified products of 7 pairs of InDel markers, it was found that in the InDel01 marker electrophoresis results, the descendants number of 34, 41 and 56 were singly banded, and these plants were identified as zygotic seedlings. Similarly, 1, 3, 23, 27, and 41 were identified as zygotic seedlings by the InDel02 marker. 1, 3 and 27 were identified as zygotic seedlings by InDel05 labeling results. 1, 3, 23, 27, 41 and 56 were identified as zygotic seedlings by InDel06 and InDel07 labeling results. Based on the results of all markers, it can be concluded that a total of 7 zygotic seedlings (1, 3, 23, 27, 34, 41 and 56) were identified in the 56 DB02 progeny, and the remaining 48 nucellar seedlings were consistent with the maternal parent, the rate of nucellar embryos is 87.5%. On the other hand, the epicotyl material required in the genetic transformation generally comes from sterile test tube. To investigate whether the nucellar embryos ratio of kumquat DB02 is the same as that of substrate seeding, the DB02 seeds were sown in sterile test tube. A total of 62 seedlings after germination were counted and numbered. According to the identification results of 7 pairs of InDel-markers, a total of 2, 3 and other 16 zygotic seedlings were identified in the 62 DB02 progeny, the remaining 46 seedlings are nucellar seedlings, and nucellar embryo rate is 74.2%.【Conclusion】Nucellar poly-embryony is of great significance for the evolution, reproduction and breeding of citrus, and the asexual offspring developed from the nucellar embryos are genetically identical to the parents, so in citrus study, the epicotyl of polyembryonic seedlings are frequently used for genetic transformation. In this study, 7 pairs of InDel markers were selected from resequencing data of the wild kumquat DB02, which can accurately screen out the asexual offspring. Meanwhile, the nucellar seedlings rate under different growth conditions was also statistical, and it was found that the nucellar seedlings rate of kumquat DB02 under substrate conditions was about 87.5% , which was at a moderate level compared with other citrus varieties, while the nucellar seedlings rate under sterile medium conditions was 74.2%. This may be due to the fact that under sterile test tube conditions, some stunted zygotic embryos have better culture conditions and increased regeneration opportunities, while under soil sowing conditions, stunted zygotic embryos are often difficult to grow, which in turn leads to an increase in the proportion of zygote seedlings in sterile conditions. However, in general, the vast majority offspring of kumquat DB02 are asexual offspring, which can be used as an alternative material for genetic transformation of F. hindsii, and this study further improves the Agrobacterium-mediated epicotyl genetic transformation system in kumquats