Application of palynology and InDel markers in germplasm identification in Fengtangli plum

Abstract: 【Objective】Fengtangli, a superior Chinese plum variety that was bred from the local landraces in Zhenning county of Anshun City, is renowned for its crisp and tender texture. In recent years, the increasing market demand for Fengtangli seedlings has resulted in a proliferation of inferior and counterfeit seedlings being marketed as high- quality products. On the market, the visually similar Siyueli has been seen falsely sold as Fengtangli, consequently disrupting the market equilibrium. Additionally, distinct lines within the Fengtangli variety have been identified, exhibiting many variations such as yellow-skinned, green-skinned, and partially red-skinned fruits upon maturation. These phenotypic variations not only affect the fruit quality but also the required cultivation and management practices. The presence of mixed lines complicates standardized orchard management, harvest scheduling and fruit grading processes, negatively impacting the overall economic yield of the orchards. Hence, it is impera-tive to accurately identify and differentiate the intrinsic variations within Fengtangli as well as between Fengtangli and other plum germplasms to advance standardization and augment the quality control in the Fengtangli industry.【Methods】Flower buds were collected from 5- year- old fruit trees in the orchard at the stage of flower bud swelling to white bell, and pollen was collected after air-drying under natural conditions. Gold-spray treatment was performed using an ion sputtering instrument under vacuum, followed by observation of pollen micro-morphology under a scanning electron microscope. Young leaves from the plum germplasms were sampled for DNA extraction via an optimized CTAB method. Targeted InDel loci were identified from resequencing data using bioinformatics tools, and primers for these loci were designed. Amplification via PCR and validation of the InDel markers were performed, with the confirmation conducted through the agarose gel electrophoresis.【Results】This study conducted a systematic analysis of the pollen micro-morphology of Fengtangli and other Chinese plum germplasms. The analysis revealed that the pollen of the materials tested exhibited isopolar and radially symmetric forms with three colporate apertures, conforming to the classification of N3P4C5 type pollen. In view of the polar point, the pollen presented a trifid circula shape, and in view of the equatorial point, it showed an elongate ellipse, reflecting a degree of morphological uniformity. However, significant differences were observed in the polar axis length and equatorial axis length among the germplasms, especially between the Siyueli and Sanyueli compared to the Fengtangli. Within the three Fengtangli lines, the partially red-skinned line had a slightly longer equatorial axis length than the yellow-skinned and greenskinned lines. Moreover, the pollen exine ornamentation of all tested germplasms displayed a“striateperforate”type, but there were significant differences in the direction of the striations, density and porosity. Among the three Fengtangli lines, differences in pollen were primarily reflected in the characteristics of the exine ornamentation. The ridge width was relatively larger in the yellow-skinned line, while the green- skinned and partially red- skinned line had similar ridge numbers, with minor differences in the distance between ridges, particularly in the partially red-skinned line, which had significantly smaller aperture and porosity than the other two lines. Cluster analysis, based on multiple morphological indexes of pollen, revealed the similarities and relatedness among the tested plum germplasms. The clustering results indicated that the plum germplasms were effectively grouped according to pollen characteristics at a certain Euclidean distance criterion, with the Siyueli forming a unique group due to its distinctive pollen morphology. Further analysis showed that the three lines of Fengtangli and several other plum germplasms exhibited high similarity in pollen morphology. These findings provided significant palynological evidence for understanding the classification and relatedness of Fengtangli and related plum germplasms. In the aspect of InDel molecular markers, based on predicted InDel sites from resequencing data, two pairs of primers successfully amplified the strips of target DNA in 12 plum germplasms. These results showed consistency across multiple PCR assays, demonstrating the effectiveness of these primer combinations and providing a molecular basis for distinguishing Fengtangli from other 21 representative plum germplasms. Concurrently, the primer P3-38 produced specific bands through PCR amplification in the three main lines of Fengtangli, effectively distinguishing them.【Conclusion】 Palynological analysis revealed that the distinctions among different species within the prunus and three main lines of Fengtangli were primarily concentrated on exine ornamentation. Based on pollen appearance and exine ornamentation data, the cluster analysis indicated significant differences between Fengtangli and both Sanyueli and Siyueli, suggesting that their relatedness may be relatively distant. Thus, palynology can serve as an auxiliary method for distinguishing them. Although palynological results can express a broader range of genetic information, but as a method for variety identification, they pos-sess certain limitations. Meanwhile, the InDel molecular markers developed by resequencing were highly effective for identifying three main lines of Fengtangli as well as its differentiation from other Chinese plum germplasms, compensating the limitation inherent in palynology.