Diversity and DNA fingerprinting construction of 30 guava (Psidium guajava) germplasm resources in Guangdong province based on AFLP markers

Abstract： 【Objective】Guava (Psidium guajava) is a foreign- introduced species, widely planted in Guangdong, Guangxi, Fujian, Hainan, Taiwan and Yunnan areas of China. The guava varieties in different regions are constantly being introduced and crossed with each other, resulting in complex genetic backgrounds in different regions. The identification of varieties by traditional biological and morphological methods cannot meet the current requirements. In this study, AFLP (Amplified Fragment Length Polymorphism) molecular marker technology was used to detect and analyze 30 guava germplasm samples from Guangdong province. The genetic differences among selected varieties were revealed by cluster analysis and the DNA fingerprint was constructed for quickly distinguishing among different guava varieties.【Methods】AFLP molecular marker technology combines the properties of both RAPD (Polymerase Chain Reaction) and SSR (Simple Sequence Repeat) technologies to provide greater information, high resolution and good stability, which possess the technical advantages for analyzing the diversity of germplasm resources and construction of DNA fingerprints of guava. Researchers from the Fruit Research Institute of Guangdong Academy of Agricultural Sciences collected and transplanted morethan 30 guava germplasm resources from different counties and cities of Guangdong province, and analyzed the diversity of guava germplasm resources through AFLP molecular marker technology. The genomic DNA extraction method of guava leaves was based on CTAB method, and the extracted DNA was detected by 0.8% agarose gel electrophoresis. Eight pairs of core primers were screened from 80 pairs of AFLP primers for guava molecular markers. UPGMA clustering was used to analyze the diversity of guava germplasm resources. The core primers E3-M6 and E4-M2 primer combinations were used to construct guava DNA fingerprinting.【Results】By screening 80 pairs of AFLP primers, 8 pairs of amplified bands with clear and polymorphic primers were obtained. Eight pairs of primers amplified a total of 1 118 bands in 30 guava samples, including 1 114 polymorphic bands, and an average of 139.25 polymorphic bands was amplified by each pair of primers. The average polymorphism ratio was 99.6%, and the overall polymorphism was rich. Primers E3-M6 had two distinct characteristic bands at 491 bp and 465 bp, respectively. Both characteristic bands were from‘Carmine guava No. 1’and‘Carmine guava No. 3’, which can be used to quickly distinguish the above-mentioned two varieties from other varieties. The genetic similarity coefficient of 30 guava germplasm samples ranged from 0.71 to 0.88, the average genetic similarity was 0.80, and the overall difference was small. Among them, the genetic similarity coefficient of‘Crystal Guava No. 2’and‘Carmine guava No. 1’was the lowest (0.71), and the genetic similarity coefficients of‘Carmine guava No. 8’and‘Crystal Guava No. 2’were the highest (0.88). At the similarity coefficient of 0.784, all tested materials can be clustered into 4 categories, among which the wild variety‘Carmine Guava No. 2’in Gaozhou city of Guangdong province was highly differentiated and clustered into one group, which indicated that its genetic differentiation was serious compared with other varieties. According to the results of UPGMA cluster analysis, we can find that although the morphological differences existed between different guava varieties in Guangdong province, the difference was not significant from the genetic perspective. Primers E3-M6 and E4-M2, chosen from 8 pairs of AFLP core primers, were used to fingerprint mapping construction. Nine bands with clear, easy-to-resolve peptide band from E4-M2 and E3-M6 were assigned number 1-9 and according to the assignment criteria, the polymorphic bands encode DNA fingerprinting number of 30 samples of guava samples were disclosed. Each of the 30 guava varieties (lines) had a unique fingerprint code. 【Conclusion】With the rapid development of biotechnology, the cost of molecular marker technology is declining, and DNA molecular marker technology has replaced traditional morphological identification as a new method of species identification. In this study, the genetic diversity of 30 guava germplasm samples from Guangdong province was analyzed by AFLP molecular marker technique and the DNA fingerprinting was established. The DNA fingerprints can successfully distinguish 30 guava samples with only two primers, but the results were just limited in Guangdong province. With the continuous development of guava industry, there will be more breeding of new guava varieties, which means that the guava fingerprint database needs to be continuously updated and completed according to the actual situation. The results of this study may lay a better foundation for the establishment of the national guava fingerprint database in China.