Construction of DNA fingerprint of macadamia germplasm based on SRR markers

Abstract:【Objective】Using molecular markers to construct DNA fingerprints is an effective strategy to identify plant germplasm. SSR (Simple Sequence Repeats) molecular markers are preferred for plant DNA fingerprinting constantly, due to their abundance, co-dominant inheritance, and reproducibility.The aim of this study was to construct macadamia DNA fingerprint using a series of SSR primers in or-der to provide technical support for macadamia nut germplasm collection and variety protection.【Methods】The genome DNA sequence of macadamia (M. integrifolia) was searched on NCBI website. The repeated base sites in the sequence were screened, and the primer 3 was used to design primers in batch. The standard primers were synthesized for subsequent test, according to the primer selection standard: the repeat unit is more than 2 bases, the number of repetitions is more than 8 times, and it is evenly dis-tributed on the chromosome. The genomic DNA of the test material was extracted by CTAB method.The SSR-PCR reaction system is: 10 × Buffer 2 μL, 2.5 mmol·L^-1 dNTP 0.4 μL, positive and negative primers 0.3 μL respectively, DNA template 2μL (20 ng·μL^-1), 5U Taq 0.2 μL, ddH2O 14.8 μL. The PCR amplification procedure was shown as fellows: Pre denaturation at 94 ℃ for 5 min; Denaturation at 94 ℃ for 30 s, renaturation at 65 ℃ to 50 ℃ for 30 s, extension at 72 ℃ for 40 s, a total of 35 cycles; The final extension was made at 72 ℃ for 3 min. 4 materials with large phenotypic differences were se-lected for primer screening. Firstly, PCR amplification products were detected by 2% agarose gel elec-trophoresis, followed by PAGE detection with good amplification effect. Secondly, the initially selected PCR products plus the buffer denatured at 94 ℃ for 10 min, and silver staining was made after 6% dena-turing polyacrylamide gel electrophoresis and primers with good amplification effect and polymor-phism were selected. Thirdly, the screened SSR primers were labeled with 6-FAM fluorescent dye. Af-ter PCR amplification, 0.3 μL PCR product, 0.5 μL internal lane standards and 9.5 μL deionized for-mamide, were mixed and added to PCR plate, denatured at 95 ℃ for 5 min, cooled at 4 ℃ and then cen-trifuged. The capillary electrophoresis was performed with 3730xl sequencer. The allele loci of each primer were counted. The homozygous loci were recorded as X/X, the heterozygous loci were recorded as X/Y, the small fragment data in the heterozygous loci were in the front, the large fragment data were on the back, and the allele variation deletion loci were recorded as 0, for forming DNA fingerprint data. Referring to the methods of Yang Wenjuan and Guo Yanchun, the alleles obtained from each pair of primers were coded with numbers + English letters, and the numbers 1-9 were used in the order of mo-lecular weight from small to large, and the parts beyond 9 were expressed with English letters A-Z, so as to construct the fingerprint code of the tested materials. DNA molecular ID cards in the form of bar code and quick response (QR) code were produced through online bar code generator and quick re-sponse (QR) code generator respectively.【Results】240 pairs of primers were initially selected, and de-tected by 2% agarose gel electrophoresis after PCR amplification. 155 pairs of SSR primers were select-ed for 6% denaturing polyacrylamide gel electrophoresis. Finally, 22 pairs of SSR primers with stable amplification and high polymorphism were selected for fluorescence labeling, and 83 macadamia germ-plasm genotypes were obtained by capillary electrophoresis. The DNA fingerprints and DNA molecular ID card of the above materials were constructed using the combination of 9 pairs of fluorescent primers.For instance, primer P202 had 13 polymorphic loci, encoded in proper order as 286 (1), 286 / 288 (2),286 / 290 (3), 286 / 292 (4), 288 (5), 288 / 290 (6), 288 / 292 (7), 288 / 294 (8), 290 (9), 290 / 292 (a),292 (b), 292 / 294 (c) and 300 (d). The 9 pairs of primers were arranged and combined in the order of P211, P127, p238, p132, p118, p163, p202, p141 and P84, the coding information of each germplasm in each primer was counted. For example, the SSR fingerprint code of Jingha 57 is H7A49A52I, the first letter“H”indicates that the amplified fragment of germplasm Jingha 57 in primer P211 were ranked 17th in the primer polymorphism fragment gradient, and the second number“7”indicated that the am-plified fragment of germplasm Jingha 57 in primer P127 were ranked 7th in the primer polymorphism fragment gradient, The third letter“A”indicated that the amplified fragment of germplasm Jingha 57 in primer P238 ranks first in the gradient of polymorphic fragment of the primer, and the other codes were analogized in turn.【Conclusion】SSR primers with high efficiency were obtained and used for rapid molecular identification of Macadamia germplasm resources.