Applications of molecular markers in genetic analysis

To overcome potential problems caused by mismatch priming and secondary DNA structure and taking advantage of high primer-template ratios used in DAF reactions, annealing temperature of 55°C were used with single short arbitrary oligonucleotide as well as mini-hairpin primers to provide high resolution DNA profiles of soybean. Initially, high annealing temperatures for three arbitrary octamer primers in polymerase chain reaction (PGR) were tested for DNA fingerprinting of two soybean cultivars, Minsoy and Noirl. Fifteen PGR programs differing in levels of annealing temperature (47, 55, and 60°C), denaturation, annealing, and extension time (30, 60, and 120 second), and presence/ absence of extension step (+/- 72°C) were tested. The number of bands (amplification products) ranged from 7 (Program 10) to 51 (Program 3). The average ramping temperature for heating and cooling were calculated 1.42 and 1.27 sec/°C, respectively. Intensity of the silver-stained bands in a 10% polyacrylamide gel was high for the most PCR programs. Program 15, DAF-15, (95°C/30 sec, 55°C/120, and 72°C/30 sec) generated a complex DNA fingerprinting profiles for tested primers in Minsoy and Noirl. These profiles contained an average of 42 sharp and highly intense bands using both octamer primers 8-4 and 8-8 for DNA amplification. Using high annealing temperature increased stringency of primer-template annealing, avoided potential mismatching and hybrid molecule formation, and consequently improved reproducibility of DNA fingerprinting.

Newly-developed high annealing temperature DAF was used successfully and detected markers linked to the enod2 gene and analyzed DNA fingerprinting of soybean cyst nematode (SCN), Mycorrhizae, aphid, centipedegrass, and bermudagrass samples. RFLP patterns of 41 homozygous F₂ individuals for enod2 gene were set into two bulks of 26 and 15 with RFLP patterns identical to their parental patterns Bragg and G. soja, respectively. Screening of the bulks B and S with 31 primers resulted in detection of four polymorphic bands using primers HpC29 and HpC30 and DAF-15 program. Due to low number of polymorphic bands in the B and S bulks, sub-pools were generated and screened. B1 and SI sub-pools were tested with total 196 primers of which 32 were used for screening of sub-pools B3 versus S2. Primers Hp30, HpC22 and HpC30 generated 1, 1 and 4 polymorphic markers, respectively, in the B3 vs. S2. The major screening was focused on the B1 versus SI sub-pools which resulted in screening of 196 mini-hairpin and unstructured primers of which a set of 9 primers detected 20 polymorphic bands. Primer HpD25 generated polymorphic bands with 920B1, 320B1, 220S1, and 185B1 base pairs which were reliable and reproducible. These bands are promising bands for further analysis such as cloning and generating SCAR markers in the region of genome containing the enod2 gene.

Key Words: nitrogen fixation, RFLP, recombinant inbred lines, integration mapping, annealing temperature, PCR, DNA fingerprinting, arbitrary primers, soybean.

Abbreviations: RFLP, restriction fragment length polymorphism; SCN, soybean cyst nematode; RJLs, recombinant inbred lines; CHS, chalcone synthase; QTL, quantitative trait locus; lbc3, leghemoglobin gene; DAF, DNA amplification fingerprinting; PCR, polymerase chain reaction.