Applications of molecular markers in genetic analysis.

摘要

Recombinant inbred lines (RILs) as well as an F2 segregating population of soybean facilitated the mapping of two expressed sequence tags (EST) involved in early nodulation and subsequent nitrogen fixation in soybean. The enod2 was mapped on the RIL map close to seed coat color gene, I, a soybean cyst nematode resistance (SCN) gene, Rhg4, and a locus for seed coat hardness. The flanking marker pA 110 and seed coat color were used to integrate and map enod2 on an F2 segregating population in the same order as on the RIL map. A microsatellite from the 5' region of enod2B was mapped in the same position, demonstrating that enod2B and not enod2A was mapped. An RFLP for lbc3 (leghemoglobin) segregated independently from enod2 locus.;DNA amplification fingerprinting (DAF) and bulked segregant analysis (BSA) were used to detect markers linked to the enod2 gene. To overcome potential problems caused by mismatch priming and secondary DNA structure in DAF reactions, annealing temperature of 55°C was developed with single short arbitrary primers to provide high resolution DNA profiles of soybean. Fifteen PCR programs differing in levels of annealing temperature, denaturation, annealing, and extension time and presence/absence of extension step were tested. The average ramping temperature for heating and cooling were calculated 1.42 and 1.27 sec/°C, respectively. Program 15, DAF-15, (95°C/30 sec, 55°C/120, and 72°C/30 sec) generated a complex DNA fingerprinting profiles containing an average of 42 sharp and highly intense bands. DAF-15 was used for DNA fingerprinting of SCN, Mychorrizae , aphid, centipedegrass, and bermudagrass samples. Using high annealing temperature increased stringency of primer-template annealing, avoided potential mismatching and hybrid molecule formation, and consequently improved reproducibility of DNA fingerprinting.;RFLP patterns of homozygous F2 individuals for enod2 gene were set into B1 and S1 subpools and tested with a total of 196 primers of which 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.