[Development and appraisement of functional molecular marker: intron sequence amplified polymorphism (ISAP)].
Molecular markers are playing an increasingly important role in map construction, QTL analysis, gene mapping and marker-assisted selection. Researchers hope the target gene and locus are as close as possible, one locus can present one gene, or linked with some important trait, then, individuals with useful trait can be selected through molecular markers selecting, and it's the functional molecular marker. PCR-based molecular markers such as RAPD, SSR, AFLP amplified non-coding regions, or the whole genome randomly, the locus is far away from the gene of targeted trait, this limit the ap-plication of these molecular markers. This study established a kind of functional molecular markers based on intron of gene sequence, trying to link loci with gene sequence to achieve the purpose of its function. It used the conservative consistent sequence of intron splicing sites as its core sequence of amplification. ISAP is a PCR-based marker system, it has two kinds of primers: forward primer and reverse primer, both primers are 18 bases. Any of the primers can be used to construct a primer combination with the other kind of primers. Seventeen primers, 9 forward and 8 reverse, were used to construct 72 primer combinations, 67 of them showed polymorphism in a G. hirsutum cv. CCRI36 x G. barbadense cv. H7124 F2 population and a total of 212 loci were obtained. Together with 164 SRAP loci, these 212 loci were used to construct a genetic linkage map. ISAP markers distributed evenly in the entire linkage group, part of the region had a high saturation, might be the coding sequence-rich region. Sequencing results of 20 fragments showed that 85% of the sequences announced homology with published EST sequence stored in the NCBI which indicated that they were amplified adjacent to expressed sequences. These results showed that ISAP marker system was simple, efficient, reliable, and had a relatively high polymorphism, furthermore, it directly targeted gene sequence, was a functional molecular marker system. ISAP was also used to amplify other plants and good results were achieved.
Lu
Cai-Rui
CR
China Cotton Research Institute, Key Laboratory of Cotton Genetic Improvement of Ministry of Agriculture of China, Anyang 455004, China. lucr99@126.com
Yu
Shu-Xun
SX
Yu
Ji-Wen
JW
Fan
Shu-Li
SL
Song
Mei-Zhen
MZ
Wang
Wu
W
Ma
Shu-Juan
SJ
D001483:Base Sequence; D015415:Biomarkers / Q000378:metabolism; D018744:DNA, Plant / Q000032:analysis*; D003368:Gossypium / Q000235:genetics*; D007438:Introns / Q000235:genetics*; D008958:Models, Molecular; D011110:Polymorphism, Genetic
D015415:Biomarkers; D018744:DNA, Plant