Isolation Of Homologous Sequences Of Dwarf Gene And Construction Of BAC Pools In Wheat

The Taigu Genie Male-Sterile Wheat is a dominant male-sterile form, which is great valuable in genetic improvement by its stable inheritance, complete male abortion, and high cross-fertilize rate. Dwarfing-Sterile Wheat is a dwarf marked male sterile material, carrying the dominant gene Ms2 for male sterility and dominant dwarf gene RhtlO. The two genes are closely linked together on the short arm of chromosome 4D, with a crossing-over value as small as 0.18%. In our research, several techniques were used such as RT-PCR, allele-specific PCR, construction and screening BAG pools. The objective of the present research was to explore the co-separate marker of dwarf gene, construct and screen BAG pools of the dwarf material from Dwarfing-Sterile Wheat.The material used in this study was the Dwarfing-Sterile Chinese Spring separated individual.Six pairs of specific oligonucleotide primers were designed according to the sequence of Rht-Dla gene, which is the wild type gene of Rht-Dlb (Rht2). These overlapping primers covered the whole gene of Rht-Dla. Using the cDNA of dwarf and high materials as templates separately, a series of 300-700bp DNA fragments were amplified by RT-PCR technology and recombinant plasmids were obtained after these DNA fragments were cloned into pUCm-T vector. Through the method of primer walking, we obtained two homologous sequences of the dwarf gene, which were named as Rh and H gene respectively. Homology comparison of the Rh gene with related sequences in GenBank showed that it shared 75%, 74%, 68%, 68% and 50% identities in amino acid sequences with of common wheat, barley, maize, rice and Arabidopsis. This result indicated that the Rh gene must be a gibberellin response modulator gene.The alignment of Rh, Rht-Blb and Rht-Dlb genes with H gene in amino acid sequences showed that there only had differences in the region I among seven functional regions ( I to VD).The Rh, Rht-Blb and Rht-Dlb mutations were all nucleotide substitutions that created stop codons. It was these stop codons that interrupted these three genes into two open reading frames (ORFs) respectively, whereas the whole sequence of H gene was still the intact open reading frame. Furthermore, region I was substantially deleted in the Rhmutant other than Rht-Blb and Rht-Dlb.A pair of allele-specific oligonucleotide primer was screened based onthe difference in the region I of Rh and H genes. The result of allele-specificPCR showed there was no specific amplification in the 200 high materials, whereas about 700-800bp specific DNA fragment was amplified in the all 200 dwarf materials, so we could take this pair of allele-specific primer as a co-separate marker of Rh gene.According to the method of Doctor Zhang and Clemson University, BAG pools of the dwarf materials were constructed, taking the pECBACl as the BAG vector. A total number of 63 BAG pools (about 20,000 recombinant clones) were obtained. Analysis of 168 randomly selected clones showed that over 90% of the clones contained an insert that ranged between 25 and 150 kb, whereas the average insert size was 87kb. The result of clone stability experiment showed that the insert could be maintained stably at least to 100 generations in the E.coli.