Cloning Of Genes Associated With Taigu Genic Male Sterility Of Wheat (Triticum Aestivum)

The Taigu genie male sterility discovered in China is conditioned by the dominant gene Ms2. It has been widely used in recurrent selections and variety improvements. The cloning of genes associated with the male sterility in Ms2-carrying wheat will help us to understand how Ms2 functions, which is greatly important to both basic and applied researches on wheat (T. eastivuni).The isogenic lines of Ms2 were used for this study. SSH (suppression subtractive hybridization) was conducted using cDNA from fertile spikelets as the driver and that from sterile spikelets as the tester. A SSH library containing 883 recombinants was constructed using the amplified subtractive products. Differential screening of the SSH library using cDNA from the fertile spikelets and sterile spikelets as probes respectively showed that over 80% of the clones were up-regulated in the sterile spikelets.Based on the restriction enzyme-digested fingerprinting patterns, 414 positive SSH clones randomly picked from the library were classified into 73 groups, one clone from each of the 12 groups with the higher frequencies was sequenced. BLAST analysis indicated that 11 of the 12 clones were homologous to ESTs from wheat spikes or anthers during meiosis. Among them, s90 was homologous to the TOM7 (Translocase of Outer Membrane subunit 7) gene from mammal, plant and fungus, s106 was part of wheat histone H3 gene. s210 encodes a putative protein homologous to the dormancy-associated protein in pea and the auxin-repressed protein from apricot and tobacco. s214 and s598 were part of the wheat sucrose: fructan 6-fructosyltransferase gene and DNA topoisomerase II gene, respectively.All the 12 clones mentioned above were single or low-copy genes in the genomes of wheat, Haynaldia villasa, rice and maize. Using C.S. Nulli-tetrasomics, s36 and s90 were assigned to group 3, s210 assigned to group 5, s211, s271, s294 and s598 to group 6.Northern dot hybridization with different tissues or organs from the fertile and sterile plants showed that s36, s90, s210 and s211 were up-regulated in the stem, spikelets and anthers in sterile plants compared to the corresponding tissues in the fertile plants. No obvious difference in their expressions at the transcription level was observed in other tissues between the fertile and sterile plants.By RACE-PCR and RT-PCR, a full-length cDNA encoding the TOM7-like subunit was isolated. Comparison of TOM7 from wheat and other species showed that the membrane-spanning region was highly conservative but the remaining portion was largely divergent among different speciesA cDNA library containing 150,000 plaque forming units was constructed using the young spkelets of sterile plants. The percentage of recombinants in this library was 98%, which had an average insert size of 1.2 kb. The cDNA library was screened using s36, s211 and s271 as the probes to isolate their homologs. The s211 homolog didn't have a decent coding region and was likely a non-coding RNA gene. The s36 homolog encoded a polypeptide of 307 amino acids with similarity to an unidentified protein from Arabidopsis. The s271 homolog isolated was a partial cDNA sequence and also encoded a polypeptide homologous to an Arabidopsis protein. During the library screening, we also obtained a clone homologous to an anther-specific protein in Arabidopsis, rice and Brassica, an EST encoding a polypeptide with similarity to a nuclear acid excise repair (NER) protein in yeast and carrot, and a clone encoding a phi-1-like protein. The former two clones were assigned to the group 7 chromosomes of Triticwn aestivum.