Detection of genetic locus of self-incompatibility(S) thioredoxin like gene in rice (Oryza sativa L.) using single nucleotide polymorphism (SNP)

47. Detection of genetic locus of self-incompatibility(S) thioredoxin like gene in rice

(Oryza sativa L.) using single nucleotide polymorphism (SNP)

T. NAKAZAIU�, K. KITAURA�, N. Txiuzi�, Y. FUKUTA2 and H. IKEHAsHI�

1) Graduate School of Agriculture, Kyoto University, Kyoto, 606-8502 Japan

2)The Hokuriku Agricultural Experiment Station, Joetsu, 943-0193 Japan

Li et a!. (1994) reported that an S-thioredoxin gene expressed as a putative self-incompatibility (SI) gene in the pollen of Phalaris coeru!escens was composed of a variable region in the N-terminal side and a thioredoxin domain in the C-terminal side. Li el a!. (1997) also indicated that PGI-2 and the SI gene were linked with recombination values ranging 0.11 to 0.29 in all diploid Poaceae species with n =7. In rice, distorted segregations of PGI isozymes encoded by Pgil and Pgi2 have been reported (Lin et al. 1992, Lu et a!. 1998). These findings suggest a possibility that the S-thioredoxin gene may be responsible for the irregular segregation of PGI genes in rice. A rice homologous gene with S-thioredoxin of Phalaris was reported to be expressed in leaf tissues (Li eta!. 1997), but there has been no report about the structure and the locus for the gene of S-thioredoxin. The objective of this study was to find a rice gene homologous to S-thioredoxin of Phalaris and to estimate its linkage with PGI isozymes.

To detect a genomic sequence of a 5-thioredoxin gene of rice, DNA fragments of

Nipponbare were amplified with PCR and were sequenced using several primers

(Fig. 1). The primers were designed after a partial sequence of a rice cDNA clone in

DDBJ, C72705, which was registered by Sasaki and Kimura as a partial sequence of cDNA

from panicle in flowering stage and was considered to code the similar amino-acid sequence with Phalaris S-thioredoxin, and from the observed sequences including intron regions. As a result, a sequence of the 1841 bp region between a site of primer #1 and that of #3 was obtained (Accession number AB024702 in DDBJ). In the observed sequence four regions were almost identical to those of C72705 though three nucleotides were different from those of C72705: a nucleotide �C� which lacked in C72705 was added, and two unknown base �N� in C72705 were detected as �A� and no-base, respectively. By the alignment analysis, the observed sequence was found to be the same structure as those in the S-thioredoxin of Phalaris, and considered to be a partial sequences of rice gene (S-tm-!) that correspond to the region between the exon II and V of the Phalaris gene (Fig.

1). In the deduced partial amino-acid sequence of S-tm-I, the active site and other amino- acid residues important for the thioredoxin structure (Li eta!. 1994) were conserved. However, it had a stop codon in the region corresponding to its exon III of Pha!aris gene, despite an appreciable level of homology in the amino-acid sequence of C-terminal side.

Comparing the sequence of Nipponbare and that of indica cultiver IR 36 in c.a. 900 bp sequences around the regions corresponding to the exon II and III of the Pha!aris gene, five bases showing single nucleotide polymorphism (SNIP) between the indica and japonica cultivars were found. To detect SNP by PCR as a polymorphic-STS (P-STS), a pair of primers, #8 and #9 was designed of which 3� ends came to one of SNP bases in Nipponbare (japonica) and IR 36 (indica) sequences, respectively. Using these primers, contrasting products each of which corresponded to the indica and japonica cultivars were indicated in the defined annealing condition of the PCR (Fig. 2). From the P-STS analysis with RILs, the S-trn-l was found to be closely linked with an RFLP marker N079 A with a distance of 0.7 cM on the short-arm end of chromosome 1 (Fig. 3). Segregation analysis with an F2 population of Nekken 2/ IR 36, the two PGI-isozyme loci on chromosome 3 and 6 were found to be independent of the S-tm-i locus which was detected with P-STS (data not shown). Thus, the unusual segregation of PGI isozymes in rice was not related to the S-trn-l gene.

The S-trn-l gene was found to have a stop codon in one of its exons as an indication of the loss of its function in rice. Nevertheless, at least a portion of the S-trn-I gene sequence is found to be transcribed in most of rice tissues after RT-PCR (data not shown). This fact leads us to further research about the function of this gene.

References