Map-based cloning is a basic method for identifying the mutated gene in plants. We selected the gametophytic mutant, named as AP-26-09, in activation-tagging pool. Mutant plant showed various kinds of pollen phenotype, such as the different number of nucleus or abnormal shapes. For the map-based gene cloning, we conducted phenotypic analysis of F2 mapping population through the screening of DAPI-stained pollen using fluorescence microscopy. Genomic DNA of F2 plants is prepared from leaves of approximately 1000 plants. In order to define chromosomal region where mutation is located, we designed SSLP markers and performed PCR amplification. In this study, we characterized gametophytic mutant and determined the chromosomal location using map-based approach.

sy-2 (Seychelles-2) is a temperature sensitive natural mutant of Capsicum chinense and native to Seychelles Island in Africa. Previously we showed that sy-2 leaves were irregularly shaped and defective in chlorophyll development at temperatures below 24℃. A segregation test revealed that the sy-2 gene is controlled by a single recessive gene. To identify the sy-2 gene, we performed a map-based cloning approach using a total 600 individual F2 plants derived from crossing sy-2 and the wild type C. chinense ‘No.3341’. Fine-mapping of the locus allowed us to position sy-2 to an approximately 170-kb region flanked by markers IN2-1-1 and SNP-3-7 on chromosome 1. Among the approximately 36 hypothetical genes in this region several candidate genes including: HSP90-like ATPase family proteins, lipid-transfer proteins, calmodulin-domain protein kinases, and zinc finger proteins (ZFPs) were identified. RT-PCR and sequencing of the hypothetical genes are under way to identify sy-2.

In the course of map-based cloning, mutant genes are identified through linkage to specific region on genetic map. Here, we demonstrated gametophytic mutant line, named as AP-28-23, in which mutant gene was mapped on chromosome 2. Based on phenotypic analysis of mature pollen, mutant phenotype of AP-28-23 was classified into three classes, wild-type showing 2-4%, moderate 35-53% and severe type 97-100% on aberrant pollen frequencies, respectively. The severe type is completely sterilized with 100% unfertilized ovules. We also revealed that the transmission was reduced through male gametophyte in the AP-28-23 line. The transmission efficiency (TE) through the male gametophyte is only 0.67%, whereas in the female gametophyte is 89.87%.

sy-2 (Seychelles-2) is a temperature sensitive mutant of Capsicum chinense and native to Seychelles Island in Africa. Previously we showed that sy-2 leaves were irregularly shaped and defective in chlorophyll development at temperatures lower than 24℃. A segregation test revealed that the sy-2 gene is controlled by a single recessive gene. To identify the sy-2 gene, we performed a map-based cloning approach using a total of 1,010 F2 plants derived from crossing sy-2 and the wild type C. chinense ‘No.3341’. sy-2 gene is located on chromosome 1, 0.3 cM and 0.1cM away from cosII markers C2_At4g29120 and C2_At1g09070, respectively. The tomato genome sequence between those two markers was compared with pepper genome sequence. We found three of pepper scaffold sequences in this region. We developed seven ingle nucleotide polymorphism (SNP) markers on the pepper scaffold sequences, among which five SNP markers were co-segregated with sy-2. To fill the gap between the scaffolds which contains co-segregating markers, we screened a bacterial artificial chromosome (BAC) library, and end-sequences of total of 22 AC clones were i. We found that five clones were overlapped to cover the gap. We fully sequenced four AC clones and found that the physical distance between C2_At4g29120 and C2_At1g09070 is 343kb. This region contains 70 putative genes such as HSP90-like ATPase family proteins, lipid-transfer proteins, calmodulin-domain protein kinases, and zinc finger proteins (ZFPs). To identify the sy-2 gene, we performed RT-PCR and found that a ZFP-like gene is differentially expressed between WT and sy-2 leaves. This result suggests that the ZFP-like gene is a strong candidate for the sy-2 gene. We are currently characterizing this candidate gene.

Internode elongation is an important agronomic trait in rice that is associated with lodging, yield, and flooding adaptation. We identified a novel rice mutant line showing shortened uppermost internode among the rice Ac/Ds insertional mutant population and named it shortened uppermost internode 4 (sui4). The phenotypes of F1 plants and F2 plants from the cross of sui4 with its original variety, Dongjin, indicated that the SUI4 gene shows incomplete dominance or semidominance. Because the Ds genotypes did not co-segregate with the sui4 phenotypes, we performed mapping of this gene with 273 F2 plants from a cross between sui4 and Milyang23. Primary mapping revealed that the SUI4 locus was located between the S07012 and S07015 markers on rice chromosome 7. Further fine mapping narrowed down the location of SUI4 to the 1.1-Mbp interval of RM1253-S07015. Using re-sequencing data of this mutant along with its original variety, Dongjin, and five other varieties, we found six sui4 specific SNPs occurred within the genic region of five genes in the fine-mapped interval. Among them, one SNP is in exon, while the other five SNPs are in intron. This SNP in exon occurred in the miR172 binding site of a gene encoding AP2 domain transcription factor, which seems to interrupt suppression of this gene activity by miR172. We isolated the genomic region of this gene from sui4 and transformed the wild type variety, Dongjin. The transgenic plants showed remarkably shortened internodes, which indicates that this AP2 domain transcription factor gene is the SUI4 gene.