Development of AFLP-derived STS markers for the selection of 5-methyltryptophan-resistant rice mutants

D. S. Kim, I. S. Lee, C. S. Jang, S. Y. Kang, H. S. Song, Y. I. Lee, Yong Weon Seo

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

To increase the specific free amino acid content in the japonica rice (Oryza sativa L.) cultivar Donganbyeo, mutant cell lines resistant to growth inhibition by 5-methyltryptophan (5MT) were selected from embryocultured callus irradiated with 50 Gy gamma-rays. Four 5MT-resistant homozygous M4 lines, MRI-40, MRI-116, MRII-8, and MRII-12, were obtained. The mean content of nine free essential amino acids were 70.1, 72.5, 31.7, and 35.4% greater than the original variety in these four mutant lines, respectively. For AFLP analysis, 8 EcoRI (+2) and 8 MseI (+3) primers used in 45 primer combinations generated a total of 3,684 bands with a mean of 82 bands, of which 361 (9.8%) were clearly polymorphic with the control cultivar, the four 5MT-resistant mutants, and five sensitive lines. The lines were grouped into three clusters through cluster analysis using unweighted pair grouping method of averages. The 36 polymorphic PCR products present only in the four homozygous 5MT-resistant lines were cloned and sequenced, and 10 of these sequenced products were converted into sequence tagged site (STS) markers. These STS primer sets were designated OSMR1-OSMR10. Six STS primer sets (OSMR1, OSMR2, OSMR3, OSMR4, OSMR5, and OSMR6) generated a single monomorphic PCR product identical in size to the original AFLP fragments. The broad applicability of these STS markers for the screening of 5MT resistance was evaluated with seven putative 5MT-resistant M2 plants (PM-1 to PM-7). Four STS markers (OSMR1, OSMR2, OSMR4, and OSMR5) out of six STS primer sets were revealed as polymorphic products between the control cultivar and the seven M2 plants. These markers can be utilized for the fine selection of 5MT resistance in rice, and this PCR-screening technique is less time-consuming, less labor-intensive, and more accurate and reliable than selection based solely on phenotypic evaluation involving soaking in 5MT solutions.

Original languageEnglish
Pages (from-to)71-80
Number of pages10
JournalPlant Cell Reports
Volume23
Issue number1-2
Publication statusPublished - 2004 Aug 1

Fingerprint

amplified fragment length polymorphism
rice
mutants
cultivars
screening
essential amino acids
soaking
free amino acids
growth retardation
gamma radiation
cluster analysis
Oryza sativa
labor
callus
cell lines
methodology

Keywords

  • 5-Methyltryptophan
  • Amplified fragment length polymorphism
  • In vitro mutagenesis
  • Marker-assisted selection
  • Sequence tagged site

ASJC Scopus subject areas

  • Plant Science

Cite this

Kim, D. S., Lee, I. S., Jang, C. S., Kang, S. Y., Song, H. S., Lee, Y. I., & Seo, Y. W. (2004). Development of AFLP-derived STS markers for the selection of 5-methyltryptophan-resistant rice mutants. Plant Cell Reports, 23(1-2), 71-80.

Development of AFLP-derived STS markers for the selection of 5-methyltryptophan-resistant rice mutants. / Kim, D. S.; Lee, I. S.; Jang, C. S.; Kang, S. Y.; Song, H. S.; Lee, Y. I.; Seo, Yong Weon.

In: Plant Cell Reports, Vol. 23, No. 1-2, 01.08.2004, p. 71-80.

Research output: Contribution to journalArticle

Kim, DS, Lee, IS, Jang, CS, Kang, SY, Song, HS, Lee, YI & Seo, YW 2004, 'Development of AFLP-derived STS markers for the selection of 5-methyltryptophan-resistant rice mutants', Plant Cell Reports, vol. 23, no. 1-2, pp. 71-80.
Kim DS, Lee IS, Jang CS, Kang SY, Song HS, Lee YI et al. Development of AFLP-derived STS markers for the selection of 5-methyltryptophan-resistant rice mutants. Plant Cell Reports. 2004 Aug 1;23(1-2):71-80.
Kim, D. S. ; Lee, I. S. ; Jang, C. S. ; Kang, S. Y. ; Song, H. S. ; Lee, Y. I. ; Seo, Yong Weon. / Development of AFLP-derived STS markers for the selection of 5-methyltryptophan-resistant rice mutants. In: Plant Cell Reports. 2004 ; Vol. 23, No. 1-2. pp. 71-80.
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abstract = "To increase the specific free amino acid content in the japonica rice (Oryza sativa L.) cultivar Donganbyeo, mutant cell lines resistant to growth inhibition by 5-methyltryptophan (5MT) were selected from embryocultured callus irradiated with 50 Gy gamma-rays. Four 5MT-resistant homozygous M4 lines, MRI-40, MRI-116, MRII-8, and MRII-12, were obtained. The mean content of nine free essential amino acids were 70.1, 72.5, 31.7, and 35.4{\%} greater than the original variety in these four mutant lines, respectively. For AFLP analysis, 8 EcoRI (+2) and 8 MseI (+3) primers used in 45 primer combinations generated a total of 3,684 bands with a mean of 82 bands, of which 361 (9.8{\%}) were clearly polymorphic with the control cultivar, the four 5MT-resistant mutants, and five sensitive lines. The lines were grouped into three clusters through cluster analysis using unweighted pair grouping method of averages. The 36 polymorphic PCR products present only in the four homozygous 5MT-resistant lines were cloned and sequenced, and 10 of these sequenced products were converted into sequence tagged site (STS) markers. These STS primer sets were designated OSMR1-OSMR10. Six STS primer sets (OSMR1, OSMR2, OSMR3, OSMR4, OSMR5, and OSMR6) generated a single monomorphic PCR product identical in size to the original AFLP fragments. The broad applicability of these STS markers for the screening of 5MT resistance was evaluated with seven putative 5MT-resistant M2 plants (PM-1 to PM-7). Four STS markers (OSMR1, OSMR2, OSMR4, and OSMR5) out of six STS primer sets were revealed as polymorphic products between the control cultivar and the seven M2 plants. These markers can be utilized for the fine selection of 5MT resistance in rice, and this PCR-screening technique is less time-consuming, less labor-intensive, and more accurate and reliable than selection based solely on phenotypic evaluation involving soaking in 5MT solutions.",
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