A Rapid Construction of a Physical Contig across a 4.5 cM Region for Rice Grain Aroma Facilitates Marker Enrichment for Positional Cloning

Samart Wanchana,^a,b Wintai Kamolsukyunyong,^a Siriphat Ruengphayak,^a Theerayut Toojinda,^a Somvong Tragoonrung,^c and Apichart Vanavichit^{a,d,e *}

ABSTRACT: The whole-genome BAC-end sequences (BES) and Fingerprint Contig (FPC) are valuable resources for map-based cloning. The rice grain aroma quantitative trait loci (QTL)located on a 4.5-cM region of the rice chromosome 8 was targeted for positional cloning. Initially, a fine-scale physical map was developed in silico by aligning selected BAC clones using BES and FPC. The total physical maps spanned 1,114 kb across the flanking markers. Forty microsatellite markers were identified from the BES derived from BAC clones existing in the attained FPC. Five polymorphic SSR markers of those, together with an EST and two
previously developed SSR markers, were genetically mapped in F9 and graphically genotyped in F11 plants derived from aromatic x non-aromatic sister-line crosses. Aroma and a tightly-linked marker were mapped within 1.1 cM region of approximately 170 kb flanked by newly develped markers, 10L03_FW and CP04133. The fine location of rice grain aroma was narrowed down from the original 1,114 kb to 170 kb by the in silico approach. The plants containing such region from the jasmine rice (KDML 105) were all aromatic and able to produce 2-acetyl-1-pyrroline (2AP). The results demonstrated the success of using the in silico approaches as an alternative to genetic approach for physical mapping and marker enrichment. Such in silico technique is more applicable to crops when genetic tools are limited.

LIST OF ABBREVIATIONS: BES, BAC-end sequence; FPC, Fingerprinting contig; KDML105, Khao Dawk Mali 105; 2AP, 2-acetyl-1-pyrroline; QTL, quantitative trait loci.

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a Center of Excellence for Rice Molecular Breeding and Product Development, National Center for Agricultural Biotechnology, Kasetsart University, Kamphaeng Saen, Nakhon Pathom, 73140, THAILAND
b Interdisciplinary Graduate Program in Genetic Engineering, Kasetsart University, Bangkok, THAILAND
c DNA Technology Laboratory, National Center for Genetic Engineering and Biotechnology, at Kasetsart University, Kamphaeng Saen, Nakhon Pathom 73140, THAILAND
d Rice Gene Discovery, National Center for Genetic Engineering and Biotechnology, at Kasetsart University, Kamphaeng Saen, Nakhon Pathom 73140, THAILAND
e Agronomy Department, Kasetsart Univerisity Kamphaeng Saen, Nakhon Pathom 73140, THAILAND

* Corresponding author, E-mail: apichart@dna.kps.ku.ac.th

Received 9 Mar 2005, Accepted 8 Jun 2005