Application of the RLGS Image Analysis Tool (RAT) tothe Construction of a Genetic Linkage Map ofRecombinant Inbred Strain SMXAYuichi Sugahara 123

sugahara@rtc.riken.go.jp

Yoshihide Hayashizaki 123
yosihide@rtc.riken.go.jp

1 Genome Science Laboratory, RIKEN Life Science Tsukuba Center
3-1-1 Koyadai, Tsukuba, Ibaraki 305, Japan.
2 CREST, Japan Science and Technology Corporation(JST)3 Laboratory for Genome Exploration Research Group, Genomic Sciences Center,RIKEN

1 Introduction
The characterization of genomic DNA is essential for establishing a genetic basis of biological properties. Recently, much e ort has been made to determine the complete genomic sequence of severalorganisms. Despite the methodological and strategic improvements of mapping and sequencing aswell as progress in automation, the genome is still so large and many species of genomes have yet to
be mapped. Under such circumstances, the restriction landmark genome scanning method (RLGS),
which was originally developed to scan the whole genome, is a promising tool as it can be e ectively
applied to any organism including mammalian species. The power of the RLGS method has been
demonstrated in various lines of genetic analysis, including the construction of a genetic linkage map,
identi cation of the methylation status of a genome, and detection of the loss of heterozygosity in thegenome of cancers [1].
In constructing a genetic map by the RLGS method, the presence or absence of hundreds of spots
must be scored in many progeny. It should be possible to conduct this scoring step automaticallywith the assistance of a computer-based image analysis system. Although there are several types ofcommercially available software for the analysis of 2-D protein electrophoresis, they are not suitable
for analysis of the RLGS pro le. Therefore, the RLGS pro le has been mainly analyzed by humanvisual observation, this being a laborious and time-consuming procedure. To overcome this drawback,we developed a novel image analysis system, RAT (RLGS Analysis Tool) [2].
In this study, we assessed the accuracy of RAT by scoring the matches and discordance between
the analyses of RAT and human observation. RAT could reproduce almost all data obtained by theobservation. We then applied RAT to practical analysis for the construction of a genetic linkage map
using the recombinant inbred strain SMXA. We were able to identify 162 RLGS spots/loci, 121 ofwhich were correctly mapped to a speci c chromosome.

2 Methods and Results
RAT was developed to fully account for the characteristics of RLGS pro le and can be used toautomatically construct a genetic linkage map. RAT consists of four processes. The rst three areused to construct the master pro le, in which all spots are classi ed into paternal-speci c polymorphic,
maternal speci c polymorphic, or non-polymorphic spots. In the fourth process, the RLGS pro le ofeach o spring is compared to the master pro le, and the presence or absence of polymorphic spots isscored to prepare the strain distribution pattern (SDP). The resultant SDP can be directly used forlinkage analysis.
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The rst analysis process of RAT is background subtraction. In this process, the spots are discriminated from the background of the x-ray lm. The x-ray lm is initially subjected to digitalizationby scanner. The digitized lm image is then transformed into a binary image, in which the spotsare represented as a white area (groups of white pixels) and the background is represented as a blackarea. In the second process, RAT compares F1 and parental binary images, and determines the spotcorrespondences between two images by using a template matching technique. In the third process,RAT recognizes the origin of spots on F1 lm. The spots on F1 are inherited from paternal and/or maternal progenitors, and RAT classi es the spots into paternal-speci c polymorphic, maternal-speci cpolymorphic, and non-polymorphic (common) spots. This process is performed basically to evaluate
the degree of overlap between the corresponding spots, and the nal results are transmitted into themaster pro le. In the last process, RAT compares the RLGS pro les of each o spring with the masterpro le, scores the presence or absence of polymorphic spots, and returns a list of SDPs.
To assess the accuracy of RAT, we compared the results from RAT and human observation byusing RLGS pro les of Syrian hamster. Without low quality part of RLGS lm, RAT could reproduce
almost all data obtained by the observation and we concluded that the RAT program was feasible for
identifying polymorphic spots and scoring them in backcross progeny. We then used RAT to construct
the genetic linkage map of the recombinant inbred strain SMXA. A total of 162 novel RLGS spots/loci(72 SM/J-speci c and 90 A/J-speci c) were identi ed. We transmitted the resultant SDPs of novel
spots to linkage analysis software Map Manager, and nally 121 of 162 polymorphic RLGS spots/lociwere chromosomally mapped.

Acknowledgements
This study has been supported by Special Coordination Funds and a Research Grant for the GenomeExploration Research Project from the Science and Technology Agency of the Japanese Government,
CREST (Core Research for Evolutional Science and Technology) of Japan Science and Technology
Corporation (JST), and a Grant-in-Aid for Scienti c Research on Priority Areas and Human Genome
Program from the Ministry of Education and Culture, Japan to Y.H.

References[1] Hayashizaki, Y. et al., Restriction landmark genomic scanning method and its various applications, Electrophoresis, 14:251{258, 1993.
[2] Sugahara, Y. et al., An automatic image analysis system for RLGS lms, Mammalian Genome,
9:643{651, 1998.

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