Computational and experimental analysis of microsatellites in rice (Oryza sativa L.): frequency, length variation, transposon associations, and genetic marker potential

Abstract

A total of 57.8 Mb of publicly available rice (Oryza sativa L.) DNA sequence was searched to determine the frequency and distribution of different simple sequence repeats (SSRs) in the genome. SSR loci were categorized into two groups based on the length of the repeat motif. Class I, or hypervariable markers, consisted of SSRs > or =20 bp, and Class II, or potentially variable markers, consisted of SSRs > or =12 bp <20 bp. The occurrence of Class I SSRs in end-sequences of EcoRI- and HindIII-digested BAC clones was one SSR per 40 Kb, whereas in continuous genomic sequence (represented by 27 fully sequenced BAC and PAC clones), the frequency was one SSR every 16 kb. Class II SSRs were estimated to occur every 3.7 kb in BAC ends and every 1.9 kb in fully sequenced BAC and PAC clones. GC-rich trinucleotide repeats (TNRs) were most abundant in protein-coding portions of ESTs and in fully sequenced BACs and PACs, whereas AT-rich TNRs showed no such preference, and di- and tetranucleotide repeats were most frequently found in noncoding, intergenic regions of the rice genome. Microsatellites with poly(AT)n repeats represented the most abundant and polymorphic class of SSRs but were frequently associated with the Micropon family of miniature inverted-repeat transposable elements (MITEs) and were difficult to amplify. A set of 200 Class I SSR markers was developed and integrated into the existing microsatellite map of rice, providing immediate links between the genetic, physical, and sequence-based maps. This contribution brings the number of microsatellite markers that have been rigorously evaluated for amplification, map position, and allelic diversity in Oryza spp. to a total of 500.

Figure 1

Observed number of microsatellites with di-, tri-, and tetranucleotide motifs in 74,127 BAC-end sequences. Lighter fields correspond to Class I simple sequence repeats (SSRs) (>20 bp), darker areas correspond to Class II SSRs (12 nucleotides ≤ n < 20 nucleotides).

Figure 1

Observed number of microsatellites with di-, tri-, and tetranucleotide motifs in 74,127 BAC-end sequences. Lighter fields correspond to Class I simple sequence repeats (SSRs) (>20 bp), darker areas correspond to Class II SSRs (12 nucleotides ≤ n < 20 nucleotides).

Figure 1

Observed number of microsatellites with di-, tri-, and tetranucleotide motifs in 74,127 BAC-end sequences. Lighter fields correspond to Class I simple sequence repeats (SSRs) (>20 bp), darker areas correspond to Class II SSRs (12 nucleotides ≤ n < 20 nucleotides).

Figure 2

Relative frequency of Class I microsatellites with different simple sequence repeat motifs in three sets of DNA sequence data.

Figure 3

Molecular linkage map of rice. The framework is based on the IR64/Azucena doubled haploid (DH) population. Short arms of chromosomes are at the top. Approximate positions of centromeres are indicated by CEN with an open box. Framework markers (those ordered at LOD score >2.0) have tick marks on chromosome bars. Cosegregating markers with absolute linkage are in the same row. Vertical lines delimit probable intervals for markers mapped with low LOD score. Markers mapped onto other populations are underlined and placed to the right side of the DH map based on their position in relation to common markers. Abbreviation RM is used for RiceMicrosatellite Markers developed in this lab (Cornell University). OSR loci correspond to microsatellite markers reported previously by Akagi et al. (1996). New RM markers derived from BAC-end sequences are shown in bold; those from fully sequenced large-insert clones are in bold and marked by asterisks. (Figure continues on following pages.)

Figure 3

Molecular linkage map of rice. The framework is based on the IR64/Azucena doubled haploid (DH) population. Short arms of chromosomes are at the top. Approximate positions of centromeres are indicated by CEN with an open box. Framework markers (those ordered at LOD score >2.0) have tick marks on chromosome bars. Cosegregating markers with absolute linkage are in the same row. Vertical lines delimit probable intervals for markers mapped with low LOD score. Markers mapped onto other populations are underlined and placed to the right side of the DH map based on their position in relation to common markers. Abbreviation RM is used for RiceMicrosatellite Markers developed in this lab (Cornell University). OSR loci correspond to microsatellite markers reported previously by Akagi et al. (1996). New RM markers derived from BAC-end sequences are shown in bold; those from fully sequenced large-insert clones are in bold and marked by asterisks. (Figure continues on following pages.)

Figure 3

Molecular linkage map of rice. The framework is based on the IR64/Azucena doubled haploid (DH) population. Short arms of chromosomes are at the top. Approximate positions of centromeres are indicated by CEN with an open box. Framework markers (those ordered at LOD score >2.0) have tick marks on chromosome bars. Cosegregating markers with absolute linkage are in the same row. Vertical lines delimit probable intervals for markers mapped with low LOD score. Markers mapped onto other populations are underlined and placed to the right side of the DH map based on their position in relation to common markers. Abbreviation RM is used for RiceMicrosatellite Markers developed in this lab (Cornell University). OSR loci correspond to microsatellite markers reported previously by Akagi et al. (1996). New RM markers derived from BAC-end sequences are shown in bold; those from fully sequenced large-insert clones are in bold and marked by asterisks. (Figure continues on following pages.)