Simple polymorphisms in ribosomal DNA repeats in the nucleolus organizer region (NOR) permitted the development of markers for the genetic mapping of the soybean NOR. The markers map to the top end of soybean linkage group F, one of either telomeric end predicted in the cytogenetic and primary trisomic studies.

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THE genetic map of the soybean [Glycine max (L.) Merr.], which is an economically important legume, is one of the most densely populated maps among plants, with >3000 published markers (CHOI et al. 2007). However, its cytogenetic studies have lagged behind those of rice (Oryza sativa L.), maize (Zea mays L.), barley (Hordeum vulgare L.), and tomato (Lycopersicon esculentum Mill.). Thus, the relationships between soybean molecular linkage groups (MLG) and chromosomes remain incompletely understood (CREGAN et al. 2001; ZOU et al. 2003). The haploid chromosome number of the soybean plant is 20 (VEATCH 1934), which is almost two times that observed in major diploid crops such as rice (n = 12), maize (10), barley (7), and tomato (12) as well as in research plants including Arabidopsis thaliana (5), Lotus japonicus (6), and Medicago trancatula (8). The soybean mitotic metaphase chromosomes evidence small size variations ranging from 1.42 to 2.84 µm and are symmetrical without karyotypically visible landmarks (SEN and VIDYABHUSAN 1960). However, the pachytene chromosome analysis of an F₁ hybrid between soybean and Glycine soja Sieb. and Zucc., a wild annual progenitor of the soybean, evidenced heterochromatin distribution on either side of the centromeres, small structural differences, and a satellite chromosome, thus allowing for the construction of chromosome maps on the basis of chromosome length and euchromatin and heterochromatin distribution, which were numbered in descending order of 1–20 (SINGH and HYMOWITZ 1988). The chromosome harboring the satellite was designated as chromosome 13 (SINGH and HYMOWITZ 1988). Fluorescent in situ hybridization (FISH) using the rDNA as a probe verified that the satellite region of chromosome 13 is the nucleolus organizer region (NOR) (GRIFFOR et al. 1991). FISH resulted in the detection of a pair of NORs on the short arm of chromosome 13 in the soybean and its progenitor, G. soja, as well as three strong fluorescent signals in the soybean that are trisomic for chromosome 13. Subsequently, MLG F was assigned to chromosome 13 with a set of primary trisomics and simple sequence repeat markers (CREGAN et al. 2001). However, the accurate genetic location of the soybean NOR on MLG F remains to be determined.

The NOR loci harbor from hundreds to thousands of 18S-5.8S-26S ribosomal RNA gene units, which are clustered into tandem repeats (FLAVELL and O'DELL 1979). Each of the repeats codes for a ribosomal RNA gene unit. An intergenic spacer (IGS) region separates the transcription units of adjacent repeats. The intergenic region has been determined to be highly variable in a range of plants: Triticum aestivum (APPLES and DVOÁK 1982), Triticum dicoccoides (FLAVELL et al. 1986), H. vulgare (SAGHAI MAROOF et al. 1984), Vicia faba (ROGERS and BENDICH 1987), and Z. mays (ZIMMER et al. 1988). The high degree of variation in the number of subrepeats in the IGS region has allowed for the development of molecular markers for the genetic mapping of these NORs in several plant species (e.g., SAGHAI MAROOF et al. 1984; SNAPE et al. 1985; ZIMMER et al. 1988; COPENHAVER and PIKAARD 1996). However, the soybean IGS appears to harbor no notable subrepeat variations (NICKRENT and PATRICK 1998) among G. max variants and between G. max and G. soja, with a GT dinucleotide repeat and several single nucleotide polymorphism (SNP) loci. In the coding repeat units, other variable regions, referred to as internal transcribed spacers (ITS), are located on either side of the 5.8 S rRNA gene, which are designated as ITS1 and ITS2. The nucleotide sequences of the ITS have proven quite useful for phylogenetic studies in many angiosperm groups, due primarily to their usefulness in resolving relationships at the species level (ARNHEIM 1983; WOJCIECHOWSKI et al. 1993; BALDWIN et al. 1995). Here, we describe the genetic localization of the soybean NOR using SNP and microsatellite markers that were developed from a SNP locus between the ITS1 nucleotide sequences and from a SNP locus and a microsatellite repeat locus between the IGS nucleotide sequences of the soybean and G. soja.

We utilized a population of 113 F₁₂ recombinant inbred lines generated via an interspecific cross between the G. max cv. "Hwangkeum" and the G. soja Siebold & Zucc. line "IT182932" (hereafter referred to as the HI population). In this HI population, which harbors >400 mapped marker loci (S.-C. JEONG, unpublished results), all 20 of the soybean MLGs have been identified. DNA sequences corresponding to ITS1 and IGS were determined from the Hwangkeum and IT182932 variants (Table 1). The ITS1 sequence of Hwangkeum (GenBank accession no. EU1183310) harbors one single-base substitution relative to that of IT182932 (GenBank accession no. EU118311). This SNP locus was genotyped via the design of a set of two specific forward primers and a common reverse primer to determine the genotype of the locus using an allele-specific PCR method, as described by JEONG and SAGHAI MAROOF (2004) (Table 2). The IGS sequence of Hwangkeum (GenBank accession no. EU118312) harbors 9 single-base substitutions, 2 single-base deletions, and one 14-base deletion due to a microsatellite repeat variation relative to that of IT182932 (GenBank accession no. EU118313). One of the single nucleotide polymorphism loci was genotyped via the design of two specific forward primers and a common reverse primer as described above for the ITS1, and the microsatellite repeat variation locus was genotyped via the design of a primer pair for PCR amplification (Table 2).

View this table: In this window In a new window   TABLE 1 Primer sequences for PCR amplification and sequencing of the soybean ITS1 and IGS regions

 

View this table: In this window In a new window   TABLE 2 Attributes of SNP and microsatellite markers used for mapping ITS1 and IGS in a recombinant inbred line population from a cross between Hwangkeum and IT182932

 
The allele-specific PCR primer sets designed from the ITS1 and IGS sequences generated allele-specific PCR bands, which evidenced the expected ratio of 1:1 (² = 2.7, P = 0.1) in the HI population as codominant alleles of a Mendelian locus. The SNP marker from the ITS1 was designated as SN307 and the SNP marker from the IGS was designated as SN314. The microsatellite marker, designated SM318, cosegregates with the SN307 and SN314 markers in the HI population. The three markers were mapped 0.4 cM away from Satt325 on the top end of MLG F (Figure 1A).

View larger version (31K): In this window In a new window Download PPT slide   FIGURE 1.— Map position of SN307, SN314, and SM318 and diversity of SM318. (A) The genetic map of the top end of the soybean molecular linkage group F, which evidences the locations of the SN307, SN314, and SM318 markers developed from the nucleolus organizer region. Mapmaker 3.0b (LANDER et al. 1987) was utilized to group and order the genetic loci. The marker loci in the HI population were grouped at a LOD 5.0 and a maximum genetic distance of 37.2 cM. Arrows indicate directions toward telomere and centromere positions proposed by SINGH and HYMOWITZ (1988) on the basis of the positions of the NOR and heterochromatic zone. (B) Polyacrylamide gel separation showing microsatellite repeat variants in Hwangkeum, IT182932, and 10 cultivars of soybean. Arrowheads indicate the bands that are slightly longer than that of Hwangkeum and shorter than that of IT182932. (C) Comparison of the GT/GC repeat portions of the IGS sequences from IT182932, Hwangkeum, Sowon, V94-5152, Williams, and Evans. Nucleotides at substitution sites and the microsatellite repeat variation site are underlined.

 
The three markers were further tested on 10 soybean (G. max) cultivars to investigate their applicability for mapping the NOR in a population made from a cross between G. max cultivars. The microsatellite marker SM318 revealed a novel allele, which is 2 bases longer than the size of the Hwangkeum allele (Figure 1B). The size variation was verified by sequencing analysis (Figure 1C). SN307 and SN314 revealed the same banding patterns as those of Hwangkeum (data not shown). The results indicated that, at least, the SM318 marker can be utilized to map the NOR locus in a wide range of soybean populations.

The genetic mapping position of the soybean NOR was reported to be a telomeric end of the short arm of chromosome 13, which corresponds to soybean MLG F, as has been demonstrated by the results of FISH and primary trisomics studies (SINGH and HYMOWITZ 1988; GRIFFOR et al. 1991; CREGAN et al. 2001). However, it could not be predicted which end of the MLG F harbors the NOR, because the genetic mapping positions of the centromere on the MLG F are not currently known. Thus, our finding that the soybean NOR is mapped 0.4 cM away from Satt325 on the top end of MLG F provides an anchor point for the integration of the cytogenetic, genetic, and physical maps of the soybean (PAGEL et al. 2004; WALLING et al. 2006).

Similarly to the soybean genome, the majority of model and crop plant genomes harbor one or two major 18S-5.8S-26S rDNA sites referred to as NORs, which are generally composed of several megabases. For example, H. vulgare ssp. spontaneum harbors two major 18S-5.8S-26S rDNA sites (TAKETA et al. 1999); O. sativa ssp. japonica contains one major site and O. sativa ssp. indica harbors two major sites (SHISHIDO et al. 2000); and A. thaliana has two major sites (COPENHAVER and PIKAARD 1996). The FISH and the genetic markers generated from the subrepeat variation in the NORs provide anchor points for the construction of cytogenetic, genetic, and physical maps of plant genomes as well as for breeding programs. The results of this study indicated that the SNP and microsatellite loci in the ITS and IGS sequences can be utilized to develop molecular markers for the genetic mapping of NORs in plants including soybeans harboring no notable subrepeats within the IGS region.

This work was supported by a grant (code no. 20050401034602) from the BioGreen 21 Program, Rural Development Administration, to S.-C.J. and partly by the Korea Research Institute of Bioscience and Biotechnology Research Initiative Program.

Sequence data from this article have been deposited with the GenBank Data Library under accession nos. EU118310–EU118313.

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