Copyright Ó 2008 by the Society of America DOI: 10.1534/genetics.107.081620

Note

Genetic Linkage Map of the Nucleolus Organizer Region in the Soybean

Kiwoung Yang*,† and Soon-Chun Jeong*,1 *BioEvaluation Center, Korea Research Institute of Bioscience and Biotechnology, Cheongwon, Chungbuk 363-883, Republic of Korea and †Molecular Biotechnology Major, Chonnam National University, Gwangju 500-757, Republic of Korea Manuscript received September 6, 2007 Accepted for publication October 30, 2007

ABSTRACT 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.

HE genetic map of the soybean ½Glycine max (L.) distribution, which were numbered in de- T Merr., which is an economically important legume, scending order of 1–20 (Singh and Hymowitz 1988). is one of the most densely populated maps among plants, The harboring the satellite was designated with .3000 published markers (Choi et al. 2007). as (Singh and Hymowitz 1988). However, its cytogenetic studies have lagged behind Fluorescent in situ hybridization (FISH) using the rDNA those of rice (Oryza sativa L.), maize (Zea mays L.), barley as a probe verified that the satellite region of chromo- (Hordeum vulgare L.), and tomato (Lycopersicon esculentum some 13 is the nucleolus organizer region (NOR) Mill.). Thus, the relationships between soybean molec- (Griffor et al. 1991). FISH resulted in the detection of a ular linkage groups (MLG) and remain pair of NORs on the short arm of chromosome 13 in incompletely understood (Cregan et al. 2001; Zou et al. the soybean and its progenitor, G. soja, as well as three 2003). The haploid chromosome number of the soy- strong fluorescent signals in the soybean that are bean plant is 20 (Veatch 1934), which is almost two trisomic for chromosome 13. Subsequently, MLG F times that observed in major diploid crops such as rice was assigned to chromosome 13 with a set of primary (n ¼ 12), maize (10), barley (7), and tomato (12) as well trisomics and simple sequence repeat markers (Cregan as in research plants including Arabidopsis thaliana (5), et al. 2001). However, the accurate genetic location of the Lotus japonicus (6), and Medicago trancatula (8). The soy- soybean NOR on MLG F remains to be determined. bean mitotic metaphase chromosomes evidence small The NOR loci harbor from hundreds to thousands size variations ranging from 1.42 to 2.84 mm and are of 18S-5.8S-26S ribosomal RNA gene units, which are symmetrical without karyotypically visible landmarks clustered into tandem repeats (Flavell and O’Dell (Sen and Vidyabhusan 1960). However, the pachytene 1979). Each of the repeats codes for a ribosomal RNA chromosome analysis of an F1 hybrid between soybean gene unit. An intergenic spacer (IGS) region separates and Glycine soja Sieb. and Zucc., a wild annual progenitor the units of adjacent repeats. The inter- of the soybean, evidenced distribution genic region has been determined to be highly variable on either side of the , small structural in a range of plants: Triticum aestivum (Apples and differences, and a satellite chromosome, thus allowing Dvorˇa´k 1982), Triticum dicoccoides (Flavell et al. 1986), for the construction of chromosome maps on the basis H. vulgare (Saghai Maroof et al. 1984), Vicia faba of chromosome length and and hetero- (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 Sequence data from this article have been deposited with the GenBank development of molecular markers for the genetic Data Library under accession nos. EU118310–EU118313. mapping of these NORs in several plant species (e.g., 1Corresponding author: BioEvaluation Center, Korea Research Institute Saghai Maroof et al. 1984; Snape et al. 1985; Zimmer of Bioscience and Biotechnology, 685-1 Yangcheongri, Ochangup, openhaver ikaard Cheongwon, Chungbuk 363-883, Republic of Korea. et al. 1988; C and P 1996). However, E-mail: [email protected] the soybean IGS appears to harbor no notable subrepeat

Genetics 178: 605–608 ( January 2008) 606 K. Yang and S.-C. Jeong

TABLE 1 Primer sequences for PCR amplification and sequencing of the soybean ITS1 and IGS regions

Target Name Specificity Sequence (59 / 39) ITS1 ITS1-F Forward CCACTGAACCTTATCATTTA ITS1-R Reverse GCAATTCACACCAAGTATC IGS IGS-F Forward GCTGCCACGATCCACTGAG IGS-R Reverse ACATGCATGGCTTAATCTTT IGS-seq-F Sequencing AAAAACCACGAAGTTGAC IGS-seq-R Sequencing CAAAAACATCCCGATTAC IGS-seq-rcF Sequencing TGCTTAAGTTTTTCCATGT IGS-seq-rcR Sequencing TAGTGGTGTCAAGTCTTGC DNA fragments corresponding to ITS1 were amplified via PCR using the ITS1-F primer, which occurs at the 39 part of 18S rDNA, and the ITS1-R primer, which occurs at the 59 portion of 5.8S rDNA, from the Hwangkeum and IT182932 variants. These priming sites were identified via the examination of a Cicer arietinum rDNA sequence (GenBank accession no. AJ577394). DNA fragments corresponding to an IGS were PCR amplified using the IGS-F primer, which occurs at the 39 portion of 18S rDNA, and the IGS-R primer, which occurs at the 59 portion of 5.8S rDNA, from the Hwangkeum and IT182932 variants. These priming sites were iden- tified via the examination of IGS sequences reported by Nickrent and Patrick (1998) and soybean genomic survey sequences, including GenBank accession nos. ED789183 and ER964634. The PCR products were then prepared for sequencing via the excision of a band of the expected size from an agarose gel, followed by purification as described by Jeong et al. (2002). The sequencing of IGS PCR products was completed using the following internal primers: IGS-seq-F, IGS-seq-R, IGS-seq-rcF, and IGS-seq-rcR, in addition to the IGS-F and IGS-R PCR primers. variations (Nickrent and Patrick 1998) among G. max genotype of the locus using an allele-specific PCR variants and between G. max and G. soja, with a GT method, as described by Jeong and Saghai Maroof dinucleotide repeat and several single nucleotide poly- (2004) (Table 2). The IGS sequence of Hwangkeum morphism (SNP) loci. In the coding repeat units, other (GenBank accession no. EU118312) harbors 9 single- variable regions, referred to as internal transcribed spa- base substitutions, 2 single-base deletions, and one 14- cers (ITS), are located on either side of the 5.8 S rRNA base deletion due to a microsatellite repeat variation gene, which are designated as ITS1 and ITS2. The nucle- relative to that of IT182932 (GenBank accession no. otide sequences of the ITS have proven quite useful for EU118313). One of the single nucleotide polymor- phylogenetic studies in many angiosperm groups, due phism loci was genotyped via the design of two specific primarily to their usefulness in resolving relationships at forward primers and a common reverse primer as de- the species level (Arnheim 1983; Wojciechowski et al. scribed above for the ITS1, and the microsatellite repeat 1993; Baldwin et al. 1995). Here, we describe the variation locus was genotyped via the design of a primer genetic localization of the soybean NOR using SNP pair for PCR amplification (Table 2). and microsatellite markers that were developed from a The allele-specific PCR primer sets designed from the SNP locus between the ITS1 nucleotide sequences and ITS1 and IGS sequences generated allele-specific PCR from a SNP locus and a microsatellite repeat locus be- bands, which evidenced the expected ratio of 1:1 (x2 ¼ tween the IGS nucleotide sequences of the soybean and 2.7, P ¼ 0.1) in the HI population as codominant alleles G. soja. of a Mendelian locus. The SNP marker from the ITS1 We utilized a population of 113 F12 recombinant was designated as SN307 and the SNP marker from the inbred lines generated via an interspecific cross between IGS was designated as SN314. The microsatellite mar- the G. max cv. ‘‘Hwangkeum’’ and the G. soja Siebold & ker, designated SM318, cosegregates with the SN307 Zucc. line ‘‘IT182932’’ (hereafter referred to as the HI and SN314 markers in the HI population. The three population). In this HI population, which harbors .400 markers were mapped 0.4 cM away from Satt325 on the mapped marker loci (S.-C. Jeong, unpublished results), top end of MLG F (Figure 1A). all 20 of the soybean MLGs have been identified. DNA The three markers were further tested on 10 soybean sequences corresponding to ITS1 and IGS were de- (G. max) cultivars to investigate their applicability for termined from the Hwangkeum and IT182932 variants mapping the NOR in a population made from a cross (Table 1). The ITS1 sequence of Hwangkeum (Gen- between G. max cultivars. The microsatellite marker Bank accession no. EU1183310) harbors one single- SM318 revealed a novel allele, which is 2 bases longer base substitution relative to that of IT182932 (GenBank than the size of the Hwangkeum allele (Figure 1B). The accession no. EU118311). This SNP locus was geno- size variation was verified by sequencing analysis (Figure typed via the design of a set of two specific forward 1C). SN307 and SN314 revealed the same banding primers and a common reverse primer to determine the patterns as those of Hwangkeum (data not shown). Note 607

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

Description of Name of Product Target polymorphism primer Primer specificity Primer sequence (59 / 39)a size (bp) ITS1 SNP, T(H)/C(I) SN307-H-F Forward specific to H CCCGCGAACTTGTTTACT 210 SN307-I-F Forward specific to I CCCGCGAACTTGTTTACC SN307-com-R Common reverse CCGAGAGTCATTGTATGTAA IGS SNP, A(H)/C(I) SN314-H-F Forward specific to H TATGGGTTTTCACCCACC 169 SN314-I-F Forward specific to I TATGGGTTTTCACCCAAA SN314-com-R Common reverse CAAAAACATCCCGATTAC GT and GC repeats, SM318-F Forward CATAACATGGCAAGACAC 115(H), GT(11)GC(5)(H)/ 129 (I) GT(7)GC(2)(I) SM318-R Reverse GGTGGTGGATCATCATGTCA H, Hwangkeum; I, IT182932. a Additional base mismatch introduced and single nucleotide polymorphic site are underlined.

The results indicated that, at least, the SM318 marker Similarly to the soybean genome, the majority of can be utilized to map the NOR locus in a wide range of model and crop plant genomes harbor one or two major soybean populations. 18S-5.8S-26S rDNA sites referred to as NORs, which are The genetic mapping position of the soybean NOR generally composed of several megabases. For example, was reported to be a telomeric end of the short arm of H. vulgare ssp. spontaneum harbors two major 18S-5.8S- chromosome 13, which corresponds to soybean MLG F, 26S rDNA sites (Taketa et al. 1999); O. sativa ssp. as has been demonstrated by the results of FISH and japonica contains one major site and O. sativa ssp. indica primary trisomics studies (Singh and Hymowitz 1988; harbors two major sites (Shishido et al. 2000); and Griffor et al. 1991; Cregan et al. 2001). However, it A. thaliana has two major sites (Copenhaver and Pikaard could not be predicted which end of the MLG F harbors 1996). The FISH and the genetic markers generated the NOR, because the genetic mapping positions of the from the subrepeat variation in the NORs provide on the MLG F are not currently known. anchor points for the construction of cytogenetic, ge- Thus, our finding that the soybean NOR is mapped netic, and physical maps of plant genomes as well as for 0.4 cM away from Satt325 on the top end of MLG F breeding programs. The results of this study indicated provides an anchor point for the integration of the that the SNP and microsatellite loci in the ITS and IGS cytogenetic, genetic, and physical maps of the soybean sequences can be utilized to develop molecular markers (Pagel et al. 2004; Walling et al. 2006). for the genetic mapping of NORs in plants including

Figure 1.—Map position of SN307, SN314, and SM318 and diversity of SM318. (A) The ge- netic map of the top end of the soybean molecu- lar linkage group F, which evidences the locations of the SN307, SN314, and SM318 markers devel- oped from the nucleolus organizer region. Map- maker 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 and centromere positions proposed by Singh and Hymowitz (1988) on the basis of the positions of the NOR and heterochromatic zone. (B) Poly- acrylamide 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 Hwang- keum and shorter than that of IT182932. 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