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Tetrasomic Linkage Mapping of RFLP, PCR, and Isozyme Loci in Lotus corniculatus L.

^a USDA-ARS, Rice Research Unit, 1509 Aggie Dr., Beaumont, TX 77713
^b National Forage Seed Production Res. Cntr., USDA-ARS, 3450 SW Campus Way, Corvallis, OR 97331
^c USDA-ARS, Plant Genetics Research Unit, Univ. Missouri, Columbia, MO 65211

View larger version (39K): [in a new window]   Fig. 1. Composite image of DNA amplification products using the CTG inter-simple sequence repeat primer. The approximated molecular weights of the 13 dominantly scored markers are shown to left, lane numbers shown on top, and molecular weight marker sizes shown to right of image. Markers CTG-1250, -850, -650, -410, and -340 originate from Moroccan male parent (G31276) and markers CTG-1500, -1200, -1100, -950, -700, -570, -460, and -360 originate from the autogamous female parent (AG-S4). Lane identities are as follows: Lane 1, G312762; Lane 2, AG-S4; Lane 3, AG-S4 x G31276 F1 hybrid; Lanes 4 to 24, F2 progeny; Lane 25, molecular weight ladder.

View larger version (37K): [in a new window]   Fig. 2. Composite autoradiograph image of restriction fragment length polymorphism (RFLP) marker 3026. Molecular weight marker is shown to left, lane numbers labeled on top, and RFLP allele positions shown on right of image. Plant identity and interpreted genotypes are as follows: Lane 1—Moroccan male parent (G31276), BBDD; Lane 2—autogamous female parent (AG-S4), AACC; Lane 3—AG-S4 x G31276 F1 hybrid, ABCD; Lanes 4 to 24—F2 progeny, AADD, ABCD, ABDD, ACCD, ABCD, AACD, BBCD, ABBC, ABCC, ABCD, ABDD, ABCD, AABD, ABCC, AABD, BBDD, AACD, AABB, ACCD, CCDD, ABCD. Note that each band does not give equal autoradiographic intensity when present as a single allele (A > B C D) and that genotypes are interpreted from intensity ratios relative to each other. Each RFLP allele has a double-banded pattern. Both bands of the A and C alleles are next to each other and the B and D alleles have a shared second band labeled in parenthesis as B,D near the top of the image.

View larger version (89K): [in a new window]   Fig. 3. Composite autoradiograph image of restriction fragment length polymorphism (RFLP) marker 2043. Molecular weight marker is shown to left, lane numbers labeled on top, and RFLP allele positions shown on right of image. Plant identity and interpreted genotypes are as follows: Lane 1—Moroccan male parent (G31276), AAAA; Lane 2—autogamous female parent (AG-S4), BBCC; Lane 3—AG-S4 x G31276 F1 hybrid, AABC; Lanes 4 to 24—F2 progeny, ABCC, AAAC, ABBC, AACC, ABBC, AAAB, AACC, AABC, AABC, AABC, AACC, AABB, AACC, ABCC, AABC, BBCC, ABBC, AABC, ABCC, AAAB, AABC. Note that each band does not give equal autoradiographic intensity when present as a single allele (C > B A) and that genotypes are interpreted from intensity ratios relative to each other.

View larger version (132K): [in a new window]   Fig. 4. Genetic map of Lotus corniculatus displaying seven linkage groups identified by molecular marker analysis. Marker alleles are labeled to the right of each linkage group and Kosambi units of genetic distances between markers are labeled to the left. Restriction fragment length polymorphism (RFLP) markers are prefixed with the digits 2 or 3. Randomly amplified polymorphic DNA (RAPD) markers are prefixed with the letters "op," followed by the Operon decamer primer used and the approximate molecular weight of the amplification product. Inter-simple sequence repeat (ISSR) markers are prefixed with three capital letters designating the 19-mer primer used (sequence shown in Materials and Methods) and followed by the approximate molecular weight of the amplification product. Isozyme markers are shown in italics and the glutathione reductase-sequence tagged site (GR-STS) is labeled gr-hae. Solid lines connect markers linked at LOD = 3.0 or greater, dashed lines connect markers linked at LOD score 2.5 to 3.0, and broken lines are expected locations of unlinked markers. Fifteen unlinked markers are not shown. Six composite linkage groups constructed from combined data of four homologous linkage groups (labeled A–D) are shown on far right. Homologous Groups A and B are derived from the rhizomatous Moroccan accession and Groups C and D are derived from the autogamous accession. Alleles of RFLP markers are suffixed with lower case letters, while loci of RFLP markers are suffixed with capital letters on composite linkage groups. Five loci co-segregating with locus 30117B/2012C are not shown separately on the Composite Group 1 map, but are shown on individual homologous linkage group maps. The RFLP locus 30107 on Groups 5 and 6 is shown as locus 30107A/B to indicate its apparent duplication and lack of separation between these two groups. Linkage Group 7, derived from the autogamous parent, has not been assigned to any composite linkage groups and is shown separately.