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Circumlineatus (cl) in common bean (Phaseolus vulgaris L.) is identified by a precipitation line in the seedcoat at the boundary of the white and colored zones. Cl is recessive and is expressed in partly colored seedcoats (t) with restricted patterns such as virgarcus. In this study, amplified fragment length polymorphism (AFLP) and single nucleotide polymorphism (SNP) markers, and the common bean genome sequence were used in combination with bulk segregant analysis and bidirectional selective genotyping to identify the genetic location of Cl. Markers were identified that cosegregated with Cl using Cl/Cl and cl/cl F3 and F5 progeny bulks from the cross t z cl G b v virgarcus BC3 5-593 · t z Cl G b v sellatus BC3 5-593. Two bands from an AFLP primer combination, which yielded unambiguous polymorphisms between the bulks, were cloned and sequenced. The two sequences were used to interrogate the common bean whole genome sequence identifying a region also found through cosegregation analysis using bidirectional selective genotyping with SNPs. Thus, the Cl gene was localized on Pv09 using cosegregating AFLP and SNP markers, and the physical location was confirmed with the whole genome sequence. The seedcoat colors and patterns of common bean cultivars are major attributes enabling consumers to identify various dry bean market classes (Ernest et al., 2005). Some of the important market classes have seedcoat patterns that are partly colored; i.e., part of the seedcoat has a non-white color, whereas the remainder is white. The expression of partly colored patterns is primarily controlled at the T locus, and the genotype t/t is required for expression of the trait. Additional genes besides t are required to express various types of partly colored patterns, viz., Cl, Z, Bip, J, and Fib (Bassett, 2007). Seed coat colors are controlled by 10 genes: P, [C R], Gy, Z, J, G, B, V, and Rk (Bassett, 2007). Prakken (1972) was the first author to report a new type of partly colored pattern from the cross ‘White J’ (genotype T cl) · ‘Soldaat K’ (genotype t Cl). This seedcoat trait was named circumlineatus because ‘‘each of the colour centres and even the smallest dots [were] bordered by a sharp precipitation-like line’’ (Prakken, 1972). Genotype t cl is required for expression of cl; hence, neither of Prakken’s parents expressed the trait, which first appeared in the F2. The data of Prakken (1972) supported the hypothesis that with t/t, the cl trait was controlled by a single additional gene that he gave the symbol Cl. There is a weak genetic linkage between the T and Cl loci of 36 cM (Prakken, 1972). Subsequently, gene T was located on linkage group B9 by McClean et al. (2002), and B9 was associated with chromosome Pv09 of common bean by Pedrosa-Harand et al. (2008). Thus, mapping of Cl has not been completed. Received for publication 27 Sept. 2013. Accepted for publication 7 Jan. 2014. This work is dedicated to an excellent scientist and exemplary person, Luwbia Aranda, a co-author in this study and a Master’s student at the University of Puerto Rico from Bolivia who died in a car accident at the end of her graduate career. These sequence data queried using the cloned AFLP products were produced by the U.S. Department of Energy Joint Genome Institute. We thank Abraham Montes and Adolfo Quiles for assistance with the field and greenhouse experiments and Drs. Linda Beaver and Damaris Santana Morant for assistance with the statistical analyses. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. Corresponding author. E-mail: Timothy.Porch@ars.usda.gov. J. AMER. SOC. HORT. SCI. 139(2):213–218. 2014. 213 Bassett (2004, 2007) summarized the interactions of three partly colored seedcoat patterns with four seedcoat colors in the presence of cl (with t) (Table 1). Bassett (2007) further clarified the anatomy of the precipitation line in partly colored seeds by describing a physical groove observed in the surface of the seed, an aspect of cl expression not reported by Prakken (1972). Expression of cl (with t) was suppressed by V in virgarcus and sellatus patterns but not with the Anasazi pattern (Table 1). Expression of cl (with t) was normal (obvious) for all combinations of seedcoat patterns and the non-black colors, except for variable expression (some seeds expressed cl, whereas others on the same plant did not) with a sellatus pattern in mineral brown color. Tagging and mapping have been completed on a number of seedcoat pattern and color genes in common bean, viz., T, Bip, C, Z, J, G, V, and Gy (Bassett et al., 1999, 2002a, 2002b; Bassett and McClean, 2000; Brady et al., 1998; McClean et al., 2002). In these studies, the polymorphisms were discovered with random amplified polymorphic DNA markers and then converted to more reliable sequence tagged site markers. These markers were then used to determine the location of loci on the common bean molecular genetic map (McClean et al., 2002). With the advent of the common bean genome sequence, SNP markers, and tools such as mutagenesis populations and TILLING (Porch et al., 2009), novel methods are available for identifying genes of interest. Although the genetics of Cl have been studied, the tagging of Cl and the determination of its genetic location have not been completed. The goal of this study was to confirm the genetics and identify the genetic map location of Cl. Materials and Methods POPULATION DEVELOPMENT AND CL GENETICS. A population segregating for cl was created from the cross, t z cl G b v virgarcus BC3 5-593 · t z Cl G b v sellatus BC3 5-593 at the University of Florida in Gainesville. A selection was made there in the BC3-F2 for a virgarcus (z/z) partly colored seedcoat type without the circumlineated border. In plot 2-44 in Gainesville, FL, the F3 generation segregated for cl (Fig. 1) but was true breeding for virgarcus. A single seed of each F3 was planted under greenhouse conditions at the U.S. Department of Agriculture, Agricultural Research Service (USDA-ARS) in Mayaguez, Puerto Rico, on 23 Feb. 2004 and all harvested seed of each plant were scored for Cl. Progeny testing was completed in the F4 generation. A total of 49 F4 progenies were planted on 3 Nov. 2004 in the field in Isabela, Puerto Rico, with an average of 10.3 plants per F4 family for a grand total of 504 plants. Evaluation of Cl was performed on all seed of each plant harvested. AFLP AND BULK SEGREGANT ANALYSIS. DNA was extracted from leaf tissue of each of the F3 lines using DNeasy extraction kits (Qiagen, Valencia, CA) at USDA-ARS in Mayaguez, Puerto Rico. AFLP analysis was performed using a AFLP pre-amplification kit (LI-COR, Lincoln, NE) and selective amplification was performed according to Vos et al. (1995) using labeled Eco RI primers (LI-COR) and non-labeled Mse I primers (Illumina, San Diego, CA). The AFLP products were separated on a sequencer (4300; LI-COR). Bulk segregant analysis (BSA) (Michelmore et al., 1991) was performed on bulks of four cl/cl or four Cl/Cl genotypes as determined by progeny testing. Two bulks of each genotype were prepared. Eco RI primers, one labeled with IR700 and the other with IR800, were amplified with a non-labeled Mse I primer. Both sets of primers contained three selective nucleotides. BSA was performed using 256 primer-pair combinations, eight Eco RI and 32 Mse I primers. Those primers revealing polymorphisms were then tested on the whole population. Cosegregating markers were identified and the genetic distance between the AFLP marker and the locus was calculated on the population derived from the 2-44 F2:3 family using the Haldane mapping function. POLYMORPHIC AFLP BAND ANALYSIS AND MAPPING. The AFLP analysis and electrophoresis were performed as described previously using labeled primers for the selected AFLP primers at the USDA-ARS in Mayaguez, Puerto Rico. The AFLP bands associated with the cl trait were located and isolated from polyacrylamide gels by interrupting electrophoresis of the sequencer (4300; LI-COR) as the bands entered the real-time gel display window. The bands were excised from their estimated position in the gels using a razor blade and the AFLP bands were extracted and then reamplified using the same AFLP protocol and primers, as described previously, and confirmed by electrophoresis of the reamplified bands Table 1. Levels of expression of the circumlineatus gene (cl) in three partly colored seedcoat patterns in four seedcoat colors of common bean; i.e., the interaction of cl with genes for pattern and color. Seedcoat colors and genotypes Seedcoat types Black Mineral brown Yellow brown Chamois Pattern Genotypes G B V G B v G b v g b v Anasazi t cl Z bip Visible Visible Visible Visible Virgarcus t cl z Bip Not visible Visible Visible Visible Sellatus t cl z Bip Not visible Variable Visible Visible Variable for groove expression, depending on background genotype for seedcoat color. Refer to circumlineatus phenotype in Figure 1. Visible is normal expression that is scorable by eye, whereas variable expression indicates that some seeds expressed cl, whereas others on the same plant did not. Fig. 1. Circumlineatus phenotype with precipitation line indicated by the arrow in representative lines from the t z cl G b v virgarcus BC3 5-593 · t z Cl G b v sellatus BC3 5-593 population of common bean. 214 J. AMER. SOC. HORT. SCI. 139(2):213–218. 2014. alongside the original AFLP products. The bands were each purified from the polymerase chain reaction (PCR) products using a PCR purification kit (Qiagen) and then ligated into the pCR 2.1 TOPO cloning vector following the manufacturer’s instructions (Invitrogen, Carlsbad, CA). Miniprepped plasmid DNA was tested for insert size, after blue/white selection, using plasmid specific M13 primers as well as a non-labeled version of the original AFLP primer

journal of the american society for horticultural science

Genetics and Mapping of the Cl Gene for Circumlineated Pattern in Common Bean Using AFLP-based Bulk Segregant Analysis and SNP-based Bidirectional Selective Genotyping