DOI: 10.25338/b8g30v
title: Evolution of SSR diversity from wild types to U.S. advanced cultivars in the Andean and Mesoamerican domestications of common bean (Phaseolus vulgaris)

description: Progress in common bean breeding requires the exploitation of genetic
 variation among market classes, races and gene pools. The present study
 was conducted to determine the amount of genetic variation and the degree
 of relatedness among 192 selected common bean advanced cultivars using 58
 simple-sequence-repeat markers (SSR) evenly distributed along the 11
 linkage groups of the Phaseolus reference map. All the lines belonged to
 commercial seed type classes that are widely grown in the USA and include
 both dry bean and snap beans for the fresh and processing markets. Through
 population structure, principal components analyses, cluster analysis, and
 discriminant analysis of principal components (DAPC), Andean and
 Mesoamerican genotypes as well as most American commercial type classes
 could be distinguished. The genetic relationship among the commercial
 cultivars revealed by the SSR markers was generally in agreement with
 known pedigree data. The Mesoamerican cultivars were separated into three
 major groups - black, small white, and navy accessions clustered together
 in a distinct group, while great northern and pinto clustered in another
 group, showing mixed origin. The Andean cultivars were distributed in two
 different groups. The kidney market classes formed a single group, while
 the green bean accessions were distributed between the Andean and
 Mesoamerican groups, showing inter-gene pool genetic admixture. For a
 subset of 24 SSR markers, we compared and contrasted the genetic diversity
 of the commercial cultivars with those of wild and domesticated landrace
 accessions of common bean. An overall reduction in genetic diversity was
 observed in both gene pools, Andean and Mesoamerican, from wild to
 landraces to advanced cultivars. The limited diversity in the commercial
 cultivars suggests that an important goal of bean breeding programs should
 be to broaden the cultivated gene pool, particularly the genetic diversity
 of specific commercial classes, using the genetic variability present in
 common bean landraces.
description: <b>Plant
 materials</b>   A
 total of 192 common bean advanced cultivars representing the most popular
 commercial industrial seed types classes in the USA and released in the
 last ca. 50 years were included in this study. These genotypes were
 obtained from 20 American public and private breeding programs and have
 all been extensively used by breeders and geneticists in the USA and
 around the world. More specifically, the sample included 21 black, 29 navy
 and two small-white cultivars with small seeds (&lt;25 g 100 seed
 weight<sup>-1</sup>), belonging to race Mesoamerica; 61 pinto
 and 27 great northern cultivars with medium -seeds (25-40 g 100 seed
 weight<sup>-1</sup>), belonging to the race Durango; 18 light
 red kidney, 11 dark red kidney, 3 white kidney, and 20 green beans with
 large-seeds (&gt;40 g 100 seed weight<sup>-1</sup>),
 belonging to the Andean race Nueva Granada. As standard genotypes, three common
 bean accessions were also included in the analysis: Midas, a typical
 Andean green bean type; BAT93, a typical Mesoamerican breeding line with
 multiple disease resistance [(Veranic 2 x Tlalnepantla 64) x (Jamapa x
 Tara)], which is also the source of the Mesoamerican genome reference
 sequence [2]; and Jalo EEP558, a typical Andean cultivar, which was
 released by a breeding program in Brazil (EPAMIG, Patos de Minas, Minas
 Gerais). BAT93 and Jalo EEP558 were also used as parents to obtain a
 recombinant inbred population that was used to develop a consensus
 molecular linkage map of common bean [26], in which over 170 SSR markers
 have been mapped [27]. A complete list of the lines, including information
 on pedigree, market class, and/or origin can be found in Table
 S1. Seeds were grown
 in a greenhouse at the University of California in Davis. After 15 days,
 the primary leaves were harvested and stored overnight at – 80
 °C.   <b>Genomic DNA extraction, PCR
 and SSR genotyping</b>   The
 frozen leaf tissue samples were lyophilized for around 48 hours using
 VirTis Sentry 2.0. and ground to a fine powder. Genomic DNA was then
 extracted from the young leaf tissue using the Qiagen DNeasy Plant Kit
 (Qiagen, Valencia, CA), and following the protocols provided by the
 manufacturer. DNA was quantified with a DyNA Quant 200 fluorometer (Hoefer
 Pharmacia Biotech, San Francisco, CA) and diluted to a concentration of
 approximately 10 ng µL<sup>–1</sup> for polymerase chain
 reaction (PCR) amplification. The amount of genetic diversity in the 192
 samples was then assessed with molecular marker analysis. A set of 58 SSR
 markers were selected based on their wide distribution over the
 <i>Phaseolus</i> genome and their high polymorphism
 information content (PIC) values [27–29]. More information about the SSR
 loci used, including the primer pair sequences, the repeat motif, and the
 chromosomal locations, can be found in Table S2. The SSRs analysis was conducted using
 an economical fluorescent tagging method described by Schuelke [30] in
 which an M13 reverse sequence tail (TGTAAAACGACGGCCAGTATGC) was added to
 the 5’ end of each forward SSR primers. The fluorescent dyes, 6-FAM, PET,
 and VIC, were attached to the 5’ end of the complementary
 (TGTAAAACGACGGCCAGT) M-13 universal primer sequence. For amplification,
 PCR reaction consisted of about 30 ng of genomic DNA, 200 µM dNTP (New
 England Biolabs), 0.04 µM forward primer with M-13 universal sequence
 tail, 0.16 µM reverse primer, 0.16 µM M-13 labeled fluorescent dye (Sigma
 Life Science), one unit of standard ThermoPol (Taq) reaction buffer with 2
 mM MgSO4, and one unit of Taq polymerase (New England Biolabs). The PCR
 program consisted of 5 minutes at 94 °C, 30 cycles of 30 seconds at 94 °C,
 45 seconds at 56 °C, and 45 seconds at 72 °C followed by 8 cycles of 30
 seconds at 94 °C, 45 seconds at 53 °C, and 45 seconds at 72 °C with a 10
 minutes final extension period at 72 °C. After dilution to a standard
 concentration, the amplified DNA samples were separated and sized in
 multiplex fashion depending on their expected size variation and were
 analyzed on Applied Biosystems 3730 DNA automatic analyzers.
 Genotypes of markers were
 determined using the GeneMarker program (version1.95; SoftGenetics). When
 markers produced more than one peak, the peaks with clearly separated size
 ranges were scored independently as a different locus. This was noticed
 for three of the SSRs (PVag004, BM188, and BMd1). For each SSR locus and
 for the whole set of accessions, the total number of alleles observed were
 recorded and reported in a data set. Missing data were recorded when there
 was no detectable peak in the target size region for the marker. The data
 set was converted into Powermarker [31] and STRUCTURE [32] input format
 using the program Convert [33].