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Microsatellites
Developed and Implemented for Common Bean
 Microsatellites
are polymerase chain reaction (PCR) based markers that detect
polymorphisms at loci with simple sequence repeats. They are
also single-locus markers that are specific to given places
in the genome. Easy to manipulate, microsatellites are useful
in marker-assisted-selection (MAS) strategies requiring relatively
high throughput. Although microsatellites have been developed
for a wide range of plant species, few were available for
common bean before we started this work.
Various techniques exist for discovering new microsatellite
markers from anonymous genomic sequences. All these techniques
rely on the availability of DNA libraries. In our first experiments,
we developed microsatellites from an enrichment method that
produced two libraries for di-nucleotide motifs (BM microsatellites)
(Gaitán et al., 2002). In a second set of experiments
we screened gene and DNA sequences and libraries to study
the frequency with which different microsatellite motifs occur
in common bean (BMd microsatellites) (Blair et al., 2003).
We also screened other common bean microsatellites and a large
number of SSR markers that were developed for soybean and
cowpeas to adapt microsatellites available for other Phaseoleae
legume crops to common bean (Blair et al., 2002). A list of
microsatellites that work well for common bean is provided
here with information
on genetic map location, repeat motif, expected fragment size
and primer sequences.
Because microsatellites can distinguish between closely related
genotypes within species, they have been useful for evaluating
the genetic diversity of Phaseolus vulgaris from different
parts of the CIAT germplasm collection. We have applied microsatellites
to the study of genetic diversity in common bean landraces
from Bolivia, Brazil, Colombia, China, Cuba, Nicaragua and
other countries of the primary and secondary centers of diversity
for the crop (Gómez et al., 2004; Díaz et al.,
2005). Given that many of the microsatellites we have developed
show cross species amplification we have applied the microsatellites
to other species of Phaseolus, especially tepary bean
(P. acutifolius), for which little variation is detected
by other methods. The genetic diversity of 150 microsatellite
loci has also been evaluated in parental surveys of common
bean that provided the basis for selecting the most polymorphic
markers (Blair et al., 2006). This information is being incorporated
into a new molecular genetics database called MOLPHAS,
which includes conditions for amplification and examples of
successful allele detection.
Microsatellite
Publications from CIAT
Blair
MW, Giraldo MC, Buendía HF, Tovar E, Duque MC, Beebe
SE (2006) Microsatellite
marker diversity in common bean (Phaseolus vulgaris
L.) Theor Appl Genet 113:100-109.
Blair
MW, Pedraza F, Buendía HF, Gaitán-Solís
E, Beebe SE, Gepts P, Tohme J (2003) Development
of a genome-wide anchored microsatellite map for common bean
(Phaseolus vulgaris L.) Theor Appl Genet 107:1362-1374.
Blair
MW, Pantoja W, Pedraza F, Cregan P, Fatokun C (2002) Legume
microsatellites in common bean. Ann. Rep. of the Bean Improv
Coop 45:242-244.
Díaz
LM, Díaz JM, Blair MW (2005) Diversidad genética
de fríjol común (Phaseolus vulgaris
L.) en Colombia. Fitotecnia Colombiana 5:28-36.
Gaitán-Solís
E, Duque MC, Edwards KJ, Tohme J (2002) Microsatellite repeats
in common bean (Phaseolus vulgaris): Isolation, characterization,
and cross-species amplification in Phaseolus ssp.
Crop Sci 42:2128-2136.
Gómez
OJ, Blair MW, Frankow-Lindberg BE, Gullberg U. (2004) Molecular
and phenotypic diversity of common bean landraces from Nicaragua.
Crop Sci 44:1412-1418.
Capacity Building
Scientists from Bolivia (3), Brazil (1), China (1), Colombia
(5), Cuba (1), Mexico (1) and USA (2) have been trained in
microsatellite marker use at CIAT and have applied this information
for their research projects. Future collaborations are welcome.
Applications
SSR markers developed at CIAT have been successfully applied
in:
- Evaluation of the CIAT core collection
- Genetic diversity studies for collections from Bolivia,
Brazil, China and Colombia
- QTL mapping of tolerance to Thrips
- QTL mapping of climbing ability
- QTL mapping of adventitious rooting
- Advanced backcross population analysis
Contact:
Matthew Blair
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The
Genetics of Micronutrient Accumulation in Common Bean
Legumes
provide essential micronutrients that are found only in low
amounts in cereals or root crops. An ongoing project has shown
that bean seeds are variable in the amount of minerals (e.g.,
iron and zinc), vitamins, and sulfur amino acids that they
contain, and that these traits are likely to be inherited
quantitatively. With the hope of selecting for higher mineral
content on a regular basis, we were interested in tagging
quantitative trait loci (QTLs) that control the accumulation
of these minerals. In our initial studies, we used two populations
representing one Andean × Andean and one Mesoamerican
× Mesoamerican cross. Phenotypic data were obtained
by analyzing both the parents and the recombinant-inbred lines
for iron and zinc content by ICP (inductively coupled plasma).
The QTLs were mapped with microsatellite and RAPD markers
that were used to construct separate genetic maps for each
population.
| Analysis
of quantitative trait loci (QTLs) for iron (Fe) and zinc
(Zn) micronutrient content in seed of an Andean population
of common bean. The vertical axis represents the positive
allele from parent A (G 21242) or parent B (G 21078) and
significance (P value) of QTL effects. Chromosomes (b)
or unidentified linkage groups (LG) are identified and
the most significant QTLs are circled. |
QTLs
were found for iron and zinc content in both populations.
The positive markers varied in their level of significance
and the proportion of variance in the mineral content that
they explained. The most significant QTLs explained up to
12% of the variance in mineral content. In some cases, the
QTLs for both minerals occurred jointly at the same marker.
In other cases, there were QTLs specific for each mineral.
The QTLs were generally found in similar locations of the
same chromosomes in both populations. Most of the positive
QTLs were associated with alleles from the high mineral parent;
hence, some QTLs for the accumulation of both minerals may
be genetically linked or pleiotropic, controlling both traits
at once. If the same QTLs contribute simultaneously to both
iron and zinc content, it may be easy to select for these
traits jointly. It also appears that high mineral content
parents provide most of the genes for high mineral content
to their progeny, while low mineral parents provide only a
few additional genes for mineral content.
We
have also begun studying the common bean genes involved in
getting iron and zinc from the root zone to the grain. Several
genes from other legumes or from model species could qualify
as candidate genes for the control of micronutrient accumulation
and storage in the bean seed. Among the most likely is phytoferritin,
which is the major storage form of iron in all tissues, including
seeds. We studied both the expression and genetics of phytoferritin
production in beans.
The
protein was identified in ground bean-seed extracts, and a
sequence characterized amplified region (SCAR) marker was
developed to map the gene or genes encoding phytoferritin
in beans. Other candidate genes that we will consider are
involved in the production and processing of nicotianamine,
a polyamine known to function in scavenging iron from the
root zone in grasses and may be involved in other iron-related
functions in plants more generally, including possibly the
transport of iron.
The
present work will, hopefully, permit us to focus on certain
parts of the genome to determine if desirable alleles for
higher mineral content are located at the same loci in additional
populations developed specifically for this purpose. We also
plan to integrate the information about the map locations
of QTLs for micronutrients with those for other agronomic
traits that we have been studying, so that we can select for
the best advanced lines from crosses with high micronutrient
lines, using marker-assisted selection.
Contact:
Matthew Blair
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Genetic
Diversity Characterized
 We
continue making progress in understanding the genetic diversity
of Phaseolus species. Specific projects have dealt
with the following aspects of Phaseolus diversity:
The CIAT core collection for P. vulgaris has been
evaluated for RAPD polymorphism and a subset of over 600 genotypes
evaluated for SSR polymorphism as part of GCP genotyping activities.
Specific National collections are being analyzed as well both
for in situ and ex situ collections. One such study used SSR
markers to compare landraces in genebank storage compared
to those in farmers' field in Nicaragua. Other studies are
analyzing the diversity of common bean collections in Bolivia,
Brazil, China, Colombia, Cuba, Rwanda and other countries.
In addition, cross species amplification of microsatellites
has been determined for wild and cultivated genotypes of common
bean, P. coccineus, P. polyanthus, P. acutifolius,
and P. lunatus. A basis was thus provided for using
microsatellites over a wide range of genetic diversity studies
and for comparing results between these.
Diversity assessments for other species within the genus
include 1) the evaluation of the core collections of P.
coccineus and P. polyanthus with AFLP markers,
demonstrating that very little structure exists in these two
species, although Mexican and Guatemalan accessions of P.
coccineus separate slightly, and an ecotype of P. polyanthus
exists in South America; 2) evaluation of diversity of the
CIAT collection of tepary bean (Phaseolus acutifolius
Asa Gray) analyzed with AFLP markers and microsatellites in
order to (i) understand the relationships with a closely related
wild species P. parvifolius Freytag, and (ii) re-assess
the status of botanical varieties var. acutifolius,
var. latifolius, and var. tenuifolius; and 3)
AFLP fingerprinting of Phaseolus lunatus and wild relatives
from South America.
A database is being created on the use of SSR markers for
diversity assessment in Phaseolus at the Phaseolus Molecular
Diversity Network (MOLPHAS)
Web Site.
Genetic
Diversity Publications from CIAT
Articles for studies of diversity in Phaseolus species
include:
Beebe,
SE, Rengifo J, Gaitán-Solis E, Duque MC, Tohme J (2001)
Diversity and origin of Andean landraces of common bean. Crop
Sci 41:854-862.
Beebe
SE, Skroch PW, Tohme J, Duque MC, Pedraza F, Nienhuis J (2000)
Structure of genetic diversity among common bean landraces
of middle American origin based on correspondence analysis
of RAPD. Crop Sci 40:264-273.
Blair
MW, Giraldo MC, Buendía HF, Tovar E, Duque MC, Beebe
SE (2006) Microsatellite
marker diversity in common bean (Phaseolus vulgaris
L.) Theor Appl Genet 113:100-109.
Caicedo
AL, Gaitán E, Duque MC, Toro Chica O, Debouck DG, Tohme
J. (1999) AFLP fingerprinting of Phaseolus lunatus
L. and related wild species from South America. Crop Sci 39:1497-1507.
Díaz
LM, Blair MW (2006) Race structure within the Mesoamerican
gene pool of common bean (Phaseolus vulgaris L.) as
determined by microsatellite markers. Theor Appl Genet (in
press).
Gaitán-Solís
E, Duque MC, Edwards KJ, Tohme J (2002) Microsatellite repeats
in common bean (Phaseolus vulgaris): Isolation, characterization,
and cross-species amplification in Phaseolus ssp. Crop
Sci 42:2128-2136.
Gómez
OJ, Blair MW, Frankow-Lindberg BE, Gullberg U. (2004) Molecular
and phenotypic diversity of common bean landraces from Nicaragua.
Crop Sci 44:1412-1418.
Muñoz
C, Duque MC, Debouck D, Blair MW (2006) Taxonomy of tepary
bean (Phaseolus acutifolius) and wild relatives as
determined by amplified fragment length polymorphism (AFLP)
markers. Crop Sci 46:1744-1754.
Contact:
Matthew Blair and Joe
Tohme
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