Genetic Diversity of the Phaseolus acutifolius A. Gray Collection of the USDA National Plant Germplasm System Using Targeted Region Amplified

Polymorphism (TRAP) Markers Designed form Genes Associated with Heat and Drought Stress

Kisha, T.J.1*, and Bhardwaj, H.L.2. 1USDA-ARS, Western Regional Plant Introduction Station; 2Virginia State University.

*(theodore.kisha@ars.usda.gov)

ABSTRACT

Molecular genetic relationships among 222 accessions of the Phaseolus acutifolius A. Gray collection were assessed using Targeted Region Amplified Polymorphic (TRAP) markers designed from sequences of genes associated with heat and drought tolerance. Genetic relationships were compared to reactions to drought stress using measurements of variable fluorescence to maximum fluorescence (Fv/Fm). Cluster analysis using NTSys-pc and STRUCTURE found 3 major groups and 12 sub-groups with an average Fst of 0.753, but there was no association of drought tolerance with any group. In fact, some plants separated by seed color within an accession tested as both drought tolerant and susceptible, though genetically similar at all marker loci tested. In other cases, plants separated by seed type clustered apart in different groups. Given the level of diversity among groups, marker production will continue along with the addition of AFLP loci for future association mapping.

Introduction

Tepary bean (Phaseolus acutifolius A. Gray), a truly Native American crop, is a short life-cycle annual desert legume indigenous to northwestern Mexico and the southwestern USA (Fig 1) and is considered drought and heat tolerant (Lazcano-Ferrat and Lovatt, 1999; Rainey and Griffiths, 2005). The Western Regional Plant Introduction Station of the National Plant Germplasm System currently holds 207 active accessions of P. acutifolius.

Fig. 1. Habitat of Phaseolus acutifolius in North America Fig. 2. Distribution of Fv/Fm among the 222 accessions tested.

Materials and Methods

Plant Material. Available accessions were grown and studied at Virginia State University along with 29 samples from the Native Seeds Organization. Accessions with mixed seed types were separated and studied independently.

Drought tolerance. Differentiation for drought tolerance was analyzed at Virginia State University using in vivo fluorescence (Baker, 2008). Accessions were placed in categories of low, medium, and high tolerance based on a normal curve from measurements of Fv/Fm (Fig 2).

TRAP Markers. TRAP markers were generated according to Hu and Vick (2003) adjusted to 10 µl reactions. Miklas et al. (2006) used TRAP markers for mapping and tagging disease resistance traits in common bean. PCR procedures and primer sequences are available from author at request. TRAP marker fragments (153) were separated on a LI-COR GeneReadIR 4300 (LI-COR Biosciences, Lincoln, NE) on 6.5% polyacrylamide gel. Printed images were scored visually, markers being either present or absent.

Population Differentiation. Relationships among accessions were depicted in a tree generated using NTSys-pc. Accessions to be compared were grouped by individual plants, without a priori classification, into K clusters using the software STRUCTURE, which identifies genetically similar populations based on genotypes in Hardy-Weinberg equilibrium (Falush et al., 2003, 2007; Pritchard et al., 2000; Pritchard and Rosenberg, 1999). The program assumes models with each run of K hypothetical populations and assigns a probability (P(X|K)) that individuals (X) are correctly assigned to each of these K populations. Each individual plant is then assigned a membership coefficient; the fraction of its genome assigned to each of the K populations. Q-plots represent each individual by a thin horizontal line partitioned into K colored segments that represent that individual’s membership fractions in the K estimated populations. Black lines separate individuals of different accessions. Estimation of the most probable K was facilitated by the technique developed by Evanno et al. (2005), which utilizes the change in the slope of the graph of (P(X|K)) and the variance of the probability (P(X|K)) at each value of K. Five replications with a burn-in length of 20,000, followed by a Markov chain Monte Carlo (MCMC) of 20,000 additional iterations were run at each assumed K until results indicated lowered and erratic values for (P(X|K)). The parameter set included the admixture model with allele frequencies correlated. Average Q-plots over all replications for the best K and the resulting graphic display of ordered Q-plots were determined using the STRUCTURE ancillary programs CLUMPP (Jakobsson and Rosenberg, 2007) and DISTRUCT (Rosenberg, 2004), respectively.

Results Cluster analysis using NTSys-pc and STRUCTURE found 3 major groups and 12 sub-groups (Fig 3.) with an average Fst of 0.753, but there was no association of drought tolerance with any group. In fact, some plants separated by seed color within an accession tested as both drought tolerant and susceptible, though genetically similar at all marker loci tested. In other cases, plants separated by seed type clustered apart in different groups.

Literature cited

Baker, N.R. 2008. Chlorophyll fluorescence: A probe of photosynthesis In vivo. Ann. Rev. Plant Biol. 59:89-113. Evanno, G., S. Regnaut, and J. Goudet. 2005. Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Mol. Ecol. 14:2611-2620.

Falush, D., M. Stephens, and J.K. Pritchard. 2003. Inference of population structure using multilocus genotype data: Linked loci and correlated allele frequencies. Genetics 164:1567-1587.

Falush, D., M. Stephens, and J.K. Pritchard. 2007. Inference of population structure using multilocus genotype data: Dominant markers and null alleles. Mol. Ecol. Notes 7:574-578.

Hu, J. and B. Vick. 2003. Target region amplification polymorphism: A novel marker technique for plant genotyping. Plant Mol. Biol. Rptr .21:289-94.

Miklas, P.N., J. Hu, N.J. Grünwald, and K.M. Larsen. 2006. Potential application of TRAP (Targeted region amplified polymorphism) markers for mapping and tagging disease resistance traits in common bean. Crop Sci 46:910-916.Jakobsson, M. and N. A. Rosenberg. 2007. CLUMPP: a cluster matching and permutation program for dealing with label switching and multimodality in analysis of population structure. Bioinfomatics 23:1801-1806.

Rosenberg, N.A. 2004. DISTRUCT: a program for the graphical display of population structure. Mol. Ecol. Notes 4:137-138.

Lazcano-Ferrat, I and C.J. Lovatt 1999. Relationship between Relative Water Content, Nitrogen Pools, and Growth of Phaseolus vulgaris L. and P. acutifolius A. Gray during Water Deficit. Crop Sci. 39:467-475.

Fig. 3. Q-plots from the molecular marker analyses within the six onion groups using the software STRUCTURE. Q-plots represent each individual by a thin horizontal line partitioned into K colored segments that represent each of the 16 individual plant’s membership fractions in the K estimated populations. Each population is assigned a unique color. Black lines separate individuals of different accessions. Predominance of a unique color within an accession(s) is an indication of genetic uniqueness within the group. Accessions highlighted in green were scored as drought resistant according to Fv/Fm (Fig 2).

K=3 K=12