Genotyping and association analysis of Gossypium hirsutum lines for resistance in Reniform Nematodes

Megha V. Sharma*, Stella Kantartzi, David Weaver, Edzard Van Santen and James McD. StewartIntroduction

Objective

References

Association mapping is used to identify chromosomal regions containing disease-susceptibility loci or loci involved in other phenotypic traits of interest. It has been advocated as the method of choice for mapping complex-trait loci. The objective of this research is to perform association mapping in allotetraploid cotton. Gossypium hirsutum accessions from the USDA collection were evaluated for resistance to reniform nematodes (Fig.1) and genotyped with SSR markers. Most of the SSR markers were polymorphic. Software STRUCTURE and TASSEL were used to analyze the data.. The results provide preliminary insight into the cotton genome and are very useful as a framework for future ‘association studies’ in cotton that will accelerate development of superior cotton cultivars through a MAS program. 

Discussion

• Agrama, H.A., Eizenga, G.C., and Yan, W. 2007: Association mapping of yield and its components in rice cultivars. Mol. Breeding 19, 341-356.• Chen, N., Hinchliffe, D.J., Cantrell, R.G., Wang, C., Roberts, P.A., and Zhang, J. 2007: Identification of Molecular markers Associated with Root-knot Nematode Escoffier, L., Laval, G. and Schneider, S. 2005: Arlequin ver 3.0:An integrated Resistance in Upland Cotton. Crop Sci. 47(3), 951-960.• Software package for population genetics data analysis. Evol. Bioinform. 1, 47-50.• Falush, D., Stephens, M., and Pitchard, J.K. 2003: Inference of population structure using multilocus genotype data: linked loci and correlated allele frequencies. Genetics 164, 1567-1587.• Kantartzi, S. K., and Stewart, McD. 2008: Association analysis of fiber quality traits in Gossypium arboretum accessions. Plant Breeding 127, 173-179.

Conclusion

Materials and Methods

Results

TX1

23

TABLE 1: Scoring for the presence of Nematodes

• The results provide preliminary insight into the cotton genome and are very useful as a framework for future ‘association studies’ in cotton that will accelerate development of superior cotton cultivars through MAS program.

• These tests between 100 markers using general linear methodology suggests that significant association between these accessions exists.

• A more detailed study about the population structure need to be done in order to find more associations amongst the accessions.

Fig. 3. Average logarithm of the probability of data likelihood, Ln P(D) for one

to ten subpopulations (K) To identify associations using Association Mapping in Reniform nematode resistant Gossypium hirsutum accesions.

Accessions BNL0834a

BNL0834b

BNL0834c

BNL0834d

BNL0946a

BNL-----

TX1 0 0 0 0 1 -----

TX5 1 0 0 0 1 -----

TX9 1 1 0 0 1 -----

TX10 1 1 1 1 1 -----

TX11 0 1 1 1 1 -----

TX16 1 1 0 1 1 -----

TX17 1 0 1 1 1 -----

TX18 1 1 0 0 1 -----

---- ---- ---- ---- ---- ---- -----

Fig 1. A gravid female reniform nematode

Ln

P(D

)

Fig.2: Histogram plot for the number of frequencies

1 2 3 4 5 6 7 8 9 10 - 1.2879 0.6350 0.7127 0.6636 0.6487 0.6878 0.6732 0.8156 0.6767

1.2879 - 0.7266 0.7687 0.7577 0.7434 0.7699 1.3105 1.2389 0.7715

0.6350 0.7266 - 0.0058 0.0016 0.0007 0.0038 0.5394 0.6338 0.0036 0.7127 0.7687 0.0058 - 0.0036 0.0046 0.0025 0.6210 0.7218 0.0042 0.6636 0.7577 0.0016 0.0036 - 0.0004 0.0009 0.5705 0.6708 0.0006 0.6487 0.7434 0.0007 0.0046 0.0004 - 0.0016 0.5528 0.6494 0.0014 0.6878 0.7699 0.0038 0.0025 0.0009 0.0016 - 0.5912 0.6924 0.0004 0.6732 1.3105 0.5394 0.6210 0.5705 0.5528 0.5912 - 0.4198 0.5815 0.8156 1.2389 0.6338 0.7218 0.6708 0.6494 0.6924 0.4198 - 0.6832

0.6767 0.7715 0.0036 0.0042 0.0006 0.0014 0.0004 0.5815 0.6832 -

K

Number of runs

TABLE 2: Allele-frequency of the divergence among populations. • 96 accessions are screened with 100 primers.

• Analysis of genetic distance and population structure provided evidence of significant population structure in the G. hirsutum accessions and identified the mean likelihood at five points i:e K=3, K=4, K=6, K=7 and K=10.

Acknowledgement

The authors acknowledge Division of Agriculture, University of Arkansas for funding this research and Dr. Hesham A. Agrama, Rice Research Ext. Center, University of Arkansas for helping with the software.

Plant material:

• A population of 96 G. hirsutum accessions from the USDA collection were used (http://www.ars-grin-gov/npgs).

DNA Isolation: • Leaf tissue samples (~200mg each) were crushed and homogenized in liquid

nitrogen and DNA was isolated using DNeasy Plant Mini Kits (Qaigen Inc., Valencia, CA, USA).

Polymerase chain reaction (PCR):

• Primers were obtained from (http://www.cottonmarker.org/projects/bnl).

• PCR reaction contained 150ng genomic DNA, 0.3µM each of the forward and reverse primers, 0.25mM of each dNTP, 2mM MgCl2 and 2 U of Τaq polymerase with 1x reaction buffer (Promega) in a 50µl reaction volume.

• PCR amplification was performed in a Hybaid® thermocycler with the following conditions: 94ºC for 5min; 35 cycles of 94 ºC for 30s, 50 ºC for 30s, and 72 º C for 1min; and a final extension at 72 ºC for 5min.

Polymorphisms at each locus were assessed by electrophoresis of the PCR products in a vertical gel system on a polyacrylamide gel.

Software used for the analysis of the data:

1) STRUCTURE: The program structure implements a model-based clustering method for inferring population structure using genotypic data consisting of unlinked markers.

2) TASSEL-Trait Analysis by aSSociation, Evolution and Linkage :TASSEL makes use of the most advanced statistical methods to maximize statistical power for

finding a QTL. Once the value of K is established this software will be used.