Determination of solid stem gene location of durum wheat using double haploid and recombinant inbred line populations

Transcription

1 Proceedings of The Fourth International Iran & Russia Conference 148 Determination of solid stem gene location of durum wheat using double haploid and recombinant inbred line populations Saadollah Houshmand 1, Ron. E. Knox 2, John. Clarke 2, Fran. Clarke 2 1 Agronomy and plant breeding Dep. ShahreKord university, ShahreKord, P.O Box 115 Iran. s_hoshmand@yahoo.com, Phone: ; 2 Agriculture and Agri-Food Canada, Semiarid Prairie Agricultural Research Centre, P.O. Box 1030, S.C, Saskatchewan, Canada S9H 3X2; Abstract Solid stems provide resistance to sawfly (Cephus cinctus Norton) that is a major insect pest of wheat, and has caused substantial yield reduction and loss of quality in durum wheat ( Triticum turgidum var durum) by larvae tunnelling within and girdling the stems. The present study was undertaken to identify a DNA marker linked to the solid stem gene of durum wheat for use in marker-assisted selection. A set of 151 doubled haploid lines was developed from the cross of Kyle*2/Biodur (solid stemmed) and Kofa (hollow stemmed) were used in this study. Parents of the population were tested with approximately 500 microsatellite (simple sequence repeat) primer pairs. The polymorphic primers between the parent genotypes were tested on the whole population lines and primers that followed a 1:1 ratio of parental bands were used in linkage analysis as did least mean square phenotypic solid stem. Three microsatellite markers Xgwm247, Xgwm181 and Xgwm114 were showed to be associated to solid stem gene. The markers Xgwm247 and Xgwm114 are linked in coupling phase were located on each side of solid stem gene. The location of these markers is on the distal of long arm of chromosome 3B. Recombinant inbred line populations with different genetic backgrounds AC Navigator/G9580B-FE1C and Golden Ball/DT379 segregating for hollow and solid stems were scored to confirm the association between the markers and the solid stem gene. Result of the Golden Ball/DT379 population tested with the WMC632 microsatellite marker showed a polymorphism associated with the solid stem gene. The genetic distance in recombinant inbred lines populations were shorter than that in doubled haploid population. The result also indicated, the solid stem trait was controlled by a single locus in both doubled haploid and recombinant inbred line populations. This is the first report on the chromosome location on solidness stem gene in durum wheat and the linkage markers can be used in breeding program for identification of solidness stem gene. Key Words: durum wheat, microsatellite marker, solid stem, Triticum turgidum Introduction Sawfly (Cephus cinctus Norton) is a major insect pest of wheat, that causes substantial yield reduction and loss of quality in wheat ( Triticum spp.). The most effective way to reduce damage from the wheat stem sawfly is through the incorporation of resistant cultivars. Solid stemmed wheats provide resistance to sawfly larvae damage (Weiss and Marrill 1992; Clarke et al. 2002). The solid stem trait in hexaploied wheat is controlled by genes located on five chromosomes of homoeologous group 3 and 5 (Larson and MacDonald, 1959). In tetraploid wheat previous studies showed that the solid stemmed trait is controlled one

2 Proceedings of The Fourth International Iran & Russia Conference 149 gene with a partially dominant effect (Putnam, 1942). Recently Clarke et al. (2002) studied the inheritance of stem solidness in eight durum wheat crosses, and a doubled haploid population, they suggested a single dominant gene controls solidness. There are reports of DNA markers in durum wheat linked to qualitative traits such as Hessian fly resistance (Dweikat et al. 1994), low cadmium uptake (Knox et al. 2003), but not to stem solidness. Although a locus was found on chromosome 3BL that controls sawfly cutting resistance in durum wheat (Houshmand et al, 2003). In durum wheat breeding, marker assisted selection is of particular interest for the development of genotypes with solid stems because current screening tests for this trait are time consuming, labor intensive, and sometimes inconclusive. Section for stem solidness is challenging because the level of expression of solid stem gene can be affected by environmental conditions, such as level of sunshine, rain, plant density and temperature (Weiss and Marrill 1992). The goal of the present study was to identify the microsatellite markers associated with the solid stemmed gene in durum wheat. MATERIAL AND METHODS Genetic Materials Three durum genetic populations were used to identify linked DNA markers to the solid stemmed character. A genetic population of 151 double haploid lines was developed from the cross of a Kyle*2/Biodur (K*2B) inbred line and Kofa and used in this study. K*2B is an F 9 solid stemmed line. Kyle is a cultivar adapted to the Canadian prairies and is a hollow stemmed cultivar. Biodur is a solid-stemmed cultivar from France. Kofa is a hollow stemmed cultivar from the United States. A second genetic population of Eightyeight recombinant inbred lines (F 7 generation) was developed from the cross AC Navigator/G9580B-FE1C. AC Navigator is a hollow stemmed line and G9580B-FE1C is a solid stemmed line with Biodur in its background. The third population was forty recombinant inbred lines (F 8 generation) derived from the cross of Golden Ball and DT379. Golden Ball is a very solid stemmed line from Africa and DT379 is a hollow stemmed line. Field experiments The three populations were evaluated in the field phenotypically. Field experiments were conducted on a Swinton loam soil at the Semiarid Prairie Agricultural Research Center, Swift Current, Canada. Parents and the K*2B/Kofa population, including 15 check lines were planted in a randomized complete block experiment with two replications in The AC Navigator/G9580B-FE1C population was grown in 2002 and 2003 and Golden Ball/ DT379 population was grown in All lines were scored for stem solidness at maturity. The primary and one other tiller of five plants in each plots field were scored by cutting the stems transversely in center of each stem. DNA extraction and PCR amplification DNA was extracted using a modified cetyltrimethylammonium bromide (CTAB) method from leaves of two-week-old seedlings of the lines. Parents of the K*2B/Kofa population were tested with approximately 500 microsatellite primer pairs with known sequences and chromosome locations. Primers polymorphic between the parent genotypes were

3 Proceedings of The Fourth International Iran & Russia Conference 150 tested on the whole population of lines. DNA amplification and PCR procedures were done as explined by Houshmand et al, Statistical and genetic analysis The significance of the association of markers to the solid stemmed trait was tested using three analytical approaches on the K*2B/Kofa population. First, after marker polymorphism of the lines was tested with the chi-square test for deviation foe a 1:1 ratio, single marker analysis was performed with a modified t-test model in SAS Proc MIXED on least square solid stem means. The second approach employed to determine the marker association to the solid stemmed gene was simple interval mapping and was done using the Multiple Quantitative Trait Loci (MQTL) computer program. The Chi-square test was used to assess independent segregation between the solid stem gene and the markers as the third approach. Markers associated with the solid stem gene in the K*2B/Kofa population, were tested on the AC Navigator/G9580B-FE1C, Golden Ball/ DT379 populations. Linkage analysis was done considering classical recombination and the Kosambi function. Also maximum likelihood using the MAPMAKER program was used to calculate linkage. Linkage and order of markers and the solid stem gene were generated using the Kosambi function on the three populations (Kosambi 1944). The logarithm of the probability of linkage between the marker and gene divided by the probability of no linkage between them was estimated as the logarithm of odds (LOD). The maximum likelihood of map distance was estimated by maximization of the log-likelihood of LOD. The MAPMAKER/EXP computer program also was used to calculate the genetic distance between the markers and solid stem gene. RESULTS The doubled haploid population, K*2B/Kofa, data follow 1:1 ratio of solid and hollow stem (Table 1). The two recombinant inbred line populations, Navigator/G9580B-FE1C and Golden Ball/DT379, solid stemmed data also did not differ from 1:1 ratio and fit a single gene model for the segregation of stem solidness(table 1). Approximately 500 microsatellite markers were screened to identify polymorphisms between the parents of the K*2B/Kofa population. Forty-eight primers generated clearly scorable polymorphisms between the parents [K*2B (solid stemmed) and Kofa (hollow stemmed)]. The polymorphic primers were applied to the 151 lines of K*2B/Kofa population. Forty-seven of the polymorphic primers fit a 1:1 ratio of parental bands and were used in t test and simple interval mapping analysis. The results of both single marker analysis and simple interval mapping analysis indicated that three makers including Xgwm247, Xgwm181 and Xgwm114 were significantly (P<0.0001) associated with the solid stemmed gene in the K*2B/Kofa population. The results of the chi-square test confirmed an association between these markers and the solid stemmed gene, when as indicated by a highly significant (P<0.0001) difference of observed from the expected random segregation of ratio 1:1:1:1 for the solid stem gene and each of the markers (Table 2). The three markers, Xgwm247, Xgwm181 and Xgwm114, were segregated in the AC Navigator/G9580B-FE1C and Xgwm247, Xgwm181 in the Golden Ball/DT379 population. The chi square for the combined segregation of the solid stemmed trait and

4 Proceedings of The Fourth International Iran & Russia Conference 151 the markers were significantly (P<0.0001) different from the expected 1:1:1:1 ratio for random associate (Table 2). Kosambi linkage analysis showed the order of these markers to be Xgwm181, Xgwm247 and Xgwm114 in the K*2B/Kofa population. The distance between Xgwm181 and Xgwm247 was 3.60 cm. The Xgwm247 and Xgwm114 markers were located on each side of solid stem gene, at a distance of 7.24 and cm, with LOD value of and respectively. In this population the estimate of distance between the gene and markers by the maximum likelihood method was slightly shorter than the Kosambi method and the flanking markers, Xgwm247 and Xgwm114, were located at a distance of 7.18 and cm far from the gene respectively. MAPMAKER generated the same order for the markers and gene. The flanking markers, Xgwm247 and Xgwm114, were located at a distance of 4.1 and 8.6 cm from the solid stemmed gene respectively. In the AC Navigator/G9580B-FE1C recombinant inbred line population, no recombination was found between Xgwm181 and Xgwm247. In this population Xgwm247 and Xgwm114 markers also flank the solid stemmed gene. Kosambi linkage analysis revealed that Xgwm247 was cm and Xgwm114 was 9.19 cm away from solid stemmed gene, with LOD value of and respectively. The maximum likelihood distance was slightly shorter than the distance calculated by the Kosambi method. MAPMAKER showed the solid stemmed gene to be located 5.9 cm from Xgwm247 and 9.8 cm from Xgwm114 respectively. In Golden Ball/DT379 population the order of the markers and the solid stemmed gene was Xgwm247, Xgwm181 and the solid stemmed gene. The distance of these two markers was 2.5 cm and Xgwm181 was 2.5 cm from the solid stemmed gene based on the Kosambi and the maximum likelihood linkage analyses. LOD values of linkage between Xgwm247 and Xgwm181 with the solid stemmed gene were 9.44 and respectively. Because of Xgwm114 band was monomorphic, then this marker was not used in linkage analysis in this population. MAPMAKER revealed that Xgwm247 was located 1.3 cm from Xgwm181, and Xgwm181 was 1.3 cm away from solid stemmed gene. DISCUSSION Solid stem phenotypic data of the three populations segregated as a prefect 1:1 ratio and indicated the solid stem trait was controlled by one gene. These result confirm result of Clarke et al. (2002) study. Also these results agree with previous studies that indicate a single gene locus for the solid stemmed trait in durum wheat (Putnam 1942). The present study demonstrated a high association between the solid stemmed gene in the K*2B/Kofa population with Xgwm181, Xgwm247 and Xgwm114 microsatellite markers. These markers are all located close to the end of the long arm of chromosome 3B. This chromosomal association was confirmed with AC Navigator/G9580B-FE1C and Golden Ball/DT379 populations. The association on chromosome 3B is consistent with other research. Larson and MacDonald (1959) reported 3B as one of the chromosomes where the solid stem trait resides in a hexaploid wheat source. The solid stem varieties are resistant to sawfly larvae damage (Weiss and Marrill 1992, Clarke et al. 2002). One would expect genetic analysis based on scores of sawfly cutting to provide similar results to analysis based on scores of stem solidness. QTL analysis of sawfly cutting in durum wheat is controlled by single gene located on 3BL using

5 Proceedings of The Fourth International Iran & Russia Conference 152 (Houshmand et al. 2003). Like our results on the location of the solid stemmed gene the sawfly-cutting resistance gene is located between Xgwm247 and Xgwm114 microsatellite markers. The relationship between sawfly resistance and stem solidness seemingly is a result of pleiotropy but close linkage between sawfly cutting resistance and stem solidness genes can not be ruled out. In the K*2B/Kofa population the distance between the solid stem gene and Xgwm181 and the solid stemmed gene and Xgwm114 calculated by the Kasombi function, was only slightly shorter than the distance between the gene and markers calculated by maximum likelihood (total map distance = and cm). When MAPMAKER was used to estimate the distance between the solid stemmed gene and Xgwm181 and Xgwm114 a much shorter distance was found (total map distance = 14.7 cm). In the AC Navigator/G9580B-FE1C and Golden Ball/DT379 populations the distance calculated by MAPMAKER was also shorter than those using the Kosambi and maximum likelihood methods. Although MAPMAKER uses the maximum likelihood method, this program calculates multipoint analysis and considers the amount of missing data (Lander et al. 1987), which could account for the differences in map distance. The order of the markers and the solid stem gene was Xgwm181, Xgwm247 and the solid stem gene in the K*2B/Kofa population. In AC Navigator/G9580B-FE1C population there was not observed recombination between Xgwm181 and Xgwm247 markers. On the other hand order of these markers and gene in and Golden Ball/DT379 population changed to Xgwm247, Xgwm181 and the solid stem gene. Inversion in a region of chromosome could be a reason of the different order in the populations. Antonio et al. (1996) compared the genetic distance and order of DNA markers in five populations of rice and showed the same linkage groups with converse linkage order and variable genetic distance between some markers among the different populations. The number of lines in the K*2B/Kofa, AC Navigator/G9580B-FE1C and Golden Ball/DT379 populations were 151, 88 and 40 respectively. The different order of markers and the gene could also be the result of the number of lines studied in each population, with a small sample size of cross overs in the smaller populations. Linkage maps based on doubled haploid and recombinant inbred lines populations are immortal and suitable for continuing genetic studies. In this study the map distance of the linkage group in the double haploid population was longer than in the recombinant inbred line populations. He et al. (2001) also reported molecular linkage maps of doubled haploid and recombinant inbred line populations of rice with the same parents and they showed the map of the recombinant inbred line population to be shorter than the map of the doubled haploid population. This is opposite of the expectations based on the way these populations were developed. Doubled haploid populations are produced with only one meiosis, while recombinant inbred line populations have the opportunity for crossover in a series of meioses from several generations of selfing. Thus differences in genetic distance between these populations can not be explained by the grater number of cross overs expected through the development of recombinant inbred line populations. However difference in distance could simply be from differences in random segregation of the populations and smaller population size fit the recombinant inbred lines. In conclusion, evidence from analysis of doubled haploid, recombinant inbred line populations, indicates that the solid stem gene is linked to flanking markers Xgwm247 and Xgwm114. These markers indicate the solid stem gene to be located on the long arm

6 Proceedings of The Fourth International Iran & Russia Conference 153 of chromosome 3B. These markers have potential for marker assisted selection for the solid stemmed trait in durum wheat breeding programs. This information may also be useful to further investigate the chromosomal location of solidness in hexaploid wheat. Acknowledgements We gratefully acknowledge the financial support of the Matching Investment Initiative of Agriculture and Agri-Food Canada and the Wheat Producer Check-Off administered by the Western Grains Research Foundation as well as competitive grant foundation. The technical assistance of Brad Meyer and Isabelle Piché and marker and map information provided by Dr. Daryl Somers is very much appreciated. Reference Clarke, F. R., Clarke, J. M. and Knox. R. E Inheritance of stem solidness in eight durum wheat crosses. Can. J. Plant Sci. 82: Dweikat, I., Ohm, H., Mackenzie, S., Patterson, F., Cambron, S. and Ratcliffe. R Association of a DNA marker with Hessian fly tesistance gene H9 in wheat. Thero. Appl. Genet. 89: Knox, R. E., Menzies, J. G., Howes, N. K., Clarke, J. M., Aung, T. and Penner, G. A Genetic analysis of resistance to loose smut and an associated DNA marker in durum wheat. Can. J. Plant Pathhol. 24: Knox, R.E., Clarke, J.M., Houshmand, S., Clarke, F. and Ames, N.A Chromosomal location of the low grain cadmium concentration trait in durum wheat. 10 th International Wheat Genetic Symposium. September 1-6 Italy. Kosambi, D.D The estimate of map distance for recombination values. Ann. Eugen. 12: He, P., Li, J. Z., Zheng, X. W., Shen, L. S., Lu, C. F., Chen, Y. and Zhu, L. H Comparison of molecular maps and agronomic trait loci between DH and RIL populations derived from the same rice cross. Crop Sci. 41: Houshmand, S., Knox, R.E., Clarke, F. and Clarke. J Microsatellite markers associated with sawfly cutting in durum wheat. 10 th International Wheat Genetic Symposium. September 1-6 Italy. Lander, E., Green, P., Abrahamson, J., Barlow, A., Daley, M., Lincoln, S. and Newburg, L MAPMAKER: An interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics. 1: Larson, R. I. and MacDonald, M.D Cytogenetics of solid stem in common wheat II. Stem solidness of monosomic lines of the variety S-615. Can. J. Bot: 37, Putnam, L. G A study of the inheritance of solid stem in some tetraploid wheat. Sci. Agric. 22: Weiss, M.J. and Morrill, W.L Wheat stem sawfly (Hymmenoptra: Cephidae) revisited. American Entomologist 38:

7 Proceedings of The Fourth International Iran & Russia Conference 154 Table 1. Data and fit a single gene model for the segregation of stem solidness in the K*2B/Kofa, AC Navigator/G9580B-FE1C and Gollden Ball/ DT379 populations. K*2B/Kofa AC Navigator/ G9580B-FE1C Golden Ball/ DT379 Solid stemmed hollow stemmed χ 2 value of 1:1 ratio* * All value are not significant Table 2. Chi square value for the solid stem (Ss) gene and each marker locus (Xgwm247, Xgwm181 and Xgwm114) in the K*2B/Kofa, AC Navigator/G9580B-FE1C and Golden Ball/DT379 populations testing the deviation from 1:1:1:1: random segregation*. K*2B/Kofa AC Navigator/ GoldenBall/ DT379 G9580B-FE1C Ss/ Xgwm Ss/ Xgwm Ss/ Xgwm * All value are significant at P<0.0001