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a Dep. of Crop Sciences, University of Illinois at Urbana-Champaign, 1101 W. Peabody, Urbana, IL 61801
b USDA-ARS and Dep. of Crop Sciences, University of Illinois at Urbana-Champaign, 1101 W. Peabody, Urbana, IL 61801
* Corresponding author (ghartman{at}uiuc.edu)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Abbreviations: RFLP, restriction fragment length polymorphism • AFLP, amplified fragment length polymorphism • RAPD, random amplified polymorphic DNA • SSR, simple sequence repeat
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Genetic resistance is one of the most effective methods of controlling Phytophthora root rot. The major sources of resistance are a series of single dominant Rps genes (Schmitthenner, 1999). There are 13 Rps genes at seven different loci that provide race-specific resistance (Schmitthenner, 1999). Of these genes, five occur at the Rps1 locus including Rps1-k, which was first identified from the cultivar Kingwa (Bernard and Cremeens, 1981). Since it was first identified, Rps1-k has become one of the most predominant resistance genes in modern soybean cultivars. Often the use of single dominant resistance genes creates selection pressure within the pathogen population. Ryley et al. (1998) found that changes in the race structure of the P. sojae population followed the deployment of specific resistance genes in Australia. Anderson and Buzzell (1992a) also believed that shifts in the race composition of P. sojae in Ontario could be partially explained by the host genotype deployed in the survey area. Leitz et al. (2000) reported new races in Illinois that are virulent on Rps1-k, which is a common source of resistance, and Rps1-d, which has not been widely used as a source of resistance. Since P. sojae was first described in 1948, more than 39 different races have been identified (Schmitthenner, 1999).
Various genetic maps of soybean have been developed by using different markers like SSRs, RFLPs, RAPDs, AFLPs, and classical markers to construct these maps (Akkaya et al., 1995; Cregan et al., 1999; Kasuga et al., 1997; Keim et al., 1997; Lark et al., 1993; Shoemaker and Specht, 1995). Twenty classical linkage groups have been identified that contain morphological, pigmentation, and disease resistance genes (Cregan et al., 1999). Several Rps genes have been located on classical genetic linkage groups in soybean. The Rps2 gene was shown to be linked with Rmd, which confers resistance to Microsphaera diffusa, and Rj2 the nodulation response gene in linkage group J (Devine et al., 1991; Lohnes et al., 1993; Polzin et al., 1994). The Rps1 locus has been linked to the genes for pod wall color (Kilen, 1979) and metribuzin insensitivity (Kilen and Barrentine, 1983) as well as Rps7 (Anderson and Buzzell, 1992b). The linkages involving Rps1 form linkage group 10. The Rps1 locus also has been identified to various molecular maps.
Using RFLP markers, Diers et al. (1992) determined that Rps1-k is on linkage group K (Keim et al., 1990). This linkage group (LG) was later designated LG-N by Shoemaker and Olsen (1993). Lohnes and Schmitthenner (1997) were able to associate LG-22 from the Clark x Harosoy isoline map and LG-10 of the classical map with LG-N of the public RFLP map. Kasuga et al. (1997) used RAPD and AFLP analyses to create a genetic map of the region containing Rps1-k. Classical linkage group 10 containing Rps1-k has been associated with linkage group N-U06 from the University of Utah (Minsoy x Noir 1) map, linkage group N-ISU from the USDA/Iowa State University (G. max x G. soja) map and linkage group N-CH22 from the University of Nebraska (Clark x Harosoy) map. In soybean, the genetic linkage map as well as the molecular based maps have not been associated with specific soybean chromosomes. In other major crop plants these classical and molecular maps have been superimposed on their respective chromosome maps.
One method of creating a soybean genome map is by determining the chromosomal location of genes, such as Rps1-k, that are found in both classical linkage and molecular maps. Determining the chromosomal location of genes can be accomplished through primary trisomic analysis. A primary trisomic has one additional complete chromosome, altering its chromosome number from 2n = 2x to 2n = 2x + 1. Such an individual is called a triplo (Singh, 1993). Primary trisomics can be used to locate a gene on a particular chromosome, verify the independence of linkage groups, and associate linkage groups with individual chromosomes. This method has been used to determine gene-chromosome relationships in several plant species (Singh, 1993). The chromosomal location of a specific locus can be determined by the altered segregation ratios in the F2 offspring of trisomics. In these offspring, genetic ratios will be modified from the expected 3:1 F2 ratio for a dominant gene. The new ratio is dependent on the genotypes of the F1 primary trisomic plants, on the type of chromosome segregation, and on the female transmission rate of the extra chromosome (Singh, 1993). The average female tansmission rate of the 20 primary soybean trisomics was 42% with a range of 27 (triplo 20) to 59% (triplo 9) (Xu et al., 2000). The pollen transmission of the extra chromosome is very low due to the inability of pollen with (n + 1) chromosomes to compete with pollen with n chromosomes.
An integrated soybean genome map would be beneficial to scientists and breeders. Such a map would have applications in functional genomics and marker assisted breeding. Identifying the placement of the Rps1-k gene on the soybean physical map will create a more complete view of the soybean genome by also associating molecular and classical linkage groups that contain the Rps1-k locus. The objective of this study was to locate the Rps1 locus on a specific soybean chromosome using primary trisomic analysis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Seedlings were inoculated by the hypocotyl plug method (Pazdernik et al., 1997). Phytophthora sojae isolates were grown on lima bean agar for 10 to 12 d. A cork borer was used to cut 3-mm-diameter plugs along the leading edge of the colonies. Plugs were placed mycelial side down on the cotyledon, touching the stem. One P. sojae culture was used per tray. The trays and the inside of the clear plastic domes were misted with water from an atomizer. The domed trays were placed under black shade cloth (80% light reduction) for 4 d. The environment during the incubation period averaged 25°C and 95% humidity. At the end of 4 d domes were removed. Trays remained under the shade cloth for an additional 2 d. Reactions of each entry were recorded 6 d after inoculation. Resistance was determined on the basis of percentage survival per entry.
Trisomic plants used for crossing were first identified by determining their chromosome number from root tip samples. The procedure used is as described by Xu et al. (1998b). Five seeds from each line were grown in vermiculite for 5 to 7 d. Seedlings were harvested, 1-cm samples were taken from young roots and collected in 1.5 mL microcentrifuge tubes containing double distilled water. After the samples were taken, seedlings were replaced in the vermiculite.
The water was removed from the tubes and root tips were pretreated in 0.05% 8-hydroxyquinoline for 5 h at 16°C in a Micro Cooler to arrest the chromosome metaphase cells. Root tips were then fixed in a 3:1 mixture of 95% ethanol and propionic acid and left overnight at room temperature. The fixative was removed from the tubes and samples were then washed twice with double distilled water. Root tips were hydrolyzed in 1N HCL at 60°C for 16 min. To stain the chromosomes, root tips were first washed once with double distilled water and then placed in Feulgen stain at room temperature for 2 h. The Feulgen stain was then removed and root tips were rinsed with cold double distilled water (1 to 4°C). After two rinses, Carbol staining solution was added to the samples. Root tips were left in this solution overnight at 1 to 4°C. After 14 to 16 h, the staining solution was removed and the root tips were washed 3 to 4 times with cold double distilled water to remove any residual stain. Root tips were then stored in cold water in the refrigerator until needed. To prepare slides, root tips were squashed in a drop of 45% acetic acid.
Seedlings that were identified as having 41 chromosomes were then transplanted to clay pots in a 1:1:1 mixture of sand, soil, and perlite. Plants were grown in the greenhouse and crosses were made between the trisomic female (rrr) and cv. Resnik male (Rr). F1 seeds were harvested from crosses and planted in vermiculite. Chromosome numbers of the F1 plants were determined in the same manner as described above. Plants identified having 41 chromosomes (Rrr) were grown in the greenhouse and allowed to self-pollinate.
F2 seeds were harvested and planted in 10-cm-deep flats containing a 1:1 mix of steam-pasturized soil and sand. All of the trays contained five seeds each of cv. Clark 63 and cv. Resnik for use as a susceptible check and a resistant check, respectively. In each tray approximately 45 F2 seeds from the trisomic x cv. Resnik crosses were planted. All seeds from each individual cross were kept separate and planted in separate trays. The inoculation method used was the same as described above. A chi square goodness-of-fit test was performed on each F2 population with the Statistical Analysis System (SAS Institute, 1992).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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The average survival of cv. Resnik (resistant check) was 94% and the average survival of cv. Clark 63 (susceptible check) was 5%. The F2 populations from triplos 1, 2, 4, 5, 10, 15, 16, 17, and 20 fit the expected 3:1 segregation ratio for a single dominant gene (Table 1). The F2 population from triplo 3 did not fit this ratio. This population segregated in a 2:1 ratio, which is based on the 50% female and 0% male transmission rate of an extra chromosome from the F1's with simplex (Aaa) genotype.
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At this time there are 20 defined classical linkage groups containing 68 classical genes (Cregan et al., 1999). All but one of these linkage groups has been associated with a molecular linkage group (Cregan et al., 1999). Most of the known genes in soybean have not been identified to specific chromosomes (Xu et al., 1998a). Almost none of the genetic linkage groups and molecular maps have been associated with specific chromosomes (Xu et al., 1998a).
Xu et al. (2000) associated two seed protein genes eu1 and lx1 and a morphological marker gene p2 to chromosomes 5, 13, and 20, respectively. By using primary trisomic analysis, it is possible to associate genes and linkage groups to specific chromosomes and thereby very quickly create a universal soybean genome map.
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ACKNOWLEDGMENTS |
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Received for publication November 9, 2000.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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This article has been cited by other articles:
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  J. J. Zou, R. J. Singh, J. Lee, S. J. Xu, and T. Hymowitz SSR Markers Exhibit Trisomic Segregation Distortion in Soybean Crop Sci., May 18, 2006; 46(4): 1456 - 1461. [Abstract] [Full Text] [PDF]
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 J. J. Zou, R. J. Singh, and T. Hymowitz Association of the Yellow Leaf (y10) Mutant to Soybean Chromosome 3 J. Hered., July 1, 2003; 94(4): 352 - 355. [Abstract] [Full Text] [PDF]
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