The goal of this proposed research is to apply molecular markers to wheat improvement. We will approach our goal by develop or adapt polymerase- chain-reaction- (PCR) based DNA markers that are efficient and economic for practical application in winter and spring wheat improvement in South Dakota.

 Wheat breeding is basically optimizing wheat genotype for better yield and/or superior quality. Identifying the existence of the gene(s) of interest in breeding materials is often a time- and labor-consuming process, especially when expression of the genes is environment dependent, such as disease resistance and QTLs.  Employing DNA marker techniques in gene (and thus trait) identification will greatly increase breeding efficiency and thus reduce the cost eventually. DNA markers can also be used as the fingerprintings for the variety protection

 Currently, we focus our efforts on development/adaption of markers for scab and/or stem rust resistance in spring wheat  We first search for polymorphic DNA markers between resistant and susceptible parents. The polymorphic markers are studied in a segregating population for identification of markers that are closely linked to the resistance or susceptibility to the target disease.  The identified markers are then used in wheat breeding programs.

 In the past two years, we successfully fulfilled all our proposed objectives. We have identified and optimized a protocol to isolated DNA and RNA from a small leaf or spike sample.  A silver-staining-based protocol was optimized and routinely used in our identification of molecular markers. We also successfully carried out an investigation of differential expression of genes between the scab-susceptible and -resistant wheat varieties with the mRNA differential display (DDRT-PCR) technology. Our preliminary data revealed several types of gene expression patterns. For example, some genes were expressed only in the spikes of either scab inoculated Wheaton (a scab-susceptible variety), scab-inoculated Wheaton and Sumai 3 (a scab resistant variety) or the water- and scab-inoculated Sumai 3.  Genes that express only in scab inoculated spikes of Sumai 3 or change their expression level at certain time after scab-inoculation were also observed.

 Our goal for the study of differential gene expression is to identify expressed sequence tags (EST) of scab related genes. Such ESTs are part of the DNA coding sequence of scab resistance or susceptibility genes. Their identification enables us to search the corresponding DNA for possible sequence differences between the resistance and susceptible alleles. Such information will lead to designing molecular markers, such as single nucleotide polymorphism marker, for wheat improvement. In FY2001, six such ESTs (namely, EST7A, EST12G, EST15AU, ESTAp4CS2, ESTAp7CS and ESTAp4CS1) have been identified, cloned and sequenced. The first three were found to express specifically in scab-inoculated Sumai 3 and the last three to express substantially more in scab- inoculated Sumai 3 than scab-inoculated Wheaton. A search of GeneBank for sequence similarity revealed that EST15AU is 94% similar to part of a wheat mRNA for polypeptide elongation factor 1b, EST7A has three homologous regions (with 86% similarity) with an EST sequence from a pathogen induced sorghum bicolor cDNA, and EST12G is 99% identical to a part of the minus strand of wheat gene for chloroplast ATP synthase CF-O subunit I and III. Our analysis also indicated that ESTAp4CS2, ESTAp7CS and ESTAp4CS1 possibly belong to the gene(s) of the fungal pathogen that interact with wheat resistance gene(s) during scab resistance development.

 In FY2001, we also initiated the application of small sequence repeat (SSR) markers in spring wheat improvement. Researchers in Minnesota and North Dakota have reported that SSR markers Xgwm389, Xgwm 533 and Xgwm 493 are linked to a major scab resistance QTL on wheat chromosome arm 3BS. Scab-resistant lines in SD spring wheat project (Fifty lines from 2000 fall cross block, 24 lines from 2000 preliminary yield trial, and three lines with unknown pedigrees) were analyzed. So far, seven were found to have the SSR marker Xgwm533, 36 lines (including SD3411, which has been widely used in SD spring wheat project as parent) have Xgwm493, and six lines have Xgwm389.

 In FY2002, we will continue our efforts in EST study and in the application of SSR to spring wheat improvement. Effort will also be made to identify molecular markers for winter wheat. Therefore, our specific objectives for FY2002 are:
 1). Continue our characterization of scab-related ESTs and try to develop PCR-based markers from them;
 2). Screen populations derived from SD3411 for SSR marker Xgwm493 and study its association with scab resistance;
 3). Molecularly characterize elite breeding and germplasm lines with publically available SSR markers.