1. Karen A. Beaubien and Kevin P. Smith* Dept of Agronomy & Plant Genetics, University of Minnesota, St Paul, MN, *Corresponding Author: PH: (612) 624-1211; E-mail: smith376@umn.edu OBJECTIVES  Develop PCR primers flanking SSRs in barley ESTs and screen for polymorphisms on a set of barley mapping parents.  Increase the number of available SSR markers in BINs with poor coverage (Table 1).  Integrate new EST-derived barley SSR markers on a barley consensus map derived from three mapping populations. Developing new SSR markers for barley derived from the EST database ACKNOWLEDGEMENTS ACKNOWLEDGEMENTS  This research was supported by the North American Barley Genome Project.  Special thanks to Charles Gustus for help in constructing the linkage maps. Method 1 (Stringent) Barley ESTs processed through TRF had e-value between 0 and 1e-35. Primers were designed for SSRs with 85-100% matches to the repeat unit. Method 2 (Relaxed) Barley ESTs processed through TRF had e-value between 0 and 1e-100. Primers were designed for SSRs with 50-100% matches to the repeat unit. Method 3 (BLAST repeats) Isolated small repeats in the Rice BACs using TRF and BLASTed only the portion of the BAC that contained the SSR motif and some flanking sequence to the barley ESTs. Barley ESTs processed through TRF had e-value between 1e-150 and 1e-2. Primers were designed for SSRs with 85- 100% matches to the repeat unit.  Method 4 (Expression specific) One-hundred –seventy-three barley ESTs were identified from a Fusarium-infected library that do not have homology to any other publicly available barley and wheat ESTs in Genbank (as of April 9, 2003). These Fusarium-specific barley ESTs were processed through TRF to locate SSR motifs. Primer pairs were designed to flank SSR motifs with 60-100% matches to the repeat unit using Primer3. MATERIALS AND METHODS We evaluated four methods of designing EST-SSR markers. Methods 1, 2, and 3 were based on identifying syntenous regions in barley using rice genome sequence. Method 4 used expression specific ESTs.  Methods 1-3: Sequenced markers (RFLP and STS) from barley BINs with poor SSR coverage were BLASTed against all Rice BAC sequences. Contiguous BAC sequences with multiple strong hits to markers from the same barley BIN were BLASTed against all barley ESTs with the low complexity filter turned off. The resulting barley ESTs were processed through the Tandem Repeats Finder (TRF) (Benson, 1999) to locate SSR motifs (1-6 bases in the repeat unit). Primer pairs were designed to flank SSR motifs using Primer3 software. All three methods used the same steps, but differed in the level of homology between the rice BAC and barley EST and the degree to which the SSR region in the EST matched the pattern of a perfect repeat unit. RESULTS  Developed and evaluated 4 methods for designing SSR markers which differed in efficiency (Table 2).  238 different primer pairs were designed and screened.  44 new markers developed that produce 2-6 alleles among 12 mapping parents (Figure 2).  31 markers mapped using the Chevron x M69, Fredrickson x Stander, and Steptoe x Morex mapping populations (Figure 1).  Markers are distributed throughout the barley genome. Most do not map to the target region for which they were designed.  16 of the 31 mapped markers map to BINs identified as having poor coverage with currently available SSR markers. REFERENCES  Benson, G. 1999. Tandem repeats finder: a program to analyze DNA sequences. Nucleic Acids Research 27: 573- 580.  Li, J.Z., T. G. Sjakste, M. S. Röder, M. W. Ganal. 2003. Development and genetic mapping of 127 new microsatellite markers in barley. Theor. Appl. Genet. 107:1021–1027.  Liu, Z.-W., R. M. Biyashev and M. A. Saghai Maroof, 1996 Development of simple sequence repeat markers and their integration into a barley linkage map. Theor. Appl. Genet. 93: 869–876.  Ramsay, L., M. Macaulay, S. degli Ivanissevich, K. MacLean, L. Cardle, J. Fuller, K. J. Edwards, S. Tuvesson, M. Morgante, A. Massari, E. Maestri, N. Marmiroli, T. Sjakste, M. Ganal, W. Powell, and R. Waugh. 2000. A Simple Sequence Repeat-Based Linkage Map of Barley. Genetics 2000:156 1997-2005.  Saghai Maroof, M. A., R. M. Biyashev, G. P. Yang, Q. Zhang and R. W. Allard, 1994 Extraordinarily polymorphic microsatellite DNA in barley: species diversity, chromosomal locations and population dynamics. Proc. Natl. Acad. Sci. USA 91: 5466–5470.  Thiel, T, W. Michalek, R.K. Varshney, and A. Graner. 2003. Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley ( Hordeum vulgare L.). Theor. Appl. Genet. 106: 411–422. CONCLUSIONS  Method 3 was the most efficient method of producing useful SSR markers.  Approximately half of the barley genome has good coverage with SSR markers.  New methods must be developed to develop markers for underrepresented regions of the genome. C/M & F/S Consensus S/M Act8D0 Act8B7 MWG50216 UMB70436 Bmac016346 Bmac030647 EBmac097048 Bmag0323UMB70549 Bmag033751 CDO348D52 MWG635G54 MWG635F55 MWG635DABG497A56 MWG635H60 MWG2227B61 MWG635E63 ABC302A64 MWG503B73 ABG497B77 ABC16878 ABC71786 Bmag081287 UMB70690 Bmag011393 UMB70197 Mad20 MWG5022 ABC4837 MWG920A9 ABG316B11 ABG705A39 ABG70844 ABG39547 CDO74948 Ubi251 ksuA3A54 WG54157 WG53060 UMB70762 WG88964 ABC32468 UMB70373 ABC302A77 CDO57B89 mSrh95 WG364100 MWG514B134 CDO504139 iEst9143 WG908145 MWG877149 ABG495A152 ABC155159 ABC482166 ABG391172 ABG390174 CDO484181 ABG463183 ABG314B187 MWG851C190 MWG851B194 CDO400B0 CDO059B15 UMB70225 MWG2227B28 ABG010D33 GMS06137 EBmatc000340 ABG495A43 GMS02744 ABG49654 ABG39167 ABG39273 MWG851B89 MWG632C GMS00190 7 (5H) ASE1B0 ABG0622 MWG6207 Nar19 ABG37814 ABC152A23 cMWG652A30 MWG2040E31 MWG221834 Bmag050038 PSR10644 ABG387B47 MWG91649 Bmg000152 ABG45856 MWG887B57 Bmag080759 ABC169B60 CDO49762 Bmag017363 Bmac001864 Bmag0003e65 Bmag0870BCD340E66 Bmag0003aMWG2227A68 ksuD1769 Bmac0218a71 Bmag061372 ABC17573 EBmac0806a74 MWG82075 EBmac060276 Bmac025178 EBmac0806b84 Nar786 Amy190 bBE54B93 MWG93499 ABC170AABG461B107 UMB602115 Bmac0040118 MWG549A120 ABG713131 MWG514134 UMB603137 UMB601138 MWG798AMWG2196140 cMWG684A149 6 (6H) UMB5020 MWG835A3 MWG9386 MWG036A11 ACT00812 MWG83716 Hor118 Bmac0144a19 MWG2077ABA00425 MWG801B30 SC4-5B-133 BCD09834 BCD73838 ABG05339 HVM04342 Bmac003244 Ica145 MWG2040D46 ABC706C47 MWG2040C48 Bmac0144i50 ABG500A51 CDO58053 UMB501b55 UMB501a57 ABC152BBmac0144h ABC164A 58 Bmag0770WG789B HVM020 60 Bmac009061 ABR33764 ABG07465 Glb168 Bmag087269 ABC16076 ABG45279 BCD09880 Bmac021387 ABG46490 ABC156B92 ABC165C95 His3B96 ABC307A98 cMWG706A107 ABC257113 cMWG733A117 AtpbA123 ABG702A125 ABC322B130 ABC261132 Cab2134 Aga7136 MWG912139 ABG387A141 5 (1H) ABG7040 MWG036B3 RISIC10ABmag07676 BCD1298 MWG851A9 MWG555A10 ABG32011 HVM00412 iEst515 His3A17 Bmag020618 WG789A21 MWG08922 MWG53023 ABC167A24 dRcs126 ABG38027 Mad1MWG56430 ABC15833 ABC151A35 KSUA1A38 ABC154A40 MWG83641 UMB10642 UMB10743 Brz48 ABC156D53 BCD09858 MWG649A61 HVM014b65 MWG911B66 BCD14767 ABC30870 ABG47671 BCD34972 ABC154BUMB10474 Bmag0110aKFP194 Bmag0321 76 Amy279 ABC25580 Bmac018781 Bmac006482 Bmag0482b88 ABG156B93 Ubi194 MWG571DTLM195 ABC310B98 KFP190100 RISP103104 bBE54E105 ABG608106 ABC305107 UMB105Bmac0156111 ABG497B114 ABG461AUMB101 UMB102 118 WG420RZ682120 ABG652A122 ABC253123 HVM005135 HVPRP1B137 MWG635B138 UMB103139 PSR106B140 1 (7H) MWG6340 WG6221 ABG313B12 MWG07715 CDO669A18 HVM04020 B32E24 BCD402B32 BCD351D33 ABG705C35 MWG635A37 UMB40141 TubA1BCD108745 Dhn648 ABG715WG1026B50 ABC30351 ABA00352 Adh4ABG48453 BCD1087a54 ABC32155 STS3B14256 MWG05857 Bmac018158 KFP19559 EBmac0906HVM00360 Bmag0808Bmag074061 WG464Bmag030663 bBE54A64 HVM06867 MWG632D68 Bmac031070 BCD453B73 EBmac077574 ABG47277 ABG319A82 EBmac063585 EBmac070188 EBmac078890 CDO02094 iHxk296 ABG49897 ABG500B98 HVMLOH1A100 bAP91101 ABG366103 ABG397106 ABG319C118 Bmag0138b120 ABC305B123 HVM067124 Bmy1136 ksuH11137 ASE1C140 ABA307A147 4 (4H) ABA307B0 MWG571C1 STS3B-942 STS3B-473 UMB3014 STS3B-656 STS3B-8013 STS3B-5215 ABC17117 CDO39523 MWG798B26 MWG58430 ABG47134 ABC171A43 Bmag082844 UMB30245 Bmag0138UMB30447 Bmag0010c49 UMB303Bmag0482a50 HVDHN7BCD82851 HVM009Bmag0131 ABG399ABmag0122 52 Bmac0067Bmac0129a Bmag0136Bmag0905 Bmag0603 53 Bmag0006Bmag0482c54 MWG68055 Bmag013156 Bmag082858 PSR156A61 CDO105CMWG571B63 MWG2227C67 ABG37768 Bmag022570 MWG555BHVM06072 ABG315 75 Bmag0010b76 ABG45377 PSR7878 MWG571A81 CDO345 82 ABG49985 HVM07089 CDO113B95 Bmag0606100 His4BBmag0482d105 ABG004 117 MWG803120 WG110122 ABC151CmPub124 BCD147B125 Bmag0013 127 ABC161128 Bmag0877MWG902132 ABG654A136 Glb4140 iBgl 142 ABG495B 144 EBmac0541146 MWG838149 ABG319B153 ABC172161 3 (3H) ASE1A0 ABG0585 ABG703B7 CDO057A9 MWG878A13 SC137316 ABG00822 UMB205aRbcS25 UMB20327 BCD351F28 ABG31829 ABG00230 ABC156AHVM03631 UMB205b33 MWG85836 ABG35840 ABC31143 ABG45944 Bmag0174c45 Pox49 ABG005MWG520A51 Adh8ABG156A Bmac0218b 52 cMWG663A57 UMB20258 MWG887A60 MWG55762 Bmac0140ABG316C64 UMB201Bmac009365 EBmac055766 HVBKASIABC167B Bmac0129bEBmac0558 67 EBmac0521bGMS003 HVM023 68 UMB204GBM1024 EBmac0521c 69 EBmac0521a70 ABC306EBmac061571 bBE54D72 Bmag0140Bmag037873 CDO588ABG014 BCD1087 74 TLM3ABC25676 ABG619MWG95077 His3C79 ABC152DBmag0003c80 Bmag0003d83 MWG86585 Rrn5S186 Vrs189 KSUF1590 Bmag012591 MWG50392 GBM106294 MWG503B95 Bmac0144g99 MWG882B102 MWG503A105 Bmac0144b106 Crg3A109 ABG072111 ABG497C112 MWG649C117 STS3B-142123 ABC252124 EBmac0415126 HVM054128 ABC157131 ABC153136 ABG317B137 ABG317A139 ABG010B140 ABG316E141 ABG010CPcr1145 cMWG720150 BG123A153 bBE54C155 GBM1036165 2 (2H) INTRODUCTION Genetic mapping of germplasm relevant to plant breeding programs with molecular markers serves to connect the wide array of valuable germplasm and genetic variation used in agriculture to the expanding frontier of plant genomics. Progress in mapping genes for important traits in barley and utilizing markers to improve the efficiency of breeding is dependent on the availability, cost, and ease of use of molecular markers. There are currently 632 SSR marker primer sets that have been developed of which 242 have been mapped in barley (Saghai Maroof et al., 1994; Liu et al., 1996; Ramsay et al., 2000). More recently, several studies have reported the development of an additional 202 SSR markers (Li et al., 2003, Thiel et al., 2003), however, most of these markers mapped to regions of the barley genome that already have good coverage with SSR markers (Table 1). There is still a need for additional publicly available SSR markers to improve coverage of the barley genome and facilitate breeding and genetics research. Table 2. Comparison of four methods for designing EST based SSR markers. PairsProduced ProductPolymorphism MethodtriedNumber% % 1442761.4613.6 2511529.435.9 3795164.53544.3 4641523.400.0 Overall23810845.34418.5 Table 1. SSR marker coverage in barley by chromosome. Chromosome Bins with good SSR coverage Bins with poor SSR coverage 11--2; 5--133--4 24--121--3;13--15 36--81--4;8--16 46--91--4;10--13 55--71--4;9--14 66--81--5;9--14 73--51--2;6--15 UMB104 UMB704 UMB603 UMB702 Figure 2. Sample gel images from 4 markers. Markers are screened on 12 mapping parents (from left): Atahualpa; M81; Chevron; M69; Fredrickson; Stander; Harrington; OUH602; Hor211; Lacey; Steptoe; Morex. Figure 1. Barley consensus maps developed from Chevron x M69 (C/M), Fredrickson x Stander (F/S), and Steptoe x Morex (S/M) mapping populations. Previously available SSR markers are in Maroon. New UMB SSR markers are in Blue. Maps and marker information are available at http://agronomy.coafes.umn.edu/barley.