Research Communications 36(4), pp. 553–560 (2008) DOI: 10.1556/CRC.36.2008.4.4

Occurrence of Three Dwarfing Rht Genes in German Winter Varieties

C. KNOPF1,3*, H. BECKER1,E.EBMEYER2 and V. KORZUN2

1Department of Crop Sciences, Georg-August University of Göttingen, D-37075 Göttingen, Germany 2KWS LOCHOW GMBH, D-29296 Bergen, Germany 3Present address: State Plant Breeding Institute, University of Hohenheim (720), D-70599 Stuttgart, Germany

(Received 4 February 2008; accepted 18 April 2008)

Ninety percent of wheat varieties grown worldwide have a semi-dwarf phenotype controlled by three major genes Rht-B1 (formerly Rht1), Rht-D1 (formerly Rht2) and Rht8. The objective of this study was to determine their frequency in modern wheat varieties grown in Germany. Ninety-five varieties that were registered in Germany in 2004 were screened with PCR-based markers for Rht-B1 and Rht-D1 and with the microsatellite WMS261 for Rht8. The Rht8 allele for plant height reduction was not found in any of the varieties, which confirms previous studies. Rht-B1b or Rht-D1b alleles were present in 44% of the screened va- rieties, but only 6% contained Rht-B1b, which predominates in northern Europe. Rht-D1b was found in 38% of the varieties, which were planted on 34% of the total area of wheat in Ger- many. Wheat varieties with Rht-D1b were shorter and higher yielding than wheat varieties without this allele, but more susceptible to Fusarium head blight, which could limit their pro- ductivity.

Keywords: wheat, semi-dwarf gene, Rht-D1b, Rht-B1b, Rht8, height reduction

Introduction During the development of current wheat varieties (Triticum aestivum L.) from wild grasses many changes in plant traits have occurred such as into unspelted spike and brittle rachis into non-brittle rachis although height has not been reduced significantly (Aufhammer and Fischbeck 1973). At the begin- ning of the 20th century as the world population started growing exponentially

* Corresponding author; E-mail: [email protected]

0133-3720/$20.00 © 2008 Akadémiai Kiadó, Budapest 554 KNOPF et al.: Occurrence of Rht Genes in German Wheat world food shortages became more likely. To address this possibility, CIMMYT (International Improvement Centre of Corn and Wheat) was established in Mex- ico by the Mexican government and the Rockefeller Foundation. Its task was to increase the yield of wheat and in order to guarantee consistent grain sup- plies (Herdt 1998). With higher yielding varieties and the increased use of mineral fertilizer, lodging of tall-strawed wheat varieties became a serious problem. Breeders re- sponded by introducing shorter-strawed genotypes into their crossing prog- ramme. This material, based on Norin 10, originated from Japan and was used in the USA in the 1940s to develop shorter higher yielding varieties (Gale and Youssefian 1985). Through the efforts of CIMMYT and international contacts the short-strawed characteristics of Norin 10 spread throughout the world, and led to the so-called “” (Borlaug 1968). Three Reduced-height (Rht) genes, namely Rht-B1 (formerly Rht1), Rht-D1 (formerly Rht2) and Rht8 enabled the development of newer higher yielding wheat varieties. Almost 90% of the world’s wheat cultivation comprises varieties with the semi-dwarf alleles Rht-B1b or Rht-D1b, derived from Norin 10 (Worland et al. 1998b). The yield advantage of wheat varieties carrying one of the two semi-dwarf alleles was proofed by Gale and Youssefian (1985) and the Rht8 allele also has a positive yield effect (Worland 1988). Rht-B1b and Rht-D1b, located on chromosomes 4BS and 4DS, respectively, make up part of a homoeologous series of alleles that also govern growth habit (Gale and Marshall 1976; Börner et al. 1996). Rht8 maps on chromosome 2DS (Korzun et al. 1998) 20.9 cM distal to the gene Ppd1 (photoperiod; Worland et al. 1998a). Due to linkage with Ppd1 most wheat varieties containing Rht8 are photo- period insensitive and early flowering (Welsh et al. 1973). This can be advanta- geous during hot summers, which start in early June in southern Europe. This adaptability improves the yield potential of wheat with Rht8 and the linked Ppd1 gene (Worland et al. 1988). Varieties with Rht8 are, therefore, grown more throughout eastern and southern Europe (Worland et al. 1988) and other regions of the world with similar climates, such as Australia. In contrast to Rht8 the Rht-B1 and Rht-D1 alleles are not linked to any photoperiod insensitive genes (Worland et al. 1998a; Worland et al. 1998b). Many short-strawed wheat varieties are grown in Germany, but little is known about the distribution of the Rht genes and the impact these genes have on German wheat cultivation. Despite the yield advantage of short-strawed wheat va- rieties they are, unfortunately, highly susceptible to Fusarium head blight (Mesterhazy 1995) and it is possible that dwarfing genes and susceptibility to Fusarium head blight are correlated.

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The objective of this work was to determine the distribution and cultivated area of three semi-dwarfing genes in recently introduced German wheat varieties and whether there is an associated affect on plant height, yield level and Fusarium susceptibility.

Materials and Methods Ninety-five wheat varieties listed in Germany by the “Department for Protec- tion and National Listing of New Plant Varieties” (BSA) and described in the 2004 “Descriptive variety list” were screened with three different molecular markers. Five young leaves were removed from each variety, all of which were grown in a growth chamber, and DNA extracted according to the protocol of Rogowsky et al. (1991). Rht-B1b, Rht-B1a, Rht-D1b and Rht-D1a were detected with PCR-based markers, which were specific for the base pair change responsible for the semi- dwarf phenotype (Ellis et al. 2002). The PCR conditions for Rht-B1a/b, Rht-D1a/b and Rht8 genes were the same as described by Ellis et al. (2002) and Korzun et al. (1998). Gel electrophoresis was used and samples scanned for the different bands of the wild and the mutant allele types of Rht-B1 and Rht-D1 under a UV-light after ethidium bromide stain- ing. To determine the allelic variation for the microsatellite marker WMS261 linked with Rht8 the protocol of Korzun et al. (1998) was used. Different sized fragments for WMS261 were detected with a 31000 Genetic Analyzer (Applied Biosystems). Data analysis was carried out with the GENOTYPER program (Ap- plied Biosystems Version 3.7 NT). In addition to the marker results, information on plant height, yield and Fusarium head blight were obtained from publicly available data (BSA 2004). Additional statistics for the cultivated area in different regions of Germany in 2004 were collected from the harvest data analysis of the Ministry of Agriculture (BMELV 2006). Plant height, yield and Fusarium head blight susceptibility were analyzed us- ing ANOVA for varieties with different Rht8, Rht-B1 and Rht-D1 alleles. Tukey’s test was used for multiple mean comparisons among varieties with different Rht8 alleles. T-tests were used for comparing plant height, yield and Fusarium head blight susceptibility of wheat varieties with Rht-B1b and Rht-D1b and those with Rht-B1a and Rht-D1a. A U-test was used for comparing the median mean culti- vated area of varieties containing different semi-dwarfing genes.

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Table 1. Occurrence of Rht-D1, Rht-B1 and Rht8 alleles in 95 German winter wheat varieties

Wheat variety Rht-B1b or WMS261 Wheat variety Rht-B1b or WMS261 Rht-D1b or fragment Rht-D1b or fragment none (=tall) Rht8 = 192bp none (=tall) Rht8 = 192bp Achat tall 165 Koch Rht-B1b 174 Akteur tall 197 Kontrast tall 197 Alidos tall 197 Kornett Rht-D1b 165 Alitis tall 165 Korund Rht-D1b 174 Altos tall 197 Limes Rht-D1b 174 Aristos tall 165 Ludwig tall 174 Aron tall 165 Magnus tall 174 Astron tall 165 Maltop tall 174 Atlantis tall 174 Mandup Rht-B1b 174 Atoll Rht-D1b 174 Manhattan tall 174 Bandit Rht-D1b 174 Maverick Rht-D1b 174 Batis tall 165 Meunier Rht-D1b 174 Biscay Rht-D1b 174 Milvus Rht-D1b 165 Borneo tall 165 Monopol tall 165 Bussard tall 174 Naturastar tall 174 Buteo tall 165 Noah Rht-D1b 165 Campari Rht-D1b 174 Novalis Rht-D1b 165 Capnor Rht-D1b 174 Olivin tall 165 Capo tall 197 Opus Rht-D1b 165 Cardos Rht-D1b 165 Ordeal Rht-D1b 165 Centrum tall 174 Paroli Rht-D1b 174 Certo Rht-D1b 174 Pegassos tall 165 Champion tall 165 Petrus tall 165 Claire Rht-D1b 165 Qualibo tall 174 Complet tall 174 Quebon Rht-D1b 174 Compliment tall 197 Redford tall 174 Contur Rht-D1b 165 Ritmo Rht-D1b 174 Creativ Rht-D1b 174 Romanus tall 165 Cubus Rht-D1b 197 Skater tall 174 Dekan Rht-D1b 165 Sobi tall 174 Dream tall 165 Sokrates tall 174 Drifter tall 174 Solitär tall 174 Ebi tall 165 Striker Rht-B1b 165 Elvis tall 165 SW Maxi tall 197 Empire tall 165 SW Topper tall 174 Enorm Rht-D1b 165 Terrier tall 165 Excellenz Rht-D1b 174 Tiger tall 174 Exsept Rht-D1b 165 Tommi Rht-D1b 165 Flair tall 165 Toras Rht-D1b 197 Florida tall 174 Tulsa Rht-B1b 174 Greif Rht-D1b 165 Türkis tall 197 Hermann Rht-B1b 174 Vergas tall 165 Heroldo Rht-D1b 174 Vivant Rht-D1b 174 Hybnos 1 Rht-D1b 174 Wasmo Rht-B1b 174 Hybnos 2B Rht-D1b 174 Wenga tall 165 Hybred Rht-D1b 174 Winnetou tall 174 Idol tall 174 Zentos tall 197 Kaltop Rht-D1b 174

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Results All wheat varieties were screened for their allelic variation of Rht-B1, Rht-D1 and Rht8 genes. The results are shown in Table 1. Only 6% of the 95 wheat variet- ies carried the semi-dwarfing allele Rht-B1b, whereas 37.6% contained Rht-D1b (Table 2). Wheat varieties with either Rht-B1b or Rht-D1b were significantly shorter than non-dwarf varieties, and higher in yield. Varieties with Rht-D1b were more susceptible to Fusarium head blight (Table 2). The combination of both semi-dwarfing alleles Rht-B1b and Rht-D1b was not found in any of the varieties (Table 1).

Table 2. Percentage of winter wheat varieties with Rht-B1 or Rht-D1 and their plant heights, yields and incidences of Fusarium head blight

Allele No. of wheat Plant height Yield Fusarium varieties (%) (1–9)+ (1–9)+ (1–9)+ Rht-B1b 6 3.7 7.8 4.8 Rht-B1a 94 4.9 5.8 4.4 Significant diff. b/a ** *** n.s. Rht-D1b 38 3.8 6.7 4.9 Rht-D1a 62 5.5 5.5 4.2 Significant diff. b/a *** *** ** + Mean values of grouping in Levels of Characteristics (APS); range from 1 = short-strawed, low yield or disease incidence, to 9 = tall-strawed, high yield or disease incidence **, *** = Significant at P = 0.01 and 0.001, respectively n.s. = non-significant

Wheat varieties with Rht-D1b were not grown in particular regions of Ger- many (data not presented). The cultivated area for varieties with Rht-B1b was not given in the BMELV (2006) statistics, because of their minor cultivation area. Only ten varieties with Rht-D1b out of the total of 122 German varieties are widely grown with a total area under cultivation of 34% (data not presented). In the present study the Rht8 allele shown by the fragment of 192bp was not found (Table 1). Almost 90% of the wheat varieties showed 165 or 174 bp frag- ments. The remaining 10% of varieties had a fragment of 197 bp for WMS261 (Table 1). The cultivated area for wheat varieties with the 197 bp fragment was significantly greater in eastern parts of Germany than in the western regions. There were no differences in the distribution of any other Rht8 fragments through- out the country (data not presented).

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Discussion The allele Rht-B1b was carried by only six varieties in our sample. The area grown to these varieties was not recorded in BMELV (2006) and is likely to be in- significant. The merit of Rht-B1b is not definite (Gale and Youssefian 1985). Ac- cording to Allan and Pritchett (1980) outrank Rht-D1b over Rht-B1b with 8% yield advantage, Gale and Youssefian (1985) observed no differences. Either Rht-B1b or Rht-D1b were present in 43.6% of the varieties in Germany compared with 90% of wheat varieties worldwide (Borlaug 1968). One reason for this difference is that both alleles are present in varieties that have been developed by CIMMYT for South America and Asia. Other possible reasons for the large differences are the diversity of 21 known genes for height reduction (McIntosh 1998), which could also be contained by Germany’s wheat varieties, or the nega- tive correlation between short straw and disease incidence. More susceptible vari- eties with Rht-D1b reinforce this assumption (Table 2). For the varieties with Rht-B1b there was no significant difference for the incidence of Fusarium head blight, because there were only six varieties with Rht-B1b for comparison with va- rieties carrying Rht-B1a (Table 2). The lack of combination of both semi-dwarfing alleles (Table 1) can be ex- plained, since the additive effects of combining these alleles (Snape et al. 1977; Allan 1989) would make the crop too short for the German environment. Worldwide use of the multiallelic marker WMS261 which is diagnostic for Rht8 (Korzun et al. 1998) can detect 21 different fragments at that locus, including four predominant fragments of 192 bp, 165 bp, 174 bp and 197 bp (Worland et al. 1998b; Chebotar et al. 2001). In this work the fragment with 192 bp coding for the Rht8 allele was not found (Table 1) probably because it is more important in southern Europe. Such early flowering and ripening wheat varieties that carry Ppd1 are unsuitable for German crop production. The majority of wheat varieties showed 165 or 174 bp fragments, which is similar to the worldwide distribution (Kobiljski, personal communication). This study has demonstrated the widespread adoption of the semi-dwarfing genes Rht-B1b and Rht-D1b in German winter wheat varieties. However, the as- sociated negative side effect of increased susceptibility to Fusarium head blight could reduce the use of varieties carrying Rht alleles in the future. Nevertheless, the yield advantage of varieties with just one Rht allele is justification for their continued cultivation.

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Acknowledgements The technical assistance of Carsten Borizki and Dennis Kusak in the labora- tory is greatly appreciated. This project was supported by KWS LOCHOW GMBH. We thank Dr. Richard Pickering (Crop & Food Research, Christchurch, New Zealand) for critical reading the manuscript and giving useful comments.

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