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a Division of Biological Resources Sciences, Chonbuk National Univ., Jeonju 561-756, Korea
b Dep. of Agronomy and Plant Genetics, Univ. of Minnesota, St. Paul, MN 55108
c Dep. of Plant Pathology, Univ. of Minnesota, St. Paul, MN 55108
d Institute of Plant Biology, Univ. of Zurich, Zollikerstr. 107, CH-8008 Zurich, Switzerland
e Dep. of Crop and Soil Science, Oregon State Univ., Corvallis, OR 97331-3002
f INIA Quilamapu, Casilla 426, Chillan, Chile
g Dep. of Agroenvironmental Sciences and Technology, Univ. of Bologna, Viale Fanin 44, 40137 Bologna, Italy
* Corresponding author (muehl003{at}umn.edu)
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: AB, advanced backcross • NTNB, net type net blotch • PCR, polymerase chain reaction • QTL, quantitative trait locus • RIL, recombinant inbred line • SSLB, Septoria speckled leaf blotch • SSR, simple sequence repeat
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Populations for AB–QTL analysis commonly use exotic or wild germplasm as the donor and elite germplasm as the recurrent parent. The QTL analysis is conducted in the BC2 or BC3 generation to reduce the frequency of deleterious donor alleles. The AB–QTL analysis has been successful in identifying and introgressing favorable QTL alleles from unadapted germplasm in many crop species (Xiao et al., 1996, 1998; Fulton et al., 2000; Poncet et al., 2000; Brondini et al., 2002; Matus et al., 2003; Von Korff et al., 2004; Pillen et al., 2004). Because many of the deleterious alleles from the donor parent have been eliminated, lines from AB–QTL analysis can be directly integrated into breeding programs. In addition, markers used to genotype the AB lines are more likely to be useful for future breeding efforts. Thus, this approach tightly couples QTL mapping with breeding.
Foliar diseases have been one of the limiting factors in barley production around the world. In the USA, leaf scald is a serious disease of barley in the Pacific Northwest, California, and the Mid Atlantic region, where yield losses can reach 40% (Mathre, 1997). In the Midwest, NTNB, spot blotch, and SSLB are important foliar diseases of barley because they result in yield reductions ranging from 5 to 35% each year (Mathre, 1997; Toubia-Rahme and Steffenson, 2004).
Numerous genetic studies have identified major genes and QTLs conferring resistance to spot blotch, NTNB, SSLB, and leaf scald (Williams, 2003). Hordeum vulgare subsp. spontaneum, the wild progenitor of barley, is a rich source of disease resistance genes (Metcalfe et al., 1977, 1978; Nevo, 1992; Sato and Takeda, 1997; Fetch et al., 2003; Genger et al., 2003; Williams, 2003). We previously identified QTLs for resistance to these foliar diseases in the wild barley accession OUH602 using OUH602/Harrington RILs (Yun et al., 2005). Six QTLs for which wild barley provided the resistance alleles were identified: one for spot blotch resistance on chromosome 1(7H); two for NTNB resistance on chromosomes 3(3H) and 4(4H); two for SSLB resistance on chromosomes 2(2H) and 6(6H); and one for leaf scald resistance on chromosome 5(1H).
In this study, we developed an AB population with the wild barley accession OUH602 as a resistance donor and Harrington as a recurrent parent to (i) validate resistance QTLs for the foliar diseases identified in our OUH602/Harrington RIL population, (ii) determine the genomic location of adult plant spot blotch resistance, (iii) assess the genetic characteristics of the AB population, and (iv) provide access to novel OUH602 alleles in an adapted genetic background for further breeding and genetics applications.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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DNA Marker Analysis
DNA was extracted from the leaves of each of the 98 BC2F6 plants using the methods described by Mesfin et al. (1999). A total of 111 polymorphic SSR markers were used to genotype the AB population. Sixteen, 16, 17, 15, 12, 17, and 18 markers from chromosomes 1(7H) to 7(5H), respectively, were selected from the previously generated linkage map for the wild barley OUH602 x Harrington RIL population (Yun et al., 2005). The polymerase chain reaction (PCR) was performed according to the procedures of Ramsay et al. (2000). Amplified products were separated on 5% (w/v) polyacrylamide gels and visualized by silver staining as described by Bassam et al. (1991). The PCR products amplified using fluorescent-labeled primers were separated and detected using the IR2 DNA analyzer (Global ed., LI-COR, Lincoln, NE).
JoinMap (v. 3.0; Van Ooijen and Voorrips, 2001) was used to develop a linkage map of SSR markers for the RIL (Yun et al., 2005) and AB (BC2F6) populations. Marker locations in both populations were compared to confirm their chromosomal locations. Linkage map positions of the markers determined for the RIL population were used for graphical genotyping and QTL analysis of the AB population. Bin information for the markers was inferred following the definitions of Kleinhofs and Graner (2001), the barley genomics website (Washington State University, 2005), and the Oregon Wolfe Barley population data (Hayes and Filichkin, 2004).
Graphical Genotype Analysis
Graphical genotyping of the AB population was conducted using GGT software (Van Berloo, 1999). The size and number of introgressions were also determined using GGT software. The parental genome ratio was calculated by determining the total genome derived from the donor parent (OUH602) as a proportion of the recurrent parent (Harrington) genome, as described by Young and Tanksley (1989).
Greenhouse Evaluations
Six seeds of each AB line were planted individually into six 8- by 8-cm plastic pots filled with Metro Mix 200 growing media (Scotts-Sierra Horticulture Products, Marysville, OH). After planting, all pots were moistened and then moved to a cold room at 4°C for 7 d to break any seed dormancy. After the 4°C exposure period, pots were moved to a greenhouse, and plants were grown at approximately 22°C (range from 18–25°C) with a 16-h photoperiod using sodium vapor lights. The plants were fertilized with a 20:20:20 (N–P–K) water-soluble fertilizer (Scotts-Sierra Horticulture Products, Marysville, OH) weekly at the recommended rate.
All greenhouse disease evaluation experiments for seedling diseases were arranged in a randomized complete block design with three replications per line. At least two evaluation experiments were conducted for each disease. Each block included the AB lines, the parents, and a set of controls that were susceptible or resistant to the evaluated pathogens. The parents and progeny from the AB population were evaluated for resistance to spot blotch, NTNB, SSLB, and leaf scald as seedlings by artificial inoculation in the greenhouse at the University of Minnesota in St. Paul. The following pathogen isolates were used for disease evaluation: isolate ND85F of spot blotch, isolate 30199013 of NTNB (from R. Dill-Macky), isolate SP97–15 of SSLB, and isolate LA94–1A of leaf scald. All isolates except P. teres were provided by B. Steffenson. The pathogen isolates used represent common virulence spectra found in the Upper Midwest region of the USA.
Three plants per line were inoculated when their second leaf was fully expanded. Inoculations were performed according to Fetch et al. (2003). Disease reactions were assessed after specific incubation periods using published assessment guides for each respective pathogen (Fetch et al., 2003). Spot blotch reactions were rated 10 to 12 d after incubation using a 1-to-9 scale (Fetch and Steffenson, 1999). The NTNB reactions were evaluated 18 d after inoculation using a 0-to-10 scale (Tekauz, 1985). Leaf scald and SSLB were evaluated 23 d after inoculation using a 0-to-4 scale for leaf scald (Jackson and Webster, 1976) and a 0-to-5 scale for SSLB (Toubia-Rahme and Steffenson, 2004). For each disease scale, the higher number indicates greater susceptibility.
Field Evaluations
For the field evaluations of adult plant spot blotch reaction, seeds of AB lines were planted in paired rows of hill plots (10–12 seeds plot–1) spaced 0.3 m apart. Two experiments were conducted in St. Paul in 2002 and 2003. Inoculations were performed according to Fetch and Steffenson (1999). The AB lines were sown in a randomized complete block design with two replications. Each block included the AB lines, the parents, and a set of reference barley cultivars susceptible or resistant to spot blotch. Disease reactions were evaluated on a 1-to-9 scale (Fetch and Steffenson, 1999) on the second and third leaf below the flag leaf.
Statistical Analysis
Analysis of variance for the disease data from the greenhouse and field evaluations were conducted using Statistix 8 (Analytical Software, Tallahassee, FL). Single marker analysis was conducted by simple linear regression (Jiang and Zeng, 1997) between the disease phenotypes and the marker genotypes for the AB lines. QTL Cartographer version 1.17 (Basten et al., 2003), Window QTL Cartographer 2.0 (Wang et al., 2004), and Statistix 8 (Analytical Software, Tallahassee, FL) were used in the analysis.
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RESULTS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Disease Response of the Parents and AB Lines
OUH602 exhibited very high levels of seedling resistance to spot blotch, NTNB, SSLB, and leaf scald, and adult plant resistance to spot blotch. In contrast, Harrington was highly susceptible to these diseases (Table 1). Disease resistance levels between the AB lines were significantly different for all of the diseases evaluated. No significant interactions between lines and experiments were detected, except for seedling spot blotch in the two greenhouse experiments (Table 1). Therefore, QTL analyses for each disease phenotype, except for seedling spot blotch resistance, were conducted using the mean of the two experiments.
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DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Wild Barley Genome is Retained Normally in the Cultivated Barley Background
The AB population developed in this study exhibited a calculated average of 11.25% donor-parent (OUH602) genome introgression into Harrington. This introgression proportion is close to the expected amount of 12.5% for a BC2 population in the absence of selection (Stam and Zeven, 1981). Similar amounts of donor chromosome introgression were reported in two wild barley x cultivated barley BC2F6 and BC2DH populations (Matus et al., 2003; Von Korff et al., 2004).
The average length of donor chromosome fragments in the AB lines was 14.5 cM. This is significantly shorter than the average of 38.6 cM introgression fragments reported in the wild barley x cultivated barley BC2F6 populations (Matus et al., 2003), where 47 SSR markers were used to generate genotypes for 1067 cM of barley genome. In our study, 111 markers were used for the barley genome covering 974 cM. Thus, the shorter introgression regions in this study are likely due to the larger number of markers used to genotype the AB lines.
Segregation distortion was not observed for most markers, except for UMB101 on chromosome 1(7H), GBM1055, EBmac0701 and GBM1003 on chromosome 4(4H), and HVM30 on chromosome 7(5H). GBM1055 showed distorted segregation toward OUH602, while the others toward Harrington. This degree of segregation distortion is similar to that reported in the AB lines of wild barley Caesarea 26–24/Harrington (Matus et al., 2003). In our population, all markers exhibited transmission of donor alleles, indicating that there was no selection against the OUH602 alleles. However, unintentional selection against gene(s) on the chromosomal regions not fully covered by markers used in this study might be undetected. Segregation distortion is a prominent feature in crosses between the cultivated and wild progenitor species in pearl millet (Pennisetum glaucum (L.) R. Br.; Poncet et al., 2000), rice (Oryza sativa L.; Brondini et al., 2002), and tomato (Lycopersicon esculentum Mill.; Foolad et al., 2002). Our results suggest that there is a high similarity in genome constitution between the accession OUH602 and Harrington. This interpretation is consistent with the consensus that malting barley and wild barley belong to the same species (Baum and Johnson, 1996).
Validation of OUH602-Derived Foliar Disease Resistance QTL
All the QTLs identified in the OUH602/Harrington RIL (Yun et al., 2005), except one NTNB QTL on chromosome 5(1H), were validated in the AB population. To maintain consistency between studies, QTL names and BIN numbers used were from the OUH602/Harrington RIL mapping study (Yun et al., 2005). All validated QTL marker positions in this study were in the same general region as detected by the OUH602/Harrington RIL mapping study. The QTLs detected in the OUH602/Harington RIL and AB populations are summarized in Fig. 2.
Spot blotch resistance QTLs have been localized on chromosomes 1(7H), 2(2H), 3(3H), 5(1H), and 7(5H) in studies of several cultivated barley mapping populations (Steffenson et al., 1996; Bilgic et al., 2005). The QTLs on chromosomes 1(7H) and 3(3H) conferred both seedling and adult plant resistance, but the QTLs on chromosomes 2(2H), 5(1H), and 7(5H) conferred adult plant resistance. Our results show that the QTL on chromosome 1(7H) confers both seedling and adult plant resistance to spot blotch, indicating that the OUH602 allele provides season-long resistance to spot blotch. The QTL Rcs-qtl-4H-4-6 conferring adult plant resistance has not been reported previously, and may be a unique allele for use in cultivated barley.
Many QTLs conferring seedling resistance to NTNB have been localized on all chromosomes, except 1(7H) and 7(5H) (Steffenson et al., 1996; Richter et al., 1998). Two QTLs, Rpt-qtl-4H-5-7 and Rpt-qtl-3H-4, identified in the OUH602/Harrington RIL population (Yun et al., 2005) were validated in the AB population in this study and located to the same chromosomal regions in other studies (Steffenson et al., 1996; Spaner et al., 1998). The QTL Rpt-qtl-1H-5-6 identified in the OUH602/Harrington RIL population was not detected in the OUH602/Harrington AB lines. This result was not surprising since Rpt-qtl-1H-5–6 is a minor effect QTL that was only detected in a single environment in the RIL population (Yun et al., 2005).
The two SSLB resistance QTLs (Rsp-qtl-2H-7-11 and Rsp-qtl-6H-10-14) identified in this study validate the QTLs identified in the OUH602/Harrington RIL population (Yun et al., 2005). Many resistance sources have been identified in both cultivated and wild barley (Fetch et al., 2003; Metcalfe et al., 1977, 1978; Legge et al., 1996; Rasmusson and Rogers, 1963; Toubia-Rahme et al., 2003; Toubia-Rahme and Steffenson, 2004). Three major SSLB resistance genes have been mapped in barley. One derived from cultivated barley was localized on chromosome 4(4H) in a H. v. subsp. vulgare x H. bulbosum RIL population (Toubia-Rahme et al., 2003). The other two mapped genes (Rsp2 and Rsp3) are very closely linked and were mapped to chromosome 5(1H) (S. Zhong, H. Toubia-Rahme, and B. Steffenson, 2004, unpublished data).
Leaf scald resistance QTLs have been localized on all chromosomes, except chromosome 7(5H) (Backes et al., 1995; Spaner et al., 1998; Garvin et al., 2000; Genger et al., 2003). Leaf scald resistance QTLs have been mapped to the same chromosomal regions as identified in this study (Rrs-qtl-1H-1-4), in cultivated and wild barley (Backes et al., 1995; Garvin et al., 2000), and in the OUH602/Harrington RIL population (Yun et al., 2005).
AB Population Did Not Detect Major New Disease Resistance Alleles
Advanced backcross–QTL analysis is a powerful approach to uncover novel alleles for traits that are masked in standard RIL populations by deleterious alleles in the donor genome. In this study, we did not identify any novel seedling-expressed disease resistance loci that were not detected in the OUH602/Harrington RIL population. Moreover, the phenotypic variation explained by the associated markers (R2) was similar in AB and RIL populations for most QTLs, except Rrs-qtl-1H-1-4 (Table 2). Markers associated with Rrs-qtl-1H-1-4 had considerably higher values in the RIL than in the AB population. This is in contrast with the predicted superiority of the AB populations compared with selfing populations for detecting QTL (Tanksley and Nelson, 1996). Many new QTL for agronomic and quality traits were identified using AB lines developed from wild barley and Harrington (Matus et al., 2003). Among the 33 agronomic and quality trait QTLs detected in AB populations, 18 QTLs were not identified in the classical QTL analyses (Pillen et al., 2004). In tomato, more QTLs for soluble-solids and fruit weight were detected in an AB population compared with a BC1 population created from the same parents, demonstrating the power of AB populations in detecting additional QTLs (Grandillo and Tanksley, 1996; Tanksley et al., 1996). These results indicate contrasting impact of wild germplasm-derived alleles on disease and agronomic traits. Therefore, our results suggest that the expression of seedling disease resistance to foliar pathogens is not constrained by the donor genome. There do not appear to be any alleles in the OUH602 genome that mask the expression of the foliar disease resistance we examined. Our interpretation is that beneficial alleles from wild germplasm that impact agronomic and quality traits can easily be masked by deleterious alleles in the wild germplasm, whereas alleles that impact foliar seedling disease resistance are in general not masked. Taken together, our results indicate that the OUH602/Harrington RIL population is equivalent to the OUH602/Harrington AB population in detecting QTL for seedling disease resistance. This suggests that while AB–QTL analysis may be very important for identifying wild genes for traits like yield in rice (Xiao et al., 1996), it may not provide a substantial advantage to identify wild genes for resistance to foliar diseases in barley.
OUH602/Harrington AB Population for Future Barley Genetics and Breeding Applications
AB lines developed in this study will be useful for transferring favorable alleles to elite lines, dissection of QTLs, fine mapping of the regions where the resistance alleles were found, and cloning genes. The utility of AB lines for transferring favorable alleles from unadapted germplasms to elite lines has been demonstrated in many crops, including barley (Ho et al., 2002; Huang et al., 2003; Talamè et al., 2004; Wu et al., 2004). For instance, introgression lines with the gene Ppd-H1 from exotic barley have a significantly shorter photoperiod response (Von Korff et al., 2004). Successful cloning of QTLs using AB lines has been reported for various traits (e.g., Fridman et al., 2000; Frary et al., 2000; Takahashi et al., 2001). Therefore, the AB lines developed in this study will have a variety of useful applications.
A major barrier to the use of exotic germplasm for crop improvement is that populations derived from wide crosses segregate for many undesirable alleles. This poses several problems. First, as was discussed earlier, donor parent alleles can mask potentially desirable alleles. This was not observed for disease resistance in our study, but has been observed for agronomic or quality traits. Also, the number of undesirable alleles segregating in wide crosses makes it unlikely to identify individuals that are fixed for the desirable allele at all or most of the important loci. This is particularly true when the size of the breeding populations is small. Lastly, the segregation for undesirable alleles can interfere with the measurement of traits important for breeding and hinder selection. For example, it is difficult to assess kernel plumpness in lines with substantial shattering. The AB lines developed in this study, therefore, provide good parents for developing breeding populations since the number of undesirable donor alleles in any cross will be reduced, each line has been genotyped with markers that are directly useful in marker-assisted selection, and several of the AB lines carry useful disease resistance genes.
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Received for publication September 1, 2005.
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REFERENCES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunit of protein kinase CK2. Proc. Natl. Acad. Sci. USA 98:7922–7927.This article has been cited by other articles:
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  R. van Berloo GGT 2.0: Versatile Software for Visualization and Analysis of Genetic Data J. Hered., March 1, 2008; 99(2): 232 - 236. [Abstract] [Full Text] [PDF]
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