Powdery Mildew Resistance Genes in Wheat : Identification and Genetic Analysis

Wheat powdery mildew, caused by Blumeria graminis f. sp. Tritici is one of the most devastating diseases of common wheat worldwide. To date, 41 loci (Pm1 to Pm45, Pm18=Pm1c, Pm22=Pm1e, Pm23=Pm4c, Pm31=Pm21) with more than 60 genes/alleles for resistance to powdery mildew have been identified and located on 18 different chromosomes in bread wheat. 29 resistance genes/alleles have been tagged with molecular markers such as restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNAs (RAPDs), amplified fragment length polymorphisms (AFLPs), sequence tagged sites (STS) and simple sequence repeats (SSRs), by using F2, back-cross populations, near-isogenic lines (NILs), doubled haploids (DH), recombinant inbred lines (RILs) or bulked segregant analysis (BSA). The detail information on chromosomal location, molecular markers linked to powdery mildew, mapping population and molecular mapping of powdery mildew resistance genes have been reviewed.


Introduction
Wheat powdery mildew, caused by Blumeria graminis (DC.)E.O.Speer f. sp.Tritici Em.Marchal (Bgt) = Erysiphe graminis DC.Ex Merat f. sp.Tritici Em.Marchal, is one of the most devastating diseases of common wheat occurs in many areas, including China, Germany, Japan, Russia, United Kingdom, South and West Asia, North and East Africa, and the Southeastern United States (Bennett, 1984).Yield losses ranging from 13 to 34% due to this disease (Griffey et al., 1993;Leath & Bowen, 1989).Growing of resistant cultivars offer effective, economically sound and environmentally safe approach to eliminate the use of fungicides and reducing crop losses caused by powdery mildew.There are two types of resistance to powdery mildew.One is called monogenic (vertical) or race specific resistance, which is effective for some isolates of powdery mildew, but ineffective for others.Race specific resistance is mainly via a hypersensitive foliar reaction directly involving single major R genes, designated as Pm (powdery mildew) genes, in a gene-for-gene interaction (Bennett, 1984;Chen and Chelkowski, 1999;Hsam and Zeller, 2002).Race-specific resistance genes are expressed in seedlings and throughout the vegetative cycle of wheat.Though race-specific resistance have been extensively used in wheat breeding programs, selection pressure exerted by cultivars with race-specific resistance genes results in the rapid build-up of isolates with matching virulence genes.Afterward, race-specific resistance breaks down when confronted by pathogen isolates with matching virulence genes and, therefore, is ephemeral.
Another type of resistance to powdery mildew is called adult plant resistance (APR), which retards infection, growth and reproduction of the pathogen in adult plants but not in seedlings.It is also called "slow mildewing" (Shaner, 1973) and "partial resistance" (Hautea et al., 1987).This type of resistance can be identified in cultivars with defeated race-specific genes or lacking known race-specific resistance genes.APR to powdery mildew is more durable than race-specific resistance.For example, APR in wheat cultivar Knox and its derivatives remained effective against powdery mildew infection during the 20 years in which these cultivars were grown commercially (Shaner, 1973).Massey, a derivative of Knox62, was released from Virginia Tech in 1981 (Starling et al. 1984), and still has effective powdery mildew resistance in adult plants.Up to now,41 loci (Pm1 to Pm45,Pm18=Pm1c,Pm22=Pm1e,Pm23=Pm4c,Pm31=Pm21) with more than 60 genes/alleles for resistance to powdery mildew have been identified and located on various chromosomes in bread wheat and its relatives (Ma et al., 2011;McIntosh et al., 2008;Luo et al., 2009;Li et al., 2009;Hua et al., 2009;He et al., 2009).However, resistances of genes are frequently overcome by new Bgt isolates, because the presence and frequency of virulence genes in the pathogen population changes continuously (Leath & Murphy, 1985;Menzies & MacNeil, 1986;Limpert et al. 1987;Namuco et al. 1987).The effective management strategy has been to replace cultivars when their resistance is no longer effective (Wolf, 1984;Leath & Heun, 1990).
Molecular markers are now widely used for gene tagging, gene mapping, and other genetics research because they are not influenced by environmental conditions and growth stage.The use of PCR based molecular markers to tag genomic regions are more efficient for marker assisted selection (MAS), due to the small amount of DNA template required and easy to handling of large population sizes.The identification of molecular markers of flanking disease resistance genes, simplifies breeding activities such as cultivar development (Bonnett et al., 2005), near isogenic line development (Zhou et al., 2005), and pyramiding resistance genes into single genotypes by marker assisted selection (MAS).Many of the recently reported of Pm genes have associated markers (Miranda et al., 2006(Miranda et al., , 2007;;Perugini et al., 2008).Recently, molecular markers such as restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNAs (RAPDs), amplified fragment length polymorphisms (AFLPs), sequence tagged sites (STS) and microsatellites, also termed simple sequence repeats (SSRs), have been widely used to tag and identify powdery mildew resistance genes in wheat by using F 2 and back-cross populations, near-isogenic lines (NILs), doubled haploids (DH), recombinant inbred lines (RILs) or bulked segregant analysis (BSA, Michelmore et al., 1991).

Chromosomal Location of Identified Pm Genes
In recent years, wheat genomics research has increased the use of genetic maps to position a gene of interest between close flanking markers (Haley & Knott, 1992).The application of molecular markers in plant systems increases the efficiency of conventional plant breeding by carrying out indirect selection through molecular markers linked to the traits of interest (Gupta et al., 1999).A linkage map gives information on the position of markers within a linkage group.The map positions are inferred from estimates of recombination frequencies between markers.The distance between these markers is expressed in centimorgan (cM) which represents the recombination rates between them (Jones et al., 1997).Chromosomal positions of several mapped powdery mildew resistance gene loci are presented in Figure 1.
The presence of Pm resistant genes is vital not only for monogenic resistance but also the defeated Pm genes often confer oligogenic and quantitative type resistance when combined together (Royer et al., 1984;Pedersen & Leath, 1988;Paillard et al., 2000).Chromosomal locations, cultivars/lines, sources and references for the 64 known powdery mildew resistance genes/alleles have been identified as major genes for vertical resistance to powdery mildew in wheat (Ma et al., 2011;McIntosh et al., 2008;Luo et al., 2009;Li et al., 2009;Hua et al., 2009;He et al., 2009) (Table 1).Thirty Pm alleles at 25 loci have been nominated for wheat powdery mildew resistance (McIntosh et al. 1995).Twenty-five alleles at 19 loci from Pm1 to Pm19, their locations at chromosomes, and their sources have been reviewed (McIntosh et al. 1995).Other Pm alleles such as Pm20, Pm21, and Pm22 have been reported by Friebe et al., 1994, Qi et al., 1995, and Peusha et al., 1996 and Pm25 has been identified by Shi et al. 1998. Host-pathogen interactions analysis, chromosomal (cytogenesis) analysis and molecular marker techniques have been utilized for determining chromosomal locations of Pm genes.These powdery mildew resistance genes are non-randomly distributed in the genome (Table 3), but form clusters in gene-rich regions (Gill et al., 1996a, b).The highest numbers of Pm loci are contained by each 6B and 7B chromosome with 5 known Pm loci.Chromosomes without known Pm genes are 3A (Pm44), 4D, and 5A (according to Hsam & Zeller, 2002).Gene loci that contain more than one resistance allele are Pm1 with 4 alleles, Pm3 with 9 alleles, Pm4 with 3 alleles, Pm5 with 4 alleles (Chen & Chelkowski, 1999;Hsam & Zeller, 2002).Hsam and Zeller (2002) stated that loci Pm10, Pm11, Pm14, and Pm15 contain individual genes for resistance to Erysiphe graminis f.sp.agropyri and are not effective against Blumeria graminis f. sp.Tritici.Eight gene loci were identified in homoeologous group 1, whereas only one gene (Pm16) was found in homoeologous group 4 (i.e.chromosome 4A; Reader & Miller, 1991).Xue et al. (2009) reported that the Chinese landraces wheat line Xiaobaidong contained a new recessive gene, mlxbd which was located on chromosome 7BL and near the locus Pm5.Powdery mildew resistance gene Mld was located on chromosome 4B in the wheat lines, Halle 13471, H8810/47 and Maris Dove.It was transferred from T. durum (McIntosh et al., 1995).Zeller et al. (1993b) reported that three wheat cultivars, Abo, Aristide and Courtot, contained a major gene, Mlar, for resistance to the German Bgt isolate no.2.Robe and Doussinault (1995) reported that the line RF714 contained a new recessive gene, mlre, for wheat powdery mildew resistance, which was derived from a cross between Aegilops squarrosa 33 and Triticum dicoccum 119.They postulated that mlre was derived from T. dicoccum.A new recessive gene, pmTD1, was identified in the wheat line NC92-8562 transferred from Ae. Tauschii ssp.Tauschii (Shi et al., 1998).Liu et al. (1989) reported that the variety Kenguia 1 contained a new gene, KG, for powdery mildew resistance, which was located on chromosome 6A.

Sources and Distribution of Resistance Genes
Common sources of Pm genes are different species within the primary, secondary and tertiary gene pools.Bread wheat is an allohexaploid species (2n=6x=42), with three distinct genomes (AABBDD).Many of the resistance genes were introduced from ancestral and other wild species related to common wheat such as Triticum monococcum, close relative of the A genome progenitor Triticum uratu, the B genome progenitor Aegilops speltoides, and the D genome progenitor Ae.Tauschii (Hsam & Zeller, 2002;Jiang et al., 1994).Chen and Chelkowski (1999) and Hsam and Zeller (2002) reported a total of 22 resistance alleles at 10 loci including Pm1, Pm2,Pm3 (3a,3b,3c,3d,3e,and 3f), Pm9, Pm18, Pm22 and Pm45 in T. aestivum indicating that Pm genes may still be found in cultivated wheat.Although Bennett (1984) reported that just a small number of Pm genes have been identified which originated in the cultivars T. aestivum.Mains (1933) identified that the wild wheat relatives T. monococcum (AA genomes), T. dicoccum (AABB), and T. timopheevi (AAGG) are the sources of resistance genes to powdery mildew as early as 1933.Screening of old wheat cultivars, land-races and related species for resistance to powdery mildew started in the 1930's (Hsam & Zeller, 2002).Pm genes were identified in many different, widely distributed wheat cultivars and landraces.Pm5a and Pm5b, followed by Pm2, Pm6, and Pm8 are the most common in Europe, Asia and Mediterranean cultivars.Pm3a is commonly found in wheat cultivars grown in diverse geographical locations including the Balkans, Japan, china and the US.Pm3c was identified in Germany, while Pm3d was found in several European countries and China.Pm4a has been used in commercial wheat cultivars in Germany and China.A number of commercially grown cultivars have been found to have Pm gene combinations (Heun & Fischbeck, 1987).The best known cultivars are Normandie with Pm1, Pm2, and Pm9, Maris Huntsman with Pm2 and Pm6, Kronjuvel with Pm4b and Pm8, and 623/65 with Pm4b and Pm8 (Liu et al., 1999).Gene transfer from species within the primary gene pool of Triticum that homologous chromosomes to wheat can be done directly by hybridization, recombination and backcrossing.

Molecular Markers Linked to Powdery Mildew Resistance Gene
Molecular markers are tools that help to locate and identify parts of DNA that are located near a gene or genes of interest.DNA markers identify locations where the sequences differ among varieties.These can be located within genes or in the DNA between genes, so long as they are unique sequences and differ between the plants of interest.Differences of this type are called polymorphisms, and there are a variety of ways to detect and use these signposts within the chromosomes (Suslow et al. 2002).Different molecular techniques have been used to characterize and manipulate resistance genes and to dissect different types of resistance.Molecular markers were used for mapping monogenic resistance, characterization of quantitative resistance in germplasms and marker-aided selection (Michelmore, 1995).Molecular identification of specific DNA sequences can be used to identify the presence or absence of Pm genes in a cultivar, their chromosomal location, the number of genes and the way in which they are transmitted to progeny (Chen & Chelkowski, 1999).With the help of molecular markers, more than 20 powdery mildew resistance genes, such as Pm30 (Liu et al., 2002), Pm31 (Xie et al., 2003), Pm33 (Zhu et al., 2005), Pm34 (Miranda et al., 2006), Pm35 (Miranda et al., 2007), PmY39 (Zhu et al., 2006), PmY201 and PmY212 (Sun et al., 2006), PmU (Qiu et al., 2005), MlZec1 (Mohler et al., 2005), Mlm2033, Mlm80 and pm2026 (Yao et al., 2007;Xu et al., 2008), PmLK906 (Niu et al., 2008) Pm43 (He et al., 2009) and Pm45 (Ma et al., 2011), have been discovered and mapped.Molecular marker techniques commonly used for identification and confirmation of Pm genes to powdery mildew are:

Amplified Fragment Length Polymorphisms (AFLP)
The AFLP technique is based on selectively amplifying a subset of restriction fragments from a complex mixture of DNA fragments obtained after digestion of genomic DNA with restriction endonucleases (Vos et al., 1995).AFLP analysis is a reliable and efficient method and a powerful technique to generate large numbers of markers for the construction of high-density genetic maps (Becker et al., 1995;Keim et al., 1997), identifying specific genes (Kasuga et al., 1997;Schwarz et al., 1999) and map-based cloning of resistance genes (Buschges et al., 1997;Wei et al., 1999).Linked AFLP markers have already been found for Pm1c and Pm4a (Hartl et al., 1999), Pm17 (Hsam et al., 2000), Pm24 (Huang et al., 2000b), Pm29 (Zeller et al., 2002) and pm42 (Hua et al., 2009).

Sequence Tagged Site (STS)
STS markers are single copy sequence amplified using specific primers that match the nucleotide sequences at the ends of a DNA fragment of an RFLP probe (Olson et al., 1989).This approach is extremely useful for studying the relationship between various species and linked to some specific traits (Bustos et al., 1999;Hartl et al., 1993a).RFLP probes specifically linked to a desired trait can be converted into PCR-based STS markers, based on nucleotide sequence of the probe giving polymorphic band pattern, to obtain specific amplification.Tedious hybridization procedures involved in RFLP analysis can be overcome using this technique.Tagged STS markers have been identified for Pm1 (Hu et al., 1997), Pm2 (Mohler & Jahoor, 1996), Pm13 (Cenci et al., 1999), Pm41 (Li et al., 2009) and pm42 (Hua et al., 2009).

Mapping Population
Both F 2 and backcross populations are easy to construct and can be produced within short time.F 2 is more powerful for detecting QTLs with additive effects, and can also be used to estimate the degree of dominance for detected QTLs.When dominance is present, backcrosses give biased estimates of the effects because additive and dominant effects are completely irritating in this design (Carbonell et al., 1993).Many markers require to be analyzed for a large number of plants when F 2 or backcross populations are used for gene mapping.Besides, some traits are difficult to score on an individual plant basis.So, alternative strategies have been used to improve the efficiency of genetic mapping such as NILs (near-isogenic lines), BSA (bulked segregant analysis) and RILs (recombinant inbred Lines) lines or DH (double haploid, Michelmore et al., 1991).
NILs that differ by the presence or absence of the target gene and flanking a small region of DNA, are useful to identify markers linked with the target gene (Young et al., 1988).Genetic markers are polymorphic between the NIL and its recurrent parent that are putatively linked to the target gene (Muehlbauer et al., 1988).Many disease resistance genes have been mapped using NILs, including powdery mildew resistance in wheat and barley (Hinze et al., 1991;Schuller et al., 1992).Pm2, Pm3, Pm4a and Pm6 have been mapped using NILs (Hartl et al., 1995;Tao et al., 2000).
Although NILs are helpful to construct gene map, often they are unavailable, and the development of NILs is time-consuming and laborious.To overcome the problems of NILs, Michelmore et al., (1991) successfully used bulked segregant analysis (BSA) to identify RAPD markers tightly linked to genes for resistance to lettuce downy mildew.Many powdery mildew resistance gene/allele such as Pm1, Pm4a, Pm8, Pm24, Pm25, Pm29, Pm30 and Pm31 have been identified using BSA (Shi et al., 1998).This strategy involves comparing two DNA samples pool of individuals from a segregating population.Within each pool, or bulk, the individuals are identical for the trait or gene of interest but are uninformed for all the other genes.All polymorphic markers between two DNA pools are putatively linked with the target gene.
Recombinant inbred lines or double haploid populations are permanent populations that can be used indefinitely for mapping.They can also be readily disseminated among labs and new data can be continuously added to a pre-existing map.Furthermore, RI lines or DH populations can be evaluated in many different environments.Since each genotype is represented by an inbred line, rather than by an individual plant, a more accurate assessment of the genetic component of variance can be made in studying quantitative traits (Burr et al., 1988).Therefore, RI lines or DH populations are more useful for analysis of quantitative traits or traits that are difficult to characterize on an individual plant basis.DH lines have been used to screen molecular markers associated with genes, Pm3a, Pm3g and Pm8 for powdery mildew resistance in wheat (Hartle et al., 1993b;Sourdille et al., 1999;Wricke et al., 1996).Pm13 has been mapped using RI lines (Donini et al., 1995)

Mapping of Powdery Mildew Resistance Genes
The development of genetic maps of wheat is now adding a new dimension for identification of molecular markers associated with powdery mildew resistance genes.Screening markers can be conducted in the two parents, by selecting several markers on each chromosome of the genetic map, and then linkage between the allele for resistance and the polymorphic markers in the two parents can be estimated by use of QTL statistical analysis based on the data from a segregating population.In plants, molecular mapping and cloning of disease resistance genes will facilitate the study of molecular mechanisms underlying and evolution of resistance and will permit marker-assisted selection in breeding programs.Several powdery mildew resistance genes have been tagged with molecular markers (Table 2).Using cultivar Chancellor as the recurrent parent, Briggle (1969) developed NILs for powdery mildew resistance genes Pm1, Pm2, Pm3 and Pm4a, respectively.Hartl et al. (1995) found that RFLP marker Whs178 was 3 cM away from gene Pm1.Hu et al. (1997) used RAPD markers to tag gene Pm1.RAPD markers UBC320420 and UBC638550 cosegregated with gene Pm1 among 244 F 2 plants.Another RAPD marker OPF12650 was 5.4 cM away from gene Pm1.Recently, Hartl et al. (1999) have used AFLP markers to map gene Pm1c.Among 96 primer combinations, 31 polymorphic AFLP fragments between the resistant and susceptible pools were in accordance with the patterns of the parents.The eight most reliable polymorphic markers were analyzed in a segregating population for the gene Pm1c.Two of them cosegregated with the gene Pm1c and the other six markers were tightly linked with the gene.One AFLP marker, 18M2, was found to be highly specific for the Pm1c gene in diverse genetic backgrounds.RFLP analysis of NILs possessing the gene Pm2 and the recurrent parent indicated that: 1) RFLP marker BCD1871 was 3.5 cM away from gene Pm2 (Ma et al., 1994); 2) RFLP marker Whs295 mapped 2.7 cM away from the gene Pm2 (Hartl et al., 1995); and 3) the gene Pm2 was also linked with RFLP marker Whs350 (Hartl et al., 1995).Ma et al., (2011) found that Pm45 on chromosome 5DS which was flanked by Xgwm205 and Xmag6176, with a genetic distance of 8.3 cM and 2.8 cM, respectively.This gene was 3.3 cM from a locus mapped by the STS marker MAG6137, converted from the RFLP marker BCD1871, which was 3.5 cM from Pm2.Using RFLP analysis of NILs possessing the gene Pm3 and the recurrent parent, Hartl et al. (1993b) found that RFLP marker Whs179 revealed polymorphism not only between the NILs with and without gene Pm3, but also among NILs possessing different alleles of the Pm3 locus.The genetic distance between probe Whs179 and Pm3 was 3.3±1.9cM.Ma et al. (1994) reported that RFLP marker BCD1434 was 1.3 cM away from Pm3a or Pm3b.Ma et al. (1994) also reported that Pm4a cosegregated with RFLP markers BCD1231-2A( 2) and CDO678-2A, and was closely flanked by BCD1231-2A(1) and BCD292-2A.Xue et al. (2009) reported that SSR marker Xgwm577 was linked to powdery mildew resistance gene mlxbd with a distance of 3.5cM. Blanco et al., (2008) found that Pm36 linked on chromosome 5BL with five AFLP markers XP43M32 (250), XP46M31(410), XP41M37(100), XP41M39(250) and XP39M32(120), three genomic SSR markers (Xcfd07, Xwmc75, Xgwm408) and one EST-derived SSR marker (BJ261635).Using F 2 population Zhang et al. (2010) also found that the temporary design powdery mildew resistance gene Ml3D232 on chromosome 5BL which was flanked by Xgwm415 and Xwmc75.Zhang et al. (2009) also found temporary Pm design gene MIW29 on 5BL chromosome and also flanked by Xgwm415 and Xwmc75, with a genetic distance of 2.5 cM and 17.6 cM, respectively.

Conclusion
Molecular markers tightly linked to economically important monogenic or oligogenic trait have potential for immediate utility in plant improvement.Efficient application of molecular markers in plant breeding will depend on the development cost-effective and automated diagnostic technologies.A major problem is when the linked marker used for selection is at a distance away from the gene of interest, leading to crossover between the marker and the gene.In future, the success of marker assisted selection may depend on the possibility of tagging the favorable alleles themselves.
Valuable lessons learnt from past research are likely to encourage more researchers to develop reliable markers and plant breeders to adopt MAS.PCR-based markers are more attractive for MAS, due to the small amount of template required and more efficient handling of large population sizes.PCR-based molecular markers are suitable for marker assisted selection (MAS), due to small amount of DNA require, more efficient managing of large population sizes and possible to map and tag almost any trait.DNA markers have facilitated the dissection of the genetic basis of complex traits and have helped in understanding their mode of action and how their functioning is modulated by the environment.AFLP, RAPD and STS markers can not be applied for differentiation of homozygous and heterozygous individuals in segregating population.Among the DNA marker systems of wheat, microsatellites are recently the optimal marker for MAS, because of their co-dominant inheritance, chromosome-specific and evenly distributed along chromosomes.A large numbers of microsatellite makers are available that offer identification and molecular mapping of powdery mildew resistance gene in wheat (Gupta et al., 2002;Guyomarc'h et al., 2002;Huang et al., 2001;Roder et al., 2004;Song et al., 2002;Stephenson et al., 1998).Already some Pm genes have been identified and mapped by specific nti-inflammat markers.pm42 (Hua et al., 2009), Pm45 (Ma et al., 2011), Pm40 (Luo et al., 2009) andPm36 (Blanco et al., 2008)

Table 1 .
Chromosomal location, cultivar/line, source and Reference of identified powdery mildew resistance genes

Table 2 .
Molecular markers linked to major powdery mildew resistance genes

Table 3 .
Distribution of powdery mildew resistance genes among homoelogous chromosomes in wheat and Rye