Genetic Diversity of the Pm 3 Powdery Mildew Resistance Alleles in Wheat Gene Bank Accessions as Assessed by Molecular Markers

Genetic resources of crop plants are essential for crop breeding. They are conserved in gene banks in form of a large numbers of accessions. These accessions harbor allelic variants of agronomically important genes and molecular tools allow a rapid assessment of this allelic diversity. Here, we have screened a collection of 1005 wheat gene bank accessions for powdery mildew resistance and a molecular characterization for functional alleles at the wheat powdery mildew resistance locus Pm3 was carried out mostly on the resistant accessions. The two analyzed sets of accessions consisted of 733 accessions originating from 20 different countries and 272 landraces originating specifically from Afghanistan. The Pm3 haplotype (indicating the presence of a Pm3-type of gene, susceptible or resistant) was found to be abundantly present in both sets. The accessions with a Pm3 haplotype were further screened for the presence of the functional Pm3a to Pm3g alleles using allele-specific molecular markers. Pm3b and Pm3c were the most frequently found alleles while the other five alleles were detected only in few accessions (Pm3d, Pm3e, Pm3f) or not detected at all (Pm3a, Pm3g). The data further showed that Pm3b is the major source of Pm3-mediated powdery mildew resistance in wheat accessions from Afghanistan. Susceptible allelic variants of Pm3 were found to be OPEN ACCESS Diversity 2010, 2 769 widespread in the wheat gene pool. The presented molecular analysis of Pm3 alleles in a diverse set of wheat accessions indicates that several alleles have defined geographical origins. Possibly, the widespread Pm3b and Pm3c alleles evolved relatively early in wheat cultivation, allowing their subsequent diffusion into a broad set of wheat lines.


Introduction
Genetic variation forms the basis for crop improvement through breeding.Genetic diversity of different crop species is well conserved in the form of wild relatives, landraces and early varieties in the gene banks worldwide [1,2].Plant breeding benefits from this diversity through identification of accessions that carry agronomically important genes and could potentially serve as parental accessions to develop new varieties.The use of molecular markers for evaluation of germplasm diversity among the gene bank accessions now represents an attractive alternative to the conventional phenotypic screens [3,4].However, it is a challenging task to develop molecular markers diagnostic for a trait in wheat due to its hexaploid and large genome.Despite these difficulties, the development and use of molecular markers in wheat has strongly increased.Microsatellites (SSR markers) have been widely used to characterize genetic diversity in wheat accessions [5][6][7][8].With the recent success in cloning of some agronomically important wheat genes, it is now possible to detect the presence of their allelic forms in a large number of germplasm accessions [9].Among the important cloned wheat genes are the ones controlling protein content (Gpc-B1) [10], flowering time (VRN1, VRN2) [11,12], a domestication trait (Q gene) [13] and disease resistance genes (Lr21, Lr10, Lr1, Lr34 and Pm3) [9,[14][15][16][17][18][19][20][21].
Powdery mildew is one of the devastating wheat diseases and is caused by the biotrophic fungus Blumeria graminis f.sp.tritici.The identification of natural sources of resistance and breeding for resistant varieties is the most effective way to control this disease [22], as chemical control is expensive.To date, more than 37 Pm resistance genes have been characterized [23,24], while only one of these genes, Pm3, has been cloned [18].Pm3 is localized on the short arm of wheat chromosome 1A [25] and is now known to occur in 15 functional allelic forms (Pm3a to Pm3g, Pm3k to Pm3r).The Pm3 alleles confer race-specific resistance to different subsets of wheat powdery mildew races [18,19,21].Pm3a to Pm3g are the seven Pm3 alleles that were known and characterized by classical genetic methods before the cloning of this locus [18].The initial cloning of the Pm3b allele allowed the isolation of all the other Pm3 alleles (Pm3a and Pm3c to Pm3g), based on the high sequence conservation between the different Pm3 alleles.On the basis of this conservation, Pm3 haplotype-specific markers were developed [18,21].These markers are diagnostic for the presence of a Pm3-type of gene (can be a resistant or susceptible allele), although they do not identify the particular allele.Additionally, functional markers for specific detection of Pm3 alleles Pm3a to Pm3g were developed [26].These markers were based on nucleotide polymorphisms of the coding and adjacent non-coding regions of the Pm3 gene and were reported to be highly diagnostic for specific Pm3 resistance alleles.These markers were validated on different varieties and breeding lines [26].The Pm3-haplotype markers and the Pm3-allele specific markers are very helpful to effectively screen large sets of accessions for the presence of Pm3 alleles.Furthermore, the long range PCR based approaches using molecular markers located in conserved regions between different Pm3 alleles have recently allowed isolation of eight additional functional alleles of Pm3 (Pm3k to Pm3r) from wheat gene bank accessions [9,20].
Here, we have studied the Pm3 allele distribution in a large set of 1005 wheat gene bank accessions which originated from 20 different countries.We have used the Pm3-haplotype marker and the Pm3 allele-specific markers (Pm3a to Pm3g) to determine the presence of specific Pm3 alleles.This revealed the widespread existence of mostly susceptible Pm3 alleles in the wheat gene pool, whereas Pm3b and Pm3c were the most abundant resistance alleles.The results also indicated Pm3b to be the dominant source of wheat powdery mildew resistance in landraces from Afghanistan.The relatively low frequency of the Pm3a, Pm3d, Pm3e, Pm3f and Pm3g alleles in gene bank accessions supports the hypothesis of a recent evolution of these alleles in the hexaploid wheat gene pool.

Plant Material
A total of 1005 accessions were screened in this study.They were divided into two sets.The first set of 733 accessions was obtained from the gene bank of IPK, Gatersleben, Germany.These accessions originated from 20 different countries, i.e., Argentina, Australia, Azerbaijan, Canada, China, Ethiopia, France, India, Iraq, Japan, Kazakhastan, Kyrgystan, Mexico, Nepal, Russia, Sudan, Switzerland, USA, Tajikistan and Uzbekistan.The second set of 272 bread wheat landraces from Afghanistan was obtained from the Australian Winter Cereals Collections, Australia.

Phenotypic Screening of the Wheat Accessions for Powdery Mildew Resistance
Detached leaf segments from seven day old plants were placed on phytagar media and were subjected to infection with powdery mildew isolates [27].The scoring was done 9-10 days after infection.The phenotypes were classified into three categories based on the percentage of infected area on leaves: resistant (R), intermediate [(I) with two further categories: Intermediate resistant (IR) and Intermediate susceptible (IS)] and susceptible lines (S).

Pm3 haplotype specific STS marker
The primer pair UP3B (5′TGGTTGCACAGACAATCC3′) and UP1A (5′GAAACCCGGCATAAGGAG3′) located in the Pm3 promoter region, 4,360 bp upstream from the Pm3 ATG start codon [18,19] was used to determine the presence or absence of the Pm3 haplotype, indicating if a Pm3-type of gene is present.

Pm3 Allele Specific PCRs
Allele specific PCR for Pm3a to Pm3g was carried out as described by Tommasini et al. [26].The PCR conditions included an initial denaturation step at 94 °C for 3 min followed by 30 cycles of 94 °C for 45 seconds, an annealing step at variable annealing temperatures (as recommended for different primer pairs for specific Pm3 alleles) for 35 seconds, an elongation step of 1 min per kb length of the amplified fragment at 72 °C; and a final extension step at 72 °C for 5 min [26].Amplification products were detected by standard gel electrophoresis on 1-1.2% agarose gels.

Isolation of the Full-Length Coding Sequence of Pm3 and Sequencing
Pm3CS and Pm3Go/Jho were amplified by using Pm3 locus-specific, long-range PCR amplification followed by a nested, long range PCR [18,21].PCR primers were based on the upstream and downstream sequence of the coding region of the Pm3b allele [18].PCR amplification of the Pm3 alleles was carried out with the Herculase-II fusion high-fidelity DNA polymerase.Amplified fragments were cloned into the multiple cloning site of expression vector pGY1 [28].DNA sequencing was performed with an Applied Biosystems Capillary Sequencer model 3730.

Detection of Pm3 Alleles in Gene Bank Accessions Using Allele-Specific Markers
In order to determine the presence and frequency of Pm3 alleles in wheat gene bank accessions, we first analyzed the set of 733 accessions obtained from IPK, Gatersleben Germany.All accessions were first phenotyped for powdery mildew resistance (Bhullar and Keller, unpublished data).The 154 resistant or intermediately resistant accessions were screened both for the presence of the Pm3 gene using Pm3 haplotype-specific markers and for the seven Pm3 resistance alleles Pm3a to Pm3g.The Pm3 haplotype-specific STS marker amplifies a 946bp fragment originating from the 5′ non-coding region of Pm3 [27].It was found that 109 accessions possessed the Pm3 haplotype and these accessions were further subjected to screening with allele-specific primers for Pm3a to Pm3g.In these 109 accessions, Pm3c was the most frequently detected allele, found in 17 accessions (Table 1) originating from Nepal (8), India (7), China (1) and Australia (1).In an earlier study that determined the presence of Pm3 alleles in landraces [27], the Pm3c allele had been found in three landraces from Iran and one from Azerbaijan (Table 1).Although Pm3c was first identified in cultivar Sonora from Mexico [29,30], the data obtained here indicate that this allele has evolved in Nepal, India or close geographic areas.
The Pm3b allele was the second most frequent allele in the screened set (Figure 1).It was found in 6 accessions with two of these accessions originating from Russia while the remaining four were from France, Kazakhastan, Uzbekistan and Tajikistan (one each).In a previous study, Pm3b had been reported in 15 landraces from Afghanistan, 6 landraces each from Russia and Iran, 2 landraces from Azerbaijan and 1 landrace from Turkey (Table 1) [27].As evident from these data, Pm3b was mostly detected in accessions that originated from the countries neighboring Uzbekistan, the country of its first identification in landrace Chul [30,31].The large number of landraces from Afghanistan with Pm3b indicates an origin of this particular allele in this geographic region (see also 3.4).
We detected the presence of Pm3d, Pm3e and Pm3f alleles in 1, 2 and 2 accessions, respectively, in the screened set.The Pm3d allele was found in an accession from Argentina while the two accessions carrying Pm3e originated from India.The two accessions carrying Pm3f were from Argentina and China.The Pm3a and Pm3g alleles were not detected in this set.The alleles Pm3d, Pm3e and Pm3f had first been described in accessions originating from Afghanistan (Hindukush), Australia and USA, respectively [32].Thus, in contrast to Pm3b, we found the alleles Pm3c, Pm3d, Pm3e and Pm3f in accessions with different and distant origins compared to the places of their first identification.It is likely that the cultivars used for first identification were derived from landraces with geographical origins near the evolutionary origin of the alleles.There are two earlier studies which determined the presence of Pm3 alleles in some elite breeding lines as well as landraces [19,27].From the Pm3a-g alleles, Pm3b and Pm3c were the only detected Pm3 alleles in 30 and 4 landraces respectively, out of a total of 1320 landraces screened for the presence of Pm3a to Pm3g alleles by Kaur et al. [27].Pm3g has been identified in one breeding line Oid HD4-219 which originated from France [19].In addition, Pm3d has been detected in breeding line Oid HD4-266 (France) while Pm3e was found in a landrace from Tajikistan (TA10381) and a breeding line (Oid 91-35) from France [19].These data support a recent evolution of at least some Pm3 alleles in hexaploid wheat breeding material as Pm3a, Pm3d, Pm3f and Pm3g have not been detected in any of the wheat landraces screened to date [27], but only in advanced breeding material.
The data presented here provide a detailed overview on the presence of Pm3 resistance alleles in the wheat germplasm.Similar studies on functional allelic diversity have also been made for Vrn locus responsible for vernalization requirements in wheat.A set of 56 spring wheat cultivars and breeding lines were assessed for the allelic composition of Vrn-1 locus and it was found that the majority of the germplasm carried the dominant allele Vrn-A1 alone or in combination with Vrn-B1, Vrn-D1 or Vrn-B3 alleles [33].In another study, 278 Chinese wheat cultivars were characterized for the vernalization genes Vrn-A1, -B1, -D1, and -B3.The dominant Vrn-D1 allele was detected with the highest frequency in the Chinese wheat cultivars (37.8%), followed by the dominant Vrn-A1, -B1, and -B3 alleles [34].

The Susceptible Pm3CS Allele is Present in Accessions From Diverse Geographical Origins
Among the set of 109 accessions that possess the Pm3 haplotype (see 3.1), the Pm3a to Pm3g alleles were detected in 28 accessions (Pm3b, Pm3c, Pm3d, Pm3e and Pm3f; see 3.1).The remaining 81 accessions from this set must either have different alleles of Pm3 which could not be detected by the allele specific markers or they carry the widespread susceptible allele Pm3CS.Pm3CS is the consensus sequence of all Pm3 alleles [18] and no Pm3CS-specific markers can be developed.Therefore, its presence can only be determined by amplification and sequencing of the complete gene from a particular accession.To test for the frequency of the Pm3CS allele in resistant germplasm having a Pm3 haplotype, but none of the classical Pm3a-g alleles, we selected from the 81 accessions described above a subset of 41 accessions and amplified the sequence of Pm3 genes.The susceptible Pm3 allele, Pm3CS was isolated from 8 different accessions originating from India (2), Australia (1), France (1), Canada (2), Ethiopia (1) and Tajikistan (1) (Table 2).Furthermore, out of the 41 accessions analysed, 15 accessions contained new Pm3 sequences [35] and 18 accessions had the Pm3Go/Jho allele (see 3.3.below).
Similar observations have been made in previous studies, where Pm3CS was isolated from accessions of very different origins.Pm3CS was found to be the most frequently amplified sequence in a set of 45 resistant hexaploid wheat landraces, where it was identified in 9 accessions [9].Yahiaoui et al. [19] also reported the isolation of Pm3CS from different breeding lines and cultivars (Table 2).Pm3CS has also been isolated from tetraploid wheat accessions, as reported in Yahiaoui et al. [20], indicating that Pm3CS is an ancient allele which was present in the wheat gene pool before wheat domestication and the evolution of hexaploid wheat.Therefore, Pm3CS has been proposed to be the ancestor of resistance alleles of Pm3 [19].
These studies demonstrate that the susceptible ancestral sequence Pm3CS is present in accessions from many and very diverse geographical origins, both in the hexaploid as well as the tetraploid wheat gene pool.It is a very frequent allele of Pm3 and has been identified in different types of wheat material, including landraces, breeding lines and cultivars (see Table 2).

Abundant Presence of the Transitional, Susceptible Pm3Go/Jho Allele in Accessions from Nepal, India and Bhutan
In addition to the amplification of Pm3CS, another previously reported susceptible allele, Pm3Go/Jho, was isolated from 18 accessions (among the 41 accessions subjected to Pm3 amplification, see 3.2).We specifically identified this allele in accessions that originated from Nepal (13 accessions), India (4 accessions) and China (1 accession) (Table 2).This indicates a widespread occurrence of Pm3Go/Jho in Asia and specifically close to the Himalayan range.Pm3Go/Jho has been previously isolated from two bread wheat landraces PI481711Go and PI481723Jho, collected from high altitude (2800m) in Bhutan [19].This allele was named Pm3Go/Jho after the name of accessions from which it was first isolated.Pm3Go/Jho encodes a protein with only one amino acid difference in comparison to PM3CS and this change at position 659 (W 659 instead of R 659 ) is identical to the one in PM3D and PM3E proteins.Pm3d and Pm3e encode proteins that are highly similar to PM3CS and have only 3 and 2 amino acid differences to PM3CS, respectively.Pm3Go/Jho is a susceptible allele but the W 659 amino acid polymorphism is essential for Pm3d dependent resistance together with the other 2 polymorphic amino acids in Pm3d [19].This indicates that Pm3Go/Jho represents a transitional allele, representing the evolutionary link between the ancestral sequence Pm3CS and the functional Pm3d and Pm3e alleles.This would suggest that the Pm3d and Pm3e alleles also originated in or near the Himalayan range.

Widespread Existence of the Pm3b Resistance Allele in Landraces from Afghanistan
The second set of genetic material analyzed in this study consisted of 272 landraces from Afghanistan.We phenotyped these accessions for powdery mildew resistance by infecting them with four different isolates.Thirty-nine out of 272 accessions were found to be resistant or intermediately resistant to at least one of the isolate tested (Figure 2a, 2c; Appendix 1).These 272 accessions were also screened for the Pm3 haplotype using a specific STS marker (see above).The Pm3 haplotype was present at a high frequency in 236 accessions out of 272 (86.7%) (Appendix 1, Figure 2a, 2b).These 236 accessions were then screened for the presence of known Pm3 alleles (Pm3a-Pm3g) using Pm3 allele specific markers.The Pm3b allele was found to be the only known functional Pm3 allele present in this subset and was detected in twelve landraces (Figure 1, Appendix 1).The Pm3b allele was found to be well distributed geographically in the wheat growing regions of Afghanistan and was detected in accessions that originated from Herat, Badghis, Vardak, Parvan and Ghazni provinces (Figure 2a).Among the 39 powdery mildew resistant accessions (Figure 2a and 2c), 12 landraces had the Pm3b allele (32.4%) (Figure 2a) and two did not possess the Pm3 haplotype (Figure 2c).These data suggest that the Pm3b allele is possibly the only active Pm3 resistance gene in Afghanistan landraces.We conclude that Pm3b is a very frequent source of the observed resistance in landraces in Afghanistan and the resistance in the remaining 27 landraces with a resistance phenotype must be caused either by genes different from Pm3 alleles, by the recently characterized Pm3k-r alleles, or by new, unknown Pm3 alleles.In a previous study, large sets of landraces originating from Turkey (420 landraces), Iran (393 landraces) and Pakistan (131 landraces) were screened for the presence of the Pm3b alleles and it was only found in 1, 6 and 0 landraces from each of these country sets, respectively (Table 1) [9,27].
The high percentage of 236 accessions being susceptible (84.3%) but having the Pm3 haplotype (Figure 2b) suggest that the Pm3 alleles in these lines do not correspond to Pm3 resistance alleles and that susceptible alleles such as Pm3CS or Pm3Go/Jho must be widespread among the landraces.Evidently, the high frequency of the Pm3 haplotype provides an ideal genetic background for the mutational development of active resistance genes with new specificities in recent evolutionary times.The accessions with susceptible and resistance alleles of Pm3 originated in geographical vicinity of each other in Afghanistan (Figure 2a, 2b).It was not possible to define particular geographic areas for Pm3 resistance and susceptible alleles (Figure 2a, 2b).

Conclusions
In this work, we have studied the Pm3 allele distribution in a diverse set of more than 1000 accessions from wheat gene banks.We found a widespread occurrence of susceptible Pm3 alleles.The Pm3CS sequence was present globally and a second susceptible allele, Pm3Go/Jho was frequent in accessions from the Himalayan region.Interestingly, Pm3Go/Jho allele is a transitional allele between Pm3CS and the resistant Pm3d allele, which was originally described in an accession from Afghanistan (Hindukush).Therefore, it is likely that Pm3d is a derivative by mutation of Pm3Go/Jho and originated somewhere in the geographical region where Pm3Go/Jho is frequent.Interestingly, the Pm3e allele, although first described in an Australian accession, was also found in the geographical area of Pm3Go/Jho i.e., in two accessions from India.As the PM3E protein differs only by one amino acid from PM3Go/Jho, we propose an origin in the Himalayan region also for Pm3e allele.From the seven functional Pm3 resistance alleles Pm3a-g, only Pm3b and Pm3c were frequently identified in the gene bank material analysed.Our data provide good evidence that Pm3b originates from a geographical region centered around Afghanistan, whereas Pm3c is frequently found in accessions from Nepal and India and possibly evolved there.Given these data on the functional alleles, it is likely that the region of the Himalaya and surrounding geographical areas have been a hotspot for the evolution of new Pm3 resistance alleles.This suggests that the search for new functional Pm3 alleles should be focused on genetic material from these regions.

Figure 1 .
Figure 1.PCR amplification of the Pm3b allele by Pm3b specific molecular makers.The arrow indicates the expected band of size 1382bp which is diagnostic for Pm3b.The numbers 1 to 28 represent the tested accessions, where 2, 21, 22, 23, 24 and 26 possess Pm3b.M stands for 1kb marker ladder.

Figure 2 .
Figure 2. Geographical origin of the entire set of 272 landraces from Afghanistan.Several accessions originated from identical geographic sites and therefore, the number of geographical identifiers is not identical to the number of accessions.(a) Accessions that carry the Pm3 haplotype and are resistant to at least one of the isolates tested are indicated.The green squares with a dot indicate the presence of Pm3b resistance allele and the green triangles indicate the accessions that possess the Pm3 haplotype but none of the known alleles Pm3a to Pm3g.(b) Yellow circles indicate the accessions that carry the Pm3 haplotype but are susceptible to the powdery mildew isolates tested.(c) The red circle marks the origin of two accessions that do not have a Pm3 haplotype but are resistant to at least one of the isolates tested.(d) Blue squares mark the accessions that do not have the Pm3 haplotype and are susceptible to powdery mildew isolates tested.

Table 1 .
Detection of Pm3 alleles in gene bank accessions by allele-specific molecular markers for Pm3a to Pm3g.Accession names and countries of origin are listed.
1 IPK: Leibniz Institute of Plant Genetics and Crop Plant Research, Germany. 2 VIR: N.I.Vavilov Research Institute of Plant Industry, Russia.3 AWCC: Australian Winter Cereals Collection, Australia.4 ICARDA: International Centre for Agricultural Research in the Dry Areas, Syria.5 INRA: French National Institute for Agricultural Research, France.6 KSU: Kansas State University, USA.

Table 2 .
Summary of accessions and countries of origin from which the susceptible alleles Pm3CS and Pm3Go/Jho were isolated.
IPK: Leibniz Institute of Plant Genetics and Crop Plant Research, Germany. 2 AWCC: Australian Winter Cereals Collection, Australia.3 ICARDA: International Centre for Agricultural Research in the Dry Areas, Syria. 4 ART: Agroscope Reckenholz-Tänikon Research Station, Switzerland.5 INRA: French National Institute for Agricultural Research, France.6 KSU: Kansas State University, USA.7 USDA/ARS: United States Department of Agriculture/Agricultural Research Service, USA.