3.1. 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 All accessions were first phenotyped for powdery mildew resistance (Bhullar and Keller, unpublished data).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.
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.
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.
Pm3 allele | Number of accessions carrying the tested
Pm3 allele | Country of origin | Accession (s) in which the particular
Pm3 allele was detected | Source of the accessions (gene bank) | Reference |
---|
Pm3b | 1 | France | TRI980 | IPK1 | This work |
| 1 | Kazakhstan | TRI7321 | IPK | This work |
| 1 | Uzbekistan | TRI17549 | IPK | This work |
| 1 | Tajikistan | TRI17561 | IPK | This work |
| 2 | Russia | TRI18263; TRI18742 | IPK | This work |
| 6 | Russia | VIR23918, VIR23922, VIR34986, VIR35021, VIR35030, VIR34984 | VIR2 | [27] |
| 15 | Afghanistan | AUS9943, AUS9948, AUS10003, AUS10033, AUS13239, AUS13297, AUS13306, AUS13307, AUS13311, AUS14504, AUS14532, AUS14840, VIR45538, VIR49005, VIR49006 | AWCC3, VIR | [27] |
| 6 | Iran | IG122348, IG122354, IG122361, IG122373, IG122502, VIR38613 | ICARDA4 | [27] |
| 2 | Azerbaijan | VIR16766, VIR31595 | VIR | [27] |
| 1 | Turkey | VIR35203 | VIR | [27] |
Pm3c | 8 | Nepal | TRI2437; TRI2439; TRI2448; TRI2748; TRI2765; TRI3255; TRI4029; TRI4091 | IPK | This work |
| 7 | India | TRI2799; TRI2804; TRI3375; TRI3542; TRI3552; TRI3986; TRI9986 | IPK | This work |
| 1 | China | TRI4088 | IPK | This work |
| 1 | Australia | TRI8320 | IPK | This work |
| 3 | Iran | IG122491, IG122372, IG122346 | ICARDA | [27] |
| 1 | Azerbaijan | VIR46301 | VIR | [27] |
Pm3d | 1 | Argentina | TRI11472 | IPK | This work |
| 1 | France | Oid HD4-266 | INRA5 | [19] |
Pm3e | 2 | India | TRI2554; TRI2782 | IPK | This work |
| 1 | Tajikistan | TA10381 | KSU6 | [19] |
| 1 | France | Oid 91-35 | INRA | [19] |
Pm3f | 1 | Argentina | TRI7521 | IPK | This work |
| 1 | China | TRI16947 | IPK | This work |
Pm3g | 1 | France | Oid HD4-219 | INRA | [19] |
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 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.
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].
3.2. 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).
3.3. 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.
Table 2.
Summary of accessions and countries of origin from which the susceptible alleles Pm3CS and Pm3Go/Jho were isolated.
Table 2.
Summary of accessions and countries of origin from which the susceptible alleles Pm3CS and Pm3Go/Jho were isolated.
Pm3 allele | Origin | Number of accessions | Accession (s) | Type | Source of accessions | Reference |
---|
Pm3CS | Hexaploid wheat | |
| India | 2 | TRI2480, TRI2739 | unknown | IPK1 | This work |
| Australia | 1 | TRI7243 | unknown | IPK | This work |
| France | 1 | TRI7345 | unknown | IPK | This work |
| Canada | 2 | TRI7736, TRI7741 | unknown | IPK | This work |
| Ethiopia | 1 | TRI15026 | unknown | IPK | This work |
| Tajikistan | 1 | TRI17510 | unknown | IPK | This work |
| Pakistan | 2 | AUS 4856, IG41554 | Landrace | AWCC2 | [9] |
| Afghanistan | 7 | AWCC9947, AWCC14695, AWCC14849, AUS13655, AUS13656, AUS13704, AUS14526 | Landrace | AWCC | [9] |
| Turkey | 2 | IG42398, IG42869 | Landrace | ICARDA3 | [9] |
| Iran | 1 | IG122584 | Landrace | ICARDA | [9] |
| China | 1 | Chinese Spring | Landrace | ART4 | [19] |
| Europe | 5 | Caribo, Greif, Obelisk, Kormoran, Monopol, | Cultivar | ART | [19] |
| Belgium | 1 | Rouquin | Cultivar | ART | [19] |
| Switzerland | 1 | Boval | Cultivar | ART | [19] |
| Germany | 1 | Kanzler | Cultivar | ART | [19] |
| France | 1 | Oid HD4-234 | Breeding line | INRA5 | [19] |
| UK | 1 | Maris Huntsman | Cultivar | ART | [19] |
| USA | 1 | Thatcher | Cultivar | ART | [19] |
| Tajikistan | 1 | TA 10384 | Landrace | KSU6 | [19] |
Pm3CS | Tetraploid wheat | |
| Turkey | 5 | PI560872, PI560874, PI428145,PI428053, IG116184 | T. dicoccoides | USDA/ARS7/ ICARDA | [20] |
| Ethiopia | 1 | PI58789 | T. dicoccum | USDA/ARS | [20] |
| Ethiopia | 1 | CItr14846 | T. durum | USDA/ARS | [20] |
Pm3 Go/Jho | Hexaploid wheat | |
| India | 4 | TRI2596, TRI3197, TRI3535, TRI3992 | unknown | IPK | This work |
| Nepal | 13 | TRI2611, TRI2889, TRI3232, TRI3628, TRI4359, TRI11131, TRI11132, TRI11133, TRI11135, TRI11136, TRI11137, TRI11139, TRI11151 | unknown | IPK | This work |
| China | 1 | TRI14752 | unknown | IPK | This work |
3.4. 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).