Introduction
Leymus species are long-lived perennial grasses with drought and salt tolerance, disease resistance, and high seed set per spike (
Dewey, 1984). Examples of disease resistance include Fusarium head blight (FHB) resistance in
Leymus racemosus (2
n = 4
x= 28, genomically JJNN) (syn.
Elymus giganteus) (
Mujeeb-Kazi et al., 1983) and barley yellow dwarf virus resistance (BYDV) in
Leymus multicaulis (Kar. Et Kie) Tzvel. (2
n = 4
x= 28, genomically XXNN), and
Leymus angustus (
Plourde et al., 1989;
1992). Dong (
1986) successfully hybridized common wheat and
L. multicaulis #R79 and developed a number of disomic addition/translocation lines. Evaluation of disease resistance in wheat-
L.
multicaulis addition/translocation lines is the first step in a breeding program to integrate these resistances into commercial wheat cultivars. DNA marker identification of the introgressed chromosomes and their homeologues in wheat also is necessary for monitoring gene transfer and germplasm evaluation.
Genomic
in situ hybridization (GISH) techniques are useful in detecting introgressed chromatin in a wheat background (
Jiang and Gill, 1994;
Friebe et al., 1996). However, GISH techniques cannot determine the homeology of introgressed chromosomes with common wheat chromosomes (
Jia et al., 2002). RFLPs have been used effectively for homeologous identification, but nonradioactive probe labeling is difficult to use in wheat, and RFLP markers are very time consuming. Microsatellite, Simple Sequence Repeat (SSR), markers have been described in several plant species and have been used extensively as molecular markers. In wheat, SSR markers are more convenient than RFLPs because they detect higher levels of polymorphism (
Röder et al., 1995;
1998;
Pestsova et al., 2000) and are quicker and less expensive. Wheat SSRs also have been used to detect polymorphism in plant species related to wheat, such as rye and barley (
Röder et al., 1995). In addition, SSRs have been applied to identify disomic
Triticum aestivum-
Aegilops markgraii addition lines (
Peil et al., 1998) and
Triticum aestivum-
Lophopyrum elongatum addition and substitution lines (
Mullan et al., 2005). The objective of this research was to identify FHB, CYDV, stem rust, and powdery mildew resistances in wheat (
Triticum aestivum)-
Leymus multicaulis genetic lines that might complement disease resistance in common wheat and use microsatellite markers to verify the authenticity of the cytogenetically developed addition/translocation lines.
Materials and methods
Plant materials
The initial cross was made with
T. aestivum cv. Chinese Spring (CS) pollinated by
L. multicaulis (register number R79) collected from Xinjiang, China. However, CS does not have many favorable plant traits, and it is not used widely in agriculture. Therefore, the initial hybrid was backcrossed to the wheat cvs. Feng Kang 7, Feng Kang 10, and Feng Kang 13. The progenies were self pollinated for five generations, and 24 lines derived from F
6 plants putatively carrying alien chromosomes or fragments were selected on the basis of phenotype, chromosome number, and meiotic chromosome pairing (
Dong, 1986;
Jia et al., 2002). In addition, the amphiploid derived from
L. multicaulis was also included in this study.
FHB evaluation
Twenty-four putative addition/translocation lines, CS, Feng Kang 7, Feng Kang 10, and Feng Kang 13, as well as the amphiploid, were evaluated for type II FHB resistance (
Shen et al., 2004) by single-floret inoculation in a greenhouse with a local virulent isolate of
F. graminearum, provided by Dr. G. Shaner, Department of Botany and Plant Pathology, Purdue University. The inoculum concentration was (5-10) × 10
4 conidia·mL
-1. A basal floret of the third or fourth spikelet from the spike-tip was inoculated with 10 µL conidium suspension, and the inoculated spikes were covered with transparent plastic bags for 72 h to maintain high humidity (
Shen et al., 2004;
Kong et al., 2005a). Three weeks after inoculation, disease severity was measured as the number of diseased spikelets in the inoculated spikes and recorded. FHB disease severity, designated as Fusarium head blight index (FHBI), was determined by the ratio of the number of diseased spikelets to the number of total spikelets in the inoculated spike. The various lines, their parents, and the amphiploid were evaluated for disease in two experiments conducted in a greenhouse during 2003 and 2004. In addition,
T. aestivum cv. Len served as susceptible check, and
T. aestivum cv. Ning 7840, the FHB type II resistance source, served as resistant check in the phenotyping experiments. Data were analyzed by an unbalanced analysis of variance using the GLM program of SAS. The mean values of the lines were compared with those of their corresponding recurrent parents, resistant (Ning 7840) and susceptible (Len) checks, and the amphiploid. Fisher’s least significance test was used as the critical difference (
Shen et al., 2004).
CYDV evaluation
Resistance to the CYDV was evaluated based on virus titers as measured by enzyme-linked immunosorbent assay (ELISA). At approximately the three-leaf stage, six plants from each line were infested with aphids that were viruliferous with CYDV-RPV. The viruliferous aphids were reared on CYDV-susceptible cultivar Clintland 64 oat seedlings in a growth chamber (18°C). Leaf samples were collected 14 d after infestation, and the virus titer was determined by ELISA using the double antibody sandwich (DAS) method (
Clark and Adams, 1977;
Hammond et al., 1983). The ρ-nitrophenol chromophore was determined by optical density measured with a microtiter plate reader (Model MR600; Dynatech Laboratories Inc., Alexandria, VA) in dual-wavelength mode at 410 and 630 nm (
Anderson et al., 1998).
To verify the CYDV resistance derived from L. multicaulis, the putative CYDV-resistant wheat-L. multicaulis addition line, Line 23, was crossed to the susceptible common wheat cultivar Chinese Spring, and the resulting F1 plants were selfed to produce F2 segregation population. There were 98 F2 individuals of Line 23 and CS, as well as the parent lines, used for CYDV bioassay to determine CYDV resistance by ELISA method, as described above.
Stem rust evaluation
A local isolate of
P. graminis Pers. f. sp.
tritici Eriks. & E Henn. was collected and provided by Dr. G. Shaner, Department of Botany and Plant Pathology, Purdue University. Six adult plants of each wheat-
Leymus line at heading and prior to flowering were inoculated by atomizing urediniospores suspended in Soltrol light mineral oil. After inoculation, the plants were covered with transparent plastic sheets for about 4 h to maintain high humidity to assure a high rate of infection. Infection types (ITs) on the flag leaf and the stem below the flag leaf were determined at 14 d after inoculation, using a scale of 0 to 4 (
Stakman et al., 1962): ITs 0 (immune), 1 (small uredinia with necrosis), 2 (small uredinia with chlorosis), and 3 (small uredinia without cholorosis or necrosis) were considered resistant, and ITs 3
+ to 4 (large uredinia without chlorosis or necrosis) were considered susceptible (
Liu and Kolmer, 1998).
Powdery mildew evaluation
Fourteen differential isolates of the pathogen were selected from single-spore progenies, and the 15 wheat lines with known powdery mildew (
Blumeria graminis f. sp.
tritici (
Bgt)) resistance genes were used for the differentiation of major powdery mildew resistance. The disease response pattern of a set of differential wheat cultivars/lines were compared to the resistance in the tested lines. This comparison and application of the gene-for-gene hypothesis (
Flor, 1955) would identify which lines were resistant to powdery mildew and if known powdery mildew resistance genes are present in these lines. Disease evaluations were conducted when all susceptible control plants showed abundant signs and symptoms of powdery mildew infection. The disease severity evaluation was on a scale from 0 to 9, as described by Leath and Heun (
1990), there were three main classes of host reactions: R= resistant (0 to 3), I= intermediate (4 to 6), and S= susceptible (7 to 9) (
Srnic et al., 2005).
DNA isolation and PCR amplification
Genomic DNA was isolated from seedling leaves using the CTAB method described by Saghai Maroof et al. (1984) with minor modification. A 1.67% CTAB extraction buffer [100 mmol·L-1 Tris-HCl buffer pH 8.0, 1.67% (w/v) hexadecyltrimethylammonium bromide, 100 mmol·L-1 Na2EDTA, and 1.4 mol·L-1 NaCl] was used. DNA was quantified on a Hoefer DyNA Quant 200 Fluorometer (Hoefer Pharmacia Biotech Inc., Dubuque, IA).
Polymerase chain reaction (PCR) for each SSR marker was performed in a PTC-100 Thermal Cycler (MJ Research, Watertown, MA, USA) at 94°C for 2 min, followed by 35 cycles of 94°C for 30 s, 50°C, 52°C, 55°C, or 60°C (based on different primer annealing temperature) for 40 s, and 72°C for 1 min, with a final extension at 72°C for 7 min before cooling to 4°C. Each PCR reaction (25 µL) consisted of 40 ng template DNA, 10 mmol·L
-1 Tris HCl, pH 8.3, 50 mmol·L
-1 KCl, 0.1% Triton X-100, 1.5 mmol·L
-1 MgCl
2, 200 mol·L
-1 of each dNTP, 0.25 mol·L
-1 of each primer, and 1 unit of
Taq DNA polymerase. The amplified PCR products were fractionated on 2.0%-3.0% agarose gels (based on the size difference of the polymorphism) using a mixture of 1∶1 Metaphor
® and Seakem
® in 0.5 × TBE buffer and photographed over a UV light source (
Kong et al., 2005b).
Simple sequence repeat (SSR) marker analysis
Wheat EST-derived microsatellite (eSSR)
The Perl script MISA (http://pgrc.ipk-gatersleben.de/misa.html) was used to identify SSRs in a wheat EST database containing approximately one half million ESTs (http://wheat.pw.usda.gov/cgi-bin/ace/search/wEST). Three or four eSSRs were chosen from both arms of all chromosomes (Table 1) using seven consensus maps (
Peng and Lapitan, 2005). All of the primers were designed by Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3) and synthesized by Integrated DNA Technologies (Coralville, Iowa).
Wheat SSR markers
Wheat microsatellite markers designated as either Gwm for Gatersleben (Germany) wheat microsatellite (
Röder et al., 1998), Gdm for Gatersleben D-genome microsatellite (
Pestsova et al., 2000), Barc for Beltsville Agriculture Research Center (http://wheat.pw.usda.gov), Wmc for Wheat Microsatellite Consortium (
Gupta et al., 2002), and Wem (eSSRs) for Wheat EST-derived Microsatellites (Table 1) were selected to cover the genetic maps of chromosomes in each homeologous group, especially both ends of the chromosome, where translocation occurs most frequently, and near the centromere on each arm where homologies may be detected. These SSRs were tested for useful polymorphisms in parents and recurrent parents (
L. multicaulis, CS, and Feng Kang 10 and Feng Kang 13), as well as the amphiploid. Polymorphic SSRs identified by amplifying genomic DNA of the above lines were then used to characterize 24 putative addition/translocation lines.
Results
FHB resistance in wheat-L. multicaulis derivatives
Analysis of variance showed that there were no significant genotype × experiment interactions (F = 1.18, P = 0.24); also, the variance between experiments was not significant (F = 2.26, P = 0.13). Thus, means for lines were averaged over experiments (Table 2). The levels of infection on the check cultivars were as expected. The spikes of the susceptible check Len were totally infected (FHBI= 0.91), and those of Ning 7840 were least infected (FHBI= 0.18). The parent, CS, is moderately resistant to FHB (FHBI= 0.42). The amphiploid showed similar FHB resistance (FHBI= 0.48) to CS. FHBI of the recurrent parents, Feng Kang 7 and Feng Kang 10, were 0.24 and 0.28, respectively, which showed high FHB resistance. However, the FHBI for another recurrent parent, Feng Kang 13, was 0.77. Wheat-L. multicaulis addition lines, Line 9 and Line 26, had the lowest disease severity (0.17). In most plants of Line 9 and Line 26, the inoculated floret showed a lesion, and most likely, the disease spread to the next spikelet but not beyond, which was similar to the FHB-resistant check, Ning 7840 (Table 2).
CYDV resistance in wheat-L. multicaulis derivatives
Twenty-four putative addition/translocation lines, CS, Feng Kang 7, Feng Kang 10 and Feng Kang 13, the amphiploid, as well as a resistant check, P961341 (
Ohm et al., 2005), were evaluated for CYDV (Table 2). The resistant check P961341 and the addition line, Line 23, had low virus titer 0.016 and 0.15, respectively, which were significantly lower than the susceptible control, CS (0.88), and other recurrent wheat parents, such as Feng Kang 7 (1.03), Feng Kang 10 (0.96), and Feng Kang 13 (0.73), indicating that P961341 and Line 23 are resistant to CYDV. The amphiploid, with virus titer 0.46, had only moderate resistance to CYDV.
In the subsequent F2 progeny test, the wheat-L. multicaulis addition line, Line 23, had low virus titer (0.108), and CS had high virus titer (1.537), as expected. However, the distribution of virus titers in F2 population ranged from as low as in the resistant parent line, Line 23, to as high as in the susceptible parent line CS (Fig. 1). The segregation ratio significantly deviated from 3 ∶ 1 of one single dominant gene.
Stem rust resistance in wheat- L. multicaulis derivatives
Twenty-four putative addition/translocation lines, CS, Feng Kang 7, Feng Kang 10 and Feng Kang 13, the amphiploid, and a check, P961341, were evaluated for stem rust resistance in the greenhouse (Table 2). P961341 and the recurrent parents, Feng Kang 7 and Feng Kang 10, were resistant to stem rust (IT 0), while CS and the recurrent parent Feng Kang 13 were severely infected (IT 3+). Among the 24 addition/translocation lines investigated, the translocation lines, Trans 2 and Trans 3, and the addition lines, Line10, Line 26, and Line 27, developed a Type 1 (IT 1) reaction, which is different from either of the recurrent parent lines, suggesting that their resistances were derived from L. multicaulis. The level of infection on the amphiploid from L. multicaulis was intermediate (IT 2).
Powdery mildew resistance in wheat-L. multicaulis derivatives
Because the addition lines, Line 9 and Line 26, and the translocation line, Trans 1, were resistant to both FHB and stem rust, resistance to powdery mildew also was examined in these lines. The disease-response pattern of a set of differential wheat cultivars/lines listed in Table 3 were compared to the resistance in these three lines, their recurrent parent Feng Kang 10, and the amphiploid. The recurrent parent Feng Kang 10 and the addition line, Line 26, exhibited the same response pattern as Kavkaz, which carries Pm8, showing that both of them most likely have the Pm8 powdery mildew resistance gene. The translocation line, Trans 1, the addition line, Line 9, and the amphiploid were susceptible to all of the Bgt isolates used in our study.
Characterization of wheat-L. multicaulis derivatives
L. multicaulis, the amphiploid, and the T. aestivum parent lines were screened with 425 SSRs from Gwm, Gdm, Wmc, Barc, and Wem, of which 19 markers showed polymorphisms between the L. multicaulis and T. aestivum parents (Table 4). Four of the 19 polymorphic markers amplified a unique L. multicaulis fragment in the amphiploid. These 19 polymorphic L. multicaulis-specific SSR markers were used to characterize the 24 L. multicaulis introgression lines. Twelve of these 19 polymorphic SSRs identified lines that contained L. multicaulis chromatin (Table 5). Of the other seven SSRs, Wmc154 generated a distinct PCR product in L. multicaulis and the amphiploid but not in any of the 24 addition/translocation lines, and the remaining six SSRs, Wem22, Wem32, Gwm291, Wem45, Wem50, and Wmc27, did not amplify L. multicaulis specific fragments in either the amphiploid or any of the addition/translocation lines (Figs. 2A, 2B and 2C). Among the 24 addition/translocation lines investigated using the 12 SSR markers, 18 lines, namely, Trans 1, Trans 2, Trans 3, Line 4, Line 6, Line 7, Line 9, Line 10, Line 11, Line 12, Line 14, Line 15, Line 16, Line 18, Line 24, Line 26, Line 27, and Line 29, contained an L. multicaulis-specific microsatellite fragment, indicating that all of these 18 addition/translocation lines most likely have an introgressed L. multicaulis chromosome(s). The other six lines including Line 5, Line 20, Line 22, Line 23, Line 25, and Line 28, did not contain any of the SSR fragments obtained with L. multicaulis.
The results from this microsatellite analysis are not sufficient to give the homeology identity of chromosomes between T. aestivum and L. multicaulis. For example, SSR markers Gwm264 and Wem26 located on group 3 generated the same L. multicaulis-specific fragments in lines 4 and 15; and Wem26, but not Gwm264, amplified the same L. multicaulis-specific fragment in addition line, line 26, and the translocation lines, Trans 1 and 2 (Figs. 3A, 3B). Interestingly, the same banding pattern is generated in some lines by SSRs located on different wheat chromosome groups. For example, the three translocation lines (Trans 1, Trans 2, and Trans 3) all showed the same L. multicaulis-specific fragments using Gwm391 (3A), Wmc48 (4B), and Barc21 (6B) primers (Table 5). These results suggest that chromosomal rearrangements had occurred during the development of these wheat- L. multicaulis introgression lines. Another point of interest is that no SSRs, specific to the D genome showed polymorphism between wheat and L. multicaulis, although about 150 SSRs specific to the D genome were tested (Table 5). It is unknown whether a closer relationship exists between the X and N genomes of L. multicaulis, and the A and B genomes of T. aestivum.
Discussion
Disease resistances in L. multicaulis
Fusarium head blight (FHB) has posed a serious threat to wheat production worldwide, and the disease not only lowers grain yield but reduces grain quality (
Bai and Shannar, 1994). Breeding wheat for resistance to FHB is one of the most effective strategies to minimize crop and grain quality losses. Mujeeb-Kazi et al. (
1983) reported that
Leymus racemosus is a potential source of resistance to wheat FHB. Dong (
1986) hybridized common wheat with
L. multicaulis and developed wheat-
L. multicaulis introgression lines. The consistent resistance detected in the addition lines, Line 9 and Line 26, across the two experiments indicates that both lines are resistant to FHB. However, the amphiploid, CS, and the recurrent parents Feng Kang 7 and 10 showed intermediate resistance to FHB. These data suggest that Line 9 and Line 26 may carry a combination of two or more FHB resistance genes from wheat parents and the alien donor
L. multicaulis. Microsatellite characterization using Wmc44, Wem26, and Barc21 demonstrated that Line 9 and Line 26 do have
L. multicaulis-specific fragments (Table 5). We cannot exclude the possibility that the original
L. multicaulis used to develop the wheat-
L. multicaulis addition lines carries FHB resistance and maybe different from the one used to create the amphiploid.
Yellow dwarf viruses (BYDV and CYDV), spread by aphid vectors, are among the most serious diseases of cereals worldwide (
Conti et al., 1990). Little resistance to the disease has been identified in wheat (
Triticum aestivum L.), so plant breeders have had to derive the BYDV resistance from wild relatives (
Brettell et al., 1988;
Ayala et al., 2001;
Zhang et al., 2004;
Ohm et al., 2005). The evaluation of BYDV resistance in derivatives of
T.
aestivum ×
L. multicaulis and the F
1 of
T. aestivum ×
L. anagustus indicated that
Leymus species may have potential to improve common wheat for BYDV resistance (
Plourde et al., 1989,
1992). In this study, among the 30 lines investigated, in addition to the control P961341, Line 23 showed low virus titer (0.15), indicating that this line has CYDV resistance. To confirm the CYDV resistance derived from
L. multicaulis, the ELISA assays in F
2 population of Line 23/CS indicated that the virus titers ranged from as low as in Line 23 to as high as in CS (Fig. 1), which did not fit the regular one-gene segregation ratio 3 ∶ 1. The previous study in wheat plants carrying leaf-rust resistance on an added
Agropyron elongatum chromosome (i.e., 21 II
wheat + I
elongatum) indicated that the univalent is transmitted to 25% of the gametes, and the presence of the
Agropyron elongatum chromosome does not impair the functioning of either the eggs or the pollens (
Sharma and Knott, 1966). Consequently, when such plants with (21 II
wheat + I
elongatum) are selfed, the segregation ratio in F
2 progeny should be deviated significantly from the expected 3 ∶ 1, as illustrated in Fig. 4. Similarly, in this study, the metaphase chromosome configuration should be 21 II
wheat + I
Leymus in F
1 plants from the cross between CS (21 II
wheat) and wheat-
L. multicaulis addition line (21 II
wheat + II
Leymus). The chromosome configurations in the consequent F
2 individuals of addition line/CS should be formed following the rule illustrated in Fig. 4. Indeed, our observed ELISA data in the F
2 population showed similar results to the previous study in
Agropyron elongatum. The arbitrary three pooled data groups, lower virus titer (<0.1), medium virus titer (0.1-0.8), and high virus titer (>0.8), were segregated in 3%, 37%, and 60%, respectively. This observed segregation data appeared that the CYDV resistance in wheat plants with an added
L. multicaulis chromosome could be different from the resistance in wheat plants with one pair of
L. multicaulis chromosome. Further considerable work will be necessary to compare the chromosome configuration and virus titer in F
3 family progeny test.
Stem rust, caused by Puccinia graminis Pers. f. sp. tritici Eriks. & E. Henn., and powdery mildew, caused by Blumeria graminis (DC.) E. O. Speer f. sp. tritici, are important diseases of wheat and barley. Stem rust evaluations indicated that the recurrent parents Feng Kang 7 and 10 both have stem rust resistance (IT 0). The amphiploid has stem rust resistance (IT 2), while CS is susceptible to stem rust (IT 3+). This stem rust resistance also was detected in some of wheat-L. multicaulis derivatives. For example, three lines, Trans 1, Line 5, and Line 9, were immune (IT 0), and five lines showed significant (IT 1) resistance to stem rust. The stem rust resistance in Trans 1 and Line 9, both with L. multicaulis chromatin characterized by SSRs, may be due to a combination of stem rust resistance genes derived from wheat and L. multicaulis or was inherited from the recurrent parents Feng Kang 7 and 10.
The powdery mildew disease response patterns of 15 wheat cultivars and lines with documented resistance genes were compared with the three wheat-L. multicaulis lines that were FHB resistant. The response pattern of Feng Kang 10 was characteristic for the presence of the gene Pm8. The response pattern of Line 26 corresponded to that of its recurrent parent Feng Kang 10. Therefore, we concluded that Line 26 inherited Pm8 from Feng Kang 10. Other lines tested, including the amphiploid, Line 9, and Trans 1, were characterized as having a susceptible and/or intermediate response to all mildew isolates used, which indicates that they do not have any powdery mildew resistance genes.
SSR Characterization of alien chromatin
Molecular genetic characterization of alien chromosome(s) carrying useful genes is an important component of introgressing chromatin from related grasses into cultivated wheat. DNA markers, especially PCR-based markers that are linked to specific loci, are convenient tools to determine whether or not a progeny has the desired gene(s). RFLP, GISH, and C-banding have been employed previously for the differentiation of wheat-
Elymus tsukushiense introgression lines (
Wang et al., 2001), wheat-
Leymus racemosus addition lines (
Qi et al., 1997), and wheat-
Leymus multicaulis derivatives (
Jia et al., 2002). Recently, microsatellites generated in cereals, such as wheat or barley, were successfully utilized for chromosomal characterization in other
Gramineae. Saghai Maroof et al. (
1994) used four barley microsatellites to identify wheat-barley addition lines, but only one of them generated additional fragments in the wheat background. Peil et al. (
1998) tested 88 wheat microsatellite markers to detect wheat-
Aegilops markgrafii addition lines, 38 of which (i.e., 43%) showed polymorphism between wheat and
Ae. markgrafii, and 20 out of the 38 SSRs were further used for distinguishing
Ae. markgrafii chromosomes. Surprisingly, the SSR fragments produced in common wheat were detected in
L. multicaulis by most of the wheat-derived SSR markers used in this study; even though
L. multicaulis is not closely related to common wheat (
Dong, 1986). However, it is unknown if the amplified fragments in
L. multicaulis do contain a microsatellite sequence. A general problem observed in the present study is the lower amplification of
L. multicaulis-specific fragments in both the amphiploid and the wheat-
L.
multicaulis addition/translocation lines, compared with the donor
L. multicaulis. The low level of polymorphism may suggest that there is competition for primer binding between
T. aestivum and
L. multicaulis DNA in the PCR amplification. The wheat-derived SSR primers matched more closely to the wheat DNA templates than to the
Leymus DNA when it is present in a wheat background. Peil et al. (
1998) also reported a competition effect for primers between hexaploid wheat and
Ae. markgrafii. Possibly, the greater the genetic distance between wheat and a related species, the more likely the primer competition would be detected as a reduction in PCR amplification. Our results showed that a low percentage of wheat-derived SSRs can be used to identify chromatin introgressed from species related to wheat but not for characterizing chromatin from unrelated species.
Chromosomal rearrangement detected by SSR
Chromosomal rearrangements were detected frequently in cross-pollinated species of Triticeae, such as rye (
Devos et al., 1993),
L. racemosus (
Qi et al., 1997), and
L. multicaulis (
Jia et al., 2002); however, few chromosomal rearrangements occurred in self-pollinated species, such as
Triticum monococcum (Dubcovsky et al., 1996) and
Triticum tauschii (
Gill et al., 1991). The genus
Leymus consists of about 30 species, all previously belonging to the genus
Elymus (
Qi et al., 1997).
Leymus multicaulis is a tetraploid (2
n = 28= XXNN), which is typical for cross-pollinated species. C-banding analysis detected a large amount of polymorphism within and between different accessions of
Leymus racemosus (
Qi et al., 1997). RFLP analyses revealed that the alien chromosomes of the 15 wheat-
L. multicaulis addition lines were from the X genome, but no N-genome chromosomes or chromosomal fragments were found in any of the 24 lines investigated (
Jia et al., 2002). Furthermore, 10 out of the 15 addition lines were suggested to be related to homeologous Group 1 or 3 (
Jia et al., 2002). In the present study, the wheat-
Leymus homeology revealed by SSRs appears to be consistent with those described above by RFLP analysis. Out of the 19 polymorphic SSR markers, 10 belonged to homeologous Groups 1 and 3 in wheat (Table 5). However, the microsatellite analysis in this study was not necessarily reliable for estimating the homeology between
T. aestivum and
L.
multicaulis chromosomes. Wheat SSR markers from the same wheat homeologous group exhibited different
L. multicaulis-specific fragments in the tested lines (Figs. 3A, 3B; Table 5). In contrast, the same banding pattern is generated by SSRs located on different homoelogous groups (Table 5). This may be due to chromosomal rearrangements that existed in the
L.
multicaulis donor line or may have occurred during the development of these wheat-
L. multicaulis introgression lines. Further work using more polymorphic SSRs in combination with other molecular markers would be required to confirm the homeologous identity in these wheat-
L.
multicaulis introgression lines.
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