Various wheat differential lines (Table 1) carrying known Yr genes (kindly provided by Chen Xianming, Washington State University) as well as the main resistance genes utilized in the region were used to establish the virulence spectrum of the stripe rust pathotypes CYR31 and CYR32 and to differentiate the resistance response.

Chuanmai107 (CM107) was pollinated with octoploid Trititrigia TAI7047, derived from the wide cross Taiyuan768/T. intermedium//76(64). Two wheat lines, Yuan24 (YU24) and Yuan25 (YU25) (Triticum aestivum, 2n = 6x = 42, AABBDD), were selected from the F₅ population of the cross (CM107/TAI7047) (Luo et al. 2009b). The two lines were characterized by a hypersensitive response to both races of P. striiformis f. sp. Tritici, CYR31 and CYR32, following natural infection. To study the genetic composition and nature of the resistance, three F₂ populations were derived from the F₁ population originating from the crosses MY11/YU24, MY11/YU25 and YU24/YU25. The F₁ hybrids were also backcrossed to both parents. The backcross to the susceptible parent (BC₁^SF₁) and the backcross to the resistant parent (BC₁^RF₁) were allowed to self-pollinate, and both sets of F₁ and F₂ populations were genetically analyzed.

Puccinia striiformis f. sp. tritici pathotypes CYR31 and CYR32, which are the predominant strains in southwest China, were used to screen and assess the resistance response of homogeneous genotypes. To test the resistance of segregating populations, plants were inoculated with CYR32 because it has the same virulent factors as CYR31 (Wan et al. 2004).

The various wheat genotypes, including differential lines, main resistance lines and parental lines, were grown in a temperature- and moisture-controlled glass enclosure (25 m x 10 m; 1.6 m in height) at the Experimental Station of Sichuan Agriculture University. At the three-leaf stage, 20 seedlings per genotype were inoculated with single isolates of both CYR31 and CYR32 according to Luo et al. (2005). The inoculated seedlings were misted through an inlet until sufficient dew was formed and incubated at 14°C in the dark for 12 h and in the light for 24 h. They were then grown under natural daylight at 16-20°C. When the stripe rust pustules were fully developed, the infection types (ITs) were recorded on a 0 to 4 scale described by Wellings et al. (1988): IT0 = no visible symptoms; IT0+ = visible necrotic flecks without uredia; IT1 = small sporulating uredia surrounded by necrotic tissue; IT2 = small-size uredia with chlorosis and necrosis; IT3 = moderately-sized sporulating uredia surrounded only by chlorotic tissue; and IT4 = abundantly sporulating uredia without chlorosis.

DNA was extracted using 1 g of fresh wheat leaves from 5-wk-old seedlings (Tai and Tanksley 1990). DNA of intermediate wheatgrass and of wheat genotypes Chinese Spring (CS), MY11, CM107, YU24, YU25 and octoploid Trititrigia TAI7047 was used to screen for the presence of foreign DNA segments using wheat-specific microsatellites markers (SSR) and random amplified polymorphic DNA (RAPD). For SSR analysis, PCR reactions were carried out in an MJ RESEARCH (PTC-200) thermocycler using publicly available Xgwm primer pairs. For each PCR reaction, the 20 µL volume mixture contained 200 nM of each primer, 0.2 mM deoxynucleotides, 50 mM KCl, 10 mM Tris-HCl, 1.5 mM MgCl₂, 1 unit Taq polymerase (Pharmacia) and 50 ng template DNA. After 3 min denaturation at 94°C, 43 cycles were performed with 1 min denaturation at 94°C, 1 min annealing at temperatures varying from 50-60°C (depending on the primer sequence), and a 2 min extension at 72°C. A final extension step of 10 min at 72°C was performed (Roder et al. 1998).

RAPD analysis was performed in 20 µL reaction volumes as described by Williams et al. (1990). In total, 520 primers were screened from Operon primer kits A-Z. The amplified fragments were run on 3% agarose (FMC brand) in 0.5X TBE at a voltage of 120 V (4V cm^‑1) and were then visualized using ethidium bromide staining methods.

Inoculation tests showed that CYR31 and CYR32 are virulent on most of the described wheat stripe rust resistance genes (Table 1). CYR31 and CYR32 are virulent to Yr1, Yr2, Yr3a, Yr3b, Yr3c, Yr4a, Yr4b, Yr6, Yr7, Yr8, Yr9, Yr11, Yr12, Yr14, Yr16, Yr17, Yr18, Yr20, Yr21, Yr22, Yr23, Yr24, Yr25, Yr26, Yr27, Yr31, YrHVII, YrMin, YrV23, Yrsp, YrH46 and YrHK. Only Yr5, Yr10, Yr15, YrCN19 and YrR212 were effective against CYR31 and CYR32, while Yr13, Yr19 and YrCN17 were partially effective (Table 1).

The parental genotypes CS, CM107, MY11, Taiyuan768 and 76(64) were susceptible, with high infection types ranging between 3 and 4. Thinopyrum intermedium wheatgrass, YU24, YU25 and TAI7047 were resistant, with low infection type IT=0+ (Table 2). Their leaves produced obvious large necrotic lesions between the veins of adult plants after inoculation (Fig. 1).

The data obtained following evaluation of the seedlings of parental lines and cross progenies with isolates CYR31 and CYR32 are summarized in Table 2. Towards the end of growth, there were still no visible symptoms on the leaves of intermediate wheatgrass. The F₁ MY11/YU24 and MY11/YU25 were also highly resistant (Table 3), indicating the presence of one or more dominant resistance genes in YU24 and YU25. The two F₂ populations (crosses MY11/YU24 and MY11/YU25) each showed 3 resistant:1 susceptible segregation of a single dominant resistance gene (Table 3). Single gene segregation was also confirmed in the two BC₁^SF₁ populations, which fitted the expected 1:1 ratio. Segregation in the backcross F₂ populations also fitted the expected segregation of a single dominant resistance gene. All F₁ and F₂ populations produced from the cross YU24/YU25 were resistant (Tables 2 and 3), suggesting that the same gene was present in both parents.

In an attempt to determine the chromosome location of the introgressed foreign chromatin, 294 pairs of wheat microsatellite primers that amplify repeat sequences distributed over all wheat chromosomes were used. The results showed that wheat genotypes YU24, YU25 and octoploid TAI7047, as well as common wheat controls MY11, CM107 and CS, amplified wheat-specific SSR products that did not occur in intermediate wheatgrass. In addition, 520 primers (Operon groups A to Z) were used in an attempt to identify amplicons specific to intermediate wheatgrass. Thirteen RAPD primers (B2, F9, I17, M8, N11, P6, Q18, R1, R10, R16, R19, U5 and Z20) produced amplified products in wheat genotypes YU24 and YU25 and octoploid Trititrigia TAI7047, and these products had the same length as those in intermediate wheatgrass. The PCR results amplified by R1 are shown in Figure 2.