Screening for new sources of resistance
Over eight years, 37 different rye genomes from distant gene pools were examined. It was carried out both under controlled conditions in the greenhouse and mostly in fields that are contaminated with the various viruses, which were characterized in several reports (Kastirr 2004; Kastirr und Ziegler 2018). However, those test sites showed great variability in the infestation of cereal plants. Of course, this made genetic analysis more difficult (see Table 2).
The results obtained over the years are summarized in Table 2. Since sometimes rye populations classified to be resistant had to be declared as susceptible at other testing locations, it was extremely difficult to select suitable populations with virus resistance for a subsequent genetic analysis.
In the case of rye, three accessions turned out to be resistant after repeated studies, i.e., the wild rye Secale montanum (PC2243), S. africanum (PC2328), and S. vavilovii (PC2364). Since the allogamous rye generally has a high degree of genetic variability, the three isolated populations were further separated and increased by self-pollination. Among the cultivated rye, S. cereale, no resistant source was identified during these studies (cf. Table 2).
Leaf symptoms (cf. Figure 1) of SBCMV could not be estimated in all plants, however, sporadically. Therefore, ELISA was used as main criterion for virus infestation of tillering plants. During ELISA testing the titre threshold was set to 0.1 to characterize infected plants. On the test site Thören, in 2019 there was little infestation for WSSMV that even the parental lines of S. cereale var. Imperial and S. montanum did not show any reaction (Table 3). It was decided to only consider the infestation with SBCMV.
The first comparison of the viral infestation of young plants showed that the WSSMV did not cause any significant symptoms at the chosen location of Thören. Even the parental genotypes PC272 and PC2243 did not differ (Table 3). The F2 analysis at the Thören location was therefore not suitable for the WSSMV study and was thus no longer considered. However, some plants were found to be susceptible to WSSMV, but not to SBCMV. This means that the susceptibility or resistance is independently determined for WSSMV and SBCMV.
The evaluation of the ELISA values shows that in 2019 19 of the 170 genotyped plants exhibit a clear infection to SBCMV and 151 none. (Table 4). When this distribution of 151r: 19s is compared to a 3r:1s expectation (= 127.5r: 42.5s), then just a slight association can be considered. However, the observed distribution matches better with a 9:3:3:1 or 15r:1s distribution (= 159.4r: 10.6s) assuming duplicate dominant epistasis, suggesting A epistatic to B and b, B epistatic to A and a.
The statistical analysis applying Chi2 test did not clearly confirm neither a 3r:1s nor a 15r:1s distribution (cf. Table 5 and 6) in 2019. By the Chi2 values of 17.32 (3r:1s) and 7.03 (15r:1s) the null hypothesis can be rejected within the confidence level of P = 0, df = 1 and P = 0.0081, df = 1, respectively.
However, when false-negative plants taken into account that occur with about 10% and more in 2019, then the Chi2 values change to Chi2 = 2.3, P = 0.1294, for a 3r:1s segregation and Chi2 = 51.84, P = 0, for 15r:1s segregation. Because of various environmental conditions and inhomogeneous Polymyxa spatial distributions false-negative plants are always found in these field trials. Thus, a Chi2 value of 2.3 (P = 0.1294, df = 1) would indicate a 3r:1s segregation rather than a 15r:1s, respectively. This suggestion fits to the replication of the study in 2020 with a more homogenous virus infestation as well as the molecular study.
Based on the ELISA scores given in Table 4 the molecular data were associated with the phenotypic result. The prerequisite for this study was a clear differentiation of the ELISA scores for SBCMV between the parents PC272 and PC2243 (cf. Figure 2).
Altogether 8,950 molecular markers were available, from which 6,362 were mapped across the seven rye chromosomes. For the study on SBCMV totally 987 cleaned and translatable markers were included: 1R = 133, 2R = 135, 3R = 133, 4R = 162, 5R = 200, 6R = 152, 7R = 72, i. e., about 141 per chromosome. With the exception of chromosome 7R, there is a balanced distribution across the genome (cf. Fig. 3).
Applying these 987 markers and composite interval mapping (CIM) a QTL on chromosome 2R was detected (cf. Table 7 and Fig. 4). In this comparison, the phenotypic results of the parents were not included because of the allele effects that are highly influenced by the A-parent (S. cereale var. Imperial). The differences for the allele frequencies between groups are larger or smaller 0.1, i.e., they are rather small (Table 7).
Taking all restrictions into account, it can be assumed that there is a genetic stretch of about 13 cM (between 114 and 127 cM) on the long arm of chromosome 2R critical for the SBCMV resistance in this particular population (Fig. 4). The markers “C9654_1947” and “isotig11640” are the peak markers within this region (Table 7).
Additional calculations, such as a reduced ABH matrix with expected segregation patterns only or transformed phenotypes, where scores larger 0.1 have been coded as 1 and smaller 0.1 as 0 to simulate the non-linearity and/ or sensitivity of the ELISA test, did not improve the significance of the results. The application of the basic local alignment search tool (BLAST) for known wheat markers to SBCMV did not show a clear overlapping with the detected region on 2R. The molecular investigation also revealed a higher heterozygosity for the donor parent S. montanum as compared to the recipient parent "Imperial" (Table 7) that is in general agreement with morphological observations.
This can be explained by the fact that the variety "Imperial" variety has been propagated over several generations through self-pollination, while the S. montanum accession resulted from a sample that was once collected with two multiplications under isolated conditions.
Variation of seed color
It is known that the color of the rye caryopses can vary from yellow, green, brown to violet (Schlegel 2013). In the present investigation, the female cross parent “Imperial” had green and the male parent, S. montanum, brownish grains. The F1 grains were dark green throughout. But in the F2 generation there was a clear segregation of the seed color.
Therefore it seemed reasonable to consider this phenotypic variation in relation to the resistance against SBCMV.
Thus, four groups of approximately the same size were created for the colors yellow, green, green-yellow, and brown (cf. Fig. 5). They were sown separately and later compared with the ELISA results. Plants susceptible to SBCMV (ELISA scores > 0.10) occur with different frequencies between the classes of seed color (Table 4) and can be distinguished if the ELISA scores of the four groups are averaged. The highest ELISA scores are found among the green-yellow and brown-colored seeds. These differences from the purely yellow and green seeds are statistically significant (Tables 8, 9 and 10). It shows that the plants with the yellow seed coat show practically no infection with SBCMV.