Disease phenotyping of pea germplasm
Forty-six pea genotypes were screened for powdery mildew disease in controlled greenhouse conditions against the Erysiphe pisi isolate, Ep01 (NCBI GenBank Acc. No. KM096758). The pea genotypes included both indigenous and exotic germplasm along with a few established sources of resistance and susceptibility which served as controls for the study. Of the seven pea genotypes selected from our previous screening studies (Bheri et al. 2016) to check for consistency in the disease phenotyping, 5 of them showed consistency in their disease reaction similar to the previous findings. However, two genotypes varied in their disease reaction which might be resulting from the molecular differences in the genetic background of those genotypes.
The pea genotypes were categorized based on the disease scoring scale adopted by Gawande and Patil (2003) with modification. Based on the percent disease index (PDI), the pea genotypes were grouped into six classes: highly resistant (HR), resistant (R), moderately resistant (MR), moderately susceptible (MS), susceptible (S) and highly susceptible (HS). The PDI values were calculated as the sum total of the products of the number of leaves showing the disease pattern assigned to each of the groups and the corresponding disease score of the group, which is computed as a percentage to reflect the actual disease incidence observed on that pea genotype. A typical graph representing the disease infection pattern in pea genotypes along with the resistant and susceptible controls are presented (Fig. 1a). Further, a frequency histogram confirmed that the observed variability among the pea genotypes followed a normal distribution as expected for a randomized population which is shown in Fig. 1b. The pea genotypes were also plotted individually based on their PDI values which showed that majority of the genotypes were moderately resistant and moderately susceptible, while the highly resistant and highly susceptible phenotypes were comapartively lower in number as observed in the extreme ends of the graph (Fig. 1c).
Likewise, significant level of correlation was observed between the disease phenotypes of the pea germplasm and the amount of mycelial growth in their leaf tissues as shown in Fig. 2. The microscopic examination of trypan blue-stained leaves, sampled randomly from the pea germplasm which were challenged with E. pisi at 3 dpi in the greenhouse conditions revealed variation among the pea genotypes. A rapid spread of mycelium with effusive sporulation was observed in highly susceptible (HS) pea genotypes while the infection was relatively lower in susceptible (S) and moderately susceptible (MS) genotypes respectively. Conversely, the highly resistant and resistant genotypes showed no infection while moderately resistant revealed limited mycelial spread in E. pisi-infected leaf tissues.
The pea genotypes were grouped into the different disease categories based on their PDI values (Table. 1). The PDI value was found in the range of 5–66% in the pea genotypes. The pea genotypes; JI2480, JI2302 and P660-4 showed lower level of powdery infection with 5, 7 and 8% disease incidence respectively and were used as resistance controls. The highest mean PDI value was recorded in the susceptible pea genotype, Arkel with 66% disease incidence. In addition to Arkel, four pea genotypes; IC424887, IC424892, IC424591 and Azad were also found to be highly susceptible while nine pea genotypes (IC424588, VKG30162, IC208368, IC424888, IP-3, IC417837, VLSM-11, IC417586 and EC387113) were susceptible and thirteen genotypes (EC398589, VLSM-14, EC507771, VLSM-15, EC499976, EC414485, IC356304, IC356391, IC417880, EC398602, IC267169, EC398611 and IC424592) were moderately susceptible. The moderately resistant group comprised of 10 genotypes (IC208364, IC356203, DMR-11, EC398612, VLSM-13, VLSM-12, EC507770, IC356318, KPMR-526 and IC398604) and the resistant group included 6 pea genotypes; EC6620, IC267181, PSM-4, DDR-61, IC267152 and EC499762 suggesting them as potential sources of resistance to powdery mildew disease. The research findings were observed in agreement with the visual scoring carried according to 0–4 scale as used by Fondevilla et al. (2006) and Katoch et al. (2010) wherein 0, 1 and 2 represent resistant groups while 3 and 4 represent susceptible groups (Vaid and Tyagi 1997) as shown in Table S1.
Screening with gene-specific markers for er1, er2, and Er3 resistance
The pea genotypes; JI2302, JI2480 and P660-4 which are reported to carry er1, er2 and Er3 genes respectively were used as the resistant controls for molecular screening while Arkel served as susceptible control. The representative gel image of the SCAR markers linked to all three resistance genes along with their expected band size is presented in Fig. 3a. The SCAR marker, Sc-OPE-161600 has been reported as a resistance marker and found to be in cis-phase with er1 gene, thereby amplifying a band of size 1600 bp in the resistance control, JI2302 and absent in the susceptible control, Arkel. However, the susceptibility marker, Sc-OPO-181200 amplied the expected band of 1200 bp in Arkel along with a 1400 bp band which has been reported to be associated with susceptibility to powdery mildew disease as shown in Fig. 3b. Similarly another susceptible genotype, IP-3 amplified both the bands, 1200 bp and 1400 bp as reported for susceptible phenotype. On the contrary a 1200 bp band along with 1400 bp was amplified by the marker, Sc-OPO-181200 in the resistant control, JI2480 while 1400 bp band alone was observed in JI2302 and P660-4 suggesting that these genotypes might be exhibiting allelic variation at the er1 locus.
Eight Er1 allele-specific SCAR markers were used for screening the pea genotypes of which ScOPT16480 amplified a band size of 480 bp in Arkel, while it was not found in the resistant controls, JI2302, JI2480 and P660-4 suggesting its association with susceptibility. Similarly this marker did not amplify in the other three resistant genotypes (EC499762, EC6620 and IC208364) indicating the marker to be tightly linked with susceptibility. Of the remaining susceptible markers, ScOPL13990 marker amplified a band size of 990 bp not only in the susceptible genotypes, IP-3 and Arkel but were found in all the three resistant control genotypes as well as other disease phenotypes indicating the lack of specific polymorphism for this marker. The susceptible marker, ScOP0061100 which amplified a band size of 1100 bp was found in the resistant control, JI2480 along with the susceptible control, Arkel. Likewise, SCAR markers ScAH1R and ScAGG.CAA125 amplified bands ranging from 250–300 bp and 125–250 bp respectively which were found in all three resistant controls along with the susceptible control indicating them as unlinked to the er1 locus. Likewise, the susceptible markers, ScOPD10650 and ScOPX04880 amplified the bands of sizes 650 bp and 880 bp in all three resistant controls along with Arkel suggesting the lack of specific polymorphism for the marker.
The SCAR marker, ScX17_1400 which is reported as er2 gene-specific marker amplified a band size of 1400 bp in the resistant control JI2480 alone indicating that it is specific to the genotype, JI2480. The SCAR marker, SCW4637 reported as specific to Er3 resistance, amplified a single band of size 637 bp in P660-4 alone indicating the presence of homozygous alleles at the Er3 locus. However, the resistant marker along with the susceptible marker, SCAB1874 appeared in heterozygous condition in majority of the pea genotypes including the susceptible control, Arkel.
Genotying pea germplasm for powdery mildew resistance
Comparing the screening of the pea genotypes for powdery mildew resistance using gene-specific markers for er1, er2 and Er3 genes and their PDI values led to the identification of the few of the pea genotypes as resistant donors which are listed in Table 2. The expected band size of 1600 bp amplified with the marker Sc-OPE-161600 was observed in 26 pea genotypes including the resistant control, JI2302. However, the susceptible marker Sc-OPO-181200, amplified a band of 1200 bp alone in two pea genotypes (EC49976 and VLSM-11), while majority of the pea genotypes showed either 1400 bp band alone or in combination with 1200 bp band. The SCAR marker, ScOPD10650, amplified a band size 650 bp in most of the pea genotypes baring two genotypes; IC417837 (S) and VLSM-11(S). Though this marker was reported as a resistant marker previously to screen pea resistant and susceptible lines (Timmerman et al. 1994), the marker was found to amplify in all the genotypes irrespective of the disease phenotype (Tiwari et al. 1998). The marker, ScOPX04880, amplified a band size 880 bp in all the pea genotypes except IC208364 (MR) and IC417837 (S) pea genotypes. The marker ScOPO061100, amplified a band size of 1100 bp in eighteen pea genotypes. The marker ScOPL13990, amplified a band size of 990 bp in 44 pea genotypes including resistant and suscetible genotypes except two of the pea genotypes, IC208364 (MR) and IC417837(S). The marker ScOPT16480, amplified a band size 480 bp in 29 pea genotypes while it was absent in all three resistant controls. The SCAR markers, ScAH1R and ScAGG.CAA125, amplified a band size of 250–350 bp and 150–250 bp in all 46 pea genotypes suggesting that it as monomorphic in all the pea genotypes.
Table 1
Disease scoring and grouping of pea germplasm into different phenotypic categories using the 0–5 scale of Gawande and Patil (2003) with modifications
S. No. Disease Score (%) | Disease Reaction | Pea genotypes | |
1 0–8 | Highly Resistance | JI2302, JI2480, P660-4 | |
2 9–15 | Resistance | EC6620, IC267181, PSM-4, DDR-61 IC267152, EC499762 | |
3 16–35 | Moderately Resistance | IC208364, IC356203, DMR-11, EC398612, VLSM-13,VLSM-12, EC507770, IC356318, KPMR526, IC398604 | |
4 36–48 | Moderately Susceptible | EC398589, VLSM-14, EC507771,VLSM-15, EC499976, EC414485, IC356304, IC356391, IC417880, EC398602, IC267169, EC398611, IC424592 | |
5 49–55 | Susceptible | IC424588, VKG30162, IC208368, IC424888, IP-3, IC417837, VLSM-11, IC417586, EC387113 | |
6 >56 | Highly Susceptible | IC424887, IC424892, IC424591, Azad, Arkel | |
Table 2
Genotyping pea germplasm by molecular marker screening for powdery mildew resistance genes; er1, er2 and Er3 using the reported allele-specific SCAR markers
S. No. | Germplasm/cultivar | er1 Gene Markers | er2 Gene Marker | Er3 gene Markers | Percent Disease Index | Disease reaction |
1. | IC208364 | Sc-OPO-181400 ScOPD10650 ScAH1R ScAGG.CAA125 | A | A | 16 ± 3.6 | Moderate Resistant |
2. | EC398611 | Sc-OPE-161600 ScOPO061100 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R Sc-OPO-181200 Sc-OPO-181400 ScAGG.CAA125 | ScX17_1400 | SCW4637 SCAB1874 | 48 ± 0.5 | Moderate Susceptible |
3. | IC424592 | Sc-OPE-161600 Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | A | SCW4637 SCAB1874 | 48 ± 2.9 | Moderate Susceptible |
4. | EC6620 | Sc-OPE-161600 Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScAH1R ScAGG.CAA125 | ScX17_1400 | SCAB1874 | 9 ± 1.5 | Resistant |
5. | IC424888 | Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | ScX17_1400 | SCAB1874 | 53 ± 6.7 | Susceptible |
6. | DDR-61 | Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | ScX17_1400 | SCW4637 SCAB1874 | 10 ± 2.0 | Resistant |
7. | IC417837 | ScAH1R ScAGG.CAA125 | A | A | 54 ± 3.1 | Susceptible |
8. | EC398602 | Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScAH1R ScAGG.CAA125 | A | SCW4637 SCAB1874 | 46 ± 1.9 | Moderate Susceptible |
9. | IC267181 | Sc-OPE-161600 Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | ScX17_1400 | SCAB1874 | 9 ± 2.2 | Resistant |
10. | EC414485 | Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | ScX17_1400 | SCAB1874 | 41 ± 3.3 | Moderate Susceptible |
11. | IC208368 | Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | A | SCAB1874 | 52 ± 4.4 | Susceptible |
12. | EC507771 | Sc-OPE-161600 Sc-OPO-181400 ScOPO061100 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | A | SCW4637 SCAB1874 | 38 ± 5.0 | Moderate Susceptible |
13. | IC267169 | Sc-OPO-181400 Sc-OPO-181200 ScOPO061100 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | A | SCAB1874 | 47 ± 2.1 | Moderate Susceptible |
14. | EC499762 | Sc-OPE-161600 Sc-OPO-181400 Sc-OPO-181200 ScOPO061100 ScOPL13990 ScOPD10650 ScOPX04880 ScAH1R ScAGG.CAA125 | ScX17_1400 | SCW4637 SCAB1874 | 11 ± 2.2 | Resistant |
15. | IC424892 | Sc-OPE-161600 Sc-OPO-181400 Sc-OPO-181200 ScOPO061100 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | ScX17_1400 | SCW4637 SCAB1874 | 58 ± 4.5 | Highly Susceptible |
16. | IC398604 | Sc-OPE-161600 Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | ScX17_1400 | SCAB1874 | 35 ± 1.5 | Moderate Resistant |
17. | IC424887 | Sc-OPE-161600 Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | A | SCW4637 SCAB1874 | 57 ± 5.7 | Highly Susceptible |
18. | IC424588 | Sc-OPE-161600 Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | ScX17_1400 | SCW4637 SCAB1874 | 51 ± 3.0 | Susceptible |
19. | IC417586 | Sc-OPE-161600 Sc-OPO-181400 ScOPO061100 ScOPL13990 ScOPD10650 ScOPX04880 ScAH1R ScAGG.CAA125 | ScX17_1400 | SCAB1874 | 55 ± 3.7 | Susceptible |
20. | EC499976 | Sc-OPE-161600 ScOPL13990 ScOPD10650 ScOPX04880 ScAH1R Sc-OPO-181200 ScAGG.CAA125 | ScX17_1400 | SCAB1874 | 40 ± 1.9 | Moderate Susceptible |
21. | IC424591 | Sc-OPO-181400 ScOPO061100 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | A | SCW4637 SCAB1874 | 60 ± 2.3 | Highly Susceptible |
22. | KPMR526 | Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | ScX17_1400 | SCW4637 SCAB1874 | 34 ± 1.1 | Moderate Resistant |
23. | VKG30162 | Sc-OPE-161600 Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | ScX17_1400 | SCW4637 SCAB1874 | 51 ± 4.3 | Susceptible |
24. | AZAD | Sc-OPE-161600 Sc-OPO-181400 Sc-OPO-181200 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | ScX17_1400 | SCW4637 SCAB1874 | 65 ± 2.4 | Highly Susceptible |
25. | EC507770 | Sc-OPE-161600 Sc-OPO-181400 ScOPO061100 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | ScX17_1400 | SCW4637 SCAB1874 | 33 ± 2.7 | Moderate Resistant |
26. | IC417880 | Sc-OPO-181400 Sc-OPO-181200 ScOPL13990 ScOPD10650 ScOPX04880 ScAH1R ScAGG.CAA125 | A | SCAB1874 | 44 ± 6.4 | Moderate Susceptible |
27. | IC356318 | Sc-OPE-161600 Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | ScX17_1400 | SCAB1874 | 33 ± 1.9 | Moderate Resistant |
28. | EC398589 | Sc-OPO-181400 ScOPO061100 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | A | SCW4637 SCAB1874 | 36 ± 2.3 | Moderate Susceptible |
29. | EC387113 | Sc-OPE-161600 Sc-OPO-181400 ScOPO061100 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | A | SCAB1874 | 55 ± 6.8 | Susceptible |
30. | PSM-4 | Sc-OPE-161600 Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1 ScAGG.CAA125 | A | SCW4637 SCAB1874 | 9 ± 1.7 | Resistant |
31. | DMR-11 | Sc-OPE-161600 Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | ScX17_1400 | SCAB1874 | 24 ± 0.8 | Moderate Resistant |
32. | IC356304 | Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | A | SCW4637 SCAB1874 | 41 ± 1.2 | Moderate Susceptible |
33. | IC267152 | Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | A | SCAB1874 | 10 ± 0.7 | Resistant |
34. | EC398612 | Sc-OPE-161600 ScOPO061100 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R Sc-OPO-181400 Sc-OPO-181200 ScAGG.CAA125 | ScX17_1400 | SCAB1874 | 27 ± 3.5 | Moderate Resistant |
35. | IC356203 | Sc-OPE-161600 Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | ScX17_1400 | SCAB1874 | 23 ± 0.8 | Moderate Resistant |
36. | IC356391 | Sc-OPO-181400 ScOPO061100 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | ScX17_1400 | SCW4637 SCAB1874 | 42 ± 0.7 | Moderate Susceptible |
37. | IP-3 | Sc-OPO-181400 Sc-OPO-181200 ScOPO061100 ScOPL13990 ScOPD10650 ScOPX04880 ScAH1R ScAGG.CAA125 | A | SCW4637 SCAB1874 | 53 ± 1.9 | Susceptible |
38. | VLSM-11 | Sc-OPE-161600 ScOPO061100 ScOPL13990 ScOPX04880 ScAH1R Sc-OPO-181200 ScAGG.CAA125 | A | SCW4637 SCAB1874 | 54 ± 1.6 | Susceptible |
39. | VLSM-12 | Sc-OPE-161600 Sc-OPO-181400 ScOPO061100 ScOPL13990 ScOPD10650 ScOPX04880 ScAH1R ScAGG.CAA125 | ScX17_1400 | SCW4637 SCAB1874 | 32 ± 1.2 | Moderate Resistant |
40. | VLSM-13 | Sc-OPO-181400 ScOPO061100 ScOPL13990 ScOPD10650 ScOPX04880 ScAH1R ScAGG.CAA125 | A | SCW4637 SCAB1874 | 30 ± 0.8 | Moderate Resistant |
41. | VLSM-14 | Sc-OPE-161600 Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScAH1R ScAGG.CAA125 | A | SCW4637 SCAB1874 | 36 ± 0.8 | Moderate Susceptible |
42. | VLSM-15 | Sc-OPE-161600 Sc-OPO181400 ScOPL13990 ScOPD10650 ScOPX04880 ScAH1R ScAGG.CAA125 | A | SCW4637 SCAB1874 | 38 ± 0.8 | Moderate Susceptible |
43. | JI2302 | Sc-OPE-161600 Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScAH1R ScAGG.CAA125 | A | SCAB1874 | 7 ± 0.8 | Highly Resistant |
44. | JI2480 | Sc-OPO-181400 Sc-OPO-181200 ScOPO061100 ScOPL13990 ScOPD10650 ScOPX04880 ScAH1R ScAGG.CAA125 | ScX17_1400 | SCAB1874 | 5 ± 0.7 | Highly Resistant |
45. | P660-4 | Sc-OPO-181400 ScOPL13990 ScOPD10650 ScAH1R ScAGG.CAA125 | A | SCW4637 | 8 ± 0.8 | Highly Resistant |
46. | Arkel | Sc-OPO-181400 Sc-OPO-181200 ScOPO061100 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | A | SCW4637 SCAB1874 | 66 ± 2.3 | Highly Susceptible |
Of the 46 pea genotypes, 23 genotypes amplified a band size of 1400 bp with the SCAR marker, ScX17_1400 showing the presence of er2 gene including the resistant control genotype, JI2480. Likewise, 26 pea genotypes amplified a band size of 637 bp with the SCAR marker, SCW4637 which is a characteristic of the domiant Er3 allele including the resistant control, P660-4. The SCAB1874 marker showed a homozygous band of size 874 bp in 18 pea genotypes including the other two resistant controls, JI2302 and JI2480 suggesting the absence of Er3 resistant allele in these genotypes. On the contrary, two pea genotypes, IC208364 and IC417837 did not amplify any of the above two bands of Er3 indicating the absence of the Er3 locus in these genotypes.
Six pea genotypes; IC267152, IC208364, IC267169, IC417880, IC417837 and IC208368, did not amplify with any of the known resistant gene markers. Of these, IC267152 and IC208364 showed resitance and moderate resistance to E. pisi despite the absence of all three resistant genes, suggesting that these genotypes might be the novel sources of resistance. Whereas, the other four genotypes which also did not amplify the resitant gene markers indicated the absence of powdery mildew resistance as evident from their disease reaction as moderately susceptible and susceptible phenotypes.