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Molecular Screening of Pea Germplasm for Rust Disease Resistance using SSR Marker.

Pea is affected by a number of fungal (rust, powdery mildew, downy mildew, root rot, alternaria blight, aschochyta blight, wilt, anthracnose, cercospora leaf spot, damping off, seedling rot etc.), bacterial (bacterial blight and brown spot), nematode (cyst nematode, lesion nematode and root-knot nematode) and viral diseases (cucumber mosaic virus, pea early browning virus, pea enation mosaic, pea mosaic, pea seed borne mosaic, pea streak and pea stunt). These diseases, under the right conditions, can significantly decrease both yield and quality. Among these, the rust of pea caused by Uromyces viciae-fabae (Pers.) J. Schrot (syn. Uromycesfabae (Pers.) de Bary) is considered the most important under warm and humid conditions (1). It has been reported from different parts of the country including eastern India (2, 3), central India (4), southern parts of India (5, 6) and from Himalayan region of Uttarakhand and Himachal Pradesh (7, 8). In the last few years, disease has been observed in almost epiphytotic form and could cause up to 20-100% losses in yield (9, 8). Screening for rust severity indicated wide range of variations for rust resistance in the germplasm lines of pea and none of the genotypes tested were found to be free from infection (4, 10, 2, 6, 11, 1). Rust severity is greatly influenced by the environment during initial infection and disease development. This is the major bottleneck in screening and selection for rust resistance. Use of molecular markers would allow indirect selection for rust resistance independent of environmental effects (12). Molecular markers associated with pea rust resistance would be useful in marker assisted selection (MAS). For the development of rust resistant varieties there is need for phenotypic as well as molecular screening of existing lines/ germplasms/cultivars, therefore the present research has been carried out.

MATERIALS AND METHODS

Screening of thirty two pea germplasm under natural epiphytotic condition was carried out in the field during Rabi season 2013-14 and 2014-15 at N.E. Borlogue Crop Research Centre (NEBCRC), G.B. Pant University of Agriculture and Technology, Pantnagar. The germplasm screening was undertaken following 'Infector row technique'. Each entry was sown with wider spacing of 30 x 10cm in 3m row with a susceptible check 'HFP-4' after every five entries and a susceptible border row for over 2 seasons (Rabi 2013-2014 and 2014 2015). The observation on rust severity was recorded when first symptoms appear and subsequent observations were recorded at ten days interval and final observations was recorded at 20 days before harvesting of entries. Disease severity was determined using 0-9 rating scale (13). The genotypes were later grouped into different categories based on 0 to 9 scale of disease severity from immune to highly susceptible according to Mayee and Datar (1986) with slight modifications (Table 1.). Thereafter, molecular screening of thirty two diverse pea genotypes which were phenotypically screened on the basis of disease reaction was evaluated using four SSR markers (Table 2.) (AA446 and AA505 flanking the major QTL Qruf; AD 146 and AA416 flanking the minor QTL, Qrufl) associated with pea rust resistance (12).

PCR procedure

DNA from each germplasm was extracted following the CTAB method (14). About 100 mg of young leaf tissue was excised from aseptically grown seedlings of each genotype. PCR amplification solution was prepared using 10 mM tris-HCl (pH 9.0; 1.5 mM Mg[Cl.sub.2]; 50 mM KCl and 0.01% gelatin), 0.5 mM Mg[Cl.sub.2], 200 mM dNTPs, 1.25 iM of primer, 20 to 25ng of DNA andlunit of Taq polymerase per 25Kl reaction volume. SSR markers AA446 and AA505 flanking the major QTL Qruf and AD146 and AA416 flanking the minor QTL, Qruf1 (12) were commercially synthesized and procured from Eurofins Inc., Bengaluru. The amplification reaction was carried out in thermo cycler (MyCycler[R], Bio-Rad Laboratories, California). After initial denaturation at 94[degrees]C for 5 min, cycle was repeated 40 times; denaturing at 94 [degrees]C for 1 min, annealing according to primer [15] for 1 min, extension at 72[degrees]C for 2 min, and the final extension segment was hold for 7 min. Thereafter, PCR products were separated electrophoretically in 2% (w/v) agarose gel. Ethidium bromide solution at a final concentration of 0.5 ig/ml was added to the agarose solution. The gel was visualized and documented using gel documentation system (Transilluminator with filter, GeNei, Benagluru).

RESULTS AND DISCUSSION

Among 32 pea genotypes screened, none of them were found resistant to rust disease during both the seasons. Further, in our search, none of the genotype was found to be completely resistant to the rust disease, which was in agreement with earlier reports (2, 16) although these reports were based on the screening of limited genotypes. Only two genotypes showed moderate reaction with 110 per cent disease severity (Pant P 244 and Pant P 42). Maximum numbers (18) of genotypes fall under susceptible category followed by moderately susceptible (13) and highly susceptible category (2). Thus, two genotypes showing moderately resistant reaction can be integrated with reduced number of fungicidal spray to obtain maximum yield with minimal rust severity. Screening for rust severity indicated wide range of variations for rust resistance in the germplasms lines of pea and none of the genotypes tested were found to be free from infection (4, 10, 2, 6, 11 1). Pal et al (11) screened a total of 292 accessions of pea (Pisum spp.) under field conditions for resistance to rust and he found only three accessions PJ207508, PJ222117, and EC109188 which was resistant to rust. Kumar et a (l6) used area under disease progress curve (AUDPC) to depict the overall disease stress that the plants were subjected to and described the pea varieties Pant P8, HUP 8063, KPMR 22 to possess good level of partial resistance. Likewise, Chand et.al (18) screened 345 accessions, out of which forty-four genotypes were evaluated for disease intensity. Wide range of variation was found for those traits. The genotypes Pant P 11, FC 1, HUDP 16, JPBB 3 and HUP 14 appeared as slow rusting genotypes. Similarly, Mishra et al (19) evaluated 107 genotypes of field pea against rust (Uromyces viciae-fabae), out of which genotypes P 9-77, P 2432; P2572 and P 2930 were found resistant, whereas 27 exhibited moderate reaction. Barilli et al (20) evaluated 2759 pea accessions for resistance against Uromyces pisi (Pers.) Wint. All accessions in his experiment displayed a compatible interaction (high infection type) both in adult plants under field conditions and in seedlings under growth chamber conditions, but with varying levels of disease reduction. The identified resistance was based on reduction of disease severity with no associated host cell necrosis, which fits the definition of Partial Resistance. No complete resistance or incomplete resistance based on hypersensitivity was observed by them.

Severity of rust is greatly influenced by the environment during infection initiation and disease development. This is the major bottleneck in screening and selection for rust resistance. Thus, use of molecular markers would allow indirect selection for rust resistance independent of environmental effects.

SSR marker AA446 flanking the major QTL Qruf amplified a fragment of 450 bp in two moderately resistant (Pant P 244 and Pant P 42), seven moderately susceptible and six susceptible genotypes with 25-30 percent disease severity. Whereas, SSR marker AA505 flanking the major QTL Qruf amplified a fragment of 140 bp in two moderately resistant (Pant P 244 and Pant P 42), six moderately susceptible, nine susceptible genotypes (25-30 percent disease severity) and one highly susceptible genotypes(HFP-4) (Table 3 and Plate 1).

SSR markers AD146 flanking the minor QTL, Qruf1 were able to identify one moderately resistant (Pant P 42), six moderately susceptible genotypes and four susceptible genotypes (25-30 percent disease severity) with amplified fragment of 430bp. Whereas, SSR markers AA416 flanking the minor QTL, Qruf1 amplified a fragment of 280 bp in four moderately susceptible genotypes, seven susceptible genotypes (25-30 percent disease severity) and one susceptible genotypes with 49.17 percent severity (HUDP1301) (Table 3 and Plate 2).

It was observed that most of the germplasm with disease severity of less than 30 percent showed the presence of Qruf and/or Qruf1 governing partial resistance against rust. Therefore, molecular screening of germplasm may conclude that these SSR markers (AA446, AA505, AD 146 and AA416) if used together, then it can be more effective in marker assisted selection (MAS) for pea rust resistance. Similarly, Singh et al (21) utilize molecular markers associated with the pea rust resistance for evaluation of 30 diverse pea genotypes using these four SSR markers. On the basis of marker allele analysis they concluded that these SSR markers can be used in MAS of pea rust resistance. Vijayalakshmi et al (22) suggested two RAPD makers, viz., SC10-82360, and SCRI-711000, flanking the rust resistance gene (Ruf) if used together, the effectiveness of marker assistant selection for rust resistance would be improved considerably. Avila et al (23) also identified random amplified polymorphic DNA (RAPD) markers linked to resistance gene (Uvf-1). This result conclude that for the development of rust resistant varieties there is need for phenotypic followed by molecular screening of existing Table 3.

REFERENCES

(1.) Chand, R., Srivastava, C.P. and Kushwaha, C. Screening technique for pea (Pisum sativum L.) genotypes against rust disease (Uromyces fabae Pers. de Bary). Indian J. Agric. Sci., 2004; 74: 166-7.

(2.) Gupta, R.P. Evaluation of pea germplasm for their reaction to powdery mildew and rust. Indian J. Pul. Res., 1990; 3: 186-8.

(3.) Chand, R., Srivastava, C.P., Singh, R.M. and Singh, R.B. Pea specific strains in Uromyces fabae. Indian J. Pul. Res, 1997; 10: 127-8.

(4.) Narsinghani, V.G., Singh. S.P. and Pal, B.S. Note on rust resistance pea varieties. Indian. J. Agric. Sci., 1980; 50: 453.

(5.) Sokhi, H.S., Sokhi. S.S. and Rawal, R.D.Vertical reaction of pea to powdery mildew (Erysiphe polygoni) and rust (Uromyces vicia fabae). Mysore J. Agril. Sci, 1974; 8: 529-532.

(6.) Kumar, T.B.A., Rangaswamy, K.T. and Ravi, K. Assessment of tall field pea genotypes for slow rusting resistance. Legume Res., 1994; 17: 79-82.

(7.) Chauhan, R.S., Sugha, S.K. and Singh, B.M. A note on the prevalence and distribution of pea rust in Himachal Pradesh. Him. J. Agric. Res., 1991; 17: 105-107.

(8.) Sharma, A.K. Epidemiology and management of rust disease of French bean. Veg. Sci., 1998; 25: 85-88.

(9.) Stavely, J.R. Compendium of Bean Diseases. APS Press, St Paul MN., 1991; pp. 24-25.

(10.) Singh, R.M. and Srivastava, C.P. Evaluation, classification and usefulness of pea germplasm lines for quantitative characters. Legume Res., 1985; 8: 68-73.

(11.) Xue, A.G. and Warkentin, T.D. Reaction of field pea varieties to three isolates of Uromycesfabae. Cana. J. Pl. Sci, 2002; 82: 253-255.

(12.) Rai, R., Singh, A.K., Singh, B.D., Joshi, A.K., Chand, R. and Srivastava, C.P. Molecular mapping for resistance to pea rust caused by Uromyces fabae (Pers.) de-Bary. Theor. Appl. Genet, 2011; 123: 803-813.

(13.) Mayee, C.D. and Datar, V.V. Phytopathometry. Technical Bulletin-1 (Special Bulletin 3), Marathwada Agric. Univ. Parbhani., 1986; p 218.

(14.) Doyle, J.J. and Doyle, J.L. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bull., 1987; 19: 11-15.

(15.) Loridon, K., McPhee, K., Morin, J., Dubreuil, P., Pilet-Nayel, M.L., Aubert, G., Rameau, C., Baranger, A., Coyne, C., Lejeune-Henaut, I. and Burstin, J. Microsatellite marker polymorphism and mapping in pea (Pisum sativum L.). Theor. Appl. Genet., 2005; 111: 1022-1031.

(16.) Anil Kumar, T.B., Rangaswamy, K.T. and Ravi, K. Assessment of tall field pea genotypes for slow rusting resistance. Legume Res., 1994; 17: 79-82.

(17.) Pal, A.B., Brahmappa, R., Rawal, D. and Ullasa, B. A. Field resistance of pea germplasm to powdery mildew (Erysiphe polygoni) and rust (Uromyces fabae). Plant Dis., 1980; 64: 1085-1086.

(18.) Chand, R., Srivastava, C.P., Singh, B.D. and Sarode, S.B. Identification and characterization of slow rusting components in pea (Pisum sativum L.). Genet. Resour. Crop. Ev., 2006; 53: 219-224.

(19.) Mishra, R.K., Pandey, K.K. and Pandey, P.K. Screening of pea (Pisum sativum) genotypes against rust caused by Uromyces fabae. Indian J. Agr. Sci, 2009; 79: 402-403.

(20.) Barilli, E., Sillero, J.C., Aparicio M.F. and Rubiales, D. Identification of resistance to Uromyces pisi (Pers.) Wint. in Pisum spp. Germplasm. Field Crop Res., 2009; 114: 198-203.

(21.) Singh, A.K., Rai, R., Singh, B.D., Chand, R., Srivastava, C.P. Validation of SSR markers associated with rust (Uromycesfabae) resistance in pea (Pisum sativum L.). Physiol. Mol. Biol. Plant, 2015; 21: 243-247.

(22.) Vijayalakshmi, S., Yadav, K., Kushwaha, C., Sarode, S.B., Srivastava, C.P., Chand, R. and Singh, B.D. Identification of RAPD markers linked to the rust (Uromyces fabae) resistance gene in pea (Pisum sativum.). Euphytica, 2005; 144: 265-274.

(23.) Avila, C.M., Sillero, J.C., Rubiales, D., Moreno, M. T. and Torres, A. M. Identification of RAPD markers linked to the Uvf-1 gene conferring hypersensitive resistance against rust (Uromyces viciae-fabae) in Vicia faba L. Theor. Appl. Genet, 2003; 107: 353-358.

Vinod Upadhyay *, K.P.S. Kushwaha and Puja Pandey

Department of Plant Pathology, G.B. Pant University of Agriculture and Technology, Pantnagar, Uttarakhand--236 145, India.

http://dx.doi.org/10.22207/JPAM.11.1.43

(Received: 30 September 2016; accepted: 03 December 2016)

* To whom all correspondence should be addressed.

Tel.: +91-9435264892; E-mail: vinodupadhyay148@gmail.com

Caption: Plate 1. PCR banding pattern of the SSR markers AA446 and AA505 flanking the major QTL 'Qruf' in selected pea germplasm

Caption: Plate 2. PCR banding pattern of the SSR markers AD146 and AA416 flanking the major QTL 'Qruf1' in selected pea germplasm
Table 1. Disease severity scale showing different types of disease
reaction

Rating   Description                       Disease reaction

0        No symptoms on leaf               Immune (I)
1        Rust pustules small,              Resistant (R)
         scattering covering 1%
         or less of leaf area
3        Rust pustules more in number      Moderately resistant
         covering 1-10% of leaf area       (MR)
5        Typical rust pustules             Moderately susceptible
         covering 11-25% of leaf area      (MS)
7        Typical rust pustules             Susceptible (S)
         covering 26-50% of leaf
         area. Leaf shedding
9        Typical rust pustules covering    Highly susceptible (HS)
         51% or more of leaf area.
         Defoliation severe

Table 2. Primers used in molecular screening (Loridon et al., 2005).

Sl.   Primer   Forward sequence     Reverse sequence     PIC *
No.   Name

1.    AA446    5' TTA GCT TGC       3' ATC CGA CCC       0.66
               AGC CCA CTC 3'       ATG GAT TTA 5'
2.    AA505    5' ATT CAC ACG       3' CAA TTA AGC CCT   0.69
               CGC CCA 3'           CAT CCA GA 5'
3.    AD146    5' TGC TCA AGT CAA   3' CAA GCA AAT AGT   0.84
               TAT ATG AAGA 3'      TGT TTT GTT A 5'
4     AA416    5' TTA CTG TTA CTT   3' ATA GTG TCG AAA   0.64
               TGC GAC ATC A 3'     TTT TCC ATC C 5'

Sl.   Primer   Tm [C]
No.   Name     ([degrees]C)

1.    AA446    55

2.    AA505    55

3.    AD146    51

4     AA416    61

* PlC-Polymorphism information content, [C] Tm-Annealing temperature

Table 3. Molecular screening of selected germplasm for rust
resistance in pea.

S.    Germplasm       Disease    Disease
No.                   severity   reaction
                        (%)

1.    Pant P 244        8.17        MR
2.    Pant P 42         8.50        MR
3.    KPMR 522         42.50        S
4.    HUVP 1           55.83        HS
5.    HFP 530          30.00        S
6.    HFP 1016         25.83        S
7.    HFP 9907         21.67        MS
8.    KPMR 925         45.00        S
9.    VL 202           43.33        S
10.   Pant P223        27.50        S
11.   VL 59            21.67        MS
12.   Pant P 222       19.17        MS
13.   Pant P 217       10.33        MS
14.   Pant P 213       12.17        MS
15.   VL 58            13.17        MS
16.   KPMR 853         47.50        S
17.   HUDP 1302        22.50        MS
18.   HUDP 1209        25.83        S
19.   RFP 2009-2       19.17        MS
20.   RFP 2009-3       39.17        S
21.   HUDP 1301        49.17        S
22.   KPMR 851         25.83        S
23.   KPM 928          25.83        S
24.   HUDP 15          20.83        MS
25.   IPFD 13-14       25.83        S
26.   IPF 10           30.83        S
27.   IPFD 5-19        23.33        MS
28.   IPFD 99-13       44.17        S
29.   IPFD 11-5        25.83        S
30.   IPFD 12-2        45.83        S
31.   IPFD 13-4        39.17        S
32.   HFP-4 (check)    65.00        HS

S.    Germplasm            (1) Qruf          (2) Qruf1
No.
                       AA446     AA505     AD146     AA416
                      (450bp)   (140bp)   (430bp)   (280bp)

1.    Pant P 244         +         +         -         -
2.    Pant P 42          +         +         +         -
3.    KPMR 522           -         -         -         -
4.    HUVP 1             -         -         -         -
5.    HFP 530            +         +         -         +
6.    HFP 1016           +         +         +         +
7.    HFP 9907           +         +         +         -
8.    KPMR 925           -         -         -         -
9.    VL 202             -         -         -         -
10.   Pant P223          -         +         +         +
11.   VL 59              +         -         +         +
12.   Pant P 222         +         +         -         -
13.   Pant P 217         -         +         +         +
14.   Pant P 213         +         +         -         +
15.   VL 58              +         -         -         -
16.   KPMR 853           -         -         -         -
17.   HUDP 1302          -         +         +         -
18.   HUDP 1209          -         +         +         -
19.   RFP 2009-2         +         -         +         +
20.   RFP 2009-3         -         -         -         -
21.   HUDP 1301          -         -         -         +
22.   KPMR 851           +         +         -         +
23.   KPM 928            +         +         +         +
24.   HUDP 15            +         +         +         -
25.   IPFD 13-14         -         +         -         +
26.   IPF 10             +         +         -         +
27.   IPFD 5-19          -         -         -         -
28.   IPFD 99-13         -         -         -         -
29.   IPFD 11-5          +         +         -         -
30.   IPFD 12-2          -         -         -         -
31.   IPFD 13-4          -         -         -         -
32.   HFP-4 (check)      -         +         -         -

(1) Qruf flanked by SSR markers AA446 and AA505 (Rai et al.,
2011), (2) Qruf1 flanked by SSR markers AD146 and AA416
(Rai et al., 2011), + indicates presence of a band and
indicates absence of the specific band
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Author:Upadhyay, Vinod; Kushwaha, K.P.S.; Pandey, Puja
Publication:Journal of Pure and Applied Microbiology
Article Type:Report
Date:Mar 1, 2017
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