Legume Research

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Identification of SSR Markers Linked to Powdery Mildew Resistance in Table Pea (Pisum sativum var. Hortense L.)

Archi Gupta1,2,*, Bijendra Singh1, Mukesh Kumar1, Ujjwal Sirohi1, S.K. Yadav3, V.R. Sharma4
1Sardar Vallabhbhai Patel University of Agriculture and Technology, Meerut-250 110, Uttar Pradesh, India.
2School of Agriculture, Lovely Professional University, Phagwara-144 411, Punjab, India.
3ICAR-National Bureau of Plant Genetic Resources, Pusa-110 012, New Delhi, India.
4CSIR-National Botanical Research Institute, Lucknow-226 001, Uttar Pradesh, India.
  • Submitted08-12-2022|

  • Accepted20-02-2023|

  • First Online 07-04-2023|

  • doi 10.18805/LR-5085

Background: Powdery mildew is one of the major diseases of pea which leads to severe crop losses and also affects the quality of pods and seeds. Therefore, it is necessary to screen for resistant sources and introduce the resistant gene into cultivars. So, for marker-assisted pea breeding, rapid screening of pea germplasm for powdery mildew resistance is required. 

Methods: A total of twenty-eight pea lines were generated using half-diallel mating design with eight pea lines (AP-3, Kashi Nandini, Arkel, VL-7, PMR-53, Kashi Uday, PC-531and AP-1). These hybrids and parental lines were screened for powdery mildew resistance phenotypically by PDI and genotypically by SSR and SCAR markers. 

Result: Based on screening for powdery mildew resistance, the genotypes PMR-53, Kashi Nandini, Kashi Uday, VL-7, AP-1 and hybrids Kashi Nandini x VL-7, Kashi Nandini x PMR-53, Kashi Nandini x Kashi Uday, Kashi Nandini x AP-1, VL-7 x PMR-53, VL-7 x Kashi Uday, VL-7 x AP-1, PMR-53 x Kashi Uday,  PMR-53 x AP-1and Kashi Uday x AP-1 were found relatively resistant to powdery mildew disease. Two SSR markers (AD237 and AD141) showed polymorphism between resistant and susceptible lines and can be used for genetic improvement of pea germplasm. 
Pea (Pisum sativum L.; Fabaceae) is a significant cool-season vegetable crop grown for green pods in temperate zones and tropical high lands around the world. (Ali et al., 1994; Azmat et al., 2010). Pea is typically grown in summer as an off-season crop in the high hills and during the winter season in the Indian plains (Rana et al., 2010; Bala et al., 2011). Powdery mildew of pea, caused by Erysiphe pisi DC, is one of the most devastating diseases, causing up to 50% yield losses globally. (Munjal et al., 1963; Singh et al., 1978; Warkentin et al., 1996; Katoch et al., 2010). Powdery mildew has been found in the majority of the pea varieties grown here. There are germplasm lines that have shown resistance to powdery mildew, but the majority of them are not agronomically superior (Ghafoor et al., 2005). As a result, it is critical to identify germplasm lines that may contain genes for both disease resistance and high yielding (Tiwari et al., 2004; Zong et al., 2008; Bing et al., 2011). This could help conventional plant breeders to overcome the challenges of linkage drag while transferring powdery mildew resistance from germplasm lines with poor agronomic performance.

Traditionally, disease screening in the field or greenhouse is to select resistant plants. This is more often a time-consuming method that also involves handling and maintaining the pathogen. Use of Molecular markers is the best alternative in screening powdery mildew resistance lines and making the breeding process more efficient. Gene specific markers that are closely linked to powdery mildew resistance must be identified for rapid screening of resistant lines using marker-assisted selection (MAS) at early stages of plant development, thereby avoiding selection through disease exposure (Rakshit et al., 2001). MAS can be useful not only for qualitative traits controlled by single genes but also for quantitative traits (Lande and Thompson, 1990). The complex genetic architecture of quantitative traits, on the other hand, may limit the efficiency of MAS for such traits (Edwards and Page, 1994). Powdery mildew resistance is achieved by transferring the disease resistant gene from a resistant donor line to a susceptible recipient line. Earlier studies have established that powdery mildew resistance is controlled by a single recessive gene, designated as er According to Gupta (1987), Kumar and Singh (1981) and Tiwari et al., (1997), two genes, er1 and er2, are involved. The current study aimed to identify powdery mildew resistant lines using simple sequence repeats (SSR) markers for genetic improvement of pea germplasm.  
This experiment was conducted at the Horticulture Research Centre (HRC) of the SVP University of Agriculture and Technology, Meerut during the rabi season, of 2018-19 and 2019-20. Twenty-eight cross combinations were generated with eight parental lines (AP-3, Kashi Nandini, Arkel, VL-7, PMR-53, Kashi Uday, PC-531and AP-1) using half-diallel mating design (Table 1) during Rabi 2018-19. Under open field conditions, all eight parents and twenty-eight F1 hybrids were screened for powdery mildew resistance and disease scoring was done according to Munjal et al., (1963) (Table 2. and Table 3). The percent disease index (PDI) was calculated using Wheeler’s formula (Wheeler, 1969).

Table 1: List of pea genotypes used as parents for resistance screening.

Table 2: Disease scale description for powdery mildew.

Table 3: List of twenty-eight cross combinations generated using half-diallel mating design with eight parental lines.

Genotyping for disease resistance using molecular markers for all the parental lines and F1 hybrids was done at Molecular Biology Lab, Department of Genetics and Plant Breeding. The leaf samples were collected and stored at -80°C. The genomic DNA was isolated based on a modified protocol of CTAB (Cetyl- Trimethyl-Ammonium Bromide) by Doyle and Doyle (1990) and stored at -20°C for further experiments.

Molecular screening was performed using 27 SSR and 3 SCAR primer pairs (Merk Bioscience, USA) (Table 4).

Table 4: Random SSR and SCAR primers used to amplify the pea genotypes in this study.

SSR and SCAR markers that were found to be linked to er gene, (Loridon et al., 2005) were tested for linkage to the candidate gene within the parental lines. PCR amplification was done in 25 µl reaction mixtures, containing 1 µl of diluted template DNA (50 ng/μl), 0.5 µl (5 μM) of each forward and reverse primer, 0.5 µl of 10 mM dNTPs, 2.5 µl of 10´ buffer, 0.8 µl (U/μl) of Taq polymerase and 19.2 µl of ddH2O. PCR reactions were carried out on a PTC Peltier Thermal Cycler (MJ Research) with Initial denaturation at 94°C for 3 min then denaturation at 94°C for 1 min, annealing at 45°C to 60°C for 1 min, extension at 72°C for 1 min (35 cycle) and then final extension at 72°C for 10 min and termination at 4°C. The Amplified PCR products were separated by ethidium bromide stained gel electrophoresis, which was further visualized using Alpha Imager 1200 TM (Alpha Innotech Corporation, USA).
A total of eight parents used in the present study, were grouped as immune, resistant, moderately resistant, moderately susceptible, susceptible and highly susceptible category based on disease score using 0-5 scale (Munjal et al., 1963). The genotypes PMR-53, Kashi nandini, Kashi Uday, VL-7 had shown relatively high powdery mildew resistance. The genotype AP-1 was found to be relatively moderately resistant.  Whereas, the genotypes AP-3 and PC-531 were found to be moderately susceptible and susceptible to powdery mildew respectively. Furthermore, the genotype Arkel was adversely affected with the disease among the screened genotypes which was scored as most susceptible line.

The above results suggested that the screened lines against powdery mildew disease provide information on new resistant varieties as well as few good sources of resistance that could be useful to researchers for developing powdery mildew resistant varieties. However, there is need for further evaluation of more numbers of lines against powdery mildew to find more resistant materials. The findings of screening is similar to Tiwari et al., (1997); Singh et al., (1988); Pandey et al., (1999); Shiwani and Sharma (2022). Similarly, Davidson et al., (2004) screened for 88 lines, of which 19 lines were showed powdery mildew resistance. Furthermore, Mishra et al., (2013) evaluated nine accessions for powdery mildew resistance and found one variety as moderately resistant, two entries were found as moderately susceptible, one entry was found susceptible and two entries were found highly susceptible. Moreover, Rana et al. (2013) screened 701 accessions of garden and field pea originating from 60 countries for powdery mildew resistance under natural epiphytotic conditions. Among them 64 accessions were found resistant in field screening. Nag et al., (2018) evaluated fifteen Pea varieties for powdery mildew resistance, among them two varieties were found resistant (R), two entries and one variety was found moderately resistant (MR), two varieties were found susceptible (S) and one variety was found highly susceptible.

It is also observed that among 28 F1 hybrids, 10 hybrids (PMR-53 × AP-1, VL-7 × PC-531, Kashi Nandini × VL-7, AP-3 × PMR-53, VL-7 × PMR-53, AP-3 × AP-1, Kashi Nandini × PC-531, PMR-53 × PC-531, Kashi Uday × AP-1, AP-3 × Kashi Nandini) were found to be resistant  to powdery mildew i.e. which were categorised as resistant on 0 to 5 scale. Six hybrids i.e. PC- 531 × AP-1, AP-3 × Kashi Uday, PMR-53 × Kashi Uday , Kashi Nandini x Kashi Uday, VL-7 × Kashi Uday, Kashi Nandini × PMR-53 were found moderately resistant to powdery mildew. Three hybrids AP-3 × PC-531, VL-7 × AP-1, Arkel × PMR-53 were found moderately susceptible. Five hybrids Kashi Uday × PC-531, AP-3 × Arkel, Arkel × AP-1, Kashi Nandini × AP-1, AP-3 × VL-7 were classified as susceptible category. Four hybrids Arkel × VL-7, Arkel × Kashi Uday, Kashi Nandini × Arkel, Arkel × PC-531 were highly susceptible for powdery mildew as they exhibited maximum PDI range. The overall results indicated that out of 28 crosses, 16 hybrids were found resistant and 12 hybrids were found susceptible to powdery mildew. Out of 16 resistant hybrids, PMR-53 × AP-1, VL-7 × PC-531 were found highly resistant for powdery mildew as they exhibited minimum PDI. Out of 12 susceptible hybrids, Arkel × PC-531 were found highly susceptible for powdery mildew. This is evident that among all the parents and hybrid combinations, we found the variety Arkel and hybrids in which Arkel is used as one of the parents showed susceptibility to powdery mildew. That indicates the susceptibility is governed by dominant gene. The similar findings were reported earlier by Janila et al., (2001), screening for powdery mildew resistance in pea in 10 crosses, involving 16 different parents for inheritance studies in natural epidemic conditions was used for disease screening.

The eight parental lines and their derived 28 hybrids of pea were further screened using 27 SSR markers and 3 SCAR markers linked to powdery mildew resistance. Out of thirty, only 2 SSR markers (AD237 and AD141) had shown polymorphism between resistant and susceptible parents (Fig 1 and Fig 2).

Fig 1: Molecular profyling of eight pea parental lines using AD237 SSR marker.

Fig 2: Molecular profyling of eight pea parental lines using AD141 SSR marker.

AD237 marker produced an amplicon of approx. 300 bp in all the susceptible/ moderately susceptible genotypes. Likewise, an amplification product of approx. 200 bp was observed in all the resistant genotypes. The AD141 primer uniformly produced an amplification product of approx. 300 bp in all the susceptible moderately susceptible genotypes. Similarly, an amplification product of approx. 200 bp was observed in all the resistant genotypes. These results were in concurrence with earlier studies by Loridon et al., 2005., Katoch et al., (2010) reported a RAPD marker OPX17 1400 (2.6 cM) linked to the er-2 gene which was successfully converted to a SCAR marker, ScX 17 1400. The ScX17 1400 marker will ensure precise introgression of er-2 gene into susceptible cultivars by permitting selection of er-2 in the backcross generation without progeny tests and resistance screening. Similarly, Pereira et al., (2010) identified DNA markers linked to induced mutated genes (er1mut1 and er1mut2) conferring resistance to powdery mildew in pea (Aziz-ur-Rahman et al., 2021). Pavan et al. (2013), Sun et al., (2014) constructed SSR genetic linkage map of pea (Pisum sativum L.) based on Chinese native varieties for powdery mildew resistance. Reddy et al., (2015) screened  parental genotypes and F2 hybrids  using  12 SCAR markers and 5 SSR markers. None of the SCAR markers could distinguish between the resistant or susceptible genotypes. The SSR marker A5 clearly differentiate the homozygous resistant and susceptible parents and F2 progeny from crosses of Arka Priya ´ IP-3, Arka Pramod × IP-3 and Arka Ajit ´Azad-Pea. Thus, even if none of the markers is close enough to the er gene, two SSR markers can considerably improve the likelihood of correct identification and may thus be successfully employed in MAS for powdery mildew resistance in pea.

In both primers, hybrids i.e. Kashi Nandini × VL-7, Kashi Nandini × PMR-53, Kashi Nandini × Kashi Uday, Kashi Nandini × AP-1, VL-7 × PMR-53, VL-7 × Kashi Uday, VL-7 × AP-1, PMR-53 × Kashi Uday,  PMR-53 × AP-1and Kashi Uday × AP-1 are resistant it means that the parents used for crossing was resistance that’s why it was heritable to the off spring while AP-3 × Arkel, AP-3 × PC-531, Arkel × PC-531 are susceptible. Moreover, some hybrids AP-3 × Kashi Nadini, AP-3 × VL-7, AP-3 × PMR-53, AP-3 × Kashi Uday, AP-3 X AP-1, Kashi Nandini × Arkel, Kashi Nandini × PC-531, Arkel × VL-7, Arkel × PMR-53, Arkel × Kashi Uday, Arkel × AP-1, VL-7 × PC-531 PMR-53 × PC-531and Kashi Uday × PC-531are heterozygous as they are showing both resistant and susceptible bands (Fig 3 and Fig 4).

Fig 3: Molecular screening of 28 hybrid combinations lines using AD237 SSR marker. (100 bp ladder).

Fig 4: Molecular screening of 28 hybrid combinations lines using AD141 SSR marker. (100 bp ladder).

Similar screening results were reported by Ek et al., (2005) who used microsatellites (SSR) to find markers linked to powdery mildew resistance, using bulked segregant analysis. Pereira et al., (2010) found that, screening of 360 decamer primers enabled the identification of two RAPD markers linked to one of the mutant resistant genes (Frilene mutant), which need further confirmation in a segregant F2 progeny in Pea. These primer pairs (AD237 and AD141) can help in rapid screening of powdery mildew resistant genotypes for pea breeding. That in turn help in identifying the QTLs linked for powdery mildew resistance for marker assisted selection and gene introgression for genetic improvement pea germplasm.
Genotypes PMR-53, Kashi Nandni, kashi Uday, VL-7, AP-1 and hybrids, Kashi Nandini x Vl-7, Kashi Nandini x PMR-53, Kashi Nandini x Kashi Uday, Kashi Nandini X AP-1, VL-7 x PMR-53, VL-7 x Kashi Uday, VL-7 X AP-1, PMR-53 x Kashi Uday, PMR-53 x AP-1and Kashi Uday x AP-1 were found resistant to powdery mildew which could be further used in identifying QTLs associated with powdery mildew for  the development of resistant genotypes using markers assisted selected (MAS). SSR Markers (AD237 and AD141) gave polymorphism between resistant and susceptible parents and hybrids and further these primers can be used for rapid screening of powdery mildew resistance.

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