Molecular Markers for Powdery Mildew in Pea (Pisum sativum L.): A Review

Field pea (Pisum sativum L.) is one of the most important pulse crops grown worldwide. It was considered to be one of the most thoroughly studied genetic systems in crops, next only to maize. However, in the recent era of genomics, there has been a distinct shift in the research priorities to cereals such as rice and wheat. Accelerating research in field pea is a pressing need as it is a rich source of protein and also is capable of fixing atmospheric nitrogen for enriching the soil like other pulses crops.
        
Field pea has several biotic and abiotic stresses related constraints in achieving potential production. Powdery mildew is one among the major constraints to pea production affecting yield and quality by causing yield reductions up to 26-47% (Munjal et al., 1963). The disease not only reduces seed yield but also seed quality (Tiwari et al., 1997a,b). Dixon (1987) identified powdery mildew as the greatest threat to dry peas. The disease is particularly damaging when the seeds are sown late or in late maturing varieties. The earlier the disease occurs the more severe is the damage (Fondvilla and Rubiales, 2012). Furthermore, release of spores of the fungus can cause allergic and breathing problems (Ek et al., 2005). Conventional method uses fungicides and cultural practice such as early planting of crop to stop the spread of disease. However, the control efficacy of chemicals and agronomic practices is limited and causes environmental pollution and health hazard. Therefore, development of powdery mildew resistant cultivars is necessary. The fungus evolves continuously to overcome host resistance and keeps plant breeder to the endless task of developing new crop varieties (Collard and Mackill, 2008). Further, introgression of genes for diseases from wild species into adapted cultivars is complicated (Foolad, 2007). For all these reasons and other problems associated with the use of conventional breeding methods, successful improvement of pea demand the employment of techniques that have higher potential for resolution like Marker Assisted Selection (MAS). Molecular markers are the key in order to implement MAS in breeding program (Choudhary et al., 2019). Identification of DNA markers linked to major genes such as many disease resistance genes have permitted the identification of tightly linked DNA tags for use as diagnostic tools in breeding (Vignesh et al., 2011). MAS has several advantageous over conventional breeding such as assisting genetic selection in early generations and accelerate the breeding process (Jha et al., 2021), allowing rapid introgression of disease resistance genes into susceptible varieties as well as the incorporation of multiple genes into individual lines for durable resistance. This review discusses to understand pea powdery mildew resistance genes and importance of use of molecular markers that may lead to the successful breeding for developing lines with broad resistance against powdery mildew.
 
Causal organism of powdery mildew
 
Erysiphe pisi causes powdery mildew in field pea (Fondevilla and Rubiales, 2012). Two other species Erysiphe trifolii (Attanayake et al., 2010) and Erysiphe baeumleri (Ondřej et al., 2005) also have been identified to infect pea plants in some regions. It is an obligate parasite and depends on the photosynthetic status of the host and cannot develop on photosynthetically inactive tissue (Carver and Jones, 1988). Erysiphe pisi overwinters on infected pea debris or on alternative hosts (Falloon and Viljanen-Rollinson, 2001) and wind-dispersed the conidia locally and over long distance (Warkentin et al., 1996). The manifestations of powdery mildew not only depend upon the inherent qualities of resistance or susceptibility of the different genotypes, but also depend upon other physical and physiological properties such as age (Dixon, 1987), temperature (Banyal and Tyagi,1997) and duration of maturity (Singh et al., 1995). The conidia of Erysiphe pisi can germinate at wide range of relative humidity and limited temperature (Singh et al., 2020). Temperature plays an important role in determining powdery mildew epidemics with an ideal temperature at 20-24°C for conidia germination (Smith, 1970).
        
Powdery mildew usually first appears on the lowest part of the plants as small, diffuse spots on leaflets and stipules. These lesions grow and became white powdery, talcum like growth over entire aerial parts of the plant (Fig 1). In severe infection, the fungus penetrates to reach seeds causing discolouration (Chupp and Shref, 1960) and may affect the entire plant leading to premature drying (Singh et al., 1995). High rate of respiration and decrease in the rate of photosynthesis occurs due to reduced exposure of leaves to light under dense mycelial growth (Singh and Singh, 1983).
 

 
Genetics of powdery mildew
 
Genotypes resistant to powdery mildew was first described by Hammarlund (1925) from his pea collections. However, the mode of inheritance of powdery mildew resistance has been reported by Harland (1948) as a monogenic recessive trait and designated the gene as er1 (Erysiphe resistance). In contrary, Heringa et al., (1969) reported the resistance gene er2 in Pervuian material that was confined to leaves of pea. Sokhi et al., (1979) observed involvement of two different recessive genes er1 and er2. Gene er1 was reported to confer a high level of protection to all plant parts while the er2 resistance was reported to express complete resistance only at 25°C or in mature leaves (Fondevilla et al., 2006, Smýka et al., 2008). Genotypes exhibiting resistance with er1 gene is reported to be complete and durable under field and controlled environments. Gene er2 alone might be unable to provide resistance in pea whereas in combination with er1 might enhance resistance during the growth cycle (Su et al., 2004). Fondevilla et al., (2007) reported a new dominant gene (Er3) in Pisum fulvum that segregates independently from er1 and er2 genes. This gene leads to high rates of cell death as a prompt response to attempted infection.
        
Resistance by er1 is the most widely used for breeding purposes because of its complete and durable resistance (Sun et al., 2016). Humphry et al., (2011) reported that resistance by er1 is due to a loss of function by mutation in PsMLO1, a MLO (Mildew Resistance Locus O) gene family. Many carriers of the powdery mildew resistance trait differ in their phenotype and expression indicating existence of multiple alleles at the locus (Sharma, 2016). To date, eleven er1 alleles (er1-1 to er1-11) have been identified and characterized in pea resistant germplasm, which were produced by natural or artificial mutagenesis (Pavan et al., 2011, Pavan et al., 2013, Sun et al., 2016, Ma et al., 2017, Sun et al., 2019). The first allele er1-1 produced by spontaneous mutation, reported by Harland (1948) has been almost exclusively used in breeding as it imparts strong and reliable resistance in all regions of the world.
 
Mechanism of powdery mildew resistance in pea
 
Conidia of Erysiphe pisi in susceptible pea genotypes are spread during cool nights and dry winter. Conidia germinates forming a germ tube with a lobed primary appressorium. A penetration peg emerges from the appressorium and penetrates the epidermal host cells through the cuticle and cell wall leading to formation of a biotrophic haustorium within the epidermal cell. Nutrient extracts from the plant cell through the haustorium supports growth of secondary hyphae that radiate across the host epidermis forming hyphal appressoria from which secondary haustoria are formed. Finally, aerial conidiophores emerge from surface hyphae producing conidia capable of initiating a new cycle of infection (Falloon et al., 1989). In resistant pea genotypes harbouring er1 gene, the vast majority of Erysiphe pisi conidia germinates and form appressoria. However, no secondary hyphae are formed (Fondevilla et al., 2006). Resistance of er1 was due to avoidance of epidermal cell penetration while er2-mediated resistance is mainly based on reduction in penetration success complemented by post-penetration cell death. It is due to the occurrence of hypersensitive response in established colonies (Fondevilla et al., 2006). A proteomic study showed that resistant genotypes has higher amount of proteins involved in defence than susceptible genotypes (Curto et al., 2006). These proteins are proteins encoded by NBS-LRR resistance genes, PR1 and PR5, Kunitz–trypsin inhibitor that inhibit extracellular fungal proteinases, proteins associated with cell wall reinforcement, proteins involved in tolerance to oxidative stress caused by reactive oxygen species and proteins implicated in the synthesis of alkaloids compounds (Fondevilla and Rubiales, 2012). In resistant genotypes conferred by Er3 gene, most conidia are able to penetrate the epidermal pea cells and form secondary hyphae but a strong hypersensitive response stopped the growth of these established colonies. In the case of other uncharacterized sources of incomplete resistance, different mechanisms can contribute to the reduction of disease severity. Complete resistance can be obtained when hypersensitive response occurs fast and in a high proportion of colonies, as in lines harbouring Er3 gene, or slower and lower proportion of established colonies may result in incomplete resistance (Fondevilla et al., 2007a, b).
 
Molecular markers for disease resistant gene
 
Development of cultivars with improved resistance to biotic stresses is a primary goal of plant breeding programs throughout the world. Disease resistant cultivars can reduce both the risk of yield loss and the dependence on pesticides, enabling a more stable crop production (Miklas et al., 2006). Advance in genome research and molecular biology has led to the use of DNA markers in plant breeding and crop improvement. Molecular markers linked to resistance genes can obviate the need for field testing of genotypes to identify resistant individuals in early generations of breeding populations and also reducing the number of progeny maintained. It helps to understand complex traits, dissect into single Mendelian components and establish their chromosomal locations using linkage maps and/or cytogenetic stocks (Torres et al., 2010). Molecular markers are specific fragments of DNA that can be identified within the whole genome found at specific locations of the genome. They are used to ‘flag’ the position of a particular gene or the inheritance of a particular character. These are considered valuable tools for crop improvement. The potential usefulness of genetic markers as screening tools in plant breeding was recognized by Sax (1923). Molecular markers command several advantages over morphological markers because of their availability in large numbers, no penetrance and expressivity problem, phenotypically neutral in nature, environmentally neutral, tissue and stage independent expression, rapid screening and applicability across the species (Chopra, 1996). Among the markers, the PCR-based markers are more desirable as it requires small amount of template DNA and can be applied efficiently to large populations. Development of markers from the gene itself is desirable as crossovers between markers and gene of interest may occur if the marker is not tightly linked to gene (Huang and Roder, 2004). RFLPs (Restriction Fragment Length Polymorphisms) are reliable and yield co-dominant data, but are time-consuming and expensive, requiring relatively large amount of highly purified DNA and they do not lend themselves to automation (Gupta et al., 2001). RAPD (Random-Amplified Polymorphic DNAs) markers are unreliable with poor replication success among laboratories (Penner et al., 1993, Hallde et al., 1996). SCAR (Sequence Characterized Amplified Regions) markers are more reliable, but are developed from RAPD markers which limit their utility (Paran and Michelmore, 1993). The dominant nature of AFLP (Amplified Fragment Length Polymorphisms), RAPD and STS (Sequence-Tagged Sites) markers limits to identify heterozygous individuals from segregating populations. SSR (Simple Sequence Repeats) markers, on the other hand, combine reliability and genomic abundance with high levels of polymorphism and allow detection of heterozygotes (Mohan et al., 1997). They do not require sophisticated DNA extraction methods and are ideally suited for high throughput automated scoring and multiplexing (Tang et al., 2002). Genotypic screening of a single gene trait in maize using SSR markers was economically profitable compared to conventional phenotypic screening methods (Dreher et al., 2003, Yu et al., 2000). To ensure optimal cost-effectiveness, molecular markers used for marker assisted selection (MAS) should both permit efficient screening of large populations and show a high degree of reproducibility across laboratories (Mohan et al., 1997). The main drawback of SSRs is the initial identification of primer sites to amplify SSR loci, a procedure which is time and resource demanding. Despite this, SSR markers had pervaded the molecular genetics and plant breeding studies until recently whose hegemony was eventually broken by SNP (Single nucleotide polymorphism) markers. SNPs were proved to be universal and most abundant forms of genetic variation among individuals of the same variation providing a dense coverage of the genome for high-resolution mapping of disease resistance (Sun et al., 2015b, Jha et al., 2016, Sun et al., 2019). Development of Next Generation Sequencing (NGS) technologies over the last decade allows rapid inexpensive SNP discovery within genes and avoids highly repetitive regions of a genome (Morozova and Marra, 2008). Two main types of SNP-based markers are CAPS (Cleaved Amplified Polymorphic Sequences) and dCAPS (derived Cleaved Amplified Polymorphic Sequences) (Parsons et al., 1997, Neff et al., 1998). CAPS are PCR-RFLP markers combined with specific primers, after the digestion of restriction enzymes to detect polymorphism that cannot be directly detected by PCR amplification (Konieczny and Ausubel, 1993, An et al., 2021). There are several widely used SNP genotyping platforms among which Kompetitive Allele Specific PCR (KASP) is one that has evolved to be a global benchmark technology (Semagn et al., 2014). Genotyping-by sequencing (GBS) has also been widely used for genotyping mapping population for GWAS and QTL analysis (Elshire et al., 2011). Other types of markers based on next generation sequencing and array hybridization such as DArT (Diversity Arrays Technology) combined with NGS (DArTseq^™) were also used for high throughput genotyping that can generate a greater number of markers at relatively low cost (Kilian et al., 2012).
        
MAS is viewed as a promising approach to resistance breeding ever since the advent of the first DNA markers. Today, the most successful applications of MAS in plant breeding have been for major disease resistant genes. Application of MAS requires selection of markers in the vicinity of the resistance genes, investigation of markers in breeding programs and their linkage with resistance genes. Another important factor in MAS program is the cost associated in screening. To ensure optimal cost-effectiveness, molecular markers used for MAS should both permit efficient screening of large populations and show a high degree of reproducibility across laboratories (Mohan et al., 1997). Using the marker maps, putative genes affecting traits of interest have been detected by testing for statistical associations between marker variants and traits (Paterson et al., 1991). Following their identification, useful genes or QTLs can be introgressed into desirable genetic backgrounds using markers which are physically located close to or even within genes of interest. The application of MAS for introgression of genes from one donor to another recurrent genotype through a backcross breeding illustrates the great advantages of the use of molecular markers for indirect selection of traits which would otherwise be difficult to select by standard procedures.
 
Molecular markers for powdery mildew in pea
 
The DNA markers linked to resistance genes provide an alternative to disease screening of powdery mildew resistance genes. DNA markers can be used to confirm the presence of multiple resistance genes in the backcross since they are not affected by epistatic interactions. As the pathogen, Erysiphi pisi is an obligate parasite, its culture and maintenance are difficult. Molecular marker technology has reduced our dependence on conventional breeding (Tanksley, 1983). Markers tightly-linked to disease resistance genes have been developed for many crops and used successfully in breeding programmes. Some examples of molecular markers linked to powdery mildew resistance gene in various crops other than pea are listed in Table 1.
 

        
In pea, RFLP (Dirlewanger et al., 1994), RAPD and SCAR (Timmerman et al., 1994, Tiwari et al., 1998, Fondevilla et al., 2008, Pereira et al., 2010, Srivastava et al., 2012) as well as SSR (Loridon et al., 2005, Katoch et al., 2010, Reddy et al., 2015, Cobos et al., 2018) markers have been linked to powdery mildew resistance and reported for the er1, er2 and Er3 genes. Functional markers corresponding to the er1 alleles were developed to aid marker assisted selection (Pavan et al., 2011, Pavan et al., 2013, Sun et al., 2019). This provide a powerful tool for breeders, overcoming limitations of previously reported er1 linked markers due to occurrence of the recombination with resistance locus and/or the lack of polymorphism between parental genotypes. Genetic markers showing linkage to er1, er2 and Er3 genes as well as functional markers of er1 alleles in Pisum sativum is presented in Table 2.
 

        
Sarala (1993) and Timmerman et al., (1994) located the powdery mildew resistance gene er1 to pea linkage group VI using morphological and molecular markers, respectively. Dirlewanger et al., (1994) located the er1 gene at 9.8 cM distance from p236, a RFLP marker. Timmerman et al., (1994) reported a RAPD marker, OPD10₆₅₀, at 2.1 cM distance from the er1 gene, which is a more closely linked marker. The RAPD marker was converted to a SCAR marker which Janila and Sharma (2004) mapped at a distance of 3.4 cM from the er1 gene. Tiwari et al., (1998) identified a RAPD/SCAR marker, Sc-OPO-18₁₂₀₀, which showed complete linkage to er1 gene (i.e. distance of the marker from er1 gene was 0.0 cM). Ek et al., (2005) developed five SSR markers linked to er1 gene covering the regions extended over 49.9 cM. The distance between the er1 gene and the most closely linked marker (PSMPSAD60) was 10.4 cM. A linkage map of the er gene region is found to be located on chromosome VI. Other closely linked SSR markers were AD60, AA374 and A5 at 10.4, 11.6 and 14.9 cM distance, respectively. However, these distances are most likely too large to use in MAS since differentiation of pea germplasm with respect to powdery mildew response would require very large screening populations (Ribaut et al., 2002). When single markers are too distant to the gene of interest, two flanking markers can be used in combination (Werner et al., 2000). er1 gene was flanked by the markers, AD60 and A5 and use of both markers for selection of resistant plants is estimated to result in 1.6% error in selection (Ek et al., 2005). Tonguc and Weeden (2010) reported the er1 locus in a position between two markers, BC210 and BA9, at a distance of 8.2 cM from the marker BC210.
        
Five markers generated the linkage map of er2 region that covered 51.9 cM and the gene was assigned to LG III, a position different from that reported for er1 (Katoch et al., 2010). The gene er2 was bracketed between the RAPD markers OPX17_1400 and OPY17_550 located at a distance of 2.6 and 21.3 cM, respectively while SSR markers AD141, AA278 and AC30 were reported to be located at a distance of 9.3, 17.9 and 30.6 cM, respectively. Molecular markers linked to Er3 gene were located in pea LG IV. SCAR marker SCW4₆₃₇ co-segregate with the resistant gene allowing detection of all the resistant individuals and SCAB1₈₇₄, in repulsion phase with Er3 was located at 2.8 cM from the gene. The combined use of SCW4₆₃₇ and SCAB1₈₇₄ allowed the accurate identification of 100% of the resistant plants and was highly efficient in discriminating homozygous and heterozygous resistant plants (Fondevilla et al., 2008). SSR markers AA349 and AD61 were linked to Er3Er3 gene and the gene was located in pea LG IV at 0.39 cM downstream of marker AD61 (Cobos et al., 2018). The gene Er3 is needed to characterize for utilization to develop pea cultivars resistant to powdery mildew to broaden the genetic horizon of pea as the gene has been reported to be successfully introduced into cultivated pea (Fondevilla et al., 2011, Fondevilla et al., 2007, Bobkov and Selikhova, 2021). Moreover, er1 might not be effective against Erysiphe baeumleri and Erysiphe trifolii (Fondevilla and Rubiales, 2011).
        
Functional markers developed for selection of er1 alleles (er1-1 to er1-11) includes sequence-tagged site (STS), high- resolution melting (HRM), CAPS and dCAPS. Pavan et al., (2011) developed CAPS marker GIM-300/SmlI on the mutation site for er1-5 which is associated with a loss-of-function G-A substitution in the PsMLO1 coding sequence. Further, Pavan et al., 2013 developed CAPS marker er1-1/ AsuHPI-B for er1-1 allele, STS marker er1-2/MGB for er1-2, derived CAPS (dCAPS) marker er1-3/XbaI for er1-3 and high resolution melting (HRM) marker er1-5/HRM54 for er1-5 for using in developing resistant cultivar. HRM is a technique developed for scanning mutations, SNP detection and genotyping to determine the dissociation behaviour of PCR amplicons (Erali et al., 2008).
        
SNP marker SNP1121 was developed using High Resolution Melting (HRM) technique whose forward and reverse primers were located at 11^th exon and 11^th intron of the PsMLO1 gene, respectively. SNP1121 successfully distinguished resistant pea landraces carrying the er1-6 allele from other er1 alleles as well as susceptible genotypes (Sun et al., 2015a). Sun et al., 2016 obtained another functional marker, InDel111-120 located in exon 1 and intron 1 of PsMLO1, specific for er1-7 allele which has a 10-bp deletion in position 111-120.
        
KASPar (Kompetitive allele-specific PCR) assay which is a SNP genotyping system based on fluorescence was deployed by Ma et al., 2017 and proved as an efficient and robust tool for pea breeding. He developed eight KASPar markers viz., KASPar-er1-1, KASPar-er1-3, KASPar-er1-4, KASPar-er1-5, KASPar-er1-6, KASPar-er1-7, KASPar-er1-10 and KASPar-er1-11 for er1 alleles namely, er1-1, er1-3, er1-4, er1-5, er1-6, er1-7, er1-10 and er1-11 and validated as markers which are breeder-friendly one. The co-dominant functional markers specific to er1-8 (InDel-er1-8 and KASPar-er1-8) and er1-9 (KASPar-er1-9) were developed by Sun et al., 2019. All these functional markers allows rapid identification and characterization of Erysiphi pisi resistant alleles at the er1 locus in pea germplasm. Therefore, such markers aid in marker assisted selection in pea breeding for developing powdery mildew resistance cultivars. Some important sources for each gene are listed in Table 3.
 

The gene er1 has been widely used in breeding programs but harboring the same resistance source could likely enhance the occurrence of new races of the same pathogen that ultimately will lead to a breakdown of the resistance. A durable resistance can be obtained by combining several major genes from different sources into a variety. If er1 gene fails to limit the colony establishment, the hypersensitive response of er2 or Er3 gene causes death of the established colonies providing a double barrier to disease development. This strategy would provide a complete resistance of pea to powdery mildew disease. This breeding strategy could be aided by the use of the available and more precise molecular markers linked to the powdery mildew resistant genes. Screening for resistance using marker assisted selection needs to be coupled with artificial disease inoculation techniques such as leaf disk assay, detached leaf assay to save time and resources for cultivar development. Moreover, advancement in the use of high throughput next generation sequencing (NGS) and genotyping technologies, the cost of development of molecular markers has been reduced considerably leading to Marker Assisted Breeding more broad, useful, efficient as well as cost-effective in future.