Eight of the selected primers showed polymorphism except primer SH20. However, these seven primers detected five different loci in our accessions. Number of amplified primers and the number of resistant loci were different because three primers SAS13, SH18 and SBB14 code for the same gene Co-42 (Young et al. 1998; Melotto and Kelly 2001) and SAZ20 and SZ04 code for gene Co-6 (Queiroz et al. 2004; Kelly et al. 2003). Locus Co-1 and Co-5 were not detected in the accessions collected from Garhwal hills of Uttarakhand Himalaya. Co-1 locus has been found in Andean gene pool of French bean while other loci (Co-4, Co-5, Co-7 and Co-9) were found in gene pool of Mexican origin (Kelly and Vellejo 2004). Our study thus gives preliminary indication towards the origin of Garhwal Himalayan French bean as Mesoamerican. Since no work has been done so far to investigate the origin of the French bean germplasm in this region, more research efforts are needed to describe the origin of French bean germplasm available in Garhwal region.
In the present study, SCAR markers were successfully used to assess the genetic diversity of anthracnose resistant loci among 100 common bean genotypes collected from Garhwal hills. These SCAR primers were specifically selected to cover all the linkage groups. Our results were close to some earlier studies carried out on common beans by other researchers. Mean PIC, GD and frequency of allele were 0.452, 0.457 and 0.55 respectively (Table 2). The findings suggest that the bean loci identified by SCAR markers have moderate level of genetic diversity. The moderate level of heterozygosity observed in these accessions might be due to predominantly autogamous habit of common bean. There are several hypotheses which explain such intra-accession variation, such as, the mixed-mating reproductive system of bean, with up to 10% outcrossing ((Ibarra-Perez et al. 1997), the small sample set tested and the self-pollinating system (Angioi et al. 2010) Perseguini et al. (2011) evaluated carioca bean cultivars by SSR markers and identified an average PIC of 0.47. Moreover, Delfini et al. (2017) analyzed a set of 39 Brazilian cultivars by using 17 SSR primers and reported a mean PIC of 0.33 with a mean number of alleles 3.4 per locus. The study therefore reflects that these markers are effective in accessing diversity for anthracnose resistant loci in French bean germplasm.
The results were in accordance with Darben et al. (2017), who found moderate level of diversity for anthracnose resistant loci for two anthracnose strains among Brazilian French bean accessions. Almeida et al. (2020) also found moderate diversity in common bean cultivars for resistance to Pseudocercospora griseola. The genetic distance and STRUCTURE analysis confirmed the moderate degree of diversity of the evaluated group, with the formation of 4 groups. Findings of the present study are in accordance with Perseguini et al. (2015) who analyzed the genetic diversity of 180 common bean accessions collected from Carioca. Resistant accessions were grouped in one group.
Co-10 gene is explained in literature as the most potential gene for marker assisted breeding programme for Brazilian French bean germplasm (Alzate marin et al. 2003) but in our collection (Garhwal Himalayan germplasm) this gene alone was not found very effective in restoring resistance against anthracnose. For example, the accessions (GFB-2, GFB-7, GFB-15, GFB-38, GFB-56, GFB-88) possess Co-10 gene but were found susceptible for anthracnose. However, Co-10 gene in the presence of Co-6, Co-3/Co-9 gene was able to induce moderate resistance in the accessions GFB-21, GFB-22, GFB-40, GFB-41, GFB-59, GFB-65, GFB-66 and GFB-67.
Single Co-42 genes was found in one accession (GFB-90) which was found susceptible to anthracnose under field trials. Various level of resistance was observed in different accessions for Co-42 allele with other genes. Co-42 allele with Co-4,Co-6, Co-3/Co-9 gene provided tolerance (moderate resistance) to anthracnose as accessions GFB-28, GFB-46 and GFB-95 were moderately resistant for anthracnose. Though, Co-42 allele is also considered as one of the best resistance source by breeders (Miklas and Kelly 2002) but in our accessions this gene alone was not found very effective against anthracnose disease. Co-3/Co-9, Co-4 and Co-6 genes were not having enough potential in restoring resistance in Garhwal Himalayan French bean accessions as most of the accessions were found susceptible having these three genes singly or in combination. In Brazil and Maxico Co-3/Co-9 gene was of little value because of frequent failure against anthracnose pathogen (Alzate-Marin et al. 2003). The original Co-4 gene was found very weak as resistance of this gene was overcome by most of the anthracnose pathogens in Maxico (Balardin et al. 1997; Kelly 2000). Co-6 gene was found effective in restoring resistance in Andean gene pool (Kelly et al. 2003) but was much less effective against Mesoamerican races (Falconi et al. 2003).
However, it was observed that the accessions having Co-10 and Co-42 gene together were found resistant for anthracnose. All the 13 accessions those were found resistant under field and polyhouse conditions possess these two genes consistently with some other genes. However, GFB-4 was found as an exception to this as it contains both the genes eventhough it is moderately resistant for anthracnose (Supplemental Table 1, 2). While accessions GFB-75 and GFB-76 were having only these two genes and were found resistant for anthracnose (Supplemental Table 1). It clearly suggests that both the genes together confer resistance for anthracnose in Garhwal Himalayan region. Banoo et al. (2020) collected 188 common bean landraces from North-West region in India and screened against five important anthracnose races. They found that the presence of Co-4 and Co-2 genes in common bean landraces was encouraging for breeding durable anthracnose resistant cultivars for the region. French beans from Harshil and Chakarata are preferred by the consumers for their taste, cooking quality and digestibility. However, the accessions from Harshil were found tolerant while from Chakrata beans were susceptible to anthracnose disease (Supplemental Table 1). It is therefore necessary to identify or develop anthracnose resistant accessions for these regions so that the economic benefit of the farmers could increase.
Correlation coefficient is important in plant breeding because it measures the degree of association between two or more factors (biotic and abiotic) (Dewey and Lu 1959). It is clearly visible in Fig. 4 where trend line showing more resistant accessions when screened under field condition. Results of controlled condition inoculation and natural filed infection were not highly correlated in this study (Table 4). Some accessions which were resistant under field conditions sowed susceptibility for anthracnose under polyhouse trial (Supplemental Table 2). This greater experimental accuracy is mainly due to sufficient amount of pathogen inoculums, controlled temperature and humidity in polyhouse, providing favorable environment for progress of disease. For disease screening of germplasm it is necessary to screen germplasm under in-vitro conditions. Our findings get support from Leite et al. (2016), who reported resistance in common bean progenies for white mold (Sclerotinia sclerotiorum Lib.) in field and greenhouse experiments. To determine the spectrum of resistance in the 14 bean genotypes, seedlings were inoculated with anthracnose inoculums under controlled condition (Gonc¸alves-Vidigal et al. 2020). The advantage of in-vitro screening methods is that the most favourable conditions for disease progression can be provided. So, if the cultivar is identified as resistant to disease, the cultivar can further be considered for breeding programmes. In addition, these methods reduce the risk of the pathogen spreading to the surrounding environments.
A large number of Co-genes have been substantiated in the anthracnose differential cultivars and accessions (Melotto et al. 2000). These genes are presented as part of a multi-allelic series or in group with other Co genes. They are most likely subsisting resistance gene clusters and have been established at the molecular level for the B4 R-gene cluster (Ferrier-Cana et al. 2013). Multiallelic series and gene cluster bound the breeders’ to choose the useful gene in breeding for anthracnose resistance. This is well known that the resistance provided by a single gene gets easily broken down. It is therefore needed to pyramid the genes for anthracnose resistance in case of French bean too. This is also suggested that while selecting for anthracnose resistance in a particular region, bean breeders should carefully choose a gene pair that, if deployed singly, would confer resistance to all known races in that region. New resistance source for anthracnose needs to be identified to expand the resistance spectrum of future bean cultivars.
Genes Co-10 and Co-42 together were found able to provide maximum resistance against anthracnose disease in French bean accessions collected from Garhwal Himalayan regions. Information generated during this study may be helpful in getting information about the resistance level of accessions from different regions. The identification of agronomically superior and anthracnose resistant accessions will be useful in minimizing the linkage drag usually breeders come across while transferring disease resistance in already available high yielding but susceptible varieties. Further, subsequent analysis of diversity using more specific molecular markers is required to explicate more information on the overall genetic diversity, origin and particular gene(s) responsible for resistance in the Himalayan gene pool.