Development of SNP based functional marker for anthracnose resistant Co-2 gene in common bean (Phaseolus vulgaris L.)

doi:10.21203/rs.3.rs-2110360/v1

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Research Article

Development of SNP based functional marker for anthracnose resistant Co-2 gene in common bean (Phaseolus vulgaris L.)

https://doi.org/10.21203/rs.3.rs-2110360/v1

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Background Anthracnose caused by the fungus Colletotrichum lindemuthianum is one of the most devastating diseases of common beans resulting in catastrophic yield loss. Among the various disease control management techniques, genetic resistance in the host is the most efficient and sustainable strategy for its control. In common bean, the single dominant Co-2 gene confers broad-spectrum resistance against a large number of Colletotrichum isolates. Functional markers (FMs) derived from polymorphic regions in gene sequences influencing phenotypic variation are very effective in marker assisted selection (MAS) of target gene for host plant resistance.

Methods and results The present study was undertaken to develop a FM from the promoter region of the Co-2 gene that could enhance its incorporation in breeding programmes. The diverse common bean genotypes were evaluated for anthracnose resistance under controlled conditions. The identified resistant and moderately resistant genotypes were further screened for the presence of the Co-2 gene, a broad-spectrum disease resistance gene using the SCAreolimarker. The promoter regions of the genes of anthracnose resistant and susceptible genotypes were amplified, cloned and sequenced. The SNPs within the regulatory motifs of the promoter region were identified and 14 out of 23 SNPs were found to be strongly associated with disease resistance using genotypic and phenotypic data. The allele-specific CAPS marker was developed and further validated in 43 common bean genotypes with varying anthracnose resistance. The genotype of the CAPS marker and the observed phenotype were perfectly correlated, thereby can be utilized in breeding projects in poorer nations where anthracnose is a common problem.

Conclusions The identified allelic marker can be used for transferring anthracnose resistance from highly resistant genotypes into susceptible cultivated varieties of common beans using MAS.

Anthracnose

Common bean

Co-2

Functional marker

SNPs

CAPS

Common bean (Phaseolus vulgaris L.) is a major source of protein for human consumption and is grown extensively around the world. Anthracnose, caused by the hemibiotrophic fungus Colletotrichum lindemuthianum (Sacc. and Magnus) Briosi and Cavara, is one of the most prevalent and recurring diseases, threatening their production worldwide [1, 2]. It is a seed-borne disease, which makes it difficult for small-scale farmers in many developing nations to conserve their seeds from year to year [3]. The pathogen uses biotrophic and necrotrophic processes to infect and colonize plant hosts [4, 5]. It attacks aerial parts of the plant and the most prominent symptoms appear on pods as a circular rust-colored sunken lesion with a black border carrying flesh-colored spores [3, 6]. A humid environment with moderate temperature stimulates disease progression, which eventually leads to the death of the plant [7]. The disease is transmitted through seed, and planting an infected seed results in a yield loss of 80 to 100 percent [6, 8].

Genetic resistance in a host is the most effective and eco-friendly strategy to control anthracnose [9, 10]. The pathogen is highly variable and 298 races have been reported across 29 countries [11]. The co-evolution of the fungus and common bean-resistant plants has resulted in the emergence of novel pathogenic races, which makes resistance implementation difficult as varieties resistant to one race might be susceptible to the other, necessitating the identification of durable anthracnose resistance varieties [12, 13, 14, 15]. Thus, it is necessary to broaden the genetic base of the common bean by uncovering new sources of resistance and incorporating resistant genes to overcome the vast pathogenic variability of C. lindemuthianum [16, 17, 18]. To date, more than 25 anthracnose resistance genes have been mapped onto seven of the 11 linkage groups (LG) and designated as Co-1-Co-17, Co-x, Co-y, Co-w, Co-Pa, Co-z, Co1^HY [19]. The Co-genes, alone or in combination, confer long-term resistance [10, 20]. The Are gene conferring anthracnose resistance was discovered in the black common bean variety 'Cornell 49–242' [21]. Later, this gene was designated as Co-2 [22]. It codes for proteins that have leucine-rich repeat (LRR) domains at their C-termini and nucleotide binding sites (NBS) [23]. The single dominant Co-2 gene showed a broad-spectrum resistance against a large number of Colletotrichum isolates and is located on chromosome 11 [24]. In Europe and North America, it has been widely employed in common bean breeding and has also proven to be beneficial in other non-tropical countries with moderate temperatures [25, 26, 27].

Advances in molecular biology, including genomics, high-throughput sequencing, and genome editing, enable increasingly faster and more precise cultivar development [28]. For crop improvement programmes, it is essential to identify the genes and molecular markers related to trait variation. Molecular markers like SSR, ISSR, AFLP and RAPD have been used to assess the genetic diversity in common beans as well as to establish genetic linkage with a phenotype [29, 30, 31, 32]. However, even if the markers are tightly linked, occasional uncoupling of the marker from the trait across numerous cycles of meiosis in a breeding programme significantly reduces the efficacy of MAS. FMs, on the contrary, are developed from polymorphic sites within genes that causally affect the phenotypic traits of interest [28, 33]. These are associated with the gene-controlling phenotypic trait and do not get lost by recombination. This enables accurate characterization of germplasm for allelic diversity [28]. The development of FM, based on single-nucleotide polymorphism (SNP) from allele sequences of functionally characterized genes allows for the identification of polymorphic functional motifs impacting plant phenotype. It also plays a massive role in marker-assisted breeding [34, 35, 36]. Cleaved amplified polymorphic sequences (CAPS) are co-dominant genetic markers that identify SNPs in a region of interest using PCR amplification followed by digestion with restriction endonuclease [37]. The CAPS marker turns into a FM when an SNP is discovered that differentiates the alleles controlling a desired trait, and it is 100% indicative of the occurrence or absence of the allele and its associated phenotype [31]. CAPS marker associated with desired traits has been routinely used in breeding programmes to identify genotypes with desirable alleles [33, 38]. Several FMs for disease resistance have been developed within species by analyzing SNPs on allele sequences in crops like wheat [39], rice [40], pea [41] etc. The RAPD (OQ04) and SCAR (SCAreoli) markers linked with the Co-2 gene have been reported in the common bean [42, 43, 44] but FM associated with Co-2 gene has yet to be developed.

In Asian countries, the Co-2 gene has not been widely utilized in the development of anthracnose-resistant varieties due to the limited availability of resistance sources. Keeping in view the importance of this disease, the present investigation was carried out to screen the released/improved genotypes, as well as indigenous and exotic collections, for anthracnose resistance. Also, the FM from the promoter region of the Co-2 gene was developed, which can be used for the improvement of varieties of the common bean by transferring anthracnose- resistant genes into susceptible varieties through MAS.

Plant material

The present study comprised of different genotypes of common bean such as local landraces, wild and improved genotypes collected from different areas of Jammu & Kashmir (Table 1). Released or improved genotypes have also been collected from the Central Pulse Research Institute, ICAR, Kanpur; National Bureau of Plant Genetic Resources (NBPGR), Shimla and Vivekananda Parvatiya Krishi Anusandhan Sansthan (VPKAS-ICAR), Almora.

Table 1

Germplasm collection from different locations
S.No.	Genotype	Location	Latitude	Longitude
1	BR 1	Bhadarwah, Doda, J&K	32°59'02" N	75°42'36" E
2	BR 2	Bhadarwah, Doda, J&K	32°59'02" N	75°42'36" E
3	BR 3	Bhadarwah, Doda, J&K	32°59'02" N	75°42'36" E
4	BR 4	Bhadarwah, Doda, J&K	32°59'02" N	75°42'36" E
5	BR 5	Bhadarwah, Doda, J&K	32°59'02" N	75°42'36" E
6	BR 6	Bhadarwah, Doda, J&K	32°59'02" N	75°42'36" E
7	BR 7	Bhadarwah, Doda, J&K	32°59'02" N	75°42'36" E
8	BR 8	Bhadarwah, Doda, J&K	32°59'02" N	75°42'36" E
9	BR 9	Bhadarwah, Doda, J&K	32°59'02" N	75°42'36" E
10	BR 10	Bhadarwah, Doda, J&K	32°59'02" N	75°42'36" E
11	BR 15	Bhadarwah, Doda, J&K	32°59'02" N	75°42'36" E
12	BR 16	Bhadarwah, Doda, J&K	32°59'02" N	75°42'36" E
13	BR 18	Bhadarwah, Doda, J&K	32°59'02" N	75°42'36" E
14	BR 22	Bhadarwah, Doda, J&K	32°59'02" N	75°42'36" E
15	BR 30	Bhadarwah, Doda, J&K	32°59'02" N	75°42'36" E
16	BR 31	Bhadarwah, Doda, J&K	32°59'02" N	75°42'36" E
17	BR 36	Bhadarwah, Doda, J&K	32°59'02" N	75°42'36" E
18	BR 39	Bhadarwah, Doda, J&K	32°59'02" N	75°42'36" E
19	PL 1	Pulwama, Kashmir, J&K	33°52'20" N	74°53'40" E
20	S 1	Shopian, Kashmir, J&K	33°42'59" N	74°50'05" E
21	S 2	Shopian, Kashmir, J&K	33°42'59" N	74°50'05" E
22	S 3	Shopian, Kashmir, J&K	33°42'59" N	74°50'05" E
23	S 4	Shopian, Kashmir, J&K	33°42'59" N	74°50'05" E
24	S 5	Shopian, Kashmir, J&K	33°42'59" N	74°50'05" E
25	S 6	Shopian, Kashmir, J&K	33°42'59" N	74°50'05" E
26	P 1	Poonch, Jammu, J&K	33°45'57" N	74°05'36" E
27	P 2	Poonch, Jammu, J&K	33°45'57" N	74°05'36" E
28	P 3	Poonch, Jammu, J&K	33°45'57" N	74°05'36" E
29	P 4	Poonch, Jammu, J&K	33°45'57" N	74°05'36" E
30	P 5	Poonch, Jammu, J&K	33°45'57" N	74°05'36" E
31	P 6	Poonch, Jammu, J&K	33°45'57" N	74°05'36" E
32	P 13	Poonch, Jammu, J&K	33°45'57" N	74°05'36" E
33	P 14	Poonch, Jammu, J&K	33°45'57" N	74°05'36" E
34	P 15	Poonch, Jammu, J&K	33°45'57" N	74°05'36" E
35	P 17	Poonch, Jammu, J&K	33°45'57" N	74°05'36" E
36	P 22	Poonch, Jammu, J&K	33°45'57" N	74°05'36" E
37	P 28	Poonch, Jammu, J&K	33°45'57" N	74°05'36" E
38	P 29	Poonch, Jammu, J&K	33°45'57" N	74°05'36" E
39	P 32	Poonch, Jammu, J&K	33°45'57" N	74°05'36" E
40	P 33	Poonch, Jammu, J&K	33°45'57" N	74°05'36" E
41	B 1	Baramulla, Kashmir, J&K	34°10'54" N	74°21'42" E
42	B 4	Baramulla, Kashmir, J&K	34°10'54" N	74°21'42" E
43	B 5	Baramulla, Kashmir, J&K	34°10'54" N	74°21'42" E
44	B 6	Baramulla, Kashmir, J&K	34°10'54" N	74°21'42" E
45	B 7	Baramulla, Kashmir, J&K	34°10'54" N	74°21'42" E
46	B 9	Baramulla, Kashmir, J&K	34°10'54" N	74°21'42" E
47	B 12	Baramulla, Kashmir, J&K	34°10'54" N	74°21'42" E
48	EC-13097	Exotic collection, NBPGR, Shimla
49	EC-121013	Exotic collection, NBPGR, Shimla
50	EC-398565	Exotic collection, NBPGR, Shimla
51	EC-398586	Exotic collection, NBPGR, Shimla
52	EC-398591	Exotic collection, NBPGR, Shimla
53	EC-398527	Exotic collection, NBPGR, Shimla
54	EC-400397	Exotic collection, NBPGR, Shimla
55	EC-400433	Exotic collection, NBPGR, Shimla
56	EC-400442	Exotic collection, NBPGR, Shimla
57	EC-405220	Exotic collection, NBPGR, Shimla
58	EC-500250	Exotic collection, NBPGR, Shimla
59	EC-500305	Exotic collection, NBPGR, Shimla
60	EC-500308	Exotic collection, NBPGR, Shimla
61	EC-500374	Exotic collection, NBPGR, Shimla
62	EC-500507	Exotic collection, NBPGR, Shimla
63	EC-530898	Exotic collection, NBPGR, Shimla
64	EC-531076	Exotic collection, NBPGR, Shimla
65	EC-755305	Exotic collection, NBPGR, Shimla
66	EC-894826	Exotic collection, NBPGR, Shimla
67	IC-043562	Indigenous collection, NBPGR, Shimla
68	IC-199277	Indigenous collection, NBPGR, Shimla
69	IC-202274	Indigenous collection, NBPGR, Shimla
70	IC-243198	Indigenous collection, NBPGR, Shimla
71	IC-258273	Indigenous collection, NBPGR, Shimla
72	IC-258276	Indigenous collection, NBPGR, Shimla
73	IC-260299	Indigenous collection, NBPGR, Shimla
74	IC-260312	Indigenous collection, NBPGR, Shimla
75	IC-260336	Indigenous collection, NBPGR, Shimla
76	IC-262837	Indigenous collection, NBPGR, Shimla
77	IC-265932	Indigenous collection, NBPGR, Shimla
78	IC-274530	Indigenous collection, NBPGR, Shimla
79	IC-328372	Indigenous collection, NBPGR, Shimla
80	IC-361884	Indigenous collection, NBPGR, Shimla
81	Amber	ICAR, Kanpur
82	PDR 14	ICAR, Kanpur
83	Hur 137	ICAR, Kanpur
84	Hur 15	ICAR, Kanpur
85	Arun	ICAR, Kanpur
86	Utkarsh	ICAR, Kanpur
87	Jawala	SKUAST Kashmir
88	VL 63	VPKAS, Almora
89	VL 125	VPKAS, Almora

Isolation And Identification Of The Pathogen

Conidia were directly isolated from infected pods of common bean from the northern Himalayas, India. Single conidial cultures were established on 2% potato dextrose agar (PDA) and 2% malt extract agar (MEA). The isolates were identified based on morphology and DNA sequence data of six loci, namely ITS, gapdh, chs-1, his3, act, and tub2. Also, pathogenicity testing was performed on Phaseolus vulgaris cv. Bhaderwah- Rajmash to identify the most aggressive strain [45].

Phenotypic Evaluation For Anthracnose Resistance

The most aggressive strain identified in our previous study was used for phenotypic evaluation of the common bean germplasm for anthracnose resistance. To enhance sporulation, a single conidial culture of C. lindemuthianum was inoculated on sterile pods partially immersed in the water agar medium plates, followed by incubation at 25°C for 14 days. The spore suspension was prepared by flooding the plates with 10 mL of 0.01% Tween 20 in distilled water and the extract was filtered. The concentration of spores was adjusted to 1.2×10⁶ spores/ mL using a haemocytometer [46]. Eighty-nine common bean genotypes were evaluated for resistance to C. lindemuthianum under green-house conditions. The inoculum was sprayed on leaves of 15–20-day old plants until runoff and inoculated plants were maintained at 22–25˚C, 95–100% humidity, and a 12-h photoperiod. The isolate was inoculated on three pots of each genotype, and each pot contained three plants in two replications. IC-328372 (anthracnose resistant) and Jawala (anthracnose susceptible) were used as controls. The disease reaction was scored on a 1–9 scale [47] after 17 days (Table 2), and the percent disease index (PDI) was determined. Based on PDI, the genotypes were categorized as resistant, moderately resistant, susceptible, or moderately susceptible [48] (Table 3).

Table 2

Disease reaction score with respective symptoms
Score	Symptoms
1	Absence of symptoms
2	Up to 1% of the leaf veins affected, visible only on the lower leaf surface
3	Up to 3% of the leaf veins affected, visible only on the lower leaf surface
4	Up to 1% of the leaf veins affected, visible on both surfaces of the leaves
5	Up to 3% of the leaf veins affected, visible on both surfaces of the leaves
6	Leaf veins affected, visible on both leaf surfaces, and the presence of some lesions on stems, branches and petioles
7	Necrotic spots on most of the leaf veins and in a large part of the adjacent mesophyll tissue, which ruptures, as well as the presence of abundant lesions on the stem, branches and petioles
8	Necrotic spots on almost all the leaf veins and very abundant on stem, branches, and petioles, leading to ruptures, leaf shedding and reduction of plant growth
9	Most of the plants are dead

Table 3

Categorization of genotypes based on percent disease index (PDI)
PDI (%)	Categories
0	Absolutely resistant (AR)
0.01–12.22	Highly resistant (HR)
12.22–33.33	Moderately resistant (MR)
34.44–55.55	Moderately susceptible (MS)
56.66–77.77	Susceptible (S)
78.88–100	Highly susceptible (HS)

PDI (%)\(=\frac{\text{S}\text{u}\text{m} \text{o}\text{f} \text{n}\text{u}\text{m}\text{e}\text{r}\text{i}\text{c}\text{a}\text{l} \text{r}\text{a}\text{t}\text{i}\text{n}\text{g} \times 100}{\text{N}\text{u}\text{m}\text{b}\text{e}\text{r} \text{o}\text{f} \text{p}\text{l}\text{a}\text{n}\text{t}\text{s} \text{s}\text{c}\text{o}\text{r}\text{e}\text{d} \times \text{M}\text{a}\text{x}\text{i}\text{m}\text{u}\text{m} \text{s}\text{c}\text{o}\text{r}\text{e} \text{o}\text{n} \text{s}\text{c}\text{a}\text{l}\text{e}}\)

Dna Isolation And Molecular Marker Analysis

The genomic DNA was isolated from the young leaves of common bean genotypes using the modified CTAB method [49]. The quality of DNA was checked on a 0.8% agarose gel and visualized under ultraviolet light. The concentration was determined on a spectrophotometer and DNA was diluted to a concentration of 50 ng µL^− 1. The resistant and moderately resistant common bean genotypes were screened with the SCAreoli marker: 5’-GGGAGACATCCATCAGACAACTCC-3’ (forward) and 5’-GTATCCATTTGAAGGAGCT-3’ (reverse) [43] for the confirmation of Co-2 gene. The PCR reaction was performed using a Thermal Cycler (Applied Biosystems, Foster City, California) in a final volume of 25 µl, which included 1 µl genomic DNA (50 ng), 0.25 mM dNTP, 0.2 µM of each primer, 1X PCR buffer, 0.5 U Taq DNA polymerase (Promega) and ddH₂O. The thermal cyclic profile includes 35 cycles of initial denaturation at 94°C for 5 min, followed by denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 1 min, and final extension at 72°C for 7 min. The restriction enzyme digestion of specific PCR products to confirm gene Co-2 was conducted at 37°C for 1 hour using Dra I (Fermentas, USA). PCR samples and their digestion products were fractionated by electrophoresis on a 1.2 percent agarose gel using 1X TAE buffer at 100 V for 1.5 hours and photographed in a gel documentation system (DNR Bioimaging system, Israel). The l kb DNA ladder (Thermo Scientific, USA) was used as a molecular weight standard.

Cloning of promoter sequence of Co-2 and sequencing

The 1500 bp region upstream of Co-2 gene was located using the Co-2 DNA sequence from NCBI. The primers specific to the promoter region, 5’-TGATGTTGGATCTGGGTTGGCT-3’ (forward) and 5’-TCGTAAGTTCAAGGCGACGCAA-3’ (reverse), were designed using Primer 3 software (http://frodo.wi.mit.edu/cgi-bin/primer3) [50]. PCR was performed in a 2720 Thermal Cycler (Applied Biosystems, Foster City, California) in a total volume of 25 µl, which included 1 µl genomic DNA (50 ng), 0.25 mM dNTP, 0.2 µM of each primer, 1X PCR buffer, 0.5 U Taq DNA polymerase (Promega) and ddH₂O. The thermal cyclic profile includes 94°C for 3 min, followed by 35 cycles of 94°C for 30 sec, 60°C for 30 sec, 72°C for 1:30 min, and a final extension of 72°C for 7 min. An amplicon of the expected size was recovered from three anthracnose resistant (B 12, IC-274530, EC-398591) as well as susceptible genotypes (IC-199277, BR 5, P 3) using a Gene Jet gel extraction kit (Thermo Scientific, K0701) and cloned into the pJET1.2/ blunt cloning vector (Thermo Scientific, K1231). The transformation was carried out into competent cells of E. coli strain DH5α [51]. Plasmid DNA was isolated using the HiPurA® Plasmid DNA Miniprep Purification Kit (Himedia, MB508) followed by sequencing by AgriGenome Labs (Infopark Road, Kakkanad, Kerala, India). The forward and reverse sequences thus obtained were aligned using Bioedit (http://www.mbio.ncsu.edu/BioEdit/bioedit. html) [52] to get a full-length sequence. The regulatory motifs in the promoter sequence were determined using Nsite (www.softberry.com). The sequences obtained were aligned in Clustal Omega and SNPs within regulatory motifs were determined. The candidate SNPs associated with disease resistance were identified using a student’s t-test.

Development Of Allele-specific Marker

The SNPs identified were converted into CAPS marker using dCAPS Finder 2.0 software (http://helix.wustl.edu/dcaps) [53]. The anthracnose resistant genotype (IC-274530) was considered a wild parent and the susceptible genotype (BR 5) was considered a mutant parent. 25 nucleotides on each side of SNP of both the parents were entered into dCAPS Finder 2.0 software and the restriction site of the Mse I enzyme was found at position 477. The primers targeting SNP were designed in Primer 3 to amplify the bases surrounding the SNP between the resistant and susceptible alleles. The primers obtained were: 5’-TGGATGATGTTTGGAACGAA-3’ (forward) and 5’-CCTTCCCACTCTCCAACAGA-3’ (reverse) respectively. To test the application of the markers, 43 genotypes of common beans with disease reaction phenotypes were genotyped with the CAPS marker. The PCR reaction was performed in a final volume of 25 µl, which included 50 ng of genomic DNA, 0.25 mM dNTP, 0.2 µM of each primer, 1X Taq buffer, 0.5 U Taq DNA polymerase (Promega) and ddH₂O. The thermal cyclic profile includes 35 cycles of initial denaturation for 4 min at 94°C, followed by denaturation for 30 sec at 94°C, annealing for 30 sec at 54°C, extension for 30 sec at 72°C, and final extension for 8 min at 72°C. The DNA fragments were amplified and restriction digestion was performed with Mse I at 37ºC for 5 minutes. Digested products were run on 3 percent agarose gel.

Evaluation for anthracnose resistance

The common bean genotypes were assessed as disease resistant or susceptible based on the severity of the infection on a 1–9 scale after 17 days. These were categorized into four reaction groups, namely highly resistant, moderately resistant, moderately susceptible, and susceptible, based on the percent disease index. Genotypes EC-398527, EC-400397, Hur 137, EC-398565, IC-258273, S 2, B 4, Utkarsh, P 2, Hur 15, EC-530898, BR 31, IC-260299, PDR 14, Amber, S 1, Arun, VL 63, IC-328372, IC-274530, B 12, B 6, S 6, EC-894826, P 32, EC-398591 were highly resistant. Moderately resistant genotypes include IC-243198, BR 8, IC-262837, BR 2, IC-361884, EC-400433, EC-531076, BR 22, P 6, BR 9, EC-500250, IC-260312, S 3, EC-500507, B 5, P 28, BR 6, IC-043562, B 7, EC-500308, BR 10, BR 1, B 9, IC-202274. Genotypes PL 1, EC-121013, BR 4, EC-398586, P 17, B 1, EC-500374, BR 15, P 29, P 15, EC-755305, P 14, P 33, EC-405220, IC-265932, S 4, EC-500305, VL 125, BR 3, S 5, P 5, BR 16, BR 30, IC-258276, BR 39, IC-260336, P 13, EC-13097, P 4 were moderately susceptible. BR 5, IC-199277, EC-400442, BR 18, BR 36, P 22, BR 7, Jawala, P 1, P 3 were susceptible (Fig. 1) (Table 4).

Table 4

Screening of common bean genotypes for anthracnose resistance
Genotype	Percent disease index	Disease reaction
IC-243198	30.58%	MR
EC-398527	10.84%	HR
BR 8	33.33%	MR
PL 1	37.03%	MS
EC-121013	55.55%	MS
IC-262837	27.77%	MR
BR 2	33.17%	MR
IC-361884	17.45%	MR
BR 5	58.32%	S
EC-400397	9.47%	HR
Hur 137	6.17%	HR
IC-199277	66.66%	S
EC-398565	1.48%	HR
IC-258273	6.17%	HR
BR 4	41.66%	MS
EC-400433	13.88%	MR
EC-398586	37.03%	MS
S 2	9.47%	HR
EC-531076	33.33%	MR
EC-400442	66.66%	S
B 4	10.84%	HR
P 17	55.55%	MS
B 1	41.66%	MS
BR 22	21.36%	MR
EC-500374	55.55%	MS
Utkarsh	1.48%	HR
BR 15	38.79%	MS
P 6	32.40%	MR
P 29	41.66%	MS
BR 9	31.7%	MR
BR 18	66.66%	S
EC-500250	32.40%	MR
P 15	37.03%	MS
P 2	1.48%	HR
IC-260312	22.22%	MR
BR 36	66.66%	S
EC-755305	37.05%	MS
Hur 15	1.48%	HR
P 14	36.10%	MS
P 33	43.20%	MS
EC-405220	54.16%	MS
IC-265932	39.15%	MS
EC-530898	10.10%	HR
S 3	12.34%	MR
S 4	37.03%	MS
BR 31	10.84%	HR
EC-500305	55.55%	MS
IC-260299	10.10%	HR
EC-500507	30.94%	MR
PDR 14	6.17%	HR
P 22	66.66%	S
BR 7	58.32%	S
P 28	20.05%	MR
Jawala	66.66%	S
VL 125	37.03%	MS
B 5	20.83%	MR
Amber	1.48%	HR
S 1	1.48%	HR
Arun	6.17%	HR
BR 6	31.74%	MR
EC-398591	6.17%	HR
VL 63	9.47%	HR
IC-043562	24.69%	MR
B 7	17.35%	MR
EC-500308	16.20%	MR
BR 3	39.68%	MS
IC-328372	9.47%	HR
S 5	37.05%	MS
P 5	39.50%	MS
BR 10	26.61%	MR
BR 16	36.50%	MS
BR 1	25.30%	MR
IC-274530	10.7%	HR
B 9	26.84%	MR
BR 30	36.55%	MS
IC-258276	55.55%	MS
P 1	58.32%	S
BR 39	47.00%	MS
IC-260336	39.68%	MS
IC-202274	31.74%	MR
P 13	41.66%	MS
B 12	6.47%	HR
EC-13097	39.50%	MS
B 6	1.48%	HR
S 6	10.7%	HR
P 3	66.66%	S
P 4	39.68%	MS
EC-894826	1.48%	HR
P 32	1.48%	HR
Where, HR: highly resistant; MR: moderately resistant, MS: moderately susceptible, S: susceptible

Evaluation of genotypes for the Co-2 gene

The phenotypically screened resistant and moderately resistant genotypes were further evaluated for the presence of the Co-2 gene using the SCAreoli marker. PCR amplification with the SCAreoli marker gives a band size of 1.3 kb in all the genotypes. On digestion with the Dra I enzyme, a band size of 1 kb was produced in EC-500250, IC-274530, S 1, S 3, EC-398527, EC-398565, BR 8, EC-398591, and B 12 genotypes (Fig. 2).

Snp Identification

DNA sequences corresponding to the promoter region of the anthracnose resistant Co-2 gene were amplified from three resistant (B 12, IC-274530, EC-398591) and susceptible (IC-199277, BR 5, P 3) genotypes. The assembled forward and reverse sequences were used to determine regulatory motifs in the promoter database and candidate SNPs were identified. Twenty-three SNPs were obtained at positions 30, 32, 38, 39, 188, 223, 228, 326, 329, 475, 477, 481, 499, 503, 1017, 1019, 1024, 1369, 1373, 1469, 1471, 1472 and 1476. Fourteen SNPs (at positions 30, 32, 38, 39, 228, 326, 475, 477, 481, 499, 1017, 1471, 1472, 1476) were found to be strongly associated with disease resistance (Fig. 3) (Table 5).

Table 5

Identification of SNPs in the regulatory motifs
SNP	Position	Significant/ non-significant
T/C	30	Significant
G/A	32	Significant
T/C	38	Significant
A/C	39	Significant
G/A	188	Non-significant
T/C	223	Non-significant
T/C	228	Significant
C/A	326	Significant
T/A	329	Non-significant
T/G	475	Significant
C/T	477	Significant
G/A	481	Significant
C/G	499	Significant
A/G	503	Non-significant
G/A	1017	Significant
T/A	1019	Non-significant
T/G	1024	Non-significant
A/G	1369	Non-significant
C/T	1373	Non-significant
C/T	1469	Non-significant
T/G	1471	Significant
C/T	1472	Significant
C/T	1476	Significant

Development And Validation Of Allele-specific Marker

On aligning the sequences of resistant (IC-274530 (45); wild type) and susceptible (BR 5 (75); mutant) genotypes in dCAPS finder 2.0, a C/T mutation at 477 nucleotide position was observed. The SNP identified was converted into a CAPS marker by designing suitable primers and screening for corresponding restriction enzymes. The DNA fragment containing the SNP of interest was successfully amplified in IC-274530, BR 18, P 1, P 33, EC-500374, Arun, P 4, EC-398586, EC-400442, PL 1, BR 7, EC-894826, P 2, BR 36, EC-121013, VL 125, BR 39, P 29, IC-199277, S 5, P 14, P 5, B 1, BR 30, BR 5, EC-530898, IC-260336, P 22, EC-398527, EC-13097, VL 63, P 15, BR 4, BR 15, BR 16, BR 3, P 32, S 4, S 2, P 17, P 3, B 4, P 13 genotypes.

The Mse I digested PCR products from susceptible and moderately susceptible genotypes i.e. BR 18, P 1, P 33, EC-500374, P 4, EC-398586, EC-400442, PL 1, BR 7, BR 36, EC-121013, VL 125, BR 39, P 13, P 15, P 29, IC-199277, S 5, P 14, P 5, B 1, BR 30, BR 5, IC-260336, P 22, EC-13097, BR 4, BR 15, BR 3, S 4, P 17, P 3, BR 16 (200bp) but not from resistant genotypes i.e. IC-274530, Arun, EC-894826, P 2, EC-530898, EC-398527, VL 63, P 32, S 2, B 4 (290 bp) (Fig. 4).

Common beans are an important grain legume grown worldwide for direct human consumption [54]. However, the yield of common beans is severely affected by fungal pathogens. Among them, anthracnose caused by C. lindemuthianum is one of the most serious fungal infections threatening agricultural sustainability worldwide [3]. The use of anthracnose-resistant cultivars is the most successful, efficient, and safe means of managing anthracnose in common beans [55, 56, 57]. However, cultivars that are resistant to one race may be susceptible to another due to the occurrence of several pathogen races as well as diversity within a single pathogen race [58, 59, 60, 61], and no single resistance gene effective against all races has been discovered yet. The screening and identification of anthracnose-resistant common bean genotypes are critical to this effort. In the present study, 89 common bean genotypes were evaluated for anthracnose resistance under controlled conditions using an isolate of C. lindemuthianum. Out of 89 genotypes, 26 were highly resistant and 24 were moderately resistant. Some of the local and exotic accessions, namely G 2333, Widusa, Cornell 49292, TO, Perry Marrow, PI 207262, Mexique 222, Kaboon, Hans and KRC- 5 have shown resistance to different races of the pathogen in Himachal Pradesh [62]. In another study, 10 lines, viz. IC-328537, IC-328538, IC-448888, IC-313294, IC-278723, IC-339645, IC-341862, EC-169813, EC-398530 and EC-500226, were identified to be resistant to different races of the pathogen [15]. Four cultivars from Turkey namely Sazova, Zulbiye, Akın and Karacasehir 90 were found to be resistant to three isolates of C. lindemuthianum [63]. Local accessions of common bean were screened from Garhwal hills under screenhouse conditions, where 13 accessions were found to be resistant and 32 moderately resistant [64]. The identified resistant genotypes in this study could be used as a source of resistant genes in breeding programmes to create cultivars with broad and long-lasting anthracnose resistance.

The Co-2 locus, situated on the LG11 gives broad-spectrum resistance to races of C. lindemuthianum worldwide [24, 65]. Thus, identifying genotypes carrying the Co-2 gene can aid in its introgression into anthracnose-susceptible cultivars through breeding programmes [25, 27, 66]. The SCAReoli marker reported in previous studies was used for introgression of the Co-2 gene in marker-assisted selection (MAS) programmes as it is polymorphic in Andean and Mesoamerican genotypes of common bean [43, 67]. The presence of the Co-2 gene in common bean genotypes was evaluated in the present study by screening fifty common bean genotypes with the SCAreoli marker. A band of 1.3 kb was amplified in all genotypes. On digestion with the Dra I enzyme, a band of 1 kb was produced in EC-500250, IC-274530, S 1, S 3, EC-398527, EC-398565, BR 8, EC-398591, and B 12 genotypes. The SCAreoli marker is beneficial for selecting genotypes with the Co-2 gene introgressed, which gives resistance to anthracnose races [24]. The Co-2 resistance gene has been discovered in the Drezden cultivar and Beslet variety of common bean and its incorporation has been recommended into breeding programmes [44]. Common bean landraces were screened against five races of C. lindemuthianum from the North-Western region and the presence of Co-4 and Co-2 genes were identified, which can be used for breeding durable anthracnose resistant cultivars [68]. The Co-2 gene has also been identified in the Sazova cultivar among 40 cultivars screened for the presence of Co resistance genes in common bean [69]. The SCAreoli marker is not gene-pool specific, hence it will be beneficial in marker-assisted selection (MAS) techniques for Co-2 introgression. The Co-2 gene in combination with other genes like Co-1, Co-10, Co-13, Co-4 and Co-6 was effective in providing resistance to different isolates of the pathogen [44, 69, 70].

MAS utilizes molecular markers associated with the gene of interest for the selection of disease resistance phenotype. Markers derived from polymorphisms within the desired gene are much more reliable in MAS as they do not get lost during recombination [28, 33]. FMs are novel molecular markers that were generated using polymorphic motifs of functional genes and influence the phenotypic traits. These markers are present in the target genes itself, and can be used with high accuracy and efficiency to detect desirable alleles. These have a wide potential for use in assisted genetic breeding [31, 71]. In the present study, SNPs identified from the promoter region of the anthracnose resistant Co-2 gene in resistant and susceptible genotypes were converted into CAPS marker. These co-dominant markers can accurately predict the functional nucleotide polymorphism which distinguishes susceptible and moderately susceptible genotypes from resistant genotypes. This indicates that the genotype of the CAPS marker and the observed phenotype were perfectly correlated, thereby can be utilized in breeding projects in poorer nations where anthracnose is a common problem. In a recent study, functional marker was developed using the single nucleotide polymorphisms of leaf rust resistant gene Lr22a in wheat. The markers developed could discriminate between the susceptible and resistant Lr22a alleles and considerably facilitates breeding programmes [39]. Functional marker associated with xa5 gene mediating resistance to bacterial blight was developed in rice, which can identify blight resistance varieties [31].

The main aim of breeding against anthracnose has been the introduction of resistance genes into cultivated varieties; this procedure necessitates the establishment of genetic markers for the resistance gene track. Resistance gene-based approaches now have the advantage of being able to create molecular markers that are related to possible functional genes and can provide information about the targeted gene [72]. The development of gene-targeted and functional marker technologies has ushered in a new era in marker-assisted selection. The newly identified genotypes are interesting breeding materials that could be employed in resistance breeding as new sources of Co-2 resistance for common beans. The newly discovered marker might be used as an alternative breeding tool to help breeders track the Co-2 gene in common beans for breeding purposes.

Fifty out of eighty-nine genotypes of common beans were resistant or moderately resistant. These can be exploited as a source of anthracnose resistance in breeding programmes to create cultivars with broad and long-lasting anthracnose resistance. The genotypes identified such as EC-500250, IC-274530, S 1, S 3, EC-398527, EC-398565, BR 8, EC-398591, and B 12 possessing the Co-2 gene can be exploited as a resistant source of anthracnose disease in future common bean breeding programmes for the development of resistant varieties. Also, the newly discovered marker might be employed as a suitable alternative to assist breeders in tracking the Co-2 gene in common beans.

Acknowledgements

CG gratefully acknowledges the financial support (San No. 09/1238(0001)/2019-EMR-I) received from the Council of Scientific and Industrial Research (CSIR), Govt. of India and the School of Biotechnology, SKUAST-Jammu for the work reported in this paper.

Author contributions

RKS conceived and designed the experiments and wrote the final manuscript. CG performed experiment and wrote the manuscripts. RAV helped in conceiving the experiment.

Conflict of interest The authors declare no conflict of interest.

Compliance with Ethical Standards

This article does not contain any studies with human participants or animals performed by any of the authors.

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Development of SNP based functional marker for anthracnose resistant Co-2 gene in common bean (Phaseolus vulgaris L.)

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Abstract

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Introduction

Materials And Methods

Plant material

Isolation And Identification Of The Pathogen

Phenotypic Evaluation For Anthracnose Resistance

Dna Isolation And Molecular Marker Analysis

Development Of Allele-specific Marker

Results

Evaluation for anthracnose resistance

Snp Identification

Development And Validation Of Allele-specific Marker

Discussion

Conclusion

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References

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