Legume Research

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Legume Research, volume 46 issue 10 (october 2023) : 1326-1331

Development of CAPS Marker Linked to H2 Locus of Fusarium Wilt Resistance in Chickpea

Sunil Subramanya1,*, Chandrakanth D. Soregaon2, Dalpat Lal3, Ramapura Laxmipathi Ravikumar1
1Gandhi Krishi Vigynana Kendra, University of Agricultural Sciences, Bengaluru-560 065, Karnataka, India.
2University of Agricultural Sciences, Dharwad-580 005, Karnataka, India.
3Jagannath University, Jaipur-302 022, Rajasthan, India.
  • Submitted02-06-2020|

  • Accepted01-12-2020|

  • First Online 06-04-2021|

  • doi 10.18805/LR-4433

Cite article:- Subramanya Sunil, Soregaon D. Chandrakanth, Lal Dalpat, Ravikumar Laxmipathi Ramapura (2023). Development of CAPS Marker Linked to H2 Locus of Fusarium Wilt Resistance in Chickpea . Legume Research. 46(10): 1326-1331. doi: 10.18805/LR-4433.
Background: Fusarium wilt is one of the widely distributed biotic stress of chickpea limiting its productivity worldwide. The major problem limiting the resistance breeding is screening of germplasm and breeding lines for disease resistance. To address such problems, identification of molecular marker closely linked to resistance locus is an effective strategy.

Methods: The RAPD marker A07C417 closely linked H2 locus of Fusarium wilt resistance locus has been converted into SCAR and the loss of initial polymorphism was recaptured by alignment of consensus sequences from SCAR amplified locus for the identification of common motifs. Further, the linkage association of this marker with Fusarium wilt resistance locus has been reaffirmed using Fusarium wilt response of RILs phenotyped for wilt reaction using wilt sick pots and also wilt sick plots.

Result: Identification of single nucleotide polymorphism at the consensus SCAR locus between susceptible and resistant cultivar, enabled development of CAPS marker through suitable restriction enzyme Aci I making it an effective codominant marker system for resistance deployment via marker assisted selection.
Fusarium wilt caused by the Fusarium oxysporum f. sp. ciceri (FOC) is one of the major yield constraining elements in chickpea. The production of chickpea is seriously abridged by Fusarium wilt in all chickpea growing areas of the world and it has been reported from at least 33 countries, causing an annual yield loss of upto 100 per cent under favourable conditions (Cortes and Hav, 2000; Chattopadhya and Mohapatra, 2015). Fusarium oxysporum f. sp. ciceri (Foc) is a soil born pathogen and it also survives in the hilum region of seed as chlamydospore-like structures (Haware et al., 1978). Till date, two pathotypes (yellowing and wilting) and eight distinct physiological races of pathogen (0, 1A, 1B, 2, 3, 4, 5 and 6) have been reported, of which six races cause wilting symptoms, whereas races 0 and 1B/C cause yellowing symptoms (Kelly et al., 1994; Del Mar Jimenez-Gasco and Jimenez-Diaz, 2003; (Jimenez-Gasco and Perez-Artes, 2001)).
The genetic analysis of wilt resistance has indicated that the resistance to Fusarium oxysporum f. sp. ciceri race 0 (FOC 0) is governed by means of one or two independent genes (Tekeoglu et al., 2000 and Rubio et al., 2003); resistance to FOC 2 is conferred by a single recessive gene (Sharma and Muehlbauer, 2007); resistance to FOC 3 by monogenic (Sharma et al., 2004) and resistance to race 4 (FOC 4) is monogenic recessive (Tullu et al., 1998 and  Sharma et al., 2005) as well as digenic recessive (Tullu et al., 1999), but resistance to race 5 (FOC 5) is governed by single gene (Tekeoglu et al., 2000 and Sharma et al., 2005). The present study, is concentrated on race 1A, which is overwhelming in peninsular India in a pestilence manner, where resistance to FOC 1 is conditioned by two to three independent loci designated as H1, H2 and H3 ( Upadhyaya et al., 1983 ) ; Singh et al., 1987 and Brinda and Ravikumar, 2005). Dominant alleles at both loci (H1HH2H2) results in early wilting reaction, homozygous recessive allele at either one of the loci (H1_ h2h2 or h1h1 H2_) produces late wilting reaction and the homozygous recessive allele at both the loci (h1h1h2h2) confer resistance to Fusarium wilt (Brinda and Ravikumar, 2005).
It is very difficult to manage this particular disease due to soil borne nature of pathogen (Chaithra et al., 2019), however, the major problem encountered for the development of high yielding wilt resistant genotypes is the divergence of the pathogen and also the development and maintenance of uniform wilt sick plots. Further the severity of disease is also affected by inoculum concentration, virulence and environment conditions (Jimenez-Gasco and Perez-Artes, 2001). To overcome all these drawbacks, deployment of host plant resistance and development of resistant cultivar through marker assisted selection (MAS) is one of the best means to combat this problem. Therefore, identification of reliable DNA markers closely linked to resistant loci increase the efficiency of selection of resistant genotypes and also for the introgression of resistance loci against Fusarium wilt.
Several molecular markers linked to Fusarium wilt resistance have been reported in chickpea. However, there are only a few attempts to specifically identify molecular marker linked to H1 or H2 loci independently (CS27700; Mayer et al., 1997 and A07C417; Soregaon et al., 2007). RAPD (Random Amplified Polymorphic DNA) markers are generally dominant, sensitive and non-stringent with poor reproducibility (Williams et al., 1990; Jones et al., 1997 and Bardakci, 2001). In the last few decades, there are growing number of cases, in which RAPD markers have been used to develop either allele-specific or locus-specific primers. SCAR (Sequence Characterized Amplified Regions)/CAPS (Cleaved Amplified Polymorphic Sequence) marker were developed by cloning and sequencing of amplified products of an RAPD marker offers more advanatges over RAPD marker as they detect only single locus, less  sensitive to changing reaction condition and they can be potentially converted into co-dominant markers (Paran and Michelmore, 1993; Zheng et al., 1999, Zhang and John, 2001 and Guiterrez et al., 2006).
In this contest, in the present study an attempt was made to convert RAPD marker A07C417 closely linked H2 locus of Fusarium wilt resistance in chickpea (Soregaon et al., 2007) into SCAR marker. However, loss of initial polymorphism among the parental genotypes in the SCAR amplified amplicons has been recovered by development of co-dominant CAPS marker and we also report our effort in ascertaining the linkage of this CAPS marker to Fusarium wilt resistance using recombinant inbred lines (RILs) screened over the seasons for wilt reaction.
Plant material and DNA extraction
The experiment was conducted in rabi session of 2016-17 at Department of Plant Biotechnology, University of Agricultural Sciences (UAS), GKVK, Bangalore, India. The experimental material consists of two parental genotypes, JG62, an early wilting genotype (H1H1H2H2), highly susceptible to Foc race 1 and WR315 genotype (h1h1h2h2) resistant to race 1A, race 2, race 3, race 4 and race 5 of Fusarium wilt (Upadhyaya et al., 1983; Mayer et al., 1997 and Sharma et al., 2005) and also 117 F12 RILs derived from the cross JG62 X WR315.  Total genomic DNA was extracted from vegetative buds and young leaves of two parental lines and individual RILs population by Cetyl Trimetyl Ammonium Bromide (CTAB) method as described in Doyle and Doyle (1987) with little modifications. The quality of DNA was assessed on 0.8 per cent agarose gel prepared in 1X TBE buffer followed by visualization and documentation using Alpha digidoc system (Alpha Innotech Corporation, USA)  and quantification was done by nanodrop (Eppendorf Bio spectrometer).
Phenotyping: Evaluation of RILs for Fusarium wilt resistance
The RILs and parental lines were phenotyped for wilt reaction in green house using wilt sick pots developed at Department of Plant Biotechnology, UAS, GKVK, Bangalore. The wilt sick pots (30 cm diameter) were developed using the single spore culture of Fusarium oxysporum race 1A by following the standard procedure (Brinda and Ravikumar, 2005). The presence of pathogen in the pots was confirmed by growing the susceptible cultivar JG-62 plants (5-6 plants per pot). The disease screening using wilt sick pots was unambiguous and reliable as the early wilting susceptible genotype JG-62 showed complete wilting within 30 days in wilt sick pots (Fig 1). The six seeds of each RILs were sown in wilt sick pot along with JG-62 as susceptible check at the centre in each wilt sick pots. The RILs were classified as susceptible if 60 per cent of the plants recorded wilting symptoms and death on 60th day and remaining as resistant (Haware and Nene, 1982) (Fig 1).  Similarly, the data on the wilt reaction of RILs in the early generation over two years (Rabi 2007 and Rabi 2008) in wilt sick plots maintained at ICRISAT, Hyderabad was also used for the present study.

Fig 1: Phenotyping of RILs for Fusarium wilt reaction in wilt sick pots.

Design and amplification of SCAR marker
The five different SCAR primers were developed on the basis of available sequence information of an RAPD marker A07C417 associated with H2 locus of wilt resistance (Soregaon et al., 2007) for the specific amplification of the target locus using Primer3 software. The primer sequence ranged from 15 bp in A07CSCAR-2 to 25 bp in A07CSCAR-3 and A07CSCAR-5 (Table 1). The primers were synthesised from Sigma-Aldrich Corporation (United Nation).

Table 1: Sequence of SCAR primers derived from polymorphic DNA fragment of an RAPD marker A07C417

CAPS marker development and analysis
SCAR amplified products produced monomorphic amplicons and did not differentiate the parental lines even after the optimization of PCR parameters. To recover the loss of initial polymorphism, SCAR amplified products from both the parental lines were sequenced and the consensus sequence obtained were analysed by means of sequence homology using BioEdit software to identify common motifs and appropriate restriction enzyme was selected for the development of CAPS marker. Restriction endonuclease digestion assay of parental lines and RILs were carried out using restriction enzyme Aci I in a 15 μl of SCAR amplified product in a reaction volume of 25 μl with 3U of restriction enzyme Aci I and 1X appropriate buffer based on supplier manual recommendation (New England Biolabs, Inc.) and restriction digested products were resolved on 3% agarose gel in 1X TBE buffer stained with 0.05 μg/ml ethidium bromide.
Statistical analysis
The segregation of CAPS marker among RIL population was tested for goodness-of-fit test (χ2) for the segregation ratio of 1:1 and Goodness-of-fit test of CAPS marker was also performed together with 60th day wilting percentage for digenic inheritance ratio of 1:1:1:1 to determine the linkage. The percentage of plants infected on the 60th days in each RIL was used to compare the two classes for marker and the difference was compared using t-test.
Development of SCAR/CAPS marker
The amplification of DNA of parental lines JG-62 (susceptible) and WR-315 (resistant) using five different
SCAR primer sets produced monomorphic bands. The SCAR primer A07CSCAR-3 producing single, specific predicted size in both susceptible (JG62) and resistant parent (WR315) was selected and amplified products from both parental lines were sequenced. Comparison of consensus sequence information of SCAR amplified products from both the parental lines JG-62 and WR-315 enabled the identification of single nucleotide polymorphism (SNP) with a restriction site mutation for restriction enzyme Aci I (C▼CGC/GGC▲G) specific to resistant parent WR-315 at 219 bp (Fig 2), which clearly distinguished the susceptible and resistant parents. The restriction enzyme digestion of monomorphic SCAR product from both the parents JG-62 and WR-315 produced an expected electrophoretic banding pattern of two bands of size 212 bp and 159 bp in resistant parent WR-315 and single band of 371 bp in susceptible parent JG-62 (Fig 3).

Fig 2: Alignment of A07CSCAR-3 amplified nucleotide sequences from susceptible parent JG62 and resistant parent WR315.

Fig 3: Electrophoretic banding pattern of CAPS marker.

The essential requirement of any MAS programme involves the identification of polymorphic markers that are closely linked to trait of interest and their validation.  RAPD markers are the first generation marker system extensively used for germplasm classification in addition to their usefullness in gene tagging and mapping studies owing to their major attributes such as use of universal primers, short period with cost effectiveness, requirement small quantity of DNA for analysis and also non requirement of cloned DNA probes and hybridization filters (Ranade et al., 2001). Although, RAPD markers provide an efficient assay for polymorphism as the differences in amplicon primarily results from the differences in their priming site or amplifying DNA segment from one parent but not in the other and used successfully to map chickpea wilt resistance loci (Soregaon et al., 2007 and Babayeva et al., 2018), the technique is dominant and sensitive to modification in PCR reaction condition, eventually resulting in poor reproducibility. These limitations has led to the successful conversion of RAPD marker linked to different traits into allele-specific or locus-specific primers (SCAR/CAPS) and it has been reported in melon, tomato, faba bean, black gram and mung been by Zheng et al., (1999); Zhang and John (2001); Gutierrz et al. (2006); Prashanti et al. (2011); Dhole and Reddy (2013) respectively.   
Segregation and linkage analysis of CAPS marker
The c2 test for monogenic inheritance of CAPS marker and wilt reaction displayed an expected 1:1 segregation ratio in RILs population. Among 117 RILs, 61 RILs produced CAPS banding pattern similar to WR-315. The digenic analysis of wilt reaction and marker was carried out to determine the linkage between markers and wilt resistance. The joint segregation of CAPS marker together with 60th day wilt reaction in all the three seasons indicated significant deviation from 1:1:1:1 ratio in the RILs suggesting linkage between CAPS marker and wilt reaction (Table 2). Further in an effort to map the CAPS marker on to the sequence based physical map of chickpea by in silico approach using BLAST search tool of NCBI showed significant similarity with LOC101491241 on chromosome Ca4 (E-value = 0.0) of the chickpea genome with a putative function of wound responsive family protein determined using chickpea transcriptome database.

Table 2: Joint segregation of CAPS marker and wilt reaction in RILs of chickpea

The first DNA markers linked to the Fusarium wilt race 1A at H1 locus was identified by Mayer et al., (1997) using allele specific associated primer (ASAP) CS27700. Similarly, RAPD marker A07C417 linked to H2 locus have been identified by Soregaon et al., (2007). Both of these allele specific markers were developed in different genetic backgrounds segregating independently for each locus. Independent segregation of markers linked to H1  (CS27700) and H2 (A07C417) locus were also confirmed using RILs of cross JG-62 x WR-315 revealing reliability of both these DNA markers (Brinda and Ravikumar, 2005; Soregaon and Ravikumar, 2010). Along with these linked markers, many SSR markers linked to wilt resistance have been identified by several researchers (Radhika et al., 2007; Gowda et al., 2009; Patil et al., 2014 and  href="#jingade_2015">Jingadae et al., 2015). However, many of these markers need to be validated in different genetic background for their association with wilt resistance and it is also necessary to develop  high density of polymorphic markers in these region, which will enable fine mapping of resistance locus (MPerez-de-Castro et al., 2012). Hence, identifying markers tightly linked to resistance loci/QTL will provide an effective solution for resistance deployment via MAS particulary in case of complex inheritance pattern of disease resistance.             
In the present investigation, SNP has been identified in the monomorphic SCAR locus of RAPD primer A07C417, which is able to differentiate between susceptible and resistant genotypes enabling the development of reproducible codominant CAPS marker. The mapping population (RILs) used were developed by hybridization of highly susceptible (JG62) and resistant parent (WR315) segregating independently for both H1 and H2 locus potentially generating a full range of variability for wilt resistance. Further, to overcome the problems associated with disease screening of segregating population in wilt sick plots, development of standardized disease screening using uniform wilt sick pots in green house enabled efficient screening of segregating population. Disease screening of RILs for wilt reaction in wilt sick pots and wilt sick plots enabled detection of linkage association of CAPS marker with H2 locus of Fusarium wilt resistance. Thus, identification of SNP and the development of codominant CAPS marker in this study may find its usefulness in precise genetic mapping and marker assisted trait introgression of resistance locus into elite cultivar.
The authors express their gratitude to Department of Science Technology (DST) - Fund for Improvement of Science and Technology infrastructure in universities and higher educational institutions (FIST), Government of India for providing research facilities.

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