Single Marker Analysis in Groundnut for Tolerance to in vitro Seed Colonization by Aspergillus flavus and Aflatoxin Contamination

Groundnut (Arachis hypogaea L.), also popularly known as peanut, is a major oilseed and food crop grown on ~27.9 m.ha across 100 countries for a global production of 47 m.t. during 2017 (FAOSTAT, 2018). The crop is consumed mainly as confectionery and in various food products in Western countries and is used for cooking oil and confectionery in the Indian subcontinent. Aflatoxin contamination caused by Aspergillus flavus is a major constraint to peanut industry worldwide due to its toxicological effects to human and animals. Aflatoxins are highly toxic and carcinogenic substances and hard to be eliminated from the contaminated materials (Kew, 2013; Khlangwiset and Wu, 2010; Patten, 1981). Peanut tend to be infected by A. flavus covering the whole industrial chain including pre-harvest, during harvest, post-harvest drying, in storage and during transport (Torres, 2014; Sugri, 2017; Al-Saad, 2017). A lot of prevention strategies for aflatoxin contamination have been implemented, including using bio-control agents, taking good agricultural practices and planting resistant varieties (Dorner, 2008; Horn and Dorner, 1998; Chulze, 2015; Bhatnagarmathur et al., 2015). Developing peanut varieties with resistance to seed infection and/or aflatoxin accumulation is the most effective and economic strategy for reducing aflatoxin risk in food chain. Breeding for resistance to aflatoxin in peanut is a challenging task for breeders because the genetic basis is still poorly understood. Further more, the trait phenotyping faces high environmental influence and variable soil microbiome across environments and locations (Bolun Yu et al., 2019).
       
Molecular markers based on DNA sequence variations are increasingly being utilized in crops for construction of genetic maps and marker-assisted selection. But, their application in groundnut enhancement is lagging behind because of limited knowledge of its genome. However, the recent genome sequencing of cultivated groundnut may certainly increase the use of genomic tools in groundnut crop improvement through marker aided selection schemes (Chen et al., 2019).
       
Association of DNA markers particularly SSRs to foliar diseases of groundnut like rust and late leaf spot are well documented (Mondal and Badigannavar 2010; Kukanur et al., 2014; Yol et al., 2016). Lei-Yong et al., (2005) identified two AFLP markers that were closely linked to the gene conferring resistance to A. flavus. Tests to verify the reliability of the markers using 20 groundnut genotypes showed a high correlation between the molecular markers and the resistance to A. flavus. Hong et al., (2009) reported that, out of 100 SSR primers, only 41 showed polymorphism with 2 to 4 alleles per locus in 12 resistant and susceptible groundnut varieties infected by Aspergillus flavus with PIC values ranging from 0.153 to 0.750. Correlation analysis of SSR markers and host resistance showed that, 5 markers were found to have a correlation with Aspergillus flavus resistance and Pearson’s correlation coefficient reached to 0.913. A marker named pPGSseq19D9 showed the highest correlation and was able to differentiate the resistant and susceptible varieties, which indicate that, pPGSseq19D9, may be linked with one major gene that controls the resistance to Aspergillus flavus infection (Hong et al., 2009). Single major QTL for resistance to percent seed infection by Aspergillus flavusand two important co-localized intervals associated with major QTLs for resistance to aflatoxin B1 and aflatoxin B2 were reported recently (Bolun Yu et al., 2019).
       
In the present study, an effort was made to ascertain the association of the SSR markers which were earlier reported to be associated with tolerance to major diseases of groundnut including Aspergillus flavus infection/aflatoxin contamination (Hong et al., 2009; Mondal and Badigannavar, 2010; Yol et al., 2016; Zongo et al., 2017) and molecular diversity in groundnut (Frimpong et al., 2015).

Experimental location and plant material          
 
The present study was conducted at the All India Co-ordinated Research Project on Groundnut, Zonal Agricultural and Horticultural Research Station (ZAHRS), Hiriyur, (University of Agricultural and Horticultural Sciences, Shivamogga, Karnataka). In the present study, a total of 66 groundnut genotypes collected from different sources and having variable reaction to in vitro seed colonization by Aspergillus flavus (IVSCAF) and aflatoxin contamination were used for the single marker analysis using 30 SSR markers to find out the marker trait association particularly for tolerance to IVSCAF and/or aflatoxin contamination.  The details of the genotypes used in the present study along with their reaction to IVSCAF and aflatoxin contamination are represented in Table 1. Screening for tolerance to in vitro seed colonization by Aspergillus flavus (IVSCAF) and identification of tolerant genotypes has been done at ZAHRS, Hiriyur, Karnataka during 2017-18. The aflatoxin quantification was carried out through outsourcing at International Crop Research Institute for Semi Arid Tropics, Patancheru, Hyderabad using indirect ELISA (Enzyme Linked Immuno Sorbent Assay) technique by using the groundnut kernel samples derived from the crop harvested during 2017-18.  The SSR marker analysis was carried out at the Department of Genetics and Plant Breeding, University of Agricultural Sciences, Bangalore.
 

 
Isolation of DNA
 
The DNA was isolated from 66 genotypes using modified CTAB method (Doyle and Doyle, 1987) by using  2-3 grams of fresh leaf sample (15-20 days) from each of the genotypes at 2-3 leaf stage. The quality and quantity of the extracted DNA were checked on agarose gel (0.8% w/v) by electrophoresis at 100 V for 60 min.
 
Polymerase chain reaction using SSR primers
 
The PCR reaction mixture consisted of 20 ng DNA, 1x reaction buffer, 1.5 mM MgCl₂, 0.2 mM of each of dNTPs, 0.5 μM of each forward and reverse primers, 0.3 IU Taq DNA polymerase. DNA amplification was performed in a Veriti® 96-Well Fast Thermal Cycler (Applied Biosystems Inc., Foster city, CA) with 10 μl reaction volume. DNA samples were denatured initially at 94°C for 3 min, then subjected to the following 20 cycles: 94°C for 30 s, 63°C for 30 s with a decrement of 0.5°C per cycle and 70°C for 1 min. This was followed by another 20 cycles of 94°C for 15 s, 55°C for 30 s and 70°C for 1 min. A 10 minutes extension was performed at 72°C as the last step. Amplified products were analyzed using 1.5% agarose gel. A total of 30 SSRs were used to find out thepolymorphism among the genotypes (Table 2). The basis for selection of these SSR primers was that, they have shown association with foliar disease resistance, tolerance to Aspergillus flavus seed infection and/or aflatoxin contamination and also effectively deciphered molecular diversity in groundnut crop. Electrophoresis was performed at 120 volts DC for 2.5 hrs in a submarine electrophoresis system (Maxi sub XL) using metaphor agarose. After electrophoresis, the banding pattern was visualized and captured by using gel documentation system (Uvitech, Cambridge, England).
 

 
Data scoring and data analysis
 
Clear and unambiguous bands were scored for their presence or absence with the score 1 indicating their presence and 0 indicating their absence to generate a binary matrix.The data matrix of binary codes thus obtained was subjected to single marker analysis using SHAN module of NTSYS version 2.0 (Rohlf, 1998). Phenotypic mean values of all the 66 genotypes were subjected to associate with corresponding marker score for its significance by using simple regression.
       
Simple linear regression method (Haley and Knott, 1992) was used to identify significant marker trait association. The linear equation formed was,
 
Where,
Y = Phenotypic trait value.
μ = Population mean.
f (marker) = Function of the molecular marker.
       
The potential relationship between the marker and trait was established considering the significance of the regression coefficient at 5 and 1 per cent probability. Adjusted R² values were used to express phenotypic variance as explained (PVE) as reported by Anitha et al., 2015.

Simple linear regression was calculated for in vitro seed colonization by Aspergillus flavus (IVSCAF) and the extent of aflatoxin contamination in the groundnut genotypes with all the 30 SSR marker classes. The potential relationship between the marker and trait was established considering the significance of the regression coefficient. The marker which is having the strongest relationship can be judged from its PVE (phenotypic variance as explained). The PVE will give the overall percentage of variability of that particular trait explained by the marker. Totally 30 SSR markers reported in groundnut were used to ascertain the association of any of the markers with two traits viz., tolerance to aflatoxin contamination and/or in vitro seed colonization by Aspergillus flavus (IVSCAF). Four markers viz., GM-1954, GM-1883, pPGPseq-2F05 and S-03 were found to be associated with in-vitro seed colonization by A. flavus. The SSR marker GM-1954 has shown a maximum R² value of 14.07 indicating that 14.07 per cent phenotypic variation for IVSCAF has been explained by this marker (F=0.002**) which happens to be the maximum among other markers used in the study. Other SSR markers, GM-1883, pPGPseq-2F05 and S-03 have recorded R² values of 11.91, 6.64 and 5.88, respectively for the trait, tolerance to IVSCAF (Table 3). Among these, GM-1883 and pPGPseq-2F05 have been reported to be linked with foliar diseases in groundnut.  The marker GM-1883 was reported to be linked with early leaf spot (Zongo et al., 2017) and pPGPseq-2F05 was found to be linked with both late leaf spot and rust in groundnut (Mace et al., 2006). These findings suggest that these markers may be linked to those genes which are involved in conferring resistance to fungal diseases of groundnut at the time of infection by the fungal pathogens.
       

 
Three markers viz., S-21, S-80 and GM-1954 were found to be associated with the tolerance to aflatoxin contamination. Marker S-21 has recorded a maximum R² value of 13.77 (F=0.002**) indicating 13.77 per cent phenotypic variation for aflatoxin tolerance has been explained by this marker. Further, the markers GM-1954 and S-80 have shown R² values of 6.21 and 6.08, respectivelyfor tolerance to aflatoxin contamination. The banding pattern upon gel electrophoresis of the SSR markers associated with tolerance to IVSCAF and/or aflatoxin contamination are depicted in Fig 1.
 

       
It is evident from the results that, the marker GM 1954 has shown association with tolerance to both IVSCAF as well as aflatoxin contamination. However, the R² value for aflatoxin contamination was less (6.21) as compared to that of IVSCAF (14.07). It was earlier reported that, the SSR marker GM 1954 was linked with rust disease in groundnut (Yol et al., 2016). In the present study, the marker GM-1954 was found to be significantly linked with tolerance to aflatoxin contamination and IVSCAF and was reported to having a PIC value of 0.58 (Nadaf et al., 2019) indicating its possible association with tolerance to both IVSCAF and aflatoxin contamination substantiating its role in deciphering the molecular variation for different traits in the groundnut genotypes used in the present study. These results are in accordance with Frimpong et al., (2015). This finding is also supported by the earlier reports wherein these markers were able to reveal the presence of variation in the present set of genotypes for tolerance to rust disease as well (Mondal and Badigannavar, 2010; Yol et al., 2016). Recently, a major QTL for resistance to seed infection by A. flavus and two important co-localized intervals associated with major QTLs for resistance to aflatoxin B1 and B2 were identified and validated by using SSR markers linked to these intervals (Bolun Yu et al., 2019). However, the SSR markers identified in the present study may be either associated with one or more major genes or QTLs associated with tolerance to IVSCAF and/or aflatoxin contamination. Generally molecular markers linked with QTL/major genes for traits of interest are routinely developed in several crops using materials derived from planned crosses such as F2, RIL, DH populations, etc. However, non-availability of mapping populations and substantial time needed to develop such populations are sometimes major limitations in the identification of molecular markers for complex traits like IVSCAF and aflatoxin contamination. Therefore, markers identified during the present study need to be subjected to validation and/or functional analysis for effective utilization in the marker assisted selection.

The SSR markers identified in the present study shows association with tolerance to aflatoxin contamination as well as in vitro seed colonization by Aspergillus flavusindicating that these markers may be either linked to one or more major genes or QTLs associated with tolerance to aflatoxin contamination as well as in vitro seed colonization by Aspergillus flavus substantiating the association of these markers with those genes involved in conferring resistance to fungal diseases of groundnut at the time of infection by the fungal pathogens. However, markers identified during the present study need to be subjected to validation and/or functional analysis for effective utilization in the marker assisted selection.

The financial assistance in the form of Student Assistantship to Mr. Hasanali Nadaf and the Staff Research Project to Dr. Harish Babu, B.N., by the Directorate of Research, University of Agriculture and Horticultural Sciences, Shivamogga, Karnataka (India) to carry out this work is gratefully acknowledged.