Molecular Mapping of a Gene Conferring Fusarium Wilt Resistance in Lentil (Lens culinaris Medikus subsp. culinaris) using Bulked-segregant Analysis

Lentil (Lens culinaris Medikus subsp. culinaris) is a diploid (2n=2x=14 chromosomes), self-pollinating, annual, cool-season grain legume crop, having haploid genome of 4,063 Mbp size (Arumuganathan and Earle 1991). From Mediterranean region, lentils has been spread to various parts of the world and ultimately evolved into six geographical groups (Cubero, 1981). Although, lentil is cultivated nearly in 52 countries, major share is occupied by countries like India, Canada, Turkey, Bangladesh, Iran, China, Nepal and Syria. Currently, the global area under lentil cultivation is about 6.10 m ha, producing 6.33 m tons of grains with an average production of 1,038 kg/ha (FAOSTAT, 2018). In India, lentil is cultivated in about 2.21 m ha with production and productivity of 1.62 m tons and 731 kg/ha, respectively (FAOSTAT, 2018).

Lentil grains are immensely valued for its richness in protein (22-35%), minerals (K, P, Fe, Zn, Se) and vitamins (A, K, E, folate, thiamin, β carotene, riboflavin, niacin, pantothenic acid and pyridoxine) for human nutrition (Sarker et al., 2018; Kiran et al., 2021). Lentil productivity is constrained by various biotic and abiotic factors of which vascular-wilt (Fusarium oxysporum Shlecht. Emend. Snyder and Hansen f. sp. lentis Vasudeva and Srinivasan) is the most important yield limiting factor in Sub-Saharan Africa, South Asia, West Asia and North Africa (WANA), causing severe economic yield losses (Erskine et al., 2011).

It is a soil-borne, host-specific fungus infecting only cultivated lentil (Lens culinaris spp. culinaris) and wild vetch (Vicia montbretii). It prefers warm and dry conditions and mostly infects the crop during reproductive crop growth stage. Its symptoms include drooping and wilting of top leaflets resembling water-deficit stress, stunting of plants, shedding of leaflets and ultimate plant death. Although, it survives on the debris of infested plants, seed transmission has also been reported (Erskine et al., 1990). In India, Fusarium wilt is the main reason which limits the production of lentil in majority of the lentil growing states including Uttar Pradesh, Madhya Pradesh, Himachal Pradesh, Bihar, West Bengal, Assam, Rajasthan, Haryana and Punjab (Chaudhary et al., 2009).

The genetics of wilt-resistance in lentil is essential to understand the number of gene(s) controlling the trait in different background for its effective deployment in the breeding programme. However, limited reports are available on the genetics of lentil wilt resistance. Based on test of allelism, Kamboj et al., (1990) reported five dominant genes governing the lentil wilt resistance, of which two showed duplicate gene action and complimentary gene action in different genetic backgrounds. Further, Abbas (1995) and Eujayl et al., (1998) reported dominant single gene inheritance for wilt resistance.

Efforts to commercially control the wilt using chemical and biological means have not succeeded much due to its high cost and complexity of incorporation into the soil during crop growth. Hence, development and deployment of resistant cultivars is most effective, economical and environmentally friendly way of managing wilt in lentil (Bayaa et al., 1995). Further, field based screening methods for the identification of donors is tedious and sometimes gives inconsistent results. Thus, more precise and efficient strategy like identification of closely linked DNA markers to the resistance gene can provide effective tool for tagging, mapping and pyramiding of resistant gene in desirable agronomic background. Based on resistance and susceptible reaction in lentil cultivars eight races of fusarium wilt pathogen identified by Hiremani and Dubey (2018)  and Chaithra et al. (2019) also isolated twelve Fusarium oxysporum f. sp. ciceri isolates from chickpea cultivars that could be help in race specific wilt resistance lentil variety. 

Marker assisted breeding research has been taken up for various biotic stresses in lentil such as ascochyta blight (Gupta et al., 2012) and for fusarium wilt  in chickpea (Pratap et al., 2017). Even for Fusarium wilt resistance, Eujayl et al. (1998) has reported a linked RAPD marker OPK15900 at 10.8 cM distance, whereas Hamwieh et al., (2005) mapped the gene on LG 6 which was found flanked by SSR59-2B and P17m30710 (AFLP) at a distance of 8.0 cM and 3.5 cM respectively. The present investigation was aimed to identify the SSR marker linked with Fusarium wilt resistance gene in lentil using bulked-segregant analysis. 

Plant material and development of mapping population

The genotypes, L9-12 and ILL10965 which was earlier identified by our group as susceptible and resistant to the Fusarium wilt respectively, are used for the development of F2 mapping population. These resistant and susceptible genotypes are identified from a set of 93 diverse genotypes which were screened against fusarium wilt in well-established sick-plot at Rafi Ahmad Kidwai (RAK) College, Sehore, India (23°12' N, 77°05 E, 502 m AMSL) and also under controlled conditions.

Hybridization  was performed during rabi 2014-15 at ICAR-Indian Agricultural Research Institute, New Delhi, India (28°63"24' N, 77°15"14' E, 218 m AMSL). A total of 11 putative F1s were raised as offseason nursery at IARI, RS Wellington during summer 2015. Then, four true hybrids were identified using polymorphic SSR markers and harvested individually to produce F₂ populations during rabi 2015-16. One of the F₂ population having 120 plants was raised in the farm of IARI, New Delhi and were harvested individually to get the F_2:3 populations.
 
Phenotyping of parents and mapping population
 
To work out the genetics of the disease resistance, the parents, their F₁’s and F_2:3 families were phenotyped against Fusarium-wilt in a well-established wilt-sick plot at RAK College of Agriculture (Sehore), which is also the hot-spot for Fusarium wilt in India, using infector row technique. The parents and 120 individuals of F_2:3 population were raised in individual rows during rabi 2015-16 and rabi 2016-17 respectively under sick-plot conditions as described by Eujayl et al., (1998). The planting of F_2:3 and parental lines were done at a row-to-row and plant-to-plant distance of 5.0 cm and 25.0 cm respectively, with the row-length of 4.0 meter.
 
DNA extraction and PCR analysis
 
Genomic DNA extraction was done using 2.0 g leaf tissue of 21 days old seedlings with CTAB method. Twenty μl PCR reaction mixture constituted of 10× buffer (100 mM Tris-HCl, 500 mM KCl, 15 mM MgCl₂ and 0.01% gelatin), 200 μM dNTPs, 0.5 μM forward and reverse primers, 1U Taq DNA polymerase (Sigma-Aldrich, USA) and nearly 40 ng DNA. The amplification was performed in a thermal cycler (Applied Biosystems, Singapore). The PCR amplification protocol comprised of denaturation at 94°C for 4 min followed by 30 cycles of 94°C for 1.0 min, annealing at 59 to 62°C for 30 seconds, extension at 72°C for 1 min and final extension at 72°C for 10 min. Electrophoretic separation of amplified products were performed in 3.0% Metaphor agarose gel (Lonza, USA) in TBE buffer at 100 V for nearly 3 h and stained using ethidium bromide. Gel documentation system (Syngene) was used to record the gel photograph using CCD camera. DNA ladder 50bp (MBI, Fermentas, Lithuania) was used as marker and more than two hundreds SSR primer pairs were used to study the parental polymorphism.
 
Bulked-segregant analysis (BSA)
 
BSA, as proposed by Michelmore et al., (1991) was performed to tag the Fusarium wilt resistance gene in lentil. The resistant and susceptible bulks were constituted from the 10 F₂ homozygous individuals each, showing extreme phenotype for the wilt disease and having 30 ng/µL DNA. The polymorphic SSR makers identified between resistant and susceptible parents were used for studying polymorphism among the resistant and susceptible bulks. The amplification of allele in resistant parent and resistant bulk or susceptible parent and susceptible bulk was the basis of association of marker with gene controlling wilt resistance. The polymorphic primers identified between resistant and susceptible bulks are used to genotype all the F₂ plants of mapping population.
 
Segregation and linkage analysis
 
The individuals of F_2:3 families which were derived from the F₂ population were screened for wilt resistance in the wilt sick plot at RAK College, Sehore (Madhya Pradesh, India). The segregating populations were classified in two distinct classes as resistant and susceptible. Chi square test (Gomez and Gomez 1984) for a fixed ratio hypothesis was used to analyze the data (tested at 5% level of significance). MapMaker ver. 3.0 (Lander et al., 1987) was used to determine linkage between resistant gene and markers, and linkage map was constructed using Kosambi mapping function at LOD 3.0 (Kosambi 1944).

Inheritance of wilt resistance
 
The key approach of controlling wilt disease in lentil is by resistance breeding which has resulted in the identification of resistant accessions in both wild and cultivated lentil (Erskine et al., 1994). These resistant lines have been extensively used by the breeders and famers throughout the world (Bayaa et al., 1997). Although, a number of resistant sources to Fusarium wilt including released varieties are known but only few reports about the inheritance of this deadly disease is published (Kamboj et al., 1990; Abbas 1995).

In this study, the genetics of inheritance of wilt resistance gene in lentil was studied in F₂ population which was derived from the cross, L9-12 × ILL10965 after the selfing of F₁ plants. The parent L9-12 exhibited high susceptibility to wilt with the score of >50.0% wilting while, ILL10965 exhibited moderate resistant reaction with the score of <2.0-10% wilting. Chi-square test for the studied F₂ population confirmed the segregation ratio of 3:1, meaning that the wilt resistance in lentil was under the control of monogenic dominant gene (Table 1). The 120 F_2:3 progeny-rows were expressed as 34 non-segregating wilt-resistant plant progeny row, 58 heterozygote segregating for wilt resistance/susceptibility and 28 non-segregating susceptible plant progeny-rows in 1:2:1 ratio (c2= 0.50; P-Value is 0.779). The results were in conformity with the previous reports of Eujayl et al., (1998) and Hamwieh et al., (2005) in which single dominant gene control of rust resistance have been reported.



On the similar note, Abbas (1995) also reported single dominant gene control of wilt resistance in the crosses studied at ICARDA, Syria; while Kamboj et al., (1990), reported five independently segregating genes controlling wilt resistance in Indian germplasms.

Mapping of wilt resistance gene in ILL10965

This study was conducted to tag/map the gene(s) controlling resistance to Fusarium wilt in lentil using SSR markers. For tagging/mapping wilt resistance gene F₂ population was used and its homozygosity for wilt resistance gene was determined by screening F_2:3 progeny rows against the wilt disease in the well-established sick-plot at Sehore (India). Ten plants each of the non-segregating resistant and susceptible plant progeny rows were used for development of resistant and susceptible bulks for the bulk segregant analysis. For the parental polymorphism survey, a total of 212 SSRs markers have been used of which 29 SSRs exhibited polymorphism between parental lines L9-12 and ILL10965. These markers were then used to study the polymorphism between the resistant and susceptible bulks of F₂ population using BSA. The BSA identified three SSRs namely PBALC233, PBALC1409 and PBALC203 which discriminated the two extreme bulks viz resistant and susceptible (Table 2). These three bulk discriminating primers were then used to screen the entire F₂ population consisting of 120 individuals. The amplification profile of parents and individuals of resistant and susceptible bulks of F2 population of wilt is presented in Fig 1. The data with respect to segregation of individual marker locus are presented in Table 3. All the three markers which differentiated the bulk, showed goodness of fit with the expected 1:2:1 ratio. Similar segregation of markers was also observed by Dikshit et al., (2016) in lentil and Chaithanya et al., (2011) in pigeonpea.







The data generated was analyzed using Map Maker_ver.3.0. The resistance gene was found flanked by SSR markers, PBALC203 and PBALC1409 at distance of 8.2 cM and 9.4 cM respectively. The map of wilt resistant locus with linked SSR markers is presented as Fig 2. Eujayl et al., (1998) reported a RAPD marker (OPK-15900) linked with the Fw locus at a distance of 10.8 cM on LG6. Similarly, Halila et al., (2009) has been mapped gene conferring fusarium wilt resistance for Fusarium oxysporum f. sp ciceris race 0, to linkage group 2 (LG2) of the chickpea genetic map.



Further, Hamwieh et al., (2005) also localized the Fusarium wilt resistance gene on LG 6 which was found flanked by SSR59-2B and p17m30710 (AFLP marker) at a distance of 8.0 cM and 3.5 cM, respectively. In recent times, a number of relatively dense linkage maps have been reported (Gupta et al., 2012; Saha et al., 2013; Kaur et al., 2014) which can assist in the execution of more precise marker assisted breeding for wilt disease resistance in lentil.

Linkage analysis revealed that the resistance gene Fw was flanked by SSR markers, PBALC203 and PBALC1409 at distance of 8.2 cM and 9.4 cM respectively. Further, PBLAC233 was also found present on the same linkage group at a distance of 10.2 cM from PBLAC1409. Identified molecular markers (PBALC 233, PBALC1409 and PBALC 203) linked to wilt resistant loci in lentil after validation can be used for transfer of the wilt resistance gene into agronomically superior but wilt susceptible cultivars. The information about the genetics of Fusarium wilt disease resistance is of immense use in lentil breeding programme intended to develop wilt resistant varieties. The linked SSRs identified in this study is expected to aid Fusarium wilt resistance breeding in lentil by incorporating gene(s) in a short span of time.