QTL mapping for heat stress tolerance in chickpea (Cicer arietinum L.)

Soybean [Glycine max (L.) Merrill] is the world’s most important seed legume, which contributes about 25 per cent of the global edible oil (Rai et al., 2016). It is commonly known as a wonder crop (Husain and Shrivastav, 2011), cow of the field, gold from soil and golden bean (Horvath, 1926) belongs to the family Leguminacae and derived from a wild progenitor Glycine ussuriensi (Robert, 2010). Sub species of soybean are Glycine gracilis and Glycine soja  they are generally considered as the undomesticated progenitors of the domesticated soybean (Glycine max) (Chen and Nelson, 2004). Cultivated soybean has a genome size of 1.1 to 1.15 Gb with chromosome pair of twenty (2n=40).
 
The remarkable progress made in plant genetic resource management in recent days has resulted in the collection of a huge set of plant germplasm that hinders the very purpose for which they exist (Odong et al., 2013). Frankel (1984) proposed the concept of core collection that could be established from an existing collection for better management and utilization of plant genetic resources. Characterization of a core set is an efficient approach for exploring and capturing the genetic diversity of large populations, as it represents the maximum genetic diversity of the whole collection and become a powerful tool for evaluation and identification of trait-specific accessions in germplasm (Gireesh et al., 2015). Therefore, an investigation was carried out to assess the variability for morpho-metric traits among germplasm accessions conserved at the University of Agricultural Sciences (UAS), Bangalore which in turn helps in understanding the limitations of the domesticated germplasm and potential use of its wild relatives in crop improvement. 

Phenotyping
 
DCP 92-3 a medium to late maturity chickpea variety was used as female parent and ICCV 92944 a heat tolerant genotype (Gaur et al., 2012) was used as male parent for developing F₁ progenies. A total of 206 individuals were obtained from the given cross in F₂. The F₂ individuals were evaluated for various breeding and physiological traits under HS by sowing them in late condition in the first week of January in the year 2016-17 under field condition in augmented design. Data on twelve different traits of breeding importance and phenological traits such as primary branch (PB), plant height (PH), days to first flowering (FF), days to pod initiation (DPI), days to pod filling (DPF), days to maturity (DM), total pods/plant (TPP), empty pods/plant (EP), yield/plant (YPP), 100 seed weight (100 SW), biological yield (BY), and harvest index (HI) were taken. Likewise, data on five traits of physiological interest such as nitrogen balance index (NBI), chlorophyll content (CHL), flavonoid content (FLV), anthocyanin content (ANTH) and membrane stability index (MSI) were taken under HS condition. These physiological traits were measured by leaf spectrometer instrument. General statistics for the traits were estimated by ‘Analyse it’ software.
 
Genotyping and construction of genetic linkage map
 
Genomic DNA was extracted from the parental genotypes and 206 F₂ individuals at seedling stage following CTAB method (Doyle and Doyle 1987). The PCR assay was carried out in a10 μl reaction mixture containing 5.9 μl of sterilized distilled water, 1.00 μl template DNA (25 ng), 0.5 μl of forward and 0.5 μl of reverse primer (5 μM), 1.00 μl 10 × PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl), 1.00 μl dNTP mix (0.2 mM each of dATP, dGTP, dCTP and dTTP) and 0.1 μl Taq polymerase (5U/ μl) (Thermo Fisher Scientific Mumbai, India, Pvt. Ltd.) by using G-40402 thermo cycler (G-STORM, Somerset, UK). A touch down PCR profile was performed for amplifications with initial denaturation at 94°C for 5 min followed by 10 cycles of touch down 61-51°C, 30 s at 94°C, annealing for 30 s at 61°C (the annealing temperature for each cycle being reduced by 1°C per cycle) and extension for 30 s at 72°C. This was accompanied by 40 cycle of denaturation at 94°C for 30 s, annealing at 51°C for 30 s, elongation at 72°C for 45 s, and 10 min of final extension at 72°C. Amplified fragments were resolved in 3% agarose gel using 0.5 × TBE running buffer and images were analyzed with Quantity one software (Bio-Rad, CA 94547, USA).  A total of   450 SSR markers reported by (Winter et al., 1999; Udupa et al., 1999; Sethy et al., 2003, Sethy et al., 2006; Choudhary et al., 2009; Choudhary et al., 2012; Gaur et al., 2011) were used for checking parental polymorphism.  Among the given SSRs only 78 SSRs yielded polymorphic fragments and these were used for genotyping of 206 F₂ individuals. To measure segregation pattern of each SSR marker against the expected ratio of 1:2:1 at 0.01 probability level Chi square test was performed. Linkage analysis was done by Join Map version 4.0 (Van Ooijen, 2006) at critical LOD scores of 3 between two markers. For identification of QTL, Win QTL cartographer 2.5 (Wang et al., 2010) was used. Composite interval mapping, with LOD score of 2.5 and Kosambi mapping function (Kosambi, 1944) was used for detection of QTLs for various breeding and physiological traits under HS condition.

Genetic variability for different traits
 
A wide range of genetic variability for various traits of both breeding and phonological importance was recorded in the parental genotypes ( Table 1) and the derived F₂ mapping population evaluated under late sown condition given in (Table 2). Importantly, considering various heat stress tolerant indicative physiological traits viz., NBI (Li et al., 2015) representing the ratio of chlorophyll to polyphenols that accurately measure canopy nitrogen concentrations, CHL, MSI, FLV and ANTH, a wide range of genetic variability was captured (Table 2). Likewise, significant genetic variability for various phenological and yield related traits in chickpea mapping population has been reported under HS (Paul et al., 2018a, 2018b).
 

 

 
QTL mapping
 
In order to elucidate genetic basis of heat tolerance, several mapping population segregating for various HS relevant traits have been reported in various crops including rice (Ye et al., 2012, Lei et al., 2013), wheat (Mason et al., 2011, Bennett et al., 2012, Paliwal et al., 2012), maize (Frova and Sari-Gorla, 1993). However, limited mapping populations have been developed for elucidating HS tolerance in chickpea (Paul et al., 2018a, 2018b). In the current study, a total of 78 SSR markers were assayed in the above F₂ mapping population. Out of 78 SSR markers only 39 markers showed the Mendelian segregation pattern of 1:2:1. And these SSR markers were assigned to all LG groups except LG8, covering 859 cM map distance. Two QTLs (Table 3), one minor QTL for PB flanked by TA142 and ICCM281a markers, with LOD value >3 (Fig 1 and Fig 2) explaining 2% phenotypic variation (PV) on LG3 and the other QTL  contributing in CHL content flanked by NCPGR206 and H3G031 with LOD value >3(Fig 3 and Fig 4) explaining 17.2% PV on LG6 were identified. Similarly QTLs controlling number of primary branches an important yield contributing trait have been reported on LG1 (Saxena et al., 2014) and on LG8 (Kale et al., 2015) under drought stress. While for chlorophyll content, five SRAP markers associated to chlorophyll content QTL with PV up to 53% was elucidated by Elshafei et al., (2013) in wheat under drought stress condition. Likewise, Talukdar et al., (2014) identified seven QTLs explaining PV up to 30.8% on chromosomes 6A, 7A, 1B and 1D in wheat for SCMR chlorophyll content under HS. Considering yield related traits viz., filled pods/plant, Paul et al., (2018b) mapped qfpod02_5 (explaining 11.57% PV) and qfpod03_6 (explaining 6.56 % PV) QTLs on Ca LG5 and Ca LG6 respectively in chickpea by employing genotyping by sequencing in ICC15614 × ICC 4567 mapping population in chickpea under HS. They also mapped another two QTLs qgy02_5 on CaLG05 and qgy03_6 on CaLG06 contributing in grain yield under HS. Thus, in the present study the investigated genomic regions could be further fine mapped for underpinning the potential candidate gene(s) contributing in HS tolerance in chickpea.
 

 

 

 

 

The present study provides preliminary information on number of primary branches an important yield contributing trait and chlorophyll content related to HS tolerance controlling genomic regions that could be fine mapped in future. Thus these genomic regions need further detailed research for utilizing them in marker assisted breeding programme for developing heat tolerant chickpea genotype.

Authors acknowledge support from Indian Council of Agricultural Research (ICAR), India. The work is based on PhD thesis of the first author. The authors also thank Dr. Swarup K. Parida, NIPGR, New Delhi, for providing SSR markers and Dr. Mahender Thudi, from ICRISAT, Patancheru, India for linkage map analysis.