Transferability of Simple Sequence Repeat Markers from other Members of Family Fabaceae to Chickpea (Cicer arietinum L.)

Chickpea (C. arietinum L.) is the third most important food legume in the world. With advent of marker technologies, DNA-based markers are being used frequently in crop improvement (Reddy et al., 2021) wherein marker linked tightly to gene of interest is being used to select the progeny plants rather than phenotype, a technique called marker assisted selection. The SSR markers are the markers of choice in chickpea for gene mapping (Barmukh et al., 2021) and marker assisted selection (Kosgei et al., 2022) owing to co-dominant nature and high reproducibility (Gupta and Gopalakrishna, 2010). The marker assisted selection in this crop is, however, limited due to paucity of polymorphism (Sharma and Muehlbauer, 2007; Gaur et al., 2012). Following sequencing of genome of chickpea (Varshney et al., 2014, Jain et al., 2013), several new microsatellite markers were generated. The majority of SSR markers developed so far (70%) are however, monomorphic (Jhanwar et al., 2012) and narrow genetic base of chickpea is the reason for limited SSR polymorphism. Low polymorphism limits the use of SSR markers in chickpea genotyping (Amina et al., 2020; Nayak et al., 2010; Gaur et al., 2012; Gujaria et al., 2011; Choudhary et al., 2012; Suman et al., 2018) due to which scientific community resorted to single nucleotide polymorphism (SNP) as an alternative to SSRs (Gaur et al., 2020). Since, SSR marker technology is within the reach of ordinary laboratories, it is highly desirable to develop new SSR markers for chickpea, so that genetic mapping of genes including quantitative trait loci (Jha et al., 2021) and marker assisted selection (Henkrar and Udupa, 2020) can become a routine procedure as is the case in crops like rice (Courtois et al., 2000).
       
Development of SSR markers is, however, expensive and time consuming process which limits the development of new markers (Gutierrez et al., 2005). A cheaper alternative to SSR marker development is cross-transferability from closely related genera and species usually termed as cross-genera and cross-species transferability (Scott et al., 2000; Zhang et al., 2005).SSR marker’s transferability within taxa of family Fabaceae is well documented (Raghu et al., 2021; Hou et al., 2012; Gupta and Gopalakrishna, 2010; Bakir, 2019; Choudhary et al., 2009).
               
The aim of the present study is to add new SSR markers to repository of already existing markers in chickpea by cross-transferability from other legume genera. In the present study, we report transferability of SSR markers from three genera of family Fabaceae viz., horsegram (Macrotyloma uniflorum), lentil (Lens culinaris) and pea (Pisum sativum) to chickpea (Cicer arietinum). The study added new SSR markers in chickpea that can be used in molecular breeding, germplasm characterization, mapping and comparative genomics in chickpea.

The present investigation was carried out at the Department of Agricultural Biotechnology, CSKHPKV, Palampur during the year 2018-20.
 
Plant material
 
Cross-transferability of SSR markers from lentil, pea and horsegram was estimated on a panel of four Cicer arietinum genotypes viz., GPF2, ICC16349, ICC10685 and ICC15614. Of these four genotypes, GPF2 is cold sensitive, ICC16349 is cold tolerant, ICC10685 is heat sensitive and ICC15614 is heat tolerant. The seeds were sown in 10² diameter pots filled with standard potting mixture (Soil: Sand: FYM:: 1: 1: 1). At 3-4 leaf stage, a small amount of leaf tissue from each genotype was harvested and transported immediately in ice to lab for DNA extraction.
 
DNA isolation
 
Genomic DNA was extracted using the CTAB method (Murray and Thompson, 1980).
 
Primers
 
A total of 292 SSR primers were used. Out of these, 94 were from horsegram (Macrotyloma uniflorum, Kaldate et al., 2017), 66 from lentil (Lens culinaris, Saha et al., 2010)  and 132 were from pea (Pisum sativum, Loridon et al., 2005).
 
PCR amplification
 
The primers from horsegram, lentil and pea were used to amplify genomic DNA of four genotypes of chickpea i.e. GPF2, ICC16349, ICC10685 and ICC15614. For polymerase chain reaction (PCR) assay, 10 μl PCR reaction mixture i.e. 6.7 μl PCR water, 1 μl 10X Taq buffer, 1.2 μl DNA (20 ng/μl), 0.3 μl dNTPs (2 mM), 0.2 μl DNA polymerase (1 U/μl), 0.3 μl of each forward and reverse primer was prepared. PCR profile was followed with initial denaturation of 5 min at 95^oC; 35 cycles with denaturation at 94^oC for 30 seconds, annealing temperature depending on primer used for 30 seconds, followed by extension at 72^oC for 1 min; and a final extension at 72^oC for 8 mins. The amplification products were resolved on 3% agarose gel (0.5X TAE Buffer) using gel electrophoresis system at 100V for 2 hours and amplified products were stained with ethidium bromide (0.5 μg/ml). Gel documentation system (Biorad, USA) was used to visualize the amplified products. The size of the amplicons was estimated by 100 bp ladder (Sigma-Aldrich). The primers which showed clear amplicons were identified. Transferability percentage and percentage polymorphism were calculated as:

Transferability of horsegram SSRs to chickpea
 
Out of 94 horse gram primers used in the present study, 14 amplified genomic DNA of chickpea with percent transferability of 14.89% (Table 1). Of these, only four were polymorphic (percentage polymorphism 4.25%). Transferable SSR markers were: MUGR601, MUGR608, MUGR613, MUGR614, MUGR621, MUGR622, MUGR623, MUGR624, MUGR625, MUGR630, MUGR635, MUGR645, MUGR646 and MGR-23. Among polymorphic markers, MUGR601 showed amplicons in all the genotypes and polymorphism was due to variable length of amplification product (Fig 1a). In other three polymorphic markers, viz., MUGR613, MUGR630 and MGR-23, polymorphism was due to presence or absence of bands in genotypes suggesting differences in SSR flanking sites in chickpea and horsegram. Among the three, MUGR613 showed amplification only at a single locus in GPF2 and ICC16349, MUGR630 in GPF2, ICC16349 and ICC15614 whereas, MUG-23 showed amplification only in GPF2 (Fig 1a). While transferability of horsegram SSR markers to chickpea was lower in present study i.e. 14.89% (Raghu et al., 2021, Sharma et al., 2015), the transferability of chickpea SSR markers to horsegram was also reported to be relatively higher (Jingade et al., 2014, Kaldate et al., 2017).
 

 

 
Transferability of lentil SSRs to chickpea
 
Of 66 lentil SSR primers tested for their transferability to  chickpea, 24 (36.36%) displayed amplification and seven (10.61%) showed polymorphism. The transferable primers were: L-48-4, L-48-6, L-48-7, L-48-9, L-48-13, L-48-14, L-48-18, L-48-22, L-48-24, L-48-25, L-48-26, L-48-27, L-48-28, L-48-31, L-48-32, L-48-33, L-48-34, L-48-35, L-48-36, L-48-37, L-48-39, L-48-41, L-48-42 and L-48-45 (Table 2). Polymorphism by three markers i.e. L-48-27, L-48-28 and L-48-22 was due to amplicon size variation and in remaining four, viz., L-48-7, L-48-13, L-48-24 and L-48-25, it was due to presence or absence of amplification in some genotypes. Primer L-48-7 showed amplification only at a single locus in genotypes GPF2, ICC16349 and ICC10685, L-48-13 showed amplification in ICC16349 and ICC10685, L-48-24 showed amplification in ICC16349, ICC10685 and ICC15614, L-48-25 showed amplification in GPF2 and ICC15614 (Fig 1b). Transferability of lentil SSRs to chickpea was highest (36.36%) among the three genera tested. Higher transferability rates of lentil SSR markers to chickpea (Bakir, 2019) and vice-versa (Agarwal et al., 2008; Rana et al., 2004; Choudhary et al., 2009) were also reported suggesting that lentil genomic resources can be exploited effectively to add new SSR markers to chickpea genome.
 

 
Transferability of pea SSRs to chickpea
 
Twenty four pea SSR primers out of one hundred thirty two were transferable to chickpea (Table 3) with per cent transferability rate of 18.18%. Transferable markers were: P-18391, P-16758, P-16534, P-16452, AC-17, AD81, AF109922, PSU81287, CHPSCPA1, P-16208, PS1AA6D, P-16697, P-17056, P-17181, P-17560, P-17122, P-17526, P-18341, P-18542, P-18938, P-18781, P-18702, P-17684 and AB53. Among transferable markers, five (PS1AA6D, AF109922, P-17560, P-17526, P-18781) were polymorphic (3.79% polymorphism). Of these five, PS1AA6D produced amplicons of variable sizes in the panel of four genotypes. Rest four polymorphic markers showed dominant polymorphism i.e. presence or absence of amplified products. Primer AF109922 showed amplification in genotypes ICC10685 and ICC15614. P-17560 showed amplification only in one genotype i.e. ICC16349, P-17526 showed amplification in ICC16349, ICC10685 and ICC15614, P-18781 showed amplification in genotypes ICC16349 and ICC10685 (Fig 1c). SSR marker transferability rates from pea to chickpea were in conformity with an earlier study (Pandian et al., 2000) whereas higher rates were also observed (Mishra et al., 2012; Gangadhar et al., 2016).
 

               
Findings of the present study are summarized in Table 4. Lentil SSR markers showed highest transferability (36.36%) to chickpea which is comparable to an earlier study on legumes (Raghu et al., 2021). SSR markers of lentil also showed maximum polymorphism in chickpea as compared to horsegram and pea SSRs. This high polymorphism in chickpea may again be attributed to higher evolutionary similarity between lentil and chickpea (Pandian et al., 2000). Limited polymorphism in chickpea by the SSR markers transferred from lentil as observed by us was also reported earlier (Bakir, 2019; Amina et al., 2020). Lack of variability in chickpea was further proved by the fact that even the intra-specific SSR markers showed limited polymorphism in chickpea (Nayak et al., 2010; Hiremath et al., 2011; Gaur et al., 2012; Gujaria et al., 2011; Choudhary et al., 2012). Hence, the polymorphism revealed in chickpea by lentil SSR markers, though, appears to be low, can be considered adequate in the context of chickpea.
 

The study added 62 new SSR markers to the existing chickpea genomic resources from three legumes lentil, horsegram and pea. Presence of few polymorphic markers in chickpea suggests that lower polymorphism remains a major bottleneck for marker assisted breeding in chickpea owing to narrow genetic base. The study further revealed higher SSR marker transferability from lentil to chickpea. Hence, emphasis should be on the exploitation of lentil genomic resources and all lentil SSR markers must be screened in chickpea to add more markers to the repository of existing chickpea markers. The new SSR markers are expected to contribute in molecular breeding, gene cloning, germplasm characterization and comparative genomics in chickpea.

None.