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A survey for QTL-linked markers revealed ten SNPs from nine loci within the QTL region (Table 7.2). The contig and scaffold sequences underpinning the QTL region were annotated by BLASTx similarity search against protein sequences of species of the Fabaceae family, returning 659 matches with an E-value < 1e-20 and percent identities ranging between 45.42 and 99.10% (Appendix 7.2). Among them, the top five annotations for each of the loci were filtered based on the highest percent identity and highest bit score. One best matching and fully characterised putative candidate gene was selected from the top five annotations for each of the nine loci (Table 7.3).

Post-confirmation of the SNP location on the CDS of each of the nine candidate loci, five markers were found positioned on the CDS and four other markers on the UTR of the putative candidate genes (Table 7.3). A comparison of the five SNPs located on the CDS with the reference protein sequence of the candidate genes revealed three non-synonymous and two synonymous mutations. The three non-synonymous mutations belonged to the putative candidate genes that are associated with stress response such as uroporphyrinogen decarboxylase (UROD; XP_003601037.1), glutathione-S-transferase DHAR3, chloroplastic (GST-DHAR3; XP_013460594.1) and protein EXECUTER 2, chloroplastic isoform X1 (P- EXE2; XP_003601283.1). The effects of the three non-synonymous mutations revealed substitution of an amino acid with the other (Threonine (T) 35 Alanine (A); Phenylalanine (F) 13 Leucine (L); Valine (V) 387 Isoleucine (I)) at specific positions on the coding sequence of the putative candidate genes. An investigation for the possible effects of the mutations on the 3D structure of putative proteins revealed that mutations T35A and F13L may affect the domain and substrate binding sites of the proteins UROD and GST-DHAR3, respectively (Figure 7.7). However, no relative crystal structure was identified for the protein P-EXE2 within the CDD and PDB of NCBI.

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Table 7.2: Details of markers and corresponding loci identified within the QTL region on linkage group 5

Marker Locus Position

(cM) SNP position REF allele ALT allele ILL 6002 ILWL 180 Scaffold14200_Locus_14227_0_59.0_FORK_762 Scaffold14200_Locus_14227_0_59.0_FORK 31.50977 762 T C 0/0 1/1 C205172_60.0_311 C205172_60.0 34.67857 311 C T 0/0 1/1 C210460_55.0_1836 C210460_55.0 35.24678 1836 G A 0/0 1/1 C199840_59.0_1368 C199840_59.0 35.24678 1368 G T 0/0 1/1 C196012_59.0_549 C196012_59.0 35.24678 549 C T 0/0 1/1 C196012_59.0_1181 C196012_59.0 35.24678 1181 A G 0/0 1/1 C197180_56.0_678 C197180_56.0 35.84922 678 A G 0/0 1/1 C207980_54.0_1302 C207980_54.0 35.84922 1302 T G 0/0 1/1 Scaffold25376_Locus_46472_0_39.2_LINEAR_2694 Scaffold25376_Locus_46472_0_39.2_LINEAR 35.84922 2694 C G 0/0 1/1 Scaffold20706_Locus_28526_0_45.3_LINEAR_1458 Scaffold20706_Locus_28526_0_45.3_LINEAR 36.44449 1458 G A 0/0 1/1

127 Table 7.3: Details of putative candidate genes and corresponding SNP effects

Locus SNP location SNP effect Candidate gene Accession number

Scaffold14200_Locus_14227_0_59.0_FORK cds_2 Non-Synonymous (T35A) uroporphyrinogen decarboxylase [Medicago

truncatula] XP_003601037.1

C205172_60.0 5'UTR N/A sedoheptulose-1,7-bisphosphatase,

chloroplastic [Medicago truncatula] XP_003600853.1

C210460_55.0 3'UTR N/A probable E3 ubiquitin-protein ligase RHC2A

[Medicago truncatula] XP_013460694.1

C199840_59.0 3'UTR N/A cytochrome c-type biogenesis ccda-like

chloroplastic protein [Medicago truncatula] XP_013445560.1

C196012_59.0 cds_3 Synonymous glutathione S-transferase DHAR3,

chloroplastic [Medicago truncatula] XP_013460594.1 C196012_59.0 cds_2 Non-Synonymous (F13L) glutathione S-transferase DHAR3,

chloroplastic [Medicago truncatula] XP_013460594.1

C197180_56.0 cds_2 Synonymous ribonucleoprotein [Pisum sativum] CAA74889.1

C207980_54.0 cds_3 Synonymous 60S ribosomal export protein nmd3-like

[Trifolium pratense] PNX76336.1 Scaffold25376_Locus_46472_0_39.2_LINEAR 3'UTR N/A anthranilate phosphoribosyltransferase,

chloroplastic [Medicago truncatula] XP_003601245.1 Scaffold20706_Locus_28526_0_45.3_LINEAR cds_3 Non-Synonymous (V387I) protein EXECUTER 2, chloroplastic isoform

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Figure 7.7: Predicted 3D model structures of proteins a) UROD (PDB ID: 5ECS) and b) GST-DHAR3 (PDB-1J93) from Arabidiopsis thaliana and Nicotinia tabacum, respectively. The green annotations indicate the positions of the mutations within the 3D structures of the proteins.

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7.5. Discussion

In the process of domestication, lentil has been estimated to have lost approximately 40% of its genetic diversity (Alo et al., 2011). Selection and breeding for higher yield and related traits in the cultivated germplasm eventually led to increased genetic similarity (Kumar et al., 2018a).Not surprisingly, breeding programs from around the world have reported limited diversity in their respective cultivated lentil gene pool (Ferguson et al., 1998; Lombardi et al., 2014; Khazaei et al., 2016). Particularly, the diversity of South Asian, Canadian and Australian germplasm is estimated as low (Ford et al., 1997; Lombardi et al., 2014; Khazaei et al., 2016; Kumar et al., 2018a). Therefore, interspecific introgression of new genes of interest such as disease resistance, abiotic stress tolerance and nutrient toxicities such as B is advocated and practiced (Gupta and Sharma, 2007; Tullu et al., 2010a; Bhadauria et al., 2017; Dadu et al., 2017). Moreover, the process is suggested to be fast-tracked through identification of trait- linked markers and marker-assisted breeding (MAS) (Bhadauria et al., 2017).

Lentil has a relatively large genome of approximately 4 Gb (Arumuganathan and Earle, 1991) and hence, genome complexity reduction-based methods were adapted in genomic studies of lentil (Sharpe et al., 2013; Kaur et al., 2014; Bhadauria et al., 2017). Likewise, genotyping of the RIL population in the current study was accessed through transcriptome sequencing, otherwise known as the GBS-t method. Particular advantages of employing a GBS-t approach include cost effective, reduction of the genome complexity, maintaining sufficient resolution of sequence assembly, conservation of gene sequences and SNP identification (Malmberg et al., 2018). Interestingly, 98.5% of reads per sample of the 694 million reads generated through the GBS-t approach in this study were successfully mapped to the reference transcriptome of lentil cultivar Cassab. In addition, a total of 671 of the 815 SNP markers identified within this RIL population were found to be common to the markers generated through transcriptome sequencing of six lentil cultivars (Kaur et al., 2014). These results show the extensive conservation for genes between L. orientalis and L. culinaris as established in previous studies (Wong et al., 2015; Ogutcen et al., 2018).

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The linkage map constructed in this study included 815 markers across eight LGs. The length of the map was shorter (488.02 cM) than many previously published maps in lentil (Eujayl et al., 1998; Rubeena et al., 2003; Duran et al., 2004; Hamwieh et al., 2005; Kahraman and Muehlbauer, 2010; De la Puente et al., 2012; Gupta et al., 2012b; Ates et al., 2016; Bhadauria et al., 2017; Ates et al., 2018b) but was very similar to other recently published maps using GBS-t (432.8 cM) and DArT (497.1 cM) derived SNP markers (Temel et al., 2015; Aldemir et al., 2017). However, as observed in these maps, the mean marker-marker distance of the map constructed was only 0.66 cM and is much shorter compared to the marker intervals of previously published maps that varied from 1.11 cM to 19.3 cM (Eujayl et al., 1998; Rubeena et al., 2003; Duran et al., 2004; Hamwieh et al., 2005; Kahraman and Muehlbauer, 2010; De la Puente et al., 2012; Gupta et al., 2012b; Sharpe et al., 2013; Gujaria-Verma et al., 2014; Ates et al., 2016; Sudheesh et al., 2016a; Bhadauria et al., 2017; Ates et al., 2018b). Nevertheless, there were a few gaps within the linkage map, potentially due to insufficient markers, low polymorphism in the regions with gaps or possibly because of genic markers that were interspersed across intergenic gaps on the genome. Also, this may be because of the under- representation of low expressed genes within the leaf tissue that was used for sequencing (Serin et al., 2017). Similarly, gaps were also evident in the maps previously constructed using GBS- t derived SNP markers (Sharpe et al., 2013; Temel et al., 2015).

QTL analysis identified one QTL on LG5 conferring leaf resistance to AB and, as expected, the resistance allele is contributed by ILWL 180 confirming previous symptomology, physical and biochemical evidences of resistance in this accession (Chapter 4; Chapter 5). Single QTL identification suggests a lack of selection pressure on ILWL 180 (L. orientalis) by A. lentis and thus a limited chance of coevolution (Madrid et al., 2014). Alternatively, potential QTLs might have remained undetected either due to incomplete map coverage or by falling below the statistically significant LOD threshold of 3. For example, two QTLs featured at exactly the same position on LG5 for the leaf lesion score at 14 and 28 dpi, however, failed to score a significant LOD threshold (2.54 and 2.90, respectively). Similarly, a QTL with a LOD score of 2.56 was found on LG3 for stem lesion score at 14 dpi. These QTLs without significant LOD scores are likely contributing minor effects to the cumulative resistance in accession ILWL 180 (Bhadauria et al., 2017).

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As one of the advantages of transcriptome sequencing, identified SNPs are most likely to be associated with the expressed trait (Kaur et al., 2014; Malmberg et al., 2018). Accordingly, ten markers from nine loci underpinning the QTL region were annotated for nine putative candidate genes following a BLASTx search for matching protein sequences. Among the nine loci, seven found best matching sequences from Medicago truncatula, a model species to which a direct relationship has been established with lentil in previous comparative mapping analysis studies (Sharpe et al., 2013; Kaur et al., 2014; Temel et al., 2015; Ates et al., 2018b). Among the nine putative candidate genes that were chosen based on the best matching annotations with highest percent identities and bit scores, five gene families showed evidence of direct involvement in the defence response. These included proteins UROD (XP_003601037.1), E3 ubiquitin-protein ligase RHC2A (E3 UPL-RHC2A; XP_013460694.1), GST–DHAR3 (XP_013460594.1), P-EXE2 (XP_003601283.1) and anthranilate phosphoribosyltransferase, chloroplastic (APRT; XP_003601245.1). Although the rest of the genes are reported to be part of various metabolic pathways and subsequently plant development, these may contribute indirectly to the defences against the pathogen (Miyagawa et al., 2001; Simon and Hederstedt, 2011; Wu et al., 2016; Ostendorp et al., 2017).

Among the defence related proteins putatively involved, UROD is involved in the pathway of chlorophyll biosynthesis and has been characterised to induce biochemical defence responses upon pathogen infection. Reduced activity of UROD in the transgenic tobacco leaves has been reported to result in antioxidative stress responses and necrotic leaf lesions (Mock et al., 1998; Mock et al., 1999). Additionally, UROD was also identified within the QTL region associated with resistance to AB caused by Peyronellaea pinodes in M. truncatula (Madrid et al., 2014). Considering the close relationship between barrel medic and lentil, it may be speculated that the candidate gene regulating the function of UROD in lentil may be an orthologue to the UROD gene of barrel medic. Therefore, a comparative mapping analysis of the linkage map constructed in this study to the genomic sequence of barrel medic may be beneficial. E3 UPL-RHC2A belongs to the family of ubiquitin ligases and is a component of the ubiquitin proteasome pathway, known for its role in plant defence responses (Craig et al., 2009). The role of E3 UPLs from recognition to downstream defence responses and cell death mechanisms is well established in Arabidiopsis thaliana (Bao et al., 2013; Zhou et al., 2015). E3 UPLs were also detected in the transcriptome of A. lentis infected lentil cultivars ILL 7537

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(AB resistant) and ILL 6002 (AB susceptible), however, showed completely opposite patterns of expression (Mustafa et al., 2009; Khorramdelazad et al., 2018). Protein APRT is considered as a branchpoint enzyme in the synthesis of tryptophan, which plays a direct role in plant defence responses through production of secondary metabolites that are anti-fungal such as indole-3-methyl-glucosinolates and indole phytoalexins (Niyogi and Fink, 1992; Asai et al., 2017).

Another defence related gene associated with the QTL region is GST-DHAR3, which belongs to a diverse protein family, gluatathione-S-transferases (GST), and has a role in pathogen-related stress responses such as oxidative stress and detoxification of a variety of compounds including microbial toxins (Ball et al., 2004). GST was up-regulated in lentil-A. lentis interactions (Sambasivam, 2011) as observed in chickpea – Ascochyta rabiei interactions (Fondevilla et al., 2011; Garg et al., 2018). P-EXE2, a plastid protein, enables higher plants to perceive the production of singlet oxygen, a reactive oxygen species (ROS) as a stress signal to activate a genetically determined programmed cell death (PCD) in response to pathogen infection (Lee et al., 2007; Kim et al., 2012). In summary, most of the candidate genes identified underpinning the QTL region on LG5 play a role in antioxidative stress and hypersensitive reaction (HR) or PCD to contain pathogen infection. This confirmed ILWL 180 as a relatively more durable resistance source to A. lentis along with previous evidences of physical and biochemical defence responses expressed during an interaction with A. lentis (Dadu et al., 2018a).

A further analysis of potential effects of SNPs, revealed three non-synonymous mutations causing a single amino acid substitution in the predicted protein sequence of the putative candidate genes including UROD (T35A), GST-DHAR3 (F13L) and P-EXE2 (V387I). These non-synonymous mutations with an amino acid substitution could potentially affect the structure and function of the corresponding protein and thereby determine the resistance of lentil to A. lentis. More recently, a non-synonymous SNP causing an amino acid substitution from arginine to lysine in a leucine rich repeat (LRR) domain was predicted based on a combination of genetic analysis to provide resistance to anthracnose in watermelon (Jang et al., 2018). The mutation of a single glycine to an arginine residue resulted in the drastic modification of the 3D protein structure of an effector AvrLm4-7 secreted by Leptosphaeria

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maculans (causal agent of stem canker disease in Brassica napus), which resulted in the loss of recognition specificity by two resistance genes (Rlm4 and Rlm7) (Blondeau et al., 2015). Likewise, the effects of mutations T35A and F13L are predicted to affect the domain and substrate binding sites of the proteins UROD and GST-DHAR3, respectively, and in turn may affect the structure and functions of the respective proteins as evidenced in other instances (Pham et al., 2011). Although it can be speculated that the origins of mutations may be from either of the parents, it is possible that the mutations might have been induced in ILL 6002 throughout the domestication process of the cultivated species and thus may have weakened the defence response system against A. lentis. However, this hypothesis may need further validation.

In conclusion, the present study has added a large number of SNPs to the long list of existing marker data available to the lentil community. Like others, these SNPs can be potentially useful in various future analyses including genetic characterisation, genetic linkage map analysis and comparative genomics. SNPs generated from transcriptome sequencing were used to construct an interspecific genetic linkage map that contained a QTL conferring resistance to AB on LG5. The markers within the QTL region and the corresponding candidate genes with functional importance in defence responses may permit development of precise diagnostic markers, which may be employed in the marker-assisted breeding (MAB) programs concerning AB resistance. To our knowledge, this is the first report of non-synonymous mutations and corresponding amino acid substitutions identified in lentil in response to A. lentis infection. However, an in-depth study is required to reveal the structural and functional changes of the prospective proteins and subsequently proposing reasons for the phenotype of either of the accessions ILWL 180 or ILL 6002 to AB resistance.

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