The present study investigated the linkage between days to flowering (DTF) and Growth Habit (GH) in pigeonpea using QTL mapping, QTL-seq, and GWAS approaches. The linkage map developed here is the largest to date, spanning 1825.56 cM with 7987 SNP markers. In total, eight and four QTLs were mapped for DTF and GH, respectively, harboring 78 pigeonpea orthologs of Arabidopsis flowering time genes. Corroboratively, QTL-seq analysis identified a single linked QTL for both traits on chromosome 3, possessing 15 genes with genic variants. Together, these 91 genes were clustered primarily into autonomous, photoperiod, and epigenetic pathways. Further, we identified 39 associations for DTF and 111 associations for GH through GWAS in the QTL regions. Of these, nine associations were consistent and constituted nine haplotypes (five late, two early, one each for super-early and medium duration). The involvement of multiple genes explained the range of allelic effects and the presence of multiple LD blocks. Further, the linked QTL on chromosome 3 was fine-mapped to the 0.24 Mb region with a LOD score of 8.56, explaining 36.47% of the phenotypic variance. We identified a 10 bp deletion in the first exon of TFL1 gene of the ICPL20338 variety, which may affect its interaction with the Apetala1 and Leafy genes, resulting in determinate GH and early flowering. Further, the genic marker developed for the deletion in the TFL1 gene could be utilized as a foreground marker in marker-assisted breeding programs to develop early-flowering pigeonpea varieties.

Pigeonpea is one of the climate-resilient grain legumes. Though the optimum temperature for cultivated pigeonpea is ~ 25–35 °C, its wild relatives grow in temperatures ranging between 18 and 45 °C. To gain insight into molecular mechanisms responsible for the heat stress tolerance in pigeonpea, we conducted time-series transcriptome analysis of one pigeonpea cultivar (Cajanus cajan) and two wild relatives, Cajanus acutifolius, and Cajanus scarabaeoides subjected to heat stress at 42 ± 2 ºC for 30 min and 3 h. A total of 9521, 12,447, and 5282 identified transcripts were differentially expressed in C. cajan, C. acutifolius, and C. scarabaeoides, respectively. In this study, we observed that a significant number of genes undergo alternative splicing in a species-specific pattern during heat stress. Gene expression profiling analysis, histochemical assay, chlorophyll content, and electrolyte leakage assay showed that C. scarabaeoides has adaptive features for heat stress tolerance. The gene set enrichment analyses of differentially expressed genes in these Cajanus species during heat stress revealed that oxidoreductase activity, transcription factor activity, oxygen-evolving complex, photosystem-II, thylakoid, phenylpropanoid biosynthetic process, secondary metabolic process, and flavonoid biosynthetic process were highly affected. The histochemical assay showed more lipid peroxidation in C. acutifolius compared to other Cajanus species inferring the presence of higher quantities of polyunsaturated fatty acids in the plasma membrane which might have led to severe damage of membrane-bound organelles like chloroplast, and high electrolyte leakage during heat stress. This study paves the way for the identification of candidate genes, which can be useful for the development of thermo-tolerant pigeonpea cultivars.