Comparative transcriptome analysis, unfolding the pathways regulating the seed-size trait in cultivated lentil (Lens culinaris Medik.)

Abstract

Market class, cooking time, quality, and milled grain yield are largely influenced by the seed size and shape of the lentil (Lens culinaris Medik.); thus, they are considered to be important quality traits. To unfold the pathways regulating seed size in lentils, a transcriptomic approach was performed using large-seeded (L4602) and small-seeded (L830) genotypes. The study has generated nearly 375 million high-quality reads, of which 98.70% were properly aligned to the reference genome. Among biological replicates, very high similarity in fragments per kilobase of exon per million mapped fragments values (R > 0.9) showed the consistency of RNA-seq results. Various differentially expressed genes associated mainly with the hormone signaling and cell division pathways, transcription factors, kinases, etc. were identified as having a role in cell expansion and seed growth. A total of 106,996 unigenes were used for differential expression (DE) analysis. String analysis identified various modules having certain key proteins like Ser/Thr protein kinase, seed storage protein, DNA-binding protein, microtubule-associated protein, etc. In addition, some growth and cell division–related micro-RNAs like miR3457 (cell wall formation), miR1440 (cell proliferation and cell cycles), and miR1533 (biosynthesis of plant hormones) were identified as having a role in seed size determination. Using RNA-seq data, 5254 EST-SSR primers were generated as a source for future studies aiming for the identification of linked markers. In silico validation using Genevestigator® was done for the Ser/Thr protein kinase, ethylene response factor, and Myb transcription factor genes. It is of interest that the xyloglucan endotransglucosylase gene was found differentially regulated, suggesting their role during seed development; however, at maturity, no significant differences were recorded for various cell wall parameters including cellulose, lignin, and xylose content. This is the first report on lentils that has unfolded the key seed size regulating pathways and unveiled a theoretical way for the development of lentil genotypes having customized seed sizes.

Morpho-biochemical characterization of a RIL population for seed parameters and identification of candidate genes regulating seed size trait in lentil (Lens culinaris Medik.)

“…The related species sequence similarity showed maximum similarity with Medicago truncatula and was followed by Cicer arietinum ( Figure S7 ). To identify the candidate genes regulating small seed size trait near this marker, we checked RNA Seq data generated by us using the same parental combinations ( Dutta et al., 2022 ) and the physical chromosomal details available at CDC Redberry Genome Assembly v2.0 ( Ramsay et al., 2021 ). Using KnowPulse browser, on the left side (0.6 Mb region) of the PBALC449 amplified region, three candidate genes namely, E3 Ubiquitin ligase (log2FC -1.582), TIFY-like protein, and hexosyltransferase gene (log2FC -2.474); while on the right side (in 0.7 Mb region), a ubiquitin carboxyl-terminal hydrolase gene was found ( Ramsay et al., 2021 ).…”

“…Further, to identify the candidate genes regulating small seed size trait near this marker (1.4 Mb region), we analyzed our RNA Seq data ( Dutta et al., 2022 ). On the left side of the PBALC449 amplified region (0.6 Mb), three genes namely, E3 ubiquitin ligase (log2FC -1.582), hexosyltransferase (log2FC -2.474), and TIFY-like protein gene were found, while on the right side (0.7 Mb) a ubiquitin carboxyl-terminal hydrolase gene was found ( Figure 6 ).…”

“…Seed size and shape are regulated by the cell wall composition in lentils ( Dutta et al., 2022 ). However, no other detailed report mentioning the relationship between the seed size and cell wall composition including cellulose, lignin, and xylose in lentils is known.…”

“…Generally, microsperma types are more common in southeast Asia, while macrosperma types in western Asia and Europe ( Barulina, 1930 ). Previous genetic studies revealed large variations for seed weight and seed diameter in lentils ( Tullu et al., 2001 ; Dutta et al., 2022 ; Tripathi et al., 2022 ).…”

“…However, these are laborious methods, especially when a large number of genotypes are involved in screening. Recently, Dutta et al. (2022) used a very easy method involving VideometerLab 4.0 instrument for the measurement of various seed parameters in lentils.…”