Identification of Heat-Related ESTs in Moth Bean Through Suppression Subtraction Hybridization

Abstract

Moth bean (Vigna aconitifolia (Jacq.) Marechal), an important grain-legume crop grown in hot desert regions of Thar, under scorching sun rays, was investigated for heat tolerance at molecular level. In the present study, we constructed a forward suppression subtractive hybridization (SSH) cDNA library of heat tolerant genotype RMO-40 to identify genes expressing under delayed response to elevated temperature. Heat induction was carried out by exposing 14-day-old seedlings to elevated temperature of 42 °C for 30 min. A total of 125 unigenes (33 contigs and 92 singletons) were derived by cluster assembly and sequence alignment of 200 ESTs; out of 125 unigenes, 21 (16 %) were found to be novel to moth bean. Gene ontology functional classification terms were retrieved for 98 (78.4 %) unigenes of which 73 (58.4 %) ESTs were functionally annotated (GO consensus) where 19 unigenes were annotated with 11 enzyme commission (EC) codes and were mapped to 25 different KEGG pathways. We have identified a majority of heat-shock proteins (constituting 35 % of the present library) aiding heat stress tolerance to moth bean. An expression level of 22 ESTs generated from the above SSH cDNA library was studied through semiquantitative RT-PCR assay simultaneously under 5 and 30 min of heat stress at 42 °C.

Physiological, biochemical, and molecular responses of thermotolerance in moth bean (Vignaaconitifolia (Jacq.) Marechal)

“…A similar pattern of gene expression was observed in tolerant and susceptible varieties of rice (Chen et al, 2014). The expression of HSPs is also observed in the moth bean heattolerant genotype RMO 40 under heat stress (42 °C for 30 min) (Gurjar et al, 2014) and these studies also validated the present observation.…”

“…However, the development of stress-tolerant varieties and their inclusion in crop improvement and breeding programs might be an optimistic approach. The genotypes RMO 40 and Jadia were considered as heattolerant in earlier studies (Gurjar et al, 2014;Sharma et al, 2014;Harsh et al, 2016), although RMO 40 was reported to be drought susceptible by Sachdeva et al (2016). Therefore, the genotypes with improved resistance and better performance under particular stress conditions need to be selected and studied for potentially important traits for use in breeding programs of moth bean.…”

Heat stress transcripts, differential expression, and profiling of heat stress tolerant gene TaHsp90 in Indian wheat (Triticum aestivum L.) cv C306

“…C306. Secondary amplification of cDNA synthesis was visualized as a smear in the range of 100 bp to 1.5 kb on 1.2% agarose gel which indicated successful library preparation, and this result was in uniformity with Gurjar et al [ 11 ]. For identifying and enriching differentially expressed genes under HS in wheat cv.…”

“…For identifying and enriching differentially expressed genes under HS in wheat cv. C306, SSH has been an efficient approach in various crops [ 11 – 13 ]. We constructed two HS responsive forward SSH libraries (37°C HS and 42°C HS) at post anthesis stage in heat tolerant bread wheat cv.…”

“…It is a low cost alternative to RNA sequencing methods for identifying differentially expressed, novel and/or uncharacterized genes/ESTs, even in organisms where sequence information available is only limited. Though SSH suffers from generation of low data resulting in poor genome coverage, yet it still continues to be one of the most popular technique for generating differentially expressed ESTs in any organism [ 11 , 12 , 13 ].…”