Development of molecular markers linked to moisture stress tolerence in onion (Allium cepa L.)

Abstract

Onion is an important vegetable crop in India, having great demand in the domestic and international market. However, onion productivity in tropical countries is strikingly low. Onion is predominantly grown as a rain fed crop hence exposed to moisture stress during different stages of crop growth, resulting in heavy yield losses. At present there is no onion varieties suited for cultivation under rain fed condition. Hence there is need for development of varieties suitable for rain fed conditions. There are several physiological, morphological, biochemical and molecular mechanisms which jointly influence the moisture stress tolerance mechanism operative in a particular crop species. Exploiting the existing genetic variability for WUE is the preliminary step towards breeding for moisture stress tolerance. Screening of large germplasm collections for moisture stress tolerance employing conventional techniques would be cumbersome and time consuming. Therefore, a more accurate and easy alternative is marker assisted selection. A wide array of molecular markers have been discovered which could be employed for developing markers associated with moisture stress tolerance. Twenty four onion genotypes were screened during kharif and rabi seasons under field and pot conditions to identify the moisture stress tolerant and susceptible genotypes. The phenotypically extreme genotypes were used as parents for developing molecular markers associated with moisture stress tolerance. The cDNA from onion leaves were used to isolate some of the major genes and regulatory elements associated with drought tolerance. Significant genetic variability was observed among the genotypes for indicators of plant water status (relative water content, osmotic potential and water potential). All these parameters reduced significantly under stress. The genotypes AFLR, AK-649, S.Local, PBR-140 and hybrid H-1 maintained higher plant water status even under moisture stress. Although all the genotypes showed an increase in WUE (instantaneous, intrinsic) under stress, the highest WUE was recorded in AK-649 and the lowest in Sel-11. ABA plays a major role in moisture stress tolerance through stomatal regulation. A three fold increase in leaf ABA was observed as a result of stress. The genotype AK-649 and hybrid H-1 showed highest increase in leaf ABA in response to moisture stress. ABA accumulation has been depicted as an important trait of adaptation to drought, thus the genotype accumulating more ABA is expected to perform better under water deficit. A strong negative correlation between ABA and genotypes was suggestive of the role of ABA in regulation of stomata. The genotypes Sel-11 and AN-650 showed lowest leaf ABA under stress indicating their susceptibility to moisture stress. genotypes the CID was reduced under stress. The genotype AK-649 had lowest CID coupled with highest WUE while AN-650 had the highest CID with lowest WUE. The strong inverse relationship between CID and WUE suggested that CID could be used as a potential tool to identify the genotypes with higher WUE. One hundred and thirty seven RAPD markers generated by 15 primers were used for estimating the genetic diversity among 24 onion genotypes. Dendrogram based on Squared Euclidian distance grouped the onion genotypes into two major clusters (northern and southern India). The maximum distance was between genotypes PBR-139 and AK-649 and the minimum between AFLR and PBR-140. The Squared Euclidian distance values indicated high genetic diversity among the genotypes. PCA also differentiated genotypes of northern region from those of southern region. The tolerant lines were placed close to each other forming a subgroup, while the susceptible lines were placed together in the adjacent subgroup, indicating that the tolerant and susceptible lines were not very distant genetically. However, these were selected for BSA as the approach depends on divergence between parents in the target region and not at the whole genome level. F2 population of the cross AN-650 x AK-649 was phenotyped based on morphological and physiological parameters and phenotypically extreme plants were identified. BSA was carried out using DNA from ten tolerant and ten susceptible F2 plants. Three hundred RAPD and 22 SSR primers were used to screen the tolerant and susceptible bulks along with parents. Amplification with RAPD primers resulted in four markers 750bp (OPV-4), 1300bp (OPV-4), 900bp (OPAN-18) and 1200bp (OPAO-15) associated with moisture stress tolerance. Fifty one primer pairs specific to selected genes were designed and used to amplify complementary sequences from onion leaf cDNA. The amplified fragments were sequenced and characterized using BLAST programme of the NCBI. Significant homology was obtained for HSP, DREB, Aquaporin, Calcium Sensor homologue, Catalase, CDPK, Ubiquitin, Hexose transporter and SAPK. These genes have a crucial role to play in moisture stress tolerance.