Assessment of intra-specific divergence among Brassica rapa germplasm based on agro-morphological, biochemical and molecular markers

Abstract

Brassica rapa is one of the important oilseed crops and commonly grown all over the world for multiple purposes. The present study was designed to study intra-specific quantitative and qualitative characters among 253 B. rapa genotypes. The two years mean morphological data were recorded for all these characters under field condition. Significant variations were recorded among genotypes for days to flower initiation, days to 50% flowering completion, days to completion of flowering, days to maturity, leaf width and length, plant height, primary branches/plant, main raceme length, pod width and length, stem thickness, thousand seed weight, seed yield/plant and pod shattering (stage IV-V). Among all qualitative traits, the seed color, branching habit, early growth vigor etc. had leading variations. Many elite lines such as Br-505, Br- 512, Br-536, Br- 547, Br-560, Br-760, etc. had excellent morphogenic responses in both years. Among all quantitative traits, the days to flower initiation, days to 50% flowering and days to flower completion was found to have leading variations. The medium levels of variation were recorded to plant height and main raceme length. The least variance was observed for 1000-seed weight. The present early flowering and maturity lines, pod shatter resistant and maximum yielding genotypes gave unique opportunity to enhance the quality and production of this important crop. Brassica rapa sub-species represent rich diversity all over the world. The three different subspecies brown, yellow and Toria have distinct qualitative and quantitative characters. In present study we have described some major characteristics of three important sub-species of B. rapa (brown sarson, yellow sarson and Toria). The brown sarson, yellow sarson and Toria genotypes showed rich diversity for many important quantitative traits like leaf shapes (vegetable and non-vegetable types), early flowering, number of pods, pod shattering, yields per plant etc. The multivariate analysis recorded maximum variations at first five principal components (PCs). A total of 66.34% variability was recorded in first five PCs. The contribution to first five PCs in overall PCs was 27.32, 15.01, 9.54, 7.90 and 6.58%, respectively. Among all characters the pod shattering stage IV and V gave maximum positive contribution in overall genetic variability in PC1 (0.58) and PC3 (0.66). The principal component analysis (PCA) further classified all 253 genotypes into different population groups on the basis of their geographic origin. Seven different geographic groups were recorded and all showed different evolutionary history. The population xvi groups 1 and 2 predominantly included the Pakistani genotypes. The population group 5 and 6 mostly contained the European genotypes. The rest of groups contained genotypes from all four countries (Pakistan, Germany, Netherlands and Sweden). Among the studied traits maximum correlation (0.99) was recorded among days to flowering initiation, days to 50% flowering, days to flower completion and pod shattering stage IV and V respectively. The phylogenetic tree further classified these genotypes into six distinct groups. All the groups are highly diverged from one another on the basis of traits and geographic origins. The seed storage protein based variability of three important Brassica rapa ecotypes is useful for the improvement and efficient utilization of this crop. In this study the electrophoretic total seed storage protein based variation was studied among diverse 110 local and exotic B. rapa genotypes. A thoroughly precise protocol was established by optimizing various factors such as proper pH level, centrifugation time, sample size, ammonium per sulphate (APS) concentrations, staining/de-staining time period etc. By optimizing these conditions maximum polymorphic proteins were noted of varying sizes (10-180 kDa). Maximum 15 protein bands were recorded, in which 14 bands (93%) were highly polymorphic while 1 band (7%) was reported as monomorphic. The maximum similarity coefficient 100% was recorded among 502/583, Br-516/Br-587, Br-516/Br-587, Br-601/Br-563 and Br-505/Br-708, Br-625/Br-635 genotypes. The lowest similarity indices 13% was recorded among genotypes Br-517/Br-716. Our results showed that maximum similarity exist within brown sarson itself and brown sarson and Toria as compared to yellow sarson. The presence/absence binary data of total soluble seed protein based variations were analyzed through Unweighted Pair Group Method with Arithmetic Mean (UPGMA), which clustered all three ecotypes into ten main groups. Cluster I-X contained 13, 23, 14, 7, 8, 15, 11, 10, 8 and 1 genotypes respectively. The group X has unique one genotype Br- 517, which is distinct from other genotypes. The 2D and 3D representation of accessions in X-Y plane further visualized genotypes. The 3D diagram revealed distribution of some unique genotypes such as Br-517, Br-609 and Br-722, etc. An efficient SDS-PAGE protocol was established; it can be used to evaluate genetic variability of some other important crop species and to widen the agriculture breeding program. Our findings will be helpful as a preliminary study on the characterization of B. rapa germplasm. xvii Salt stress is one of key abiotic stresses that directly affect normal morphobiochemical and physiological properties of Brassica rapa. In this study the effect of different levels of salt stress was studied on three important sub-species of B. rapa (brown sarson, yellow sarson and Toria). The young germinated plants were subjected to four different NaCl concentrations (0, 50,100 and 150 mmol). The salt effects was recorded on important characters like shoot and root length, shoot fresh and dry weight, root fresh and dry weight, leaf relative water content (RWC), proline and chlorophyll a, b, a+b contents. The high salt stress significantly decreased the shoot and root length, shoots fresh and dry weight, and root fresh/dry weight of all three ecotypes. The germplasm 22861 and 25007 (brown type) showed improve morphogenic performances followed by Toria. The accession 26158 (yellow sarson) showed very poor performance and stunted growth. The RWC and chlorophyll a, b and total chlorophyll contents were decreased many fold at elevate salt concentrations (50, 100 and 150 mmol), while proline amount was maximum at high levels. The brown sarson and Toria showed more tolerance to high salt events than yellow sarson. These salt tolerant germplasm might be useful for salt affected areas. In third section of experiment 65 simple sequence repeats (SSRs) markers were used to study genomic difference in 65 diverse B. rapa germplasm. Most of these primers amplified fragments that ranged from 100-300bp. Majority of primers showed maximum polymorphic banding patterns (97.56%) except for one monomorphic marker (2.44%). Fifty six primers (87.5 %) detected only one allele in all tested B. rapa genotypes. The genetic similarity values (39-88%) were recorded among different B. rapa genotypes. The least similarity coefficient value 0.39 (39%) was recorded between genotypes Br-501 (brown) and Br-547 (brown) followed 0.40 (40%) between Br-501 and Br-726. The highest similarity value 0.88 (88%) was noted between Br-563 (yellow) and Br-696 (brown) and TS-1 (Toria). All the genotypes from all three ecotypes and from different origins were divided into five different groups by using UPGMA similarity method. The group I and II included majority of Pakistani genotypes as compared to group III European type. The other two groups consisted of genotypes from all four countries (Pakistan, Netherlands, Germany and Pakistan). The genetic structure study further justified the relationship between both local and exotic germplasm at ΔK=8 through STRUCTURE software package. Our results may be used as a baseline for future Brassica species breeding program in Pakistan.