Genetic diversity analysis of mustard ( Brassica spp . ) germplasm using molecular marker for selection of short duration genotypes

1 Scientific Officer, Plant Breeding Division, Pulses Research Centre, Bangladesh Agricultural Research Institute, Ishwardi, Pabna, Bangladesh. 2 Assistant Professor, Department of Plant Pathology, Bangladesh Agricultural University, Mymensingh, Bangladesh. 3 Scientific Officer, Substation, Bangladesh Institute of Nuclear Agriculture, Comilla, Bangladesh. 4 Professor, Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh, Bangladesh.


INTRODUCTION
Oilseed Brassicas occupy an important position in the rainfed agriculture throughout the world.In most of the regions of the world, its cultivation has increased drastically during the last decades and, by now; it is the third largest contributor of the world supply of vegetable oil.Rapeseed is one of the most important oil and protein rich annual crops in the world.Seed provides oil both for industrial and culinary purposes.The oils extracted from mustard contain high protein (37%), and feed concentration which is highly palatable to livestock.In Bangladesh, the seed yield of mustard/rapeseed is about 1000 kg/ha, which is very low in comparison to other developed countries.On the other hand, the area of cultivation of mustard in Bangladesh is lower due to rice based cropping system which is difficult to change.Most of the released mustard cultivars are long day duration (105 to 115 days) and thus, did not fit well for cultivation between Aman and Boro rice cropping system.Where a long duration mustard crop is grown after Aman and prior to the Boro, however, the transplanting of Boro may be pushed into February, resulting in a later harvest and greater exposure to early flood risk.So, if we can develop the short duration (75 to 80 days) lines, which would be successfully cultivated between Aman and Boro rice rotation without affecting this popular cropping pattern.So, it is urgent to analyze the genetic diversity and its response for the selection of short duration mustard genotypes for increasing our cropping intensity.
Diversity at marker loci is currently the most feasible strategy for characterizing diversity in mustard.Molecular markers provide the best estimate of genetic diversity, since these are independent of the confounding effects of environmental factors.In mustard, various marker systems have been used for assessing the genetic diversity.There is increasing number of reports where molecular markers like restriction fragment length polymorphism (RFLP) (Diers and Osborn, 1994;Hallden et al., 1994), random amplified polymorphic DNAs, (RAPDs) (Ghosh et al., 2009;Yildirim et al., 2010;Khan et al., 2011), amplified fragment length polymorphism (AFLP) (Sun et al., 2001;Zhao et al., 2005) and microsatellites or simple sequence repeats (SSRs) (Abbas et al., 2009;Wang et al., 2009) have been used to study genome organization, varietal differences and diversity analysis in Brassicas.Among molecular marker RAPDs are increasingly being employed in genetic research owing to their speed and simplicity (Williams et al., 1990;Welsh and McClelland, 1990).In mustard, RAPD method can be used as fast and effective approaches for genetic variation and relationship (Ananga et al., 2008), detecting polymorphism at the DNA level, genetic diversity analysis (Chen et al., 2000), measurement of genetic distance.It is important particularly for variety selection for breeding purpose, hybridization evaluation and conservation of their diverse gene pool.Iqbal et al. (2014) conducted an experiment entitled genetic diversity analysis of mustard germplasm based on phenotypic traits for selection of short duration genotypes in the same laboratory with same genotypes.In the present experiment, a comparative discussion was done between the diversity among the genotypes based on phenotypic traits and diversity obtained from molecular level.Keeping in mind the present study was undertaken to determine the genetic variability and diversity among different mustard genotypes.

MATERIALS AND METHODS
The experiment was conducted at the Molecular and Biotechnology Laboratory, Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh, to characterize 16 genotypes of mustard (Table 1) at molecular level with markers.DNA from 16 mustard genotypes were isolated from actively growing fresh leaves of 20 to 25 days old seedlings using Cetyl Trimethyl Ammonium Bromide (CTAB) method as described by Murray and Thompson (1980), and later modified by Doyle and Doyle (1990).DNA was precipitated with 800 μl of absolute ethanol where DNA became visible as white strands by flicking the tube several times with fingers.DNA was pelleted by centrifugation and re-precipitation of the DNA solution was done by adding 400 μl of 70% ethanol.The pellets were then air dried and dissolved in 50 μl of TE buffer (10 mM Tris.HC1, 1 mM EDTA, pH = 8.0).DNA quality was checked by electrophoresis in 0.8% agarose gel and quantified using a spectrophotometer at 260 nm wave length (Spectronic ® GENESIS TM ).The DNA samples were evaluated both quantitatively and qualitatively using spectrophotometer and agarose gel electrophoresis, respectively.The primers used in RAPD analysis were found to discriminate different genotypes (two randomly chosen individuals from two different genotypes to evaluate their suitability for amplification of the DNA sequences) effectively.Among the 12 primers (Table 2) initially tested, 3 primers (GLA-11, OPB-04 and OPD-02) yielded comparatively maximum number of amplification products with high intensity and minimal smearing, good resolution and also clear bands which were selected for further analysis.

DNA amplification by PCR and electrophoresis
The amplification conditions originally recommended by Williams et al. (1990) were applied with slight modifications.The following components were used to prepare PCR cocktail (Table 3).The total volume of PCR cocktail was 8 µl per sample.2 µl genomic DNA was added with 8 µl PCR cocktail and finally, total volume was 10 µl.DNA amplification was performed in an oil-free thermal cycler.The PCR tubes were set on the wells of the thermocycler plate.Then, the machine was run according to the following setup: Initial denaturation at 94°C for 3 min; denaturation at 94°C for 1 min;

RAPD data analysis
All distinct bands or fragments (RAPD markers) were thereby given identification numbers according to their position on gel and scored visually on the basis of their presence (1) or absence (0), separately for each individual and each primer.The scores obtained using all primers in the RAPD analysis were then pooled to create a single data matrix.This was used to estimate polymorphic loci, Nei's (1973), gene diversity, population differentiation, (Gst), gene flow (Nm), genetic distance (D) and to construct a UPGMA dendrogram among populations using a computer program, POPGENE (Version 1.31) (Yeh et al., 1999).

RESULTS AND DISCUSSION
Twelve (12) primers were initially employed on 16 mustard genotypes for their ability to produce polymorphic bands and 3 primers (GLA-11, OPB-04 and OPD-02) which gave reproducible and distinct polymerphic amplified products were selected.DNA amplification from all the primers tested in this study was not consistently reproducible and is a very common feature of RAPD technique.The present findings agree with those of Hadrys et al. (1992) and Williams et al. (1993).
Technical problems from amplification of the RAPD technique in the field of genetic population research have also been reported by many authors Schierwater and Ender, 1993;Lynch and Milligan, 1994).A total of 40 RAPD bands were scored of which 38 (94.87%) polymorphic amplification products were obtained by using these arbitrary primers (Table 4).The size of the amplification products ranged from 100 to 1000 bp.A total of 254 clear and repeatable bands were amplified from three RAPD primers (Table 5).The primers GLA-11, OPB-04 and OPD-02 produced 74, 85 and 95, respectively polymorphic bands in 16 mustard genotypes.This proportion of polymorphism is higher compared to some previous molecular analysis in mustard genotypes in primers are confirmed in higher percentage of polymorphism.The present experiment produced 13.33 scorable bands per primer and 12.67 polymorphic bands per primer.The reasons of the considerable number of average scorable and polymorphic bands consist of 60 to 70% GC content.Fukuoka et al. (1992) observed an increase in the number of bands with increasing GC content of the primer.The explanation for this correlation between the GC content of the primer and the number of bands is that the stability of base complementation is higher when G is pairing with C by three hydrogen bonds than that of the complementation of A with T by two hydrogen bonds.The DNA polymorphisms were detected according to the presence and absence of band.Absence of band may be caused by failure of primer to anneal a site in some individuals due to nucleotide, sequences difference or by insertions or deletions between primer sites Clark and Lanigan, 1993.Frequencies of maximum number of polymorphic loci were found to be high with the exception of GLA-11(0.750),OPB-04(0.875)and OPD -02 (0.750) (Table 6).The values of Nei's (1973) genetic diversity and Shannon's information index for different accessions of 16 mustard genotypes across all loci are shown in Table 7.The estimate of Nei's genetic diversity for entire genotypes of mustard was 0.3596 and Shannon's information index was 0.535.There was a high level of genetic variation among the studied mustard genotypes Figure 1.RAPD profile of 16 mustard genotypes using primer GLA-11.M, Molecular weight marker (100 bp DNA ladder in size).1, BARI Sarisha-14; 2, Tori-7; 3, BARI Sarisha-8; 4, NAP-0763; 5, BINA Sarisha-4; 6, NAP-0721-1; 7, BARI Sarisha-4; 8, BINA Sarisha-3; 9, BARI Sarisha-15; 10, NAP-0838; 11, NAP-0762-2; 12, NAP-0848-2; 13, BARI Sarisha-9; 14, Sampad; 15, BARI Sarisha-6; 16, NAP-0758-2.
Considering the genetic distance values, the results indicated that the genotypes were genetically different from each other which could be used in breeding programme to have potential genetic gains.Sixteen ( 16) mustard genotypes of the experiment were used to make dendrogram based on Nei's genetic distance using UPGMA.In this study, 16 mustard genotypes had been differentiated into three main clusters: BARI Sarisha-14, BARI Sarisha-9, BARI Sarisha-15, BINA Sarisha-4, BINA Sarisha-3, BARI Sarisha-8, Sampad and Tori-7 in Cluster A, NAP-0763, NAP-0721-1, BARI Sarisha-4, BARI Sarisha-6, NAP-0762-2 and NAP-0848-2 in cluster B and NAP-0838 and NAP-0758-2 were grouped in cluster C (Figure 4).Genotypes included in cluster A were totally different from the genotypes included in cluster B and cluster C. So, genetic relationship was not present between cluster A genotypes with cluster B and cluster C. The cluster A was divided into two sub-clusters.In sub-cluster II of cluster A, there was Tori-7 alone whereas, sub-cluster I was divided in two sub-sub clusters.In sub-sub cluster II, there was BARI Sarisha-8 and Sampad whereas sub-sub cluster I was divided in two sub-sub-sub clusters.In subsub-sub cluster II, there was BINA Sarisha-3 and BINA Sarisha-4 whereas sub-sub-sub cluster I was divided in two sub-sub-sub-sub clusters.Sub-sub-sub-sub cluster I included BARI Sarisha-14 and BARI Sarisha-14 and Subsub-sub-sub cluster II included only BARI Sarisha-15.The cluster B was divided into two sub-clusters.In subcluster I of cluster B, there was NAP-0763 and NAP-0721-1 whereas, sub-cluster II was divided in two subsub clusters.Sub-sub cluster I included BARI Sarisha-4 and BARI Sarisha-6 and Sub-sub cluster II included NAP-0762-2 and NAP-0838-2.Finally, the cluster C included NAP-0838 and NAP-0758-2.According to Ghosh et al. (2009), the 9 accessions were capable of being classified into 2 major groups.One group consists of BARI Sharisha-12, Agrani, Sampad, Daulot, Rai-5, Alboglabra and another group contained BINA Sharisha-4, BINA Sharisha-5, and BARI Sharisha-13.Genotypic variations based on molecular characterization indicated that genotypes belonging to different clusters depend on their genetic components itself, but not at geographical origin at all.Therefore, it could be concluded that for further research program, especially for hybridization, genotype could be selected from different clusters, to achieve maximum heterosis regarding yield.Iqbal et al. (2014) conducted an experiment entitled genetic diversity analysis of mustard germplasm based on phenotypic traits for selection of short duration genotypes in the same laboratory with same genotypes.They suggested that, on basis of lowest days to mature groups included Tori-7, BARI Sarisha-14, BARI Sarisha-9, BARI Sarisha-15 can be crossed with higher seed yield group which included NAP-0838, NAP-0758-2, NAP-0762-2, NAP-0848-2 and BARI Sarisha-4 for developing short durated high yielding varieties.Now after the comparison between the results of phenotypic traits and molecular markers, it can be revealed that genotypes that belongs to lowest days to mature groups represents the genotypes of cluster I of molecular markers and genotypes that belongs to clusters III and II of molecular markers represents most of the genotypes of higher seed yield group.Therefore, it could be concluded that the next crossing combination might be between the genotypes: Tori-7, BARI Sarisha-14, BARI Sarisha-9, BARI Sarisha-15 and Sampad with the genotypes: NAP-0838, NAP-0758-2, NAP-0762-2, and NAP-0848-2 for developing short duration high yielded variety which are genotypically and phenotypically significantly differentiated.

Figure 4 .
Figure 4. Unweighted pair group method of arithmetic mean (UPGMA) dendrogram based on Nei's genetic distance; summarizing data on differentiation in 16 mustard genotypes according to RAPD analysis.

Table 1 .
List of the mustard genotypes used in the experiment.

of the genotypes Name of the species Sources of the genotypes
The genotypes of BARI and BINA are the varieties and the genotypes of RARS, Jamalpur are the advanced lines.

Table 2 .
Parameters of the random primers used in the present study for screening *Selected for RAPD analysis of all samples of the 16 mustard genotypes.
annealing at 35°C for 1 min; elongation or extension at 72°C for 2 min; Cycle to step 2 for 40 more time; incubation at 72°C for 7 min; completion of cycling program (45 cycles), reactions were held at 4°C.The amplified products were separated electrophoretically on

Table 4 .
RAPD primers with corresponding bands score and their size range together with polymorphic bands observed in 16 mustard genotypes.
Ghosh et al. (2009)reported 59 reproducible DNA bands generated by four arbitrary selected primers of which 58 (98.03%) bands were proved to be polymorphic and these bands ranged from 212 to 30686 bp in size.The selected 3 primers produced comparatively maximum number of high intensity band with minimal smearing, good technical resolution and sufficient variation among different variants.The dissimilar numbers of bands were generated by primer GLA-11, OPB-04 and OPD-02 (Table4).Besides, the primer OPD-02 amplified maximum number of polymorphic bands (100.00%) while the primer GLA-11 and OPB-04 generated the least (92.31%) polymorphic bands which were minimal in number.The banding patterns of 16 mustard genotypes using primers GLA-11, OPB-04 and OPD-02 are shown in Figures 1, 2 and 3 respectively.Number of RAPD markers scored for each individual 16 mustard genotypes for each primer are presented in Table4.
from the proportion of polymorphic loci point of view.Considering the genetic distance values, the result indicate that some mustard genotypes were genetically different from each other and some were tend to be

Table 6 .
Frequencies of polymorphic RAPD markers in mustard genotypes.

Table 7 .
Summary of genetic diversity and Shanon information index statistics for all loci.