III

Materials and methods

The investigation on Assessment of Induced Genetic Variability in Jatropha curcas L. through Molecular markers was carried out at Department of Agricultural Biotechnology, Anand Agricultural University, Anand. 3.1 Ex erimental Material! Seeds of Jatropha curcas . were e!posed to various doses of gamma rays i.e. ", #, $, %, #", #%, &", &% and %" '(. )opulation of plants grown from treated seeds was studied and mutants were selected on *asis of morphological +height, variations from e!perimental plantations raised at the Jatropha farm of Anand Agricultural University, Anand.+Ta*le $.#,. The plants grown from seeds without treatment of gamma rays were ta-en as .ontrol. /our mutants for each dose were ta-en for the investigation. $.& 0lass1war .able 3.1! List of Mutants! "r. #o. $ose %&r' "r. #o. .ontrol " # # #2 & # #3 $ # #4 5 # &" % $ &# 6 $ && 2 $ &$ 3 $ &5 4 % &% #" % &6 ## % &2 #& % &3 #$ #" &4 #5 #" $" #% #" $# #6 #" $& 3.( Glass)*are+ ,lastic)*are and -eagents! $ose %&r' #% #% #% #% &" &" &" &" &% &% &% &% %" %" %" %"

The glass1wares and plastic1wares used were from Schott Duran, 0ermany and A!ygen, respectively and all the chemicals and reagents used in the present study were of *iotechnology or molecular *iology grade 7uality. 3.3 $#A Extraction !
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Total genomic D8A was e!tracted from the leaves of standing crop *y .etyl trimethyl ammonium *romide +.TAB, method +Da-shinamoorthy and selvara9,&""", with minor modifications. 3.3.1 ,re aration of stock solutions for reagents and buffers for $#A extraction The reagents and *uffers for D8A isolation were prepared as per Sam*rooet al., +#434,. The composition and procedure for preparation of various stoc- solutions and *uffers are given in Ta*le $.&.

.able 3.(! ,re aration of stock solutions for $#A extraction and electro horesis

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"r. #o

"olution

Method of re aration #&.## g Tris *ase +;imedia, was dissolved in 3" ml distilled water. The p; was ad9usted to 3." *y adding concentrated ;.l and the total volume was ad9usted to #"" ml. <t was dispensed into reagent *ottle and sterili=ed *y autoclaving. #3.6" g >DTA di Sodium salt +;imedia, was

#: Tris ;.l +p; 3.", #"" ml

&

".%: >DTA +p; 3.", #"" ml

Dissolved in 3" ml distilled water. The p; was ad9usted to 3." *y adding 8a?; pellets. The total volume was ad9usted to #"" ml and dispensed into a reagent *ottle and sterili=ed *y autoclaving. &4.&& g 8a.l +;imedia, was ta-en in to *ea-er@ %" ml of distilled water was added and mi!ed well. Ahen the

%: 8a.l, #"" ml

salts got completely dissolved, the final volume was ad9usted to #"" ml. <t was dispensed into a reagent *ottle and sterili=ed *y autoclaving. 3" ml of ethanol was ta-en and &" ml of distilled water

3"B >thanol, #"" ml

was added, mi!ed well and dispensed into a reagent *ottle and stored at 5".. 46 ml of chloroform +Dualigens, and 5 ml of isoamyl alcohol +Dualigens, were measured, mi!ed well and stored in a reagent *ottle at room temperature. #" mg >thidium Bromide +;imedia, was added to #." ml of distilled water and it was -ept on magnetic stirrer to ensure that the dye has dissolved completely. <t was dispensed into an am*er colored eppendorf tu*e and stored at 5".. #." ml of Tris ;.l +#:,, &"" Gl of >DTA +".%:, were ta-en and distilled water was added to ad9ust the final volume of #"" ml, mi!ed thoroughly, autoclaved and stored at room temperature.

.hloroformC <soamyl alcohol +&5C#,, #"" ml

>thidium Bromide +#" mgEml,, #." ml

#F T> *uffer, #"" ml #"m: Tris ;.l 2 +p; 3.", ".#m: >DTA +p; 3.",

%5.% g of Tris *ase, &2.% g of Boric acid +Dualigens, 3 TB> *uffer %F +# liter, p; 3." #2 / ) a g e $: Sodium acetate, &" ml p; 2." were ta-en, &" ml of ".%: >DTA +p; 3.", was added. The /inal volume of # liter was ad9usted *y adding distilled water and the p; was ad9usted to 3.". 3.#6 g Sodium acetate trihydrate salt +:erc-, was dissolved in distilled water and the final volume was made up to &" ml. The p; was ad9usted to 2.".

.able 3.3! ,re aration of Extraction 0uffer 0uffer Method of re aration #." ml of #: Tris ;.l +p; 3.",, &.3 ml of %: 8a.l, # ml of ".% : >DTA +p; 3.", ,".% gm of .TAB +wEw, , ".& gm .TAB e!traction *uffer of )H) +)olyvinyl pyrrolidine,+wEw, and %.& ml of distilled water were ta-en in to a flas- and mi!ed well.The *uffer was -ept incu*ated #t 6%". for the .TAB and )H) to get dissolved. ".#% ml +#.%B, I1 mercaptoethanol +Dualigens, was added into the mi!ture 9ust *efore use.

+%B,, #" ml

3.3.( ,rotocol for Genomic $#A extraction! Total D8A was e!tracted from the leaves *y .etyl trimethyl ammonium *romide +.TAB, method +Da-shinamoorthy and Selvara9, &""", with some modifications as followsC The leaves of Jatropha germplasm were directly collected from field and Aeighing leaf sample from each accession, $"" mg was grinded in li7uid

utili=ed for D8A e!traction. nitrogen using mortar and pestle. .TAB *uffer %B +# ml, containing #.%B +vEv, I1mercaptoethanol +added

fresh in e!traction *uffer, and #% Kl )roteinase - +/ermentas, USA, was 7uic-ly added to each microcentrifuge tu*e +& ml, and vorte! to mi!. The tu*e was incu*ated at 6%L. for #1#E& hr with fre7uent swirling. An e7ual volume of chloroform C isoamylalcohol +&5C#, +chilled, was added and centrifuged at #",""" rpm +>ppendorph %5#2(, and 5L. for #" min to separate the phases. The supernatant was carefully decanted and transferred to a new tu*e.

The a*ove steps, *eginning with the addition of chloroformC isoamylalcohol +&5C#, and ending with decanting of supernatant, were repeated twice. The supernatant was precipitated with dou*le volume of a*solute ethanol or ".6 volume of iso1propanol along with #"" Gl of ".$ : sodium acetate for overnight at 5".. The precipitated nucleic acids were collected and washed once with the 2"B ethanol and once with 3"B ethanol. +The tu*es should not #3 / ) a g e

*e sha-en vigorously, *ecause D8A is very vulnera*le to fragmentation at this step,. The pellets were air dried and resuspended in #"" Gl of T> *uffer +#" m: D8ase free (8ase A +/ermentas, USA, %Kl was added to the dissolved

Tris1;.l@ p; 3.", ".#m: >DTA, p; 3.",. D8A stoc- and incu*ated in a water *ath at $2 ". for # hour followed *y 6"". for #" minutes for en=yme inactivation. The samples were stored at 1&"". deep free=e for long1term usage. 3.3.3. ,urity and 1uantification test of $#A Spectrophotometry was performed to determine D8A concentration *y using 8anodrop 8.D.#""" +Software H.$.$.", Thermo Scientific, USA, at a*sor*ance ratio &6"E&3" nm and the 7uality of o*tained D8A was chec-ed on ".3B agarose gel. Dilution of &" ngEGl wor-ing solution was prepared from the stoc- solution of the isolated D8As.

3.3.3.1 ,rocedure! The #.% Kl of D8A sample was loaded into the well of 8anodrop Spectrophotometer +Thermo Scientific, U.S.A., and the concentration of D8A and a*sor*ance at &6" nm and &3" nm were measured and the A &6"EA&3" ratio was automatically calculated *y the software. .able 3.2! ,re aration of *orking solution of $#A%(3ng45l+ 1335l' for -A,$ and $AM$ analysis !

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"r. #o . # & $ 5 % 6 2 3 4 #" ## #& #$ #5 #% #6 #2 #3 #4 &" &# && &$ &5 &% &6 &2 &3 &4

Mutants

"tock %5l'

solution

taken 6ater%#uclease free' added %5l' 43.6 4#.% 43.2 42.4 42.4 42.3 43.% 42.& 4".2 45.4 4%.% 46.5 45.6 4%.3 4%.& 4$.# 4%.3 45.$ 4&.3 42.& 4%.& 4%." 45.% 45.2 42.% 43.$ 44." 43.$ 44.# 4%.&

.ontrol #'r# #'r& #'r$ #'r5 $'r# $'r& $'r$ $'r5 %'r# %'r& %'r$ %'r5 #"'r# #"'r& #"'r$ #"'r5 #%'r# #%'r& #%'r$ #%'r5 &"'r# &"'r& &"'r$ &"'r5 &%'r# &%'r& &%'r$ &" /)age &%'r5 %"'r#

#.5 3.% #.$ &.# &.# &.& #.% &.3 4.$ %.# 5.% $.6 %.5 5.& 5.3 6.4 5.& %.2 2.& &.3 5.3 %." %.% %.$ &.% #.2 #." #.2 ".4 5.3

3.2 -andomly Am lified ,olymor hic $#A %-A,$'! Amplification of (A)D fragments was performed according to

Ailliams et al., +#44", with some modifications using decamer ar*itrary primers +:A0 Biotech, 0ermany, +Ta*le $.6,.

3.2.1. -A,$ ,7- 7om onents The reagents and list of )rimers +:A0 *iotech,0ermany, used for (A)D1).( Sr.8o (eagents # ).( Ta7 Buffer with :g.l& +Bangalore 0enei,<ndia, & $ 5 % % )rimer +#"p molesE Gl,+:A0 *iotech,0ermany, d8T)s +&.%m: each,+/ermentas,USA, Ta7 )olymerase +$UE Gl, +Bangalore 0enei,<ndia, Template D8A +&"ngE Gl, 8uclease free water +Amresco,USA, Total amplification of D8A were as follows+Ta*le $.%,$.6, .able 3.8! 7om onents of ,7- reaction mixture Holume &.% Gl #.% Gl ".% Gl ".% Gl &.% Gl #2.% Gl &% Gl

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.able 3.9! List of -A,$ rimers used for the resent study Sr. 8o. # & $ 5 % 6 2 3 4 #" ## #& #$ #5 #% )rimer name ?)A"4 ?)A1#3 ?)B#" ?)B## ?).#3 ?)D"% ?)/"5 ?)/#" ?);#& ?);#$ ?)<"4 ?)<#" ?)<#$ ?)<#% ?)'#6 Se7uence +%M $M, 0. content +B, 2" 6" 6" 6" 6" 6" 6" 2" 6" 2" 6" 6" 2" 6" 6"

000TAA.0.. A00T0A..0T 0T0A.AT0.. AA0A....T. .A0.T.A.0A A..A00TT00 00AT0A0A.. T00A..00T0 A.0.0.AT0T 0A.0..A.A. T00A0A0.A0 A.AA 0.0A0 .T0000.T0A T.AT..0A00 0A0.0T.0AA

&& / ) a g e

3.2.( 7ocktail re aration! As per the a*ove coc-tail, :illipore sterili=ed water was added first followed *y addition of a*ove mentioned ).( reagents in se7uence and finally the template D8A. The reaction mi!ture was prepared in &"" Gl ).( tu*es +A!ygen, USA,. The reagents were mi!ed gently *y tapping against the tu*e followed *y a short spinning +N$,""" rpm for 6" seconds,. The tu*es were then placed in the thermal cycler +Biometra,T10radient 0ermany, for cyclic amplification with the following ).( reaction conditions + /igure $.#, ).( conditions +Ailliams et al, #44",.

<nitial denaturation 45" 5 min

Denaturation

/inal >!tension 2&" & min Annealing $3" # min 5& cycles : 2&" 2 min ;old ; 5" old e!tension 45" # min

;igure 3.1 ! "te s in ,7- am lification reaction conditions

3.2.3 Electro horesis of the am lified roduct! The amplified products of (A)D were analysed on #.%B agarose gel. 3.2.3.1 7hemicals used! Agarose + ow >>?,Bangalore 0enei,<ndia, %F Tris Borate >DTA +TB>, Buffer ,p; 3." >thidium Bromide +>tBr, +#" mgEml,+Amresco,USA, 0el oading Dye +6F, +/ermentas,USA, #"" *p D8A ladder +fermentas,USA,

&$ / ) a g e

Agarose gel of #.% B concentrations was prepared in #F TB> +#.% g agarose in #"" ml #F TB> and &.% Gl >thidium *romide from #" mgEml stoc-,. ).( amplified products +4 Gl and # Gl 6F loading dye, were loaded into the wells. The #"" *p standard D8A ladder +# Gl, +mar-er, was also run along with the samples. The electrophoresis was conducted at #&" H current +constant, for $ hrs. to separate the amplified *ands. The separated *ands were visuali=ed under UH transilluminator +Biometra,0ermany, and photographed using gel documentation system +Bio1rad,.alifornia,.

3.8! $irect Am lification <f Minisatellite $#A %$AM$ markers' The amplification of 0enomic D8A was carried out using four universal DA:D primers which were utili=ed *y (anade et al., +&""2, in their studies related to diversity assessment in Jatropha curcas.The amplification was carried out according to the method followed *y ;eath et al.,+#44$, with some modifications. The reaction components + ta*le $.2, and the list of primers used in the present study +ta*le $.#", are as follows. .able 3.=C ,7- reaction com onents Sr.8o # & $ 5 % % (eagents ).( Ta7 Buffer with :g.l& +Bangalore 0enei,<ndia, )rimer +#"p molesE Gl,+:A0 *iotech,0ermany, d8T)s +&.%m: each,+/ermentas,USA, Ta7 )olymerase +$UE Gl, +Bangalore 0enei,<ndia, Template D8A +&"ngE Gl, 8uclease free water +Amresco,USA, Total Holume &.% Gl #.% Gl ".% Gl ".% Gl &.% Gl #2.% Gl &% Gl

.able 3.>C List of $AM$ rimers +(anade et al.,,&""2,

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All the ).( reactions were carried out in ".& ml capacity thin walled ).( tu*es. ,rimer #ame HBV HVR 33.6 M13 ,rimer "e?uence GGTGTAGAGAGGGGT, CCTCCTCCCTCCT GGAGGTTTTCA, GAGGGTGGCGGTTCT, G7 content %@' 60 69.2 45.5 66.7 .m Aalue%37' 50.6 44.0 32.0 53.3

+ Tarson,, <ndia,. As per the a*ove coc-tail, :illipore sterili=ed water was added first followed *y addition of ).( reaction components, primer in se7uence and finally the template D8A. The reagents were mi!ed gently *y tapping against the tu*e followed *y a short spinning +N$,""" rpm for 6" seconds,. The tu*es were then placed in the Thermal .ycler +Biometra T10radient, 0ermany, for cyclic amplification +/igure $.&,.
<nitial denaturation

Denaturation

>!tension

/inal e!tension

45" 5 min

45" # min Annealing 54",5&",$6",%"" #."% min

2&" & min

2&" 2 min ;old 5" :

;igure 3.(! ,7- reaction conditions for $AM$ am lification

3.2.1C Electro horesis of ,7- am lified roducts The amplified products of <SS( were analy=ed using #.3 B agarose gel. 3.2.1.(! 7hemicals used &% / ) a g e

 Agarose + ow >>?,Bangalore 0enei,<ndia, %F Tris Borate >DTA +TB>, Buffer ,p; 3." >thidium Bromide +>tBr, +#" mgEml,+Amresco,USA, 0el oading Dye +6F, +/ermentas,USA, #"" *p D8A ladder +/ermentas,USA, Agarose gel of &." B concentration was prepared in #F TB> +&." g agarose in #"" ml #F TB> and &.% Gl >thidium *romide from #" mgEml stoc-,. ).( amplified products +#& Gl and #.% Gl 6F loading dye, were loaded into the wells. The #"" *p standard D8A ladder +# Gl, +mar-er, was also run along with the samples. The electrophoresis was conducted at #&" H current +constant, for $ hrs. to separate the amplified *ands. The separated *ands were visuali=ed under UH transilluminator +Biometra,0ermany, and photographed using gel documentation system +Bio1rad,.alifornia,. 3.8C $ata analysis .lear and distinct *ands amplified *y (A)D and DA:D and primers were scored for the presence and a*sence of the corresponding *and among the genotypes. The scores # and " indicates the presence or a*sence of *ands respectively. The softwares used for the analysis of the scored data were 8TSTSpc version &."& +(holf #443,, )opgene $& +Oeah et al.,#444, and S)SS .The ma9or part of the analysis was carried out using 8TSOSpc version &."& +(holf,#443, e!cept for the calculation of the Shannon inde!, o*served and effective num*er of alleles which were calculated using )opgene $&. 3.8.1 Genetic similarity and cluster analysis .oefficients of similarity were calculated *y using JaccardMs similarity coefficient *y S<:DUA function and cluster analysis was performed *y agglomerative techni7ue using the U)0:A +Un1weighted )air 0roup :ethod with Arithmetic :ean, method *y SA;8 clustering function of 8TSOS1pc. (elationships among the Jatropha curcas genotypes and among and *etween the species of 9atropha genus were e!pressed in the form of dendrograms and genetic similarity matri!. . 3.8.( 7o henetic correlation and mantel tests The cophenetic correlation analysis was carried out using the .?); function of 8TSOS1pc. <n this method dendrogram and similarity matri! were correlated to find the &6 / ) a g e

goodness1of1fit of the dendrogram constructed *ased on similarity coefficients. The correspondence *etween (A)D and DA:D *ased on similarity coefficient matrices was tested using cophenetic correlation analysis and :antel matri! correspondence test. The :antel matri! correspondence test was carried out using the :F.?:) function in the 8TSOSpc version &."&. 3.8.3 ,rinci al 7om onent Analysis )rincipal component analysis was carried out using the ><0>8 module of 8TSOSpc &."&.The results were graphically e!pressed in the form of &D and $D plots generated *y graphics module after the calculation of ><0>8 values. /irst three eigen values which showed ma!imum variation were e!tracted as principal components and &D and $D plots were generated on that *asis. 3.8.2 !7alculation of arameters of genetic Aariability

Harious components were calculated which included, 8o of monomorphic and polymorphic loci , )olymorphism <nformation .ontent +)<.,,>ffective :ultiple! ratio +>:(,, )olymorphism B. )olymorphism <nfromation content +)<., was calculated according to formula descri*ed *y Smith et al +#442, ,Bootstein et al.,+#43", 0arcia et al +&""5,. ,I7B11Pf( *here f is the fre?uency of ith allele. All the a*ove mentioned varia*les were calculated individually for *oth the mar-ers as well as the com*ined values for (A)DQDA:D were calculated for comparing a*ility of the mar-ers for D8A polymorphism assessment.

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