MATERIALS AND METHODS 94

The development of SCARs consists of various steps, depending on the technique which identified the original polymorphism. SCARs are developed to create afaster, reliable test for the presence orabsence ofa specific polymorphism. The steps in development include (Figure 5.1): (I) isolation ofa polymorphic fragment (from AFLP or RAPD products), (ii) cloning of the fragment and verification of the insert, (iii) sequencing of the insert or in the case of using the RFLP technique, sequencing of the RFLP probe

"

detecting a polymorphism in the genomic DNA, (iv) design ofoligonucleotide primers, and (v) verification by PCR of genomic DNA. (vi) In a case where no polymorphism is amplified, the PCR products can be digested with a range of restriction enzymes to screen for polymorphisms.

RFLP AFLP

- I-

Polymorphism

- detected

1

Sequence probe X Design primers Oligosynthesis

-.

1

Digested withE Southern Blot Probe with X

A B

E

!

x ^E

I

E

I Genotype A Genotype B

E I

M E

I I

1

Digested with E and M Pre-amplification Selective peR

A B

I I - L

Polymorphism

_ I

^detected

1

Isolate and clone fragment

Sequence fragment Design primers / Oligosynthesis

Primers 5'AGTCA CTG3' 5'GTCTA TAG3'

1

Amplify genomic DNA

/

I-

Polymorphism detected

_ _ _ _ _-J

A B

OR

A B

I-

Polymorphism detected

_ _ _ _ _-J

A B

1-

_ _ _ _-_----ll-NO polymorphism

1

Restriction digestion ofpeRproducts

A B

- I-

Polymorphism

_ detected

_ _ _ _---J

Figure 5.1 Protocol for the development of a SCAR.

5.2. 1 Materials

The pGEM®-Y-Easy vector was obtained from Promega Corporation, Madison, WI, USA. Ingredients ofLB- medium - Bacto-triptone, yeast extract and Bacto-agar were supplied by Difco Laboratories, Detroit, Michigan,USA. Ampicillin, IPTG (Isopropyl-~-D-thioga lactopyranoside) and X-Gal (5-bromo-4-chloro-3- indolyl-~-D-galactoside)were obtained from Roche Boehringer Mannheim,Randburg,SouthAf~ca. The pB212 probe was supplied by Biogenetic Services, Inc.,Brookings,SO. Primers were synthesised by GibcoBRL - LifeTechnologies, Glasgow, United Kingdom. The Wizard® PCR preps DNA purification system and Wizard® Plus SV miniprep DNA purification system were obtained from Promega Corporation, Madison,WI, USA.

5.2.2 Isolation and cloning ofAFLP fragments

The silver stained AFLP polyacrylamide gels (Chapter 4.2.2.2) were air-dried. The putative marker fragments were excised from the gel with ascalpel blade and the piece ofgel rehydrated in 10^ul,distilled water for easy removal from the glass plate (CHO,etal., 1996). The piece ofgel was transferred to a0.5 mL Eppendorf tube and overlaidwith 10 \-lL ofdistilled water, and stored at-20°C. The fragment was reamplified directly from the piece of gel without any purification, using the same conditions as for the original AFLP reaction. An aliquot of1 IJL was electrophoresed on asequencing gel for determination of purity, with a possible second and third round ofisolation and amplification where necessary.

The PCR products were purified using the Wizard® PCR preps DNA purification system (Promega Corporation) and cloned into the pGEM®-Y-Easy vector following the manufacturers recommendations.

Ligation reactions were set up with a positive control containing control insert DNA and a background control containing no insert. Ligation were left at 4°C overnight to obtain the maximum' number of transformants. The ligated plasmidswere transformed into high efficiency competent JM 109 bacterial cells, supplied by Promega, and plated onto selective LB-plates (10 g L-¹Bacto-triptone,5 g L-¹yeast extract,10 g L-¹NaCI,15 g L-¹Bacto-agar containing 100 \-lg mL-¹ampicillin, 0.5 mM IPTG, 80 IJg mL-¹X- Gal). The plates were incubated overnight at3rC. Whitecolonies were selected and cultured overnight in 100 ul, LB-medium containing 100 \-lg mL-¹ampicillin. The cultured cells (4 ut) were tested for the presence ofthe desired insert DNA by using the bacterial cells directly as template in aPCR reaction. The PCR profile and reaction components were the same as for the original AFLP reaction. The size of the

PCR products were verified by electrophoresis of 1_~Lsamples on a denaturing polyacrylamide gel as described for the AFLP reactions (Chapter 4.2.2.2).

5.2.3 Sequencing offragments and design ofprimers

The pB212 probe, cloned in a pBS vector, was used for the development of a SCAR (Figure 5.1). The probe was kindly sequenced by the Institute for Plant Biotechnology, University of Stellenbosch, South Africa, with the use ofT3 (5'_ATT AAC CCT CAC TAA AG-3') and T7 (5'-AATACG ACT CAC TAT AG-3')' promoter primers. Two 17-mer primers were designed from these data with aGC content ofapproximately 50%. Primers were synthesised by GibcoBRL.

Plasrnidscontaining the cloned AFLP fragments were cultured overnight in 10 mL LB-mediumcontaining 100~gmL-¹ampicillin and purifiedusing the Wizard® Plus SV miniprepDNA purification system (Promega Corporation) according to the manufacturers instructions. The relevant AFLP fragments, cloned into the pGEM®-f-Easy vector,were sequenced by the Institute for Plant Biotechnology,University ofStellenbosch, South Africa, with the use ofM13 sequencing primers. All sequences contained the EcoRI adapter at one end and the Msel adapter at the other end. Two 17-mer oligonucleotides (Table5.1)internal to the 5' and 3' ends of the fragment were designed using the NetPrimer program of PREMIER Biosoft International (www.PremierBiosoft.com).Primers were synthesised by GibcoBRL. .

Table 5.1 SCAR primers designed for AFLP fragments

FRAGMENT FORWARD PRIMER REVERSE PRIMER

NAME SOURCE SIZE(BP) 5'-3' 5'-3'

SOJA-1 E-AAC/M-CAC5 177 TGAGATACTTAGAGATG CAAAAAGTTTCACAAGA SOJA-3 E-AAG/M-CTA5 371 ATAGCCAATAGAAAACA ATGCCTATCTACTAACG SOJA-4 E-AAG/M-CTA6 369 GTCTATGTACTAACCGA GTTCGAATTGGCTTGTC SOJA-6 E-ACC/M-CTC2 244 CATGGGCCATCCTAGAG TTGTACCAAATCAGCTC SOJA-7 E-AAC/M-CAT1 246 TTTGAGATCACTTGGCT GATCCTAAATCACCTAA SOJA-9 E-AGC/M-CTG5 83 GTAGGAGAGGAAAGACC GCAAATGAAGGAAGGCA

5.2.4 Verification byPCR analysis

Amplification conditions for the designed primers were determined empirically, varying annealing temperature and MgClzconcentration. Approximately 500 ng genomic DNA was amplified with the SCAR primers and the products electrophoresed on 1.5% to 2% (m/v) agarose gels.

The PCR reaction with the designed B212 primers was optimized for MgClzconcentration (between 1.5 mM and 5 mM), DNA concentration (100 ng - 1 000 ng), Taq DNA polymerase (1-2 U) and annealing temperature (50°C, 55°C, 60°C, 62°C and 65°C). Reactions were carried out in 25IJL containing 250-500 ng genomic DNA, 3 mM MgClz,0.21JM each ofprimers B212F (5'-AGT CTT TGT CGC CGC AT-3') and B212R (5'-GCC TCA GGC ATT TGG TC-3'), 100 IJM each ofdATP, dCTP, dGTP and dTTP, 10 mM Tris- HCI (pH 9.0), 50 mM KCI, 1%(v/v) Triton X-100, 1.5 UTaq DNA polymerase. Amplification was done in a Hybaid Thermal Cycler (Hybaid Limited, United Kingdom) with a denaturation step at 94°C for 5 min, followed by 40 cycles, each cycle consisting of94°C for 1min, 60°C for 1 min and

noc

^{for 1.5min. A}

final elongation step of 5 min at

noc

was included in the program.

Primers were designed for 6 cloned AFLP fragments (Table 5.1). The amplification reactions were optimized for annealing temperature (40°C, 45°C, 50°C, 55°C and 60°C) and MgClz(2 mM and 3 mM) concentration. Reactions were carried out in 25IJL containing approximately 500 ng genomic DNA, 2or 3 mM MgClz,OAIJM each ofthe forward and reverse primers, 100 IJM each ofdATP, dCTP, dGTP and dTTP, 10 mM Tris-HCI (pH 9.0), 50 mM KCI, 1% (v/v) Triton X-100, 1 U Taq DNA polymerase.

Amplification was done in aHybaid Thermal Cycler (Hybaid Limited, United Kingdom) with adenaturation step at 94°C for 5 min, followed by 40 cycles, each cycle consisting of94°C for 1min,xoC for 1min and

noc

for 1.5 min. A final elongation step of 5 min at

noc

was included in the program.

5.2.5 Restriction digestion of peRproducts

The B212 SCAR primers did not produce linked polymorphisms between the parent genomic DNA and 20 IJL ofthe PCR products were subjected to digestion with awide range ofrestriction enzymes - Accl, Alul, BamHI, BglII, Clal, Oral, fcoRl, fcoRV, fclXl, Hae/ll, Hindlll, Kpnl, Mspl, Mvnl,Nael, Pstl, Pvull, Rsal, Sacl, Sail, Sau3A, Seal, Sful, Smal,Spel, Sspl, Stul, Styl, Xbal and Xhol at 3rC; BssHII, BstXl and Bstfll at 50°C; Taql at 65°C. The restricted fragments were electrophoresed in 2% (m/v) agarose gels

at 80 Vfor 2.5 h oralternativelyin 3% (m/v) Metaphor agarose gels (0.5 x TAE running buffer) at 80 Vfor 5 h.

5.2.6 Verification bySouthern analysis

Southern analysis was conducted with the cloned AFLP fragments as probes on genomic DNA of the parents.DNA from individuals from the F²population was hybridised to fragments whichwere successfully converted to SCARs. Samples of DNA (1 0_~g) were digested with fcaRI and treated as in Chapter 3.2.3.2. The cloned AFLP fragments were labelled as described in Chapter 3.2.3.2(iii), using the appropriate AFLP primers and used for hybridisation with the genomic DNA blots (Chapter 3.2.3.2(iv))at 40-45°C. Detection was done as described in Chapter 3.2.3.2(v). These analyses gave an indication of the copy number and heritabilityof the fragments.