5.4.1 Sequeneing data
Seven AFLP markers, identified in Chapter 4.3.2.1 ,which were significantly linked to gall index variation, were cloned and sequenced. Sequencing data ofsix ofthe markers isgiven in Figure 5.4.
pSOJA1 : AAC·CAC5 (177bp)
GATTGACTGC GTACCAATTC AACTGTTGAG ATACTTAGAG ATGAAGGTTA 27
CTAACTGACG CATGGTTAAG TTGACAACTC TATGAATCTC TACTTCCAAT
TATATAGAAT AAGCmGAA GAAACAAGAC ACGAATCACC TATGTGAATC 77
ATATATCTTA TTCGAAACTT CTTTGTTCTG TGCTTAGTGG ATACACTTAG
ATTCTTTCAT TATTTCTTGT GAAACTTTTT GTAAATTCTT GTAAAGTAAA 127
TAAGAAAGTA ATAAAGAACA CTTTGAAAAA ~ATTTAAGAA CATTTCATTT
GATACAAAGC TTTCAAAACG CCTTATATAC CTTGAGAGM AAAACTAAAA 177 CTATGTTTCG AAAGTTTTGC GGMTATATG GMCTCTCTT TTTIGATTTT GTGTTACTCA GGACTCATCA ATCACTAGTG MTT
CACAATGAGT CCTGAGTAGT TAGTGATCAC TTM pSOJA3 :AAG-CTA5 (371bp)
TTCGATTGAT GAGTCCTGAG TAACTAGTTA GMTTGGCTT GmACAGCT 24 MGCTMCTA CTCAGGACTC ATTGATCMT CTTMCCGM CAAATGTCGA TTGCACGTTT GATTTAGATA GCCMTAGM MCMTTTTT TTTCATCTGA 74 AACGTGCAAA CTAMTCTAT CGGTTATCTT TTGTTAAAM AAAGTAGACT GACATGTCTG CTTATGTGGT GGACATACAT CCTCATTCAT TGCACACGCA 124 CTGTACAGAC GMTACACCA CCTGTATGTA GGAGTAAGTA ACGTGTGCGT AGAGACAAAA GGAGAGCCAT ATGGAAAATA TTTTTTTTTT TTTTTGGTAT 174 TCTCTGTTTT CCTCTCGGTA TACCTTTTAT AAAMAAAAA AAAAACCATA AAGCCGAAAA CATAGACTCA GACATACAAC AGATACTGTA CGAGTACACA 224 TTCGGCTTTT GTATCTGAGT CTGTATGTTG TCTATGACAT GCTCATGTGT TGTGACAGCA ATGACACCM CGCCAAATCC ACTMGCTCC CCCCATCATA 274 ACACTGTCGT TACTGTGGTT GCGGTTTAGG TGATTCGAGG GGGGTAGTAT CAAAGCAAAT ATGAGGGGCC AAAMTTAGA CGCCTTTGCT GCTGCTGCTC 324 GTTTCGTTTA TACTCCCCGG TTTTTMTCT GCGGAAACGA CGACGACGAG TATGCMCAT GMCCTAGM ATCGTTAGTA GATAGGCATG GATGGATCTT 371 ATACGTTGTA CTTGGATCTT TAGCMTCAT CTATCCGTAC CTACCTAGM GAATTGGTAC CAGTCMTC ACTAGTGMT
CTTAACCATG CGTCAGTTAG TGATCACTTA pSOJA4:AAG-CTA6 (369bp)
GGCGGCCGCG GGMTTCGAT TGACTGCGTA CCAATTCAAG ATCCATTCAT 10 CCGCCGGCGC CCTTMGCTA ACTGACGCAT GGTTMGTTC TAGGTAAGTA GTCTATGTAC TAACCGA TTT CTAGGCTCAT GTTGCATAGA GCAGCAGCAG 60 CAGATACATG ATTGGCTAAA GATCCGAGTA CMCGTATCT CGTCGTCGTC CAAAGGCGTC TMTTTTTGG CCCCTCATAT TTGCTCTGTA TGATGGGGGG 110 GTTTCCGCAG ATTAAAMCC GGGGAGTATA MCGAGACAT ACTACCCCCC AGCTTAGTGG ATTTGGCGTT GGTGTCATTG CTGTCACATG TGTACTCGTA 160 TCGMTCACC TAAACCGCM CCACAGTMC GACAGTGTAC ACATGAGCAT CAGTATCTGT TGTATGTCTG AGTCTATGTT TTCGGCTTGT ACCAAAAAAA 210 GTCATAGACA ACATACAGAC TCAGATACM MGCCGMCA TGGTTTTTTT MGGATATTT TCCATATGGC TCTCCTTTTG TCTCTTGCAT GTGCMTGM 260 TTCCTATAAA AGGTATACCG AGAGGAAAAC AGAGMCGTA CACGTTACTT TGGGGATGTA TGTCCACCAC ATMGCAGAC ATGTCTCATA TGAAAAAAM 310 ACCCCTACAT ACAGGTGGTG TATTCGTCTG TACAGAGTAT ACTTTTTTTT TTGTCTTCTA TTGGCTATCT AAATCAAACG TGCAAAGTTG TAGACMGCC 360 AACAGMGAT AACCGATAGA TTTAGTTTGC AGCTTTCMC ATCTGTTCGG AATTCGMCT AGTTACTCAG GACTCATCAA TCACTAGTGA ATT 369 TTAAGCTTGA TCAATGAGTC CTGAGTAGTT AGTGATCACT ATT
pSOJA6 :ACC-CTC2 (244bp)
ATCATTACG..Q 10 GGCGGCCGCG GGAATTCGAT TGACTGCGTA CCAATTCACC
CCGCCGGCGC CCTTAAGCTA ACTGACGCAT GGTTAAGTGG TAGTAATGCG ATGGGCCATC CTAGAGAAAA GCAATTATAA CACTGAGATA TTATGGAACA 60 TACCCGGTAG GATCTCTTTT GCTTAATATT GTGACTCTAT AATACCTTGT CATGGAAAGT GTACTCACAT TGTGAAATAT GTAGCGTAKA CACTCTGGCC 110 GTACCTTTCA CATGAGTGTA ACACTTTATA CATCGCAT?T GTGAGACCGG TGAAACGAAC ATTGGACGCT TCACCGTAAA CTATGTTTTC GTCCCAAGCA 160 ACTTTGCTTG TAACCTGCGA AGTGGCATTT GATACAAAAG CAGGGTTCGT
CTTGCACCAT GATTTGKAAT TCTTGAAGAG ATTTTCCATC GCTGAATCM 210 GMCGTGGTA CTMACMTTA AGAACTTCTC TAAAAGGTAG CGACTTAGTT CAGCCCAATC ATCGAGCTGA TTTGGTACAA CCGAGAGTIA CTCAGGACTC 244 GTCGGGTTAG TAGCTCGACT AMCCATGTT GGCTCTCAAT GAGTCCTGAG ATCMTCACT AGTGAATTCG CGG
TAGTTAGTGA TCACTTMGC GCC pSOJA7 :AAC-CAT1(246bp)
GATTGACTGC GTACCAATIC AACTTTGAGA TCACTTGGCT TGATAGGAGA 27 CTMCTGACT CATGGTTAAG TTGMACTCT AGTGMCCGA ACTATCCTCT TCGATTGTTT TAGATCCCM ATCTTGATGT TTCTTTCCTC CCTTCCACTG 77 AGCTAACAAA ATCTAGGGTT TAGMCTACA MGAAAGGAG GGAAGGTGAC TAGCTGTTCA AAAACTTTAC AGATAMGCT TGTGATAATT TCTGTTTGTA 127 ATCGACAAGT TTTTGAAATG TCTATTTCGA ACACTATTM AGACAAACAT AAATCACAGT AACMGAAGA TTTTACCATA MTGMGTTG TGMCMTAT 177 TTTAGTGTCA TTGTTCTTCT AAAATGGTAT TTACTTCAAC ACTTGTTATA ATCCTACATC ATGATATTTT TATGCMTCA MGAGAATTA TTAGGTGATT 227 TAGGATGTAG TACTATAAAA ATACGTTAGT TTCTCTTMT MTCCACTM TAGGATCAGT ACATCATTTA TGTTACTCAG GACTCATCM TCACTAGTGA 246 ATCCTAGTCA TGTAGTAAAT ACAATGAGTC CTGAGTAGTT AGTGATCACT PSOJA9 :AGC-CTG5 (83bp)
ATTCGATTGA CTGCGTACCA ATTCAGCTM GCTACATGTA AGAGAGGMT 23 TAAGCTAACT GACGCATGGT TAAGTCGATT CGATGTACAT TCTCTCCTTA GACCAGGCAT CAAAATGCGA CTTCATGCM GGAATGTACT .GCCTTCCTTC 73 CTGGTCCGTA GTTTTACGCT GMGTACGTT CCTTACATGA CGGMGGAAG ATTTGCTACC CAGTTACTCA GGACTCATCA ATCACTAGTG AATTCGCGGC 83 TAAACGATGG GTCAATGAGT CCTGAGTAGT TAGTGATCAC TTMGCGCCG
Figure 5.4 Sequencing data ofcloned AFLP fragments. feaRI and Msel primer sequences are indicated in bold type. SCAR primers are double underlined.
Fragment E-AGC/M-CTG6 was not cloned successfully.
5.4.2 peRofindividual F2plants
After optimization ofthe reactionconditions with genomic DNA from the two parents, the segregation of the polymorph isms was analysed in the F2progeny. pSOJA1derived primers(Table 5.1) did not amplify the expected polymorphism in the range of 100 bp between Prima and Gazelle at the optimum reaction conditions (annealing temperature of 40°C and 3 mM MgCI2) . Three polymorph isms were amplified between 1 000 bp and 1 300 bp. Two of these fragments were present in Gazelle and one in Prima.
Reactions performed with 38 F2plantsindicated no significant linkage of either of these fragments with variation in gallindex(P>0.05). Banding patterns scored as similar to parent A, parent Borheterozygous, also did not reveal any significant linkage. No polymorphisms linked to gall index was amplified at higher temperatu res.
Amplification of fragments with primers derived from pSOJA3 (Table 5.1) occurred at temperatures between 40°C and 55°C at 2 mM or3 mM MgCI2. Atotal of5 fragments were amplified with polymorphic fragments of >1 000 bp at 50°C and 45°C. These fragments were not significantly linked to gall index variation (P>0.05). A ±500 bp fragment was polymorphic between Prima and Gazelle at 55°C (3 mM MgCI2) , but was not linked significantly to the resistance trait. Similarly, pSOJA4 primers amplified 4 fragments at 40°C (3 mM MgCI2),with two polymorphic fragments between the two parents. Analysis of the progeny did not reveal any significant linkage ofthese fragments with the resistance trait (P>0.05).
Amplification ofgenomicDNA ofthe parents and progeny with primersofpSOJA6 revealedacodominant segregation pattern linked significantly (P=0.0001) to gall index variation (Figure 5.5). A total of 5 fragments were amplified at 55°C (2 mM MgCI2) ,ofwhich two were monomorphic and three polymorphic between the two parents. The three polymorphic fragments were all smaller than 560 bp. Two ofthese amplified in Gazelle and one'in Prima, and segregated according to a codominant pattern. The codominant SCAR fragments explained 41 % of variation in gall index in the mapping population, and segregated in a 1:2:1 relationship.
564bp>
564bp>
F2 individuals
F2 individuals
Figure 5.5: Amplification of genomic DNA from parents, Gazelle (G) and Prima (P), and 38 F2progeny with pSOJA6 primers. M=Lambda DNAEcoRI/Hindlll.
A total of 9 fragments were amplified with the sequence specific pSOJA7 primers (Table 5.1). Three polymorphisms were amplified between Gazelle and Prima at an annealing temperature of 45°C with 3 mM MgCI2(Figure 5.6A). Two bands between 500 and 1 500 bp segregated in th~F plants,but no significant linkage with gall index variation could be established (P>0.05). A polymorphic band at the predicted 240 bp showed significant linkage with gall index variation (P=O.OOO) and explained 42% of variability in the trait. The fragment was present in Prima and therefore linked in repulsion. The band did not segregate in a 1:1 relationship, and it can therefore not be concluded that it is a single locus - a postulate that is also observed in the multiple bands amplified by the sequence specific primers.
Six fragments were amplified with the designed primers for pSOJA9 at 50°C (2 mM MgCI2), with a polymorphism between Gazelle and Prima at <500 bp. The polymorphism segregated in the F2progeny, but was not linked significantly togall index variation (P>O.05).
A
A F2 individuals M
<
B
Figure 5.6: (A) Amplification ofgenomic DNA from parents,Gazelle (G) and Prima (P),and 38 progeny with pSOJA7 primers. M=Lambda DNAfcoRI/Hindlll.
(B) Segregation of pSOJA7 in an RFLP blot with fcoRI digested genomic DNA of Gazelle (G), Prima (P) and 38 F2progeny.
5.4.3 Verification bySouthern analysis
The cloned pSOJA6 and pSOJA7 fragments were labelled with DIG and used as probes to hybridize to fcoRI digested genomic DNA of the parent and F2progeny plants. Fragment pSOJA6 hybridised to 6 distinct bands, with two fragments (5800 bp and 950 bp) polymorphic between the two parents. These two fragments displayed codominant Mendelian'segregationbetween the F2progeny (results not shown).
Three distinct bands could be distinguished using the pSOJA7 fragment as probe, with one band (3 700 bp) polymorphic between the parents,and present only in Prima (dominant polymorphism) (Figure 5.6B).
This fragment segregated between the F2progeny with exactly the same pattern as the peR product.
pSOJA1 gave rise to 20 distinct bands,whereas pSOJA3 and pSOJA4 led to 4 to 9 bands with a background smear. pSOJA9 gave rise to 3distinct bands on the Southern Blot. No polymorph isms were identified by any ofthese fragments.