GENETIC FINGERPRINT ESTABLISHED
FOR THE SELECTED ALFALFA
GENOTYPES USING MOLECULAR
MARKERS
OBJECTIVES
Assessment intra- and interspecific variability at molecular level
The work methods used were:
•
Molecular techniques of genetic variability detection (extraction of
genomic DNA, enzymatic amplification through the PCR reaction using 13
RAPD, 3 SSR and 5 ISSR primers, analysis of the reaction products through
agarose gel electrophoresis);
•
Statistical-mathematical methods of result processing and
interpretation.
Assessment of interspecific variability using ISSR molecular markers
Polymorphism rate for the alfalfa genotypes using ISSR primers
No. Primer
crt.
1
A-12
2
A-13
3
A-17
4
A-21
5 UBC-818
Nucleotide
sequence
(GA)6CC
(GT)6CC
(GTG)3GC
(CA)6AC
(CA)7G
Bands No. Polymor- Bands
PIC
PI
x  s
Total Polymor ph (%)
(pb)
25
20
84,00 220-1650 0,210+0,040 3,367
27
19
70,37 150-1510 0,292+0,028 5,544
31
31
100,00 240-2090 0,370+0,019 11,462
15
9
60,00 280-1320 0,324+0,052 2,918
19
16
84,21 300-2120 0,358+0,031 5,731
x
UPGMA clustering of alfalfa genotypes using the ISSR primers
Satelit
F 1109-99
Cosmina
Granat
F 105-90
Sigma
F 1822-06
Super
F 1615-04
F 1206-00
F 1306-01
F 270-91
F 1413-02
F 1310-01
F 1111-99
Pastoral
Magnat
Alina
Stolo 13
Viking
Cristal
Dorina
Opal
F 219-91
Coral
Selena
MF 42-96
Saturn
Venus
F 907-97
1.00
0.95
0.90
0.85
0.80
0.75
Coeficient de similaritate genetica
0.70
0.65
0.60
No.crt
Specific alleles
No.ct
Specific alleles
1
2
3
4
5
6
7
8
9
10
11
A 12 -220
A 12 -280
A 12 -650
A 12 -820
A 12 -950
A 13 -330
A 13 -440
A 13 -550
A 13 -600
A 13 -1017
A 13 -1190
12
13
14
15
16
17
18
19
20
21
22
A 13 -1259
A 13 -1510
A 21 -280
A 21 -410
A 21 -520
A 21 -750
A 21 -1080
A 21 -1180
UBC 818 -300
UBC 818 -510
UBC 818 -760
We identified 22 alleles present in all the 30 genotypes studied. These
alleles may be considered ISSR specific markers for alfalfa
Assessment of intergenotypical variability using RAPD molecular markers
Polymorphism rate for the alfalfa genotypes using RAPD primers
No.
Primer
Nucleotide
Sequence (5’-3’)
crt.
1
2
3
4
5
6
7
8
9
10
11
12
13
B -07
G -03
G -04
G -06
G -10
G -16
G -17
G -18
G -19
L -03
L -07
L-12
L -14
GGTGACGCAG
GAGCCCTCCA
AGCGTGTCTG
GTGCCTAACC
AGGGCCGTCT
AGCGTCCTCC
ACGACCGACA
GGCTCATGTG
GTCAGGGCAA
CCAGCAGCTT
AGGCGGGAAC
GGGCGGTACT
GTGACAGGCT
Bands No.
Polymor
Size
PIC
Total
Polymorf
(%)
bands (pb)
x  sx
9
7
12
10
15
11
14
12
13
10
21
15
11
8
4
10
7
12
9
9
9
13
8
21
15
11
88,88
57,14
83,33
70,00
80,00
81,80
64,28
75,00
100,00
80,00
100,00
100,00
100,00
190-1250
320-900
300-1250
220-1000
300-1500
300-1000
180-1100
190-1250
270-1350
260-750
170-1590
280-1300
230-1130
0,188+0,053
0,121+0,039
0,260+0,042
0,308+0,057
0,196+0,036
0,310+0,059
0,282+0,049
0,221+0,040
0,337+0,048
0,212+0,044
0,277+0,032
0,250+0,037
0,345+0,048
PI
1,504
0,484
2,600
2,153
2,353
2,789
3,100
2,429
4,382
1,698
5,824
3,751
3,796
UPGMA clustering of alfalfa genotypes using RAPD primers
Satelit
F 105-90
Sigma
Granat
F 1615-04
F 1109-99
F 1822-06
Cosmina
F 1306-01
Super
F 270-91
F 1206-00
F 1310-01
F 1111-99
Pastoral
Magnat
MF 42-96
Selena
Alina
Viking
Cristal
Opal
Stolo 13
Dorina
Coral
F 219-91
Saturn
F 907-97
Venus
F 1413-02
1.00
0.95
0.90
0.85
0.80
0.75
Coeficient de similaritate genetica
0.70
0.65
0.60
Specific RAPD alleles identified in studied alfalfa genotypes
No.crt Specific alleles
1
2
3
4
5
6
7
8
9
10
B 07 – 1250 pb
G 03 – 400 pb
G 03 – 800 pb
G 03 – 900 pb
G 04 – 500 pb
G 04 – 700 pb
G 06 – 220 pb
G 06 – 360 pb
G 06 – 500 pb
G 10 – 330 pb
Nr.crt Specific alleles
11
12
13
14
15
16
17
18
19
20
G 10 - 380 pb
G 10 - 750 pb
G 16 - 480 pb
G 16 - 950 pb
G 17 - 180 pb
G 17 - 400 pb
G 17 - 700 pb
G 18 - 670 pb
L 03 - 480 pb
L 03 -750 pb
The 20 alleles identified in all the 30 genotypes studied may be considered RAPD
specific markers for alfalfa
The results of the analyses performed using two primer categories proved that we
identified, in some genotypes from the collection studied, specific DNA fragments
These bands may be used as DNA markers in the identification of alfalfa genotypes
No.crt
Genotype
Specific
No.crt
Genotype
alleles
1
2
3
4
5
6
7
8
9
10
Pastoral
MF 42-96
F 1111-99
F 1306-01
F 1206-00
A 12 - 1320
G 18 - 1000
L 07 - 540
L 07 - 580
G 04 - 600
G 06 - 1000
A 12 - 300
G 10 - 650
G 17 - 1100
G 19 - 900
Specific
alleles
11
12
13
14
15
16
17
18
19
20
Stolo 13
Sigma
Cosmina
Granat
F 907-97
Saturn
Dorina
Coral
F 1413-02
G 03 - 500
G 18 - 1250
A 12 - 490
A 12 - 744
A 21 - 780
A 21 - 1150
G 10 -350
G 10 - 1300
L 14 - 1000
L 12 -450
In order to assess the intraspecific genetic diversity, we tested 16
ISSR primers and used only three of them: AFca 11, AFct 32 and
AFct 45.
Cod
AFct32
AFct45
AFca 16
AFca 11
AFca1
B14B03
MTLEC2A
MTIC185
MTIC124
MTIC135
MTIC14
MTIC169
MTIC237
MTIC247
MTIC248
MTIC250
Secvenţă- forward
TTTTTGTCCCACCTCATTAG
TAAAAAACGGAAAGAGTTGGTTAG
GGTCGAACCAAGCATGT
CTTGAGGGAACTATTGTTGAGT
CGTATCAATATCGGGCAG
GCTTGTTCTTCTTCAAGCTC
CGGAAAGATTCTTGAATAGATG
AGATTTCAATTCTCAACAACC
TTGTCACGAGTGTTGGAATTTT
GCTGACTGGACGGATCTGAG
CAAACAAACAACACAAACATGG
TCAAAACCCTAAAACCCTTTCTC
CCCATATGCAACAGACCTTA
TTCGCAGAACCTAAATTCAT
TATCTCCCTTCTCCTTCTCC
GCCTGAACTATTGTGAATGG
Secvenţă - reverse
TTGGTTAGATTCAAAGGGTTAC
GCCATCTTTTCTTTTGCTTC
TAAAAAACATTACATGACCTCAAA
AACGTTTCCCAAAACATACTT
TGTTATCAGAGAGAGAAAGCG
ACCTGACTTGTGTTTTATGC
TGGTTCGCTGTTCTCATG
TCTATGATGGATACGATACGG
TTGGGTTGTCAATAATGCTCA
CCAAAGCATAAGCATTCATTCA
CCCATTGATTGGTCAAGGTT
GCGTGCTAGGTTTGAGAGGA
TGGTGAAGATTCTGTTGTTG
TGAGAGCATTGATTTTTGTG
GGATTGTGATGAAGAAATGG
CGTTGATGATGTTCTTGATG
Estimated heterozygoty, effective number of alleles and diversity index in alfalfa genotypes
studied using SSR primers
No
crt
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Genotype
SATELIT
F 1109-99
F105-90
GRANAT
COSMINA
SIGMA
F1615-04
F1206-00
F1306-01
F1822-06
SUPER
F270-91
F1413-02
F1310-01
F1111-99
He
Ne
x  sx
x  sx
0,467+0,077
0,407+0,047
0,440+0,050
0,407+0,047
0,513+0,066
0,180+0,007
0,167+0,093
0,253+0,129
0,267+0,141
0,513+0,058
0,353+0,033
0,413+0,118
0,407+0,047
0,373+0,097
0,267+0,141
2,95+0,26
2,71+0,13
2,81+0,15
2,71+0,13
3,14+0,32
2,22+0,01
2,23+0,14
2,41+0,22
2,46+0,27
3,12+0,27
2,56+0,08
2,83+0,32
2,71+0,13
2,66+0,22
2,46+0,27
No
crt
0,729 1
0,619 2
0,638 3
0,635 4
0,676 5
0,461 6
0,444 7
0,511 8
0,530 9
0,685 10
0,557 11
0,632 12
0,588 13
0,598 14
0,530 15
J
Genotype
PASTORAL
MAGNAT
ALINA
SELENA
STOLO-13
MF 42-96
VIKING
CRISTAL
F219-91
CORAL
DORINA
SATURN
OPAL
VENUS
F907-97
He
Ne
x  sx
x  sx
0,373+0,053
0,260+0,080
0,247+0,127
0,333+0,093
0,253+0,129
0,413+0,052
0,273+0,146
0,307+0,070
0,300+0,151
0,380+0,100
0,433+0,057
0,380+0,106
0,260+0,080
0,300+0,151
0,527+0,047
2,62+0,15
2,39+0,17
2,40+0,21
2,56+0,23
2,41+0,22
2,73+0,15
2,49+0,29
2,47+0,15
2,55+0,28
2,69+0,23
2,80+0,17
2,71+0,28
2,39+0,17
2,55+0,28
3,16+0,24
J
0,566
0,503
0,479
0,567
0,558
0,609
0,520
0,607
0,546
0,602
0,618
0,595
0,560
0,523
0,681
•CONCLUSIONS
-Genomic DNA amplification from 30 genotypes with 5 ISSR primers, generated
117 amplicons, of which 95 were polymorphic markers, the mean – 18.2;
-22 ISSR markers, present in all the 30 genotypes studied, may be considered
specific for alfalfa
Variance analysis for the alfalfa studied genotypes, from the viewpoint of the
bands generated by the ISSR primers, recorded high and significant values in the
cultivars: Cosmina, Sigma, Super and the: F 1615-04, F 1109-99, F 105-90 lines etc
The results obtained with the help of the five ISSR primers, prove the existence of
a high genetic variability among the genotypes analyzed, which could be efficiently
explored in alfalfa breeding programs.
-20 fragments amplified in all the 30 genotypes studied may be considered specific
RAPD markers for alfalfa;
The RAPD analysis with the help of the 13 primers shows the existence of a high
genetic variability among the genotypes studied, which could be efficiently
explored in alfalfa breeding programs;
The results of the genetic diversity assessment at loci level showed the highest
frequent heterozygosity in F 907-97 (52.70 %), Cosmina (51.30 %), F 1822-06
(51.30 %), Satelit (46.70 %) and Dorina (43.30 %) genotypes;
The highest frequent allele homozygosity at loci level was observed in F 1615-04
(83.30 %), Sigma (82.00 %), Alina (75.30 %), Stolo 13 (74.70 %) and F 1206-00
(74.70 %) genotypes;
Also, in the studied genotypes, we observed the existence of a direct correlation
between the estimated heterozygosity and the effective number of alleles, so that
the genotypes having several alleles/locus record a higher frequency of
heterozygosity, too;
THANK YOU !