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.

No. Primer Nucleotide Bands No. Polymor- Bands PIC PI crt. sequence Total Polymor ph (%) (pb) x

sx 1 A-12 (GA)6CC 25 20 84,00 220-1650 0,210+0,040 3,367 2 A-13 (GT)6CC 27 19 70,37 150-1510 0,292+0,028 5,544 3 A-17 (GTG)3GC 31 31 100,00 240-2090 0,370+0,019 11,462 4 A-21 (CA)6AC 15 9 60,00 280-1320 0,324+0,052 2,918 5 UBC-818 (CA)7G 19 16 84,21 300-2120 0,358+0,031 5,731

Polymorphism rate for the alfalfa genotypes using ISSR primers

Assessment of interspecific variability using ISSR molecular markers

F 270-91

F 1310-01

PastoralF 1111-99

MagnatAlina

Stolo 13VikingCristal

OpalDorina

F 219-91Coral

Selena

SaturnMF 42-96

Satelit

Venus

Cosmina

F 907-97

F 105-90

F 1615-04Super

F 1306-01

F 1413-02

1.00 0.95

Granat

0.90

F 1206-00

0.85 0.80 0.75 0.70 0.65

F 1109-99

0.60

F 1822-06Sigma

Coeficient de similaritate genetica

UPGMA clustering of alfalfa genotypes using the ISSR primers

No.crt Specific alleles No.ct Specific alleles

1 A 12 -220 12 A 13 -1259 2 A 12 -280 13 A 13 -1510 3 A 12 -650 14 A 21 -280 4 A 12 -820 15 A 21 -410 5 A 12 -950 16 A 21 -520 6 A 13 -330 17 A 21 -750 7 A 13 -440 18 A 21 -1080 8 A 13 -550 19 A 21 -1180 9 A 13 -600 20 UBC 818 -300

10 A 13 -1017 21 UBC 818 -510 11 A 13 -1190 22 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

No. Primer Nucleotide Sequence (5’-3’)

Bands No. Polymor Size PIC PI

crt. Total Polymorf (%) bands (pb) x

sx

1 B -07 GGTGACGCAG 9 8 88,88 190-1250 0,188+0,053 1,504 2 G -03 GAGCCCTCCA 7 4 57,14 320-900 0,121+0,039 0,484 3 G -04 AGCGTGTCTG 12 10 83,33 300-1250 0,260+0,042 2,600 4 G -06 GTGCCTAACC 10 7 70,00 220-1000 0,308+0,057 2,153 5 G -10 AGGGCCGTCT 15 12 80,00 300-1500 0,196+0,036 2,353 6 G -16 AGCGTCCTCC 11 9 81,80 300-1000 0,310+0,059 2,789 7 G -17 ACGACCGACA 14 9 64,28 180-1100 0,282+0,049 3,100 8 G -18 GGCTCATGTG 12 9 75,00 190-1250 0,221+0,040 2,429 9 G -19 GTCAGGGCAA 13 13 100,00 270-1350 0,337+0,048 4,382

10 L -03 CCAGCAGCTT 10 8 80,00 260-750 0,212+0,044 1,698 11 L -07 AGGCGGGAAC 21 21 100,00 170-1590 0,277+0,032 5,824 12 L-12 GGGCGGTACT 15 15 100,00 280-1300 0,250+0,037 3,751 13 L -14 GTGACAGGCT 11 11 100,00 230-1130 0,345+0,048 3,796

Polymorphism rate for the alfalfa genotypes using RAPD primers

F 1206-00

MagnatPastoral

F 1111-99

MF 42-96

AlinaSelena

VikingCristal

OpalStolo 13

DorinaCoral

F 219-91Saturn

F 1413-02

Satelit

Venus

Sigma

F 907-97

Cosmina

F 1615-04

F 1306-01

F 270-91

F 1310-01

1.00 0.95

Granat

0.90

F 1822-06

0.85 0.80 0.75 0.70 0.65

F 105-90

0.60

Super

F 1109-99

Coeficient de similaritate genetica

UPGMA clustering of alfalfa genotypes using RAPD primers

No.crt Specific alleles Nr.crt Specific alleles

1 B 07 – 1250 pb 11 G 10 - 380 pb 2 G 03 – 400 pb 12 G 10 - 750 pb 3 G 03 – 800 pb 13 G 16 - 480 pb 4 G 03 – 900 pb 14 G 16 - 950 pb 5 G 04 – 500 pb 15 G 17 - 180 pb 6 G 04 – 700 pb 16 G 17 - 400 pb 7 G 06 – 220 pb 17 G 17 - 700 pb 8 G 06 – 360 pb 18 G 18 - 670 pb 9 G 06 – 500 pb 19 L 03 - 480 pb

10 G 10 – 330 pb 20 L 03 -750 pb

The 20 alleles identified in all the 30 genotypes studied may be considered RAPD specific markers for alfalfa

Specific RAPD alleles identified in studied alfalfa genotypes

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

alleles

No.crt Genotype Specific

alleles

1 Pastoral A 12 - 1320 11 Stolo 13 G 03 - 500 2 G 18 - 1000 12 G 18 - 1250 3 MF 42-96 L 07 - 540 13 Sigma A 12 - 490 4 L 07 - 580 14 Cosmina A 12 - 744 5 F 1111-99 G 04 - 600 15 Granat A 21 - 780 6 G 06 - 1000 16 F 907-97 A 21 - 1150 7 F 1306-01 A 12 - 300 17 Saturn G 10 -350 8 G 10 - 650 18 Dorina G 10 - 1300 9 F 1206-00 G 17 - 1100 19 Coral L 14 - 1000 10 G 19 - 900 20 F 1413-02 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 Secvenţă- forward Secvenţă - reverse AFct32 TTTTTGTCCCACCTCATTAG TTGGTTAGATTCAAAGGGTTAC AFct45 TAAAAAACGGAAAGAGTTGGTTAG GCCATCTTTTCTTTTGCTTC AFca 16 GGTCGAACCAAGCATGT TAAAAAACATTACATGACCTCAAA AFca 11 CTTGAGGGAACTATTGTTGAGT AACGTTTCCCAAAACATACTT AFca1 CGTATCAATATCGGGCAG TGTTATCAGAGAGAGAAAGCG B14B03 GCTTGTTCTTCTTCAAGCTC ACCTGACTTGTGTTTTATGC MTLEC2A CGGAAAGATTCTTGAATAGATG TGGTTCGCTGTTCTCATG MTIC185 AGATTTCAATTCTCAACAACC TCTATGATGGATACGATACGG MTIC124 TTGTCACGAGTGTTGGAATTTT TTGGGTTGTCAATAATGCTCA MTIC135 GCTGACTGGACGGATCTGAG CCAAAGCATAAGCATTCATTCA MTIC14 CAAACAAACAACACAAACATGG CCCATTGATTGGTCAAGGTT MTIC169 TCAAAACCCTAAAACCCTTTCTC GCGTGCTAGGTTTGAGAGGA MTIC237 CCCATATGCAACAGACCTTA TGGTGAAGATTCTGTTGTTG MTIC247 TTCGCAGAACCTAAATTCAT TGAGAGCATTGATTTTTGTG MTIC248 TATCTCCCTTCTCCTTCTCC GGATTGTGATGAAGAAATGG MTIC250 GCCTGAACTATTGTGAATGG CGTTGATGATGTTCTTGATG

Estimated heterozygoty, effective number of alleles and diversity index in alfalfa genotypes studied using SSR primers

No crt

Genotype He

xsx

Ne x

sx J No crt

Genotype He

xsx

Ne x

sx J

1 SATELIT 0,467+0,077 2,95+0,26 0,729 1 PASTORAL 0,373+0,053 2,62+0,15 0,566 2 F 1109-99 0,407+0,047 2,71+0,13 0,619 2 MAGNAT 0,260+0,080 2,39+0,17 0,503 3 F105-90 0,440+0,050 2,81+0,15 0,638 3 ALINA 0,247+0,127 2,40+0,21 0,479 4 GRANAT 0,407+0,047 2,71+0,13 0,635 4 SELENA 0,333+0,093 2,56+0,23 0,567 5 COSMINA 0,513+0,066 3,14+0,32 0,676 5 STOLO-13 0,253+0,129 2,41+0,22 0,558 6 SIGMA 0,180+0,007 2,22+0,01 0,461 6 MF 42-96 0,413+0,052 2,73+0,15 0,609 7 F1615-04 0,167+0,093 2,23+0,14 0,444 7 VIKING 0,273+0,146 2,49+0,29 0,520 8 F1206-00 0,253+0,129 2,41+0,22 0,511 8 CRISTAL 0,307+0,070 2,47+0,15 0,607 9 F1306-01 0,267+0,141 2,46+0,27 0,530 9 F219-91 0,300+0,151 2,55+0,28 0,546 10 F1822-06 0,513+0,058 3,12+0,27 0,685 10 CORAL 0,380+0,100 2,69+0,23 0,602 11 SUPER 0,353+0,033 2,56+0,08 0,557 11 DORINA 0,433+0,057 2,80+0,17 0,618 12 F270-91 0,413+0,118 2,83+0,32 0,632 12 SATURN 0,380+0,106 2,71+0,28 0,595 13 F1413-02 0,407+0,047 2,71+0,13 0,588 13 OPAL 0,260+0,080 2,39+0,17 0,560 14 F1310-01 0,373+0,097 2,66+0,22 0,598 14 VENUS 0,300+0,151 2,55+0,28 0,523 15 F1111-99 0,267+0,141 2,46+0,27 0,530 15 F907-97 0,527+0,047 3,16+0,24 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 !