Analysis of genetic diversity by SSR markers of date palm in the Al-Jufra

Ministero Affari Esteri

ISTITUTO AGRONOMICO PER L’OLTREMARE

LIBIA

“ IMPROVEMENT AND DEVELOPMENT OF DATE PALM

IN THE OASIS OF AL JUFRA”

GENETIC UNIT ___________

Analysis of genetic diversity by SSR markers of date palm in the Al-Jufra oasis Andrea Bove and Milvia Luisa Racchi

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Analysis of genetic diversity by SSR markers of date palm in the Al-Jufra oasis12

Date palm (Phoenix dactylifera L., 2N = 36), a perennial monocotyledonous fruit plant, is the most

important arbocultural crop oasis for that reason it has been recently object of many studies both at

phylogenetic and at molecular level and the first draft sequence is started in 2009 (Weill Cornell

Medical College in Quatar) where a predicted genome size of 550 Mb is available on the web.

Identification of date palm cultivars is principally based on fruit morphology. However, morphological

traits are often variable or imprecise indicators of plant genotype, being influenced by environmental

conditions or varying with the developmental stage of plant. Consequently, discrimination among

closely related cultivars and clones based on morphometric descriptors is often difficult. Nowadays,

molecular markers, based on polymorphisms at DNA level, are increasingly used to address species

delimitation problems where morphological methods are unreliable or inconclusive often providing

greater resolution between closely related taxa and hybrids.

Among molecular markers microsatellites, or simple-sequence repeats (SSRs) because of their

particular features represent a convenient tool for genotyping. They are tandem repeat of short (1-6

nucleotides) DNA segments that are highly variable in number due to slippage of polymerase during

DNA replication. SSRs are intersperse in the genome (both organelle and nuclear genome), show no

environmental or developmental influence and present simple codominant inheritance, this mean

that in a diploid organism as date palm, both alleles at a SSR locus should be visible in the

heterozygote condition.

1 Andrea Bove and Milvia Luisa Racchi

Dipartimento Biotecnologie agrarie, sezione di Genetica Università degli studi di Firenze

via Maragliano 77, 50144 Firenze

2 The present study has been conducted in the framework of the Italy - Lybia bilateral cooperation project "Improvement

and Valorisation of Date Palm in Al Jufrah Oasis", carried out by Istituto Agronomico per l'Oltremare (Italy) and the Board

of Improving and Developing Olive and Palm Trees (Tripoli, Libya).With the general objective of supporting and

strengthening the date palm production through the improvement and valorisation of production, transformation and

commercialisation of local varieties, the project aims to contribute to the social and economic development of the Al

Jufra Region. In this framework, the characterisation and description of local varieties, performed through the sampling

and description of genetic, morphometric and chemical characteristics, is accompanied by an integrated study of the

palm dates cropping system in the Al Jufrah Region. Such study has been carried out for the five oasis of Waddan, Hun,

Sokna, Zellah and Al Fugha.

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Because of their high mutation rates and the ease of the analysis we used SSR as molecular markers

to perform the genetic fingerprinting of the Libyan date palm’s resources. in fact this methodology

was proved useful and effective for genetic fingerprinting, cultivar identification and phylogenetic

studies among different accessions.

Materials and Methods

Plant materials

Samples were collected in palm date groves, both of recent and ancient constitution in the locality of

Sawkanah, Hun, Waddan, Zillah and Al Faqqah.

The names of 18 varieties commercially relevant sampled in farms of the different localities are listed

in Table1.

Plant materials consists of young leaves of adult trees randomly samples from the mentioned

localities of Al-Jufra oasis.

DNA extraction

The dry leaf material was ground into a fine powder using bead-mill homogenizer TissueLyser

(Qiagen, Italy ). The leaf powder was then subjected to DNA extraction using both DNeasy Plant

Maxi/Mini Kits (Qiagen, Milano Italia) or E-Z 96 Plant kit (Omega), according to manufacturer’s

instructions and the resulting DNA solutions were stored at –20°C. After purification, DNA

concentration and quality were determined on 1% of agarose gel electrophoresis.

Microsatellites amplification and Genotyping

We have tested a total of 16 date-palms specific primer pairs selected for their polymorphic

information content among SSR loci developed by Billote et al. (2004) and Akkak et al.(2009). PCR

reactions were performed in a total reaction mixture of 14 µL containing: 20 ng of total genomic

DNA, 1X/PCR buffer (Promega Corp. Madison, USA), 0.2 mM of dNTP (Promega), 0.05 U of Taq DNA

polymerase (Promega), 0,07 µM of the forward primer with M13 tail, 0,2 µM of the reverse primer

and 0,2 µM of M13 primer-fluorescent dye (Invitrogen).

For a given locus, the forward SSR primer was 5’-end labeled with M13 extension (5’-

TGTAAAACGACGGCCAGT-3’) for incorporate, via PCR step, a fluorochrome (6-FAM, VIC or NED)

necessary for detect the PCR products on the sequencer.

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Amplifications were performed in Applied Biosystem Thermocycler (AB System, Germany) with the

following conditions: for Billote’s primer, a initial denaturation at 95°C for 1 min, then 35 cycles of

94°C for 30 sec, 52°C for 1 min and 72°C for 2 min and a final elongation step at 72°C for 8 min; for

Akkak’s primer a initial denaturation at 95°C for 9 min, then 28-35 cycles of 94°C for 30 sec, 55°C for

45 sec and 72°C for 1 min and a final elongation step at 72°C for 45 min. A negative control, with the

reaction mixture excluding DNA, was also included in each experiment.

Amplification products were checked on 1,5 % agarose gel to verify the presence of a band of the

expected size.

PCR products were resolved on an MegaBace 1000 (GE Healthcare, USA) sequencer. Data were

analyzed using the software Fragment profiler ver. 2.1 (Amersham Biosciences)

Genetic Data Analysis

The total number of alleles, number of genotype, alleles frequencies and the measured of genetic

diversity and differentiation were calculated for all individuals at all loci, using the opensourse

software GenAlEx 6 (Peakall, R. and Smouse P.E.; 2006).

Barcode set

Barcode has been developed using the software opensource “Barcode Label Generator Plus”

(www.scansecretary.com)

Using that software DNA data obtained by SSR genotyping at 12 loci were transformed in a numeric

code consisting of 24 characters giving to each allele a progressive number from 0 to 9.

Results and discussion

In a preliminary analysis 18 SSR marker were tested and afterward the 12 more polymorphic were

used to perform the analysis. All the markers selected gave successful amplification in the condition

used in all the samples evaluated.

The SSR marker gave rise to a electropherogram profile that revealed the nature homozygote or

heterozygote of the locus analyzed. In figure 1 the profiles of some representative markers were

reported.

Genetic diversity evaluated as number of alleles and genotypes for each SSR locus was presented in

Table 2.

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The total number of alleles found at 12 SSR loci, considering the three localities more represented in

the collection was 75, with a weighted average on 6,25 alleles per each marker. It is interesting to

note as this number was enough to allow the unambiguous identification the cultivars. The

distribution of alleles frequencies for the SSR loci in the five locality of Al-Jufra was presented in

Figure 2.

The multilocus genotypes of the cultivars analyzed were presented in Table 3. The 12 SSR profiles

obtained allow to indentify clearly each cultivar with the exception of three varieties indicated by an

asterisk. In that cases variability was observed both among localities as for Bamour and Noyat Meka

or as in the case of Sokeri within farm. This result can be related to the small number of trees

sampled for the former and to the practice of seed reproduction instead of the clonal propagation for

the latter (Al Sanusse personal communication).

In the whole the results evidenced that the clonal propagation of the cultivar was a practice common

to the majority of the farmers and that in general they had a good skill, based on a long habit, in the

classification of the cultivars even if some mistakes were noted. The cases of misclassification could

due both to errors in the sampling or during propagation because of the difficulty, in some cases, to

identify some cultivars on the base of the morphology.

Since each variety was identified by a unique profile, it has been possible to generate an individual

barcode using the multilocus genotype. In figure 3 the barcodes for the date palms of Al Jufra oasis

are presented. The barcode represents an important tool for the genetic identity and the traceability

of the products consequently its availability can represent an additional value for Libyan dates.

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Table 1 Names of 18 varieties commercially relevant sampled in farms of the different localities

Name of cultivar Locality

N° of samples

analysed

Abel Sokna 7

Hon 8

Waddan 10

Bamour Sokna 1

Waddan 7

Berni Sokna 5

Hon 7

Waddan 9

Bestian Sokna 8

Hon 10

Waddan 9

Zillah 3

Al Fuqqah 4

Deglet Sokna 10

Hon 7

Waddan 7

Zillah 1

Halima Sokna 7

Hon 3

Waddan 6

Hamria Sokna 8

Hon 9

Waddan 7

Zillah 5

Al Fuqqah 5

Kathari Sokna 12

Hon 10

Waddan 6

Zillah 6

Noyat Meka Hon 1

Waddan 8

Omglaib Sokna 1

Hon 4

Waddan 6

Saiedi Sokna 4

Hon 7

Waddan 6

Zillah 4

Saila Sokna 3

Hon

8

7

Sokeri Sokna 8

Hon 2

Waddan 9

Tagiat Sokna 7

Hon 10

Waddan 11

Zillah 6

Al Fuqqah 5

Talis Sokna 8

Hon 6

Waddan 6

Zillah 5

Tameg Sokna 6

Hon 9

Waddan 7

Tasferit Sokna 4

Hon 4

Waddan 4

Zebur Sokna 6

Hon 6

Waddan 2

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Table 2 Genetic diversity measured by the number of alleles/genotypes for each marker in the

different localities of Al-jufra oasis.

Markers name

Locality

Number

of alleles

detected

Number

of

genotypes

DM 1 Sokna 5 11

Hon 5 11

Waddan 5 10

Zillah 5 5

Al Fuqqah 3 3

DM 2 Sokna 5 7

Hon 5 6

Waddan 5 6

Zillah 5 5

Al Fuqqah 3 3

DM 3 Sokna 7 12

Hon 7 11

Waddan 7 13

Zillah 6 8

Al Fuqqah 3 2

DM 4 Sokna 7 11

Hon 7 11

Waddan 7 13

Zillah 6 5

Al Fuqqah 4 3

DM 5 Sokna 7 10

Hon 5 9

Waddan 6 11

Zillah 5 6

Al Fuqqah 3 3

DM 6 Sokna 8 13

Hon 7 12

Waddan 8 14

Zillah 5 7

Al Fuqqah 4 3

DM 7 Sokna 9 15

Hon 7 13

Waddan 9 17

Zillah 5 5

Al Fuqqah 4 2

DM 8 Sokna 7 11

Hon 6 9

Waddan 7 10

Zillah 5 5

Al Fuqqah 3 2

9

DM 9 Sokna 6 8

Hon 6 8

Waddan 6 9

Zillah 5 6

Al Fuqqah 3 3

DM 10 Sokna 6 14

Hon 7 13

Waddan 6 12

Zillah 6 9

Al Fuqqah 4 3

DM 11 Sokna 5 7

Hon 5 7

Waddan 9 10

Zillah 4 5

Al Fuqqah 3 3

DM 12 Sokna 4 7

Hon 4 6

Waddan 5 7

Zillah 3 5

Al Fuqqah 3 5

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Figure 1 Allelic profiles at the microsatellite loci of the principal date palm varieties in the five

localities of Al-Jufra oasis. Numbers on X axis refer to allele size (bp) and on Y axis to fluorochrome

intensity.

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Figure 2 Distribution microsatellites alleles frequencies in five localities of Al-Jufra oasis date palms Allele Frequency for locus DM 1

0,000

0,200

0,400

0,600

142 144 146 151 158

Alleles

Fre

qu

en

cy

sokna

hon

w addan

zillah

faqqah

Allele Frequency for locus DM 2

0,000

0,200

0,400

0,600

309 310 316 318 321

Fre

qu

en

cy sokna

hon

w addan

zillah

faqqah


0,000

0,1000,200

0,3000,400

0,500

181 186 188 191 192 193 198

Alleles

Fre

qu

en

cy

sokna

hon

w addan

zillah

faqqah


0,000

0,100

0,200

0,300

0,400

0,500

186 192 196 198 199 206 209

Alleles

Fre

qu

en

cy

sokna

hon

w addan

zillah

faqqah


0,000

0,1000,200

0,3000,400

0,500

143 150 156 158 166 168 172

Alleles

Fre

qu

en

cy

sokna

hon

w addan

zillah

faqqah


0,000

0,100

0,200

0,300

0,400

0,500

123 133 139 141 143 145 147 149

Alleles

Fre

qu

en

cy

sokna

hon

w addan

zillah

faqqah


0,000

0,100

0,200

0,300

0,400

205 212 213 215 217 219 223 225 227

Alleles

Fre

qu

en

cy

sokna

hon

w addan

zillah

faqqah


0,000

0,200

0,400

0,600

222 225 246 252 254 256 258

Alleles

Fre

qu

en

cy

sokna

hon

w addan

zillah

faqqah


0,000

0,200

0,400

0,600

0,800

131 141 143 147 151 157

Alleles

Fre

qu

en

cy

sokna

hon

waddan

zillah

faqqah


0,000

0,200

0,400

0,600

154 158 159 163 167 175 177

Alleles

Fre

qu

en

cy

sokna

hon

waddan

zillah

faqqah


0,000

0,200

0,400

0,600

0,800

141 143 145 147 149 151 155 157 163

Alleles

Fre

qu

en

cy

sokna

hon

waddan

zillah

faqqah


0,000

0,200

0,400

0,600

106 108 120 123 127

Alleles

Fre

qu

en

cy

sokna

hon

waddan

zillah

faqqah

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Figure 3 Barcodes of the 18 date palm varieties belonging to Al-Jufra oasis

BESTIAN ABEL BAMOUR BERNI

DEGLET HALIMA HAMRIA KATHARI

NOYAT MEKA OMGLAIB SAIEDI SAILA

SOKERI TAGIAT TALIS TAMEG

TASFERIT ZEBUR

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Table 3 Multilocus genotypes of 18 commercial date palm clones at twelve nuclear microsatellite loci clone locality

‡ DM 1 DM 2 DM 3 DM 4 DM 5 DM 6 DM 7 DM 8 DM 9 DM 10 DM 11 DM 12

Abel S ,H, W 142 146 309 316 191 198 196 209 150 158 123 145 217 219 222 252 147 147 163 175 141 141 123 123

Bamour* W 146 158 316 321 198 198 186 196 158 158 123 145 217 225 252 252 147 147 159 175 149 151 123 127

Berni S, H, W 146 151 309 316 191 191 196 198 150 158 145 147 212 217 246 252 147 147 159 175 141 141 123 123

Bestian S, H, W, Z, F 144 146 309 316 191 191 206 206 156 156 149 149 217 225 222 246 141 147 159 163 141 155 108 108

Deglet S, H, W, Z 144 158 310 318 191 193 196 209 156 168 141 149 213 213 246 256 147 147 167 167 155 155 123 123

Halima S, H, W 142 151 316 321 191 193 186 209 150 156 147 149 213 225 246 254 141 147 159 175 155 155 123 123

Hamria S, H, W, Z, F 144 158 316 316 181 198 199 206 150 158 145 149 217 225 246 252 147 157 163 175 143 155 108 123

Kathari S, H, W, Z 142 146 309 316 191 198 186 209 156 158 123 149 213 215 222 246 147 147 163 163 141 143 123 123

Noyat Meka* W 146 146 309 309 181 191 186 206 150 156 149 149 212 217 246 246 147 147 159 159 141 155 108 108

Omglaib S, H, W 142 144 309 316 198 198 206 209 158 158 123 145 205 219 246 252 147 147 163 175 155 155 123 123

Saiedi S, H, W, Z 144 146 316 316 192 198 192 196 156 172 139 149 213 215 252 258 143 151 154 175 143 163 123 123

Saila S, H 142 142 309 316 191 193 196 209 150 156 133 149 213 217 246 246 141 141 159 159 141 155 108 108

Sokeri*** S 151 158 316 318 186 198 196 206 150 158 133 145 219 219 246 252 147 147 154 177 155 155 106 106

Tagiat S, H, W, Z, F 146 158 309 321 181 198 186 209 150 156 123 141 205 215 246 252 141 157 154 163 155 155 123 127

Talis S, H, W, Z 146 158 309 309 188 198 186 209 150 156 123 145 213 215 252 252 157 157 163 177 155 155 127 127

Tameg S, H, W 142 151 316 321 181 193 186 196 156 158 133 149 213 225 246 246 141 141 159 167 155 155 106 106

Tasferit S, H, W 158 158 309 321 188 188 199 209 158 172 145 149 219 225 254 254 141 147 163 177 157 157 123 123

Zebur S, H, W 158 158 316 316 186 198 186 186 150 150 145 149 215 217 246 252 131 147 154 154 141 143 106 106

localities‡

S, Sokna; H, Hon; W, Waddan; Z, Zillah; F, Al Faqqah;

* : the profil refers to Waddan only

**: the profil refers to Sokna only. In Waddan the Sokeri variety is seed propagated and then selected on the base of morphometric important traits therefore cannot be

considered as a clone..

Documents

Analysis of genetic diversity by SSR markers of date palm in the Al-Jufra