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
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. 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. 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 eterozygote 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. 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 (El Senusi 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.
Table 1 Names of 18 varieties commercially relevant sampled in farms of the different localities Name of cultivar Locality N째 of samples analysed Abel
Bamour Berni
Bestian
Deglet
Halima
Hamria
Kathari
Noyat Meka Omglaib
Saiedi
Saila Sokeri
Tagiat
Talis
Sokna Hon Waddan Sokna Waddan Sokna Hon Waddan Sokna Hon Waddan Zillah Al Fuqqah Sokna Hon Waddan Zillah Sokna Hon Waddan Sokna Hon Waddan Zillah Al Fuqqah Sokna Hon Waddan Zillah Hon Waddan Sokna Hon Waddan Sokna Hon Waddan Zillah Sokna Hon Sokna Hon Waddan Sokna Hon Waddan Zillah Al Fuqqah Sokna Hon Waddan Zillah
7 8 10 1 7 5 7 9 8 10 9 3 4 10 7 7 1 7 3 6 8 9 7 5 5 12 10 6 6 1 8 1 4 6 4 7 6 4 3 8 8 2 9 7 10 11 6 5 8 6 6 5
Name of cultivar Tameg
Tasferit
Zebur
Locality Sokna Hon Waddan Sokna Hon Waddan Sokna Hon Waddan
N째 of samples analysed 6 9 7 4 4 4 6 6 2
Table 2 Genetic diversity measured by the number of alleles/genotypes for each marker in the different localities of Al-jufra oasis. Number Number Markers name Locality of alleles of detected genotypes Sokna 5 11 DM 1 Hon 5 11 Waddan 5 10 Zillah 5 5 Al Fuqqah 3 3 Sokna 5 7 DM 2 Hon 5 6 Waddan 5 6 Zillah 5 5 Al Fuqqah 3 3 Sokna 7 12 DM 3 Hon 7 11 Waddan 7 13 Zillah 6 8 Al Fuqqah 3 2 Sokna 7 11 DM 4 Hon 7 11 Waddan 7 13 Zillah 6 5 Al Fuqqah 4 3 Sokna 7 10 DM 5 Hon 5 9 Waddan 6 11 Zillah 5 6 Al Fuqqah 3 3 Sokna 8 13 DM 6 Hon 7 12 Waddan 8 14 Zillah 5 7 Al Fuqqah 4 3 Sokna 9 15 DM 7 Hon 7 13 Waddan 9 17 Zillah 5 5 Al Fuqqah 4 2
Markers name DM 8
DM 9
DM 10
DM 11
DM 12
Locality Sokna Hon Waddan Zillah Al Fuqqah Sokna Hon Waddan Zillah Al Fuqqah Sokna Hon Waddan Zillah Al Fuqqah Sokna Hon Waddan Zillah Al Fuqqah Sokna Hon Waddan Zillah Al Fuqqah
Number of alleles 7 6 7 5 3 6 6 6 5 3 6 7 6 6 4 5 5 9 4 3 4 4 5 3 3
Number of genotypes 11 9 10 5 2 8 8 9 6 3 14 13 12 9 3 7 7 10 5 3 7 6 7 5 5
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.
Figure 2 Distribution microsatellites alleles frequencies in five localities of Al-Jufra oasis date palms Allele Frequency for locus DM 2
0,600
0,600 sokna
0,400
hon
0,200
w addan
0,000
zillah
142
144
146
151
158
sokna
Frequency
Frequency
Allele Frequency for locus DM 1
0,400
hon
0,200
w addan zillah
0,000
faqqah
faqqah
309
Alleles
310
sokna hon w addan zillah
188
191
192
193
198
sokna hon w addan zillah
186
faqqah
192
196
sokna hon w addan zillah
158
166
168
172
w addan zillah
133
139
141
143
145
Alleles
Allele Frequency for locus DM 7
Allele Frequency for locus DM 8
147
149
faqqah
0,600 sokna
0,200
hon
0,100
w addan
0,000
zillah
212
213
215
217
219
223
225
227
Frequency
Frequency
faqqah
hon
Alle les
0,300
205
sokna
0,400
hon
0,200
w addan zillah
0,000
faqqah
222
225
246
Alleles
252
254
256
258
faqqah
Alleles
Allele Frequency for locus DM 9
Allele Frequency for locus DM 10
0,800
0,600
0,600
sokna
0,400
hon
0,200
waddan
0,000
zillah
131
141
143
147
151
157
Frequency
Frequency
209
sokna
123
faqqah
0,400
sokna
0,400
hon
0,200
waddan
0,000
zillah
154
faqqah
158
159
Alleles
163
167
175
177
faqqah
Alleles
Allele Frequency for locus DM 12
Allele Frequency for locus DM 11
0,600
0,600
sokna
0,400
hon
0,200
waddan
0,000
zillah
141
143
145
147
149 Alleles
151
155
157
163
faqqah
Frequency
0,800 Frequency
206
0,500 0,400 0,300 0,200 0,100 0,000
Frequency
Frequency
0,500 0,400 0,300 0,200 0,100 0,000 156
199
Allele Frequency for locus DM 6
Allele Frequency for locus DM 5
150
198 Alleles
Alleles
143
321
0,500 0,400 0,300 0,200 0,100 0,000
Frequency
Frequency
0,500 0,400 0,300 0,200 0,100 0,000 186
318
Allele Frequency for locus DM 4
Allele Frequency for locus DM 3
181
316
sokna
0,400
hon
0,200
waddan zillah
0,000 106
108
120 Alleles
123
127
faqqah
Figure 3 Barcodes of the 18 date palm varieties belonging to Al-Jufra oasis ABEL
BERNI
BESTIAN
HALIMA
HAMRIA
KATHARI
OMGLAIB
SAIEDI
SAILA
TAGIAT
TALIS
TAMEG
BAMOUR
DEGLET
NOYAT MEKA
SOKERI
TASFERIT
ZEBUR
Table 3 Multilocus genotypes of 18 commercial date palm clones at twelve nuclear microsatellite loci locality‡
clone
DM 1
DM 2
DM 3
DM 4
DM 5
DM 6
DM 7
DM 8
DM 9
DM 10
DM 11
DM 12
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
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
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
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
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
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
Abel Bamour* Berni
Noyat Meka* Omglaib
Sokeri***
‡
locality S, Sokna; H, Hon; W, Waddan; Z, Zillah; F, Al Faqqah; Bamour* il profilo principale vale solo per la località di W poichè in S ottenuto profilo diverso ma solo un individuo campionato (non significativo) Noyat Maka** il profilo principale vale solo per la località di W poichè a H ottenuto profilo diverso su un solo individuo (non significativo) Sokari*** il profilo principale vale solo per la località S, esclusa la farm SJS dove ottenuto un altro profilo; nella località di H ottenuto un altro profilo ma solo 2 individui campionati (non significativo); nella località di W ottenuta una variabilità massima: su 9 individui saggiati 9 profili diversi (indice che tali varietà sono state propagate per seme e poi selezionate sulla base di caratteri morfologici importanti)