Molecular variance of the Tunisian almond germplasm assessed by simple sequence repeat (SSR) markers

The genetic variance analysis of 82 almond ( Prunus dulcis Mill . ) genotypes was performed using ten genomic simple sequence repeats (SSRs). A total of 50 genotypes from Tunisia including local landraces identified while prospecting the different sites of Bizerte and Sidi Bouzid (Northern and central parts) which are the most important locations of almond diversity in Tunisia were included. Analysis of molecular variance (AMOVA) was performed for 11 populations from these different regions and foreign countries to examine the distribution of genetic variation of the accessions studied. Results show that the major variation occurred within populations in each geographic site. Additionally, this analysis demonstrates that the genetic diversity within local almond cultivars was important, with a clear geographic distinction between the Northern and the Southern Tunisian cultivars. The value of prospecting new sites, preserving genetic diversity and encouraging on farmers almond collections is also discussed.


INTRODUCTION
In Tunisia, the almond Prunus dulcis (Miller) D.A. Webb, syn.P. amygdalus Batsch sector, plays an important social economic role with approximately 22 millions of trees dispersed on more than 200,000 ha (FAOSTAT, 2010).In fact, the Tunisian almond plantations located throughout all the country under different climatic conditions offered stable incomes for rural farmers.About 90% of the land devoted to this fruit crop is in the Central and Southern agricultural area of the country under arid and semi-arid conditions.Bizerte (37°16'N, 9°52'E), Sidi Bouzid (35°04'N, 9°49'E) and Sfax (34°44'N, 10°46'E) are the main producing regions (Figure 1).
While preserving genetic resources of fruit trees in gene banks is generally difficult to handle and might not be exhaustive, precise identification of landraces in farm is highly recommended.Furthermore, the correct evaluation of relatedness is capital for efficient genetic resource management and for maintaining enough variability for breeding programs.In the last five years, more than three million of almond trees were lost because of the long period of drought.This has increased the need to preserve as much as possible the Tunisian almond genetic diversity, in order to reduce genetic erosion.For this, a prospecting effort was carried out during these last few years through the Northern and Central part of Tunisia and an important genetic diversity resulting from chance seedlings or human selections was found.
As few information is available about the genetic origin of the existing diversity for the Tunisian almond cultivars and their relationship with almonds from the northern border of the Mediterranean, the aims of this work were to identify the SSR analysis of the population arrangement of the accessions collected directly from different sites of the country (Sidi Bouzid and Bizerte), to deter-mine their relatedness to European and American cultivars and to examine the distribution of the genetic variability.
The 50 Tunisian local genotypes were from the region of Bizerte, Nefta, Sfax and Tozeur that are either preserved in the National Collection of Ettaous, or originated from recent prospection undertaken in the regions of Sidi Bouzid and Bizerte (Figure 1).All the local genotypes assayed were early flowering, self incompatible and had sweet kernels.

Genomic DNA extraction
From all accessions, young leaves were collected for DNA extraction.Total genomic DNA was isolated using the procedure described by Doyle and Doyle (1987).DNA concentration and dilutions for polymerase chain reaction (PCR) amplification were carried out as described by Bouhadida et al. (2007).

DNA amplification
DNA was amplified by PCR using 10 primer pairs of microsatellite (Table 2), nine pairs derived from a library enriched for AG/TC motifs, constructed with the almond cultivar 'Texas' (Mnejja et al., 2004) and one pair previously cited by Joobeur et al. (2000).
Amplification reactions were carried out in a final volume of 15 l containing 10 ng of template DNA and PCR reagents as described in Gouta et al. (2010) with a Gene Amp 2700™ thermocycler (Applied Biosystems, CA, USA) using the following temperature cycles: 1 cycle of 3 min at 95°C; 35 cycles of 1 min at 94°C, 45 s at the corresponding annealing temperature (Table 2) and 1 min at 72°C.The last cycle was followed by a final incubation for 7 min at 72°C and the PCR products were stored at 4°C before analysis.Two independent SSR reactions were performed for each DNA sample.PCR products were loaded on 5% polyacrylamide sequencing gels, silver-stained according to the protocol described by Bassam et al. (1983).Fragment sizes were estimated using 30-330 bp AFLP ladder (Invitrogen, Carlsbad, CA, USA) DNA sizing markers, and analyzed by the quantity one program (Bio Rad, Hercules, CA, USA).

Diversity parameters
Allelic composition of each accession and total number of alleles were scored for each SSR locus from gel profile analysis.Putative alleles were indicated by the estimated size, in bp.Wright's fixation index [F (1/1 -Ho/He) (Wright, 1951)], was calculated for accessions with one or two bands per microsatellite where (Ho) represented the observed heterozygosity, and (He) the expected heterozygosity.
F statistics relative to each component (that is, F CT among groups, F SC among populations within groups, F ST within populations) were computed.Pairwise F ST values between regions were used to construct an unweighted pair group method with arithmetic mean (UPGMA) dendrogram using NTSYSpc 2.11 (Rohlf, 2000).

RESULTS
The fixation index average (F=0.13)shows a deficit of heterozygosity and a significant divergence over Hardy-Weinberg expectation (P  0.01) for nine of the 10 loci studied (Table 3).
AMOVA calculations were performed (Table 4) with 11 populations grouped by their geographic origin into three groups as described in the material and methods and divided into 11 populations: The G1 with local cultivars from South (Tozeur and Nefta), Center (Sidi Bouzid): Ben Aoun, Ouled Haffouz and Regueb; G2 grouped the American cultivars in addition to the Tunisians from Bizerte that were also added to this group according to the results of the dendrogram (Figure 2), which surprisingly allocated this population in the same cluster with the American cultivars and G3 including cultivars from France, Italy, Spain.
The results of the AMOVA analysis (Table 4) showed that although the great majority of the variation (90.54%) estimated with the ten SSR markers occurred within populations, a small but significant proportion was attributed to differences among groups (6.48%) and among populations within groups (2.98 %).F values at different levels were significant (F CT = 0.06484, F SC = 0.03187, F ST = 0.09464) with P  0.001.Similar variation percentage within populations (88.7%) was also noted by Bouhadida et al. (2011) when studying genetic variability of introduced and local Spanish peach cultivars.
The dendrogram (Figure 2) based on population pair wise genetic distance (F ST ) between regions, showing the distribution of genetic diversity for all accessions, differentiates two main groups (A and B).Group A includes the foreign populations and cultivars from North of Tunisia (Bizerte) while in group B are included the rest of the Tunisian populations from the central (Sidi Bouzid) and Southern (Sfax, Tozeur and Nefta) part of Tunisia.

DISCUSSION
In this work, we studied with 10 SSRs, the main Tunisian almond cultivars preserved in the National Collection  for many fruit trees is mainly due to their self incompatibility characteristic.As a consequence, the UPGMA dendrogram (Figure 2) based on population pairwise genetic distance (F ST ) bet-ween regions, clearly differentiates two main groups (A and B).Group A included all the European and North American populations in addition to cultivars from Bizerte (North of Tunisia).Group B which includes all the rest of   the Tunisian populations with the four of unknown origin reveals further information.In fact the different populations of Sidi Bouzid (Ben Aoun, Ouled Haffouz and Regueb) seemed to cluster differently.The most distant is the population of Regueb, followed by Ouled Haffouz which is very close to extreme southern (Tozeur and Nefta) while Ben Aoun population is almost identical to Sfax.In fact this can be explained by the easy exchange of almond seeds by farmers and the habitual trade between Sfax (as the 'capital' of the South of Tunisia) and the other southern parts.However, it should be noted that the unknown cultivars population is represented with a low number of individuals; consequently its position within this group should be taken cautiously.The clear distinction between the northern from one side and the central and southern populations from the other side is definitely due to the natural selection.These results, reveal the high diversity and the distinct origin of the Tunisian almond germplasm and can be considered as another statement in favor of the hypothesis advanced by Gouta et al. (2010Gouta et al. ( , 2012) ) regarding a distinct parental and origin of our local cultivars.Thus, prospecting new sites and helping farmers to preserve on farm large diversity will guaranty a sustainable and valueble source of traits for future breeding goals at an international level especially with the actual threats of global warming and its negative effects on biodiversity.
In conclusion, SSRs and AMOVA analyses have been successfully used to examine the crop origin, the degree of parentage and the population distribution of the main Tunisian landraces.In fact, the Northern landraces from Bizerte were genetically related to the European and American cultivars, in the second position were those from Regueb (Sidi Bouzid) while all the others were the most distant.As these last have proved a good adaptation to severe agroecological conditions, they can provide potential new genes for drought tolerance which is of great interest for developing new cultivars.
In summary, the great diversity found inside the Tunisian almond germplasm supports the idea that Tunisia has a valuable source of almond genes to be preserved and exploited in further international breeding programs, although further investigation have to be done for population structure and pedigree analyses

Figure 1 .
Figure 1.Location of the different geographic sites in Tunisia cited in our study: Bizerte, Sidi Bouzid, Sfax, Tozeur and Nefta.

Figure 2 .
Figure 2. UPGMA dendrogram of population pairwise genetic distances (F ST ) among regions derived from AMOVA calculations after amplification with 10 SSRs of 82 almond genotypes.

Table 1 .
List of origin, location and description of the 82 almond genotypes studied.

Table 2 .
SSRs used to study the 82 almond genotypes.

Table 3 .
Locus name, size range of the amplified fragments, expected heterozygotie (He), observed heterozygotie (Ho), Wright's fixation index (F) and probability test for divergence from Hardy-Weinberg equilibrium (HWE) calculated for 10 SSRs markers in 82 almond cultivars.

Table 4 .
Analysis of molecular variance (AMOVA) partitioning genetic variability within and among 11 populations and three groups after amplification of 82 almond genotypes using 10 SSRs.

of variation df Sum of squares Variance component Proportion of variation (%)
Probabilities were derived from 10,100 permutations tests and represent the probability of observing larger variance components at random.