MOLECULAR GENETIC MARKERS FOR ROOT-KNOT NEMA- TODE RESISTANCE IN SOME PEACH ROOTSTOCKS

Six peach rootstocks were collected according to their susceptibility to infestation of root-knot nematodes; Meloidogyne incognita and M. javanica. These rootstocks were Okinawa, Nemaguard and Nemared (nematode resistant) and Hegazy, Shami and Sultani (nematode  susceptible),  which  screened by randomly amplified polymorphic DNA (RAPD)  markers  using  31  arbitrary  10-mer primers and two SSR primer pairs. Twenty molecular markers related to root-knot nematode have been detected (using 12 primers), 13 of which were negative since they appeared in susceptible root-stocks  and  were  absent  in  the  resistant ones, and seven were positive markers which were present in the resistant root-stocks. Dendrogram tree generated across RAPD  and  SSR  analysis  demonstrated that the highest similarity was scored between Shami and  Sultani (94.1%) while the lowest similarity was scored between Okinawa and Sultani (82.3%). Because of the differences in the rootstocks traits and the molecular genetic diversity of these rootstocks, marker-assisted selection (MAS) in peach rootstocks breeding programs, under biotic stresses (Meloidogyne incognita and M. javanica), is recommended as an efficient tool for improving peach rootstocks resistance.


Fruit Breeding
each (Prunus persica (L.) Batsch) is a member of the Rosaceae, which contain many important fruit, nut and ornamental species. Peach is a diploid plant with chromosome haploid number (n=8), and has a comparatively small genome; 5.9 x 10 8 bp or 0.61 pg/diploid nucleus, with a haploid size of 300 Mb (Baird et al., 1994). Rootstocks (The below-ground portions of fruit tree) play a major role in modern orchards. The most important agricultural traits of the tree as a biotic unit, such as vigor, blossom initiation, fruit set, fruit size and fruit flavor, etc., may be, substantially, influenced by the rootstock (Dozier et al., 1984). The rootknot nematodes Meloidogyne spp. especially (Meloidogyne incognita and M. javanica) cause severe damage to several species of Prunus, damage can seriously affect early stages of plant development in the nursery or when rootstocks are transplanted into the field, also the extent of growth reduction caused by these pathogens has been documented for many Prunus rootstocks used for peach and nectarine varieties (Pinochet et al., 1997). Some cultivars or selections, such as ' 'Nemaguard'', ''Nemared'', and ''Okinawa'' etc., showed various levels of resistance (Lu et al., 1996;Scorza and Sherman, 1996). Genetic studies of resistance to root-knot nematodes show that inheritance patterns vary from simple to complex. Lu et al. (1996) proposed a tow-gene model for resistance to root-knot nematodes. Genetic analysis indicates that resistance to M. incognita and M. javanica are controlled by tow dominant genes (Mi or Mij; and Mj or Mij respectively), where the shared gene (Mij) may be required for resistance to both species. Because it is very difficult to observe morphological traits of rootstocks after grafting, so that DNA markers greatly facilitate rootstock identification. Molecular markers are interest to plant geneticists and breeders as a source of new genetic information on plant genomes and for use in trait selection. Randomly Amplified Polymorphic DNA analysis (RAPD) has tremendous potential for use in cultivar identification; it has been used to study genetic relationships in peach varieties (Chaparro et al., 1994;Warburton and Bliss, 1996) and in peach rootstocks (Lu et al., 1996;Casas et al., 1999). RAPD markers have been used in peach genetics and breeding programs (Dirlewanger and Bodo, 1994;Rajapakse et al., 1995). Microsatellite or Simple Sequence Repeats (SSRs) are the best available choice of markers for peach genetics and breeding, they are generally codominant, highly polymorphic, and can be found in large numbers covering the whole genome of any species (Sosinski et al., 2000). Many SSRs have been developed in peach (Testolin et al., 2000;Aranzana et al., 2002;Dirlewanger et al., 2002). The objectives of this study were to: 1. Characterize these used rootstocks through RAPD and SSR-PCR techniques.
2. Find some molecular genetic markers (RAPD and SSR) of these used rootstocks to nematode resistance.

Plant material
This study included six peach rootstocks (P. persica (L.) Batsch) 2n = 16, which were supplied by Deciduous Fruits Trees Department, Horticulture Research Institute (HRI), ARC, Giza, Egypt. Code numbers, rootstocks names, chromosome number and their relative resistance to nematodes are shown in (Table 1). These rootstocks were propagated by seeds, and their seedlings were planted at the orchard of HRI in Ali-Moubarak village, South Eltahrir, El-Behera Governorate.

Genomic DNA extraction
Young and fresh leaf samples were collected separately from 10 seedlings for each peach rootstock genotype then bulked DNA extraction was performed using DNeasy Plant Mini Kit (QIAGEN).
DNA concentration was quantified spectrophotometrically (Gene Quant, Amersham Pharmacia Biotech) and DNA quality was examined by electrophoresis in 0.8% agarose.

DNA amplification
RAPD-PCR reactions were conducted using 31 arbitrary 10-mer primers. Their codes and sequences are shown in (Table 2). Amplification reactions were performed in 30 µl total volumes according to Williams et al. (1990). The amplification procedures were carried out in a DNA thermocycler (MWG-BIO TECH Primuse) programmed as follows: initial pre-denaturation step at 94C for 5 min, followed by 45 cycles of 1 min at 94C, 90 sec 36C and 2 min at 72C followed by 7 min incubation period at 72C. The amplification products were stored at 4C before analysis.

DNA electrophoresis
The amplified products were separated in 1.2% agarose gel electrophoresis using 1 x Tris-Boric acid-EDTA buffer, (Ethidium bromide, (5 µl) was added to the melted gel after the temperature became 55C).

DNA amplification
RAPD-PCR reactions were conducted using two primers pairs according to Wang et al. (2002). Their codes and sequences are shown in (Table 3). The amplification procedures were carried out in a DNA thermocycler (MWG-BIO TECH Primuse) programmed as follows: initial pre-denaturation step at 94C for 5 min, followed by 45 cycles of 30 sec at 94C, 45 sec 62C and 2 min at 72C followed by 7 min incubation period at 72C. The amplification products were stored at 4C before analysis.

DNA electrophoresis
The amplified products were separated in 2.5% agarose gel electrophoresis using 1x Tris-Boric acid-EDTA buffer, (Ethidium bromide (5 µl) was added to the melted gel after the temperature became 55C.)

RAPD markers
Thirty one arbitrary 10-mer random primers were used to obtain some molecular genetic markers for root-knot nematode resistance, out of these used thirty one random primers only twelve gave twenty molecular markers related to root-knot nematode as grouped in Table  (4

) and (Figs. 1 & 2).
Primer OP-A01 (Fig. 1A) indicated the appearance of three negative molecular markers for root-knot nematode resistance, at molecular sizes 750, 330 and 250 bp in the three susceptible rootstocks Hegazy, Shami and Sultani. At the same time, primer OP-A11 (Fig. 1C) showed three negative markers at 745, 516 and 255 bp, which were present only in the susceptible rootstocks, while they were absent in the resistant ones. Another three negative RAPD markers at 970, 865 and 755 bp were obtained using primer OP-C12 (Fig. 1E), which were absent in the resistant rootstocks, while were present in the three susceptible rootstocks. Moreover, primers OP-B08 (Fig. 1D), OP-C19 ( Fig. 2A Previous review confirmed that Okinawa, Nemaguard and Nemared are nematode resistant rootstocks (Malo, 1967;Ramming and Tanner, 1983). While Hegazy, Shami and Sultani are considered nematode susceptible (Abdel-Aziz et al., 1985). Di Vito et al. (2002) indicated that resistance of Prunus to root-knot nematodes (Meloidogyne spp.) is controlled by several different genes confirming previous findings (Lecouls et al., 1997;Pinochet, 1997). On the other hand, these positive and negative molecular markers using RAPD-PCR analysis must be confirmed when F 2 and F 3 segregants obtained through bulked segregant analysis technique which is beyond, the scope of this study.

SSRs molecular markers
The results of the two primer pairs Pchgms 26-1 and Pchgms 26-2 for SSRs technique were used to get molecular markers for root-knot nematode resistance. Primer Pchgms 26-1 (Fig. 3A) exhibited three fragments at molecular sizes of 310, 165, 130 bp. The fragment at 310 bp was present in all rootstocks except Nemaguard rootstock (resistant), while the fragment at 165 bp was present in the two resistant rootstocks Okinawa and Nemaguard, therefore this fragment may be considered as a SSR molecular marker for nematode resistance (appeared in two resistant rootstocks out of three). However, primer pairs Pchgms 26-2 exhibited two fragments only at 360 and 336 bp (Fig. 3B) which did not show any relation with root-knot nematode resistance. These results agreed with the findings of Wang et al. (2002) who found that most SSRs are not specifically linked to gene loci of immediate interest and developing an SSR map is very time consuming and expensive. Either these results were in agreement with Testolin et al. (2000) who gave some examples to the utility of SSRs for pedigree determination of several peach cultivars. Aranzana et al., (2003) reported that the available SSRs on the Prunus saturated map allowing them to develop a resource useful for map comparison or MAS in fruit crops.  and Ahmed et al. (2004) reported that SSR molecular markers could be used to identify and characterize peach cultivars.

Genetic similarity based on RAPD markers
Results of similarity index among the six peach rootstock cultivars based on RAPD-PCR with the twelve primers using UPGMA computer analysis is shown in (Table 5). The highest similarity value recorded was (0.930), which was observed between Shami and Sultani rootstocks, while the lowest similarity value recorded was (0.818) between Okinawa and Sultani rootstocks. A dendrogram for the genetic relationships among the six peach rootstocks across the twelve primers results was carried out and is shown in (Fig. 4). The six peach rootstocks were separated into two clusters; cluster 1 included Hegazy, Shami, and Sultani, while cluster 2 comprised Okinawa, Nemared and Nemaguard. Within cluster 1, two subclusters appeared; one comprised Hegazy and Shami rootstocks, while the second subcluster contained Sultani only.
Cluster 2 was also divided into two subclusters; the first one contained the two rootstocks Okinawa and Nemared, while the remaining subcluster contained Nemaguard.

Similarity and relationships based on combined data of RAPD-PCR and SSR-PCR analyses
Cluster analysis based on RAPD-PCR and SSR-PCR analysis as shown in (Table 6) was carried out using UPGMA computer program. The highest similarity index recorded was (94.1%) between the two rootstocks Shami and Hegazy, while the lowest similarity index (81.8%) was observed between the two rootstocks Okinawa and Sultani. Dendrogram for the genetic relationships among the six peach rootstocks across the two techniques results were carried out and are shown in (Fig. 5), it was similar to the previous dendrogram based on 12 RAPD primers.
In this study of genetic diversity the use of RAPD-PCR seemed to be a satisfactory tool and could discriminate among the six peach rootstocks. Our results were in partial agreement with of Casas et al. (1999) who confirmed that RAPD-PCR results appear to play an important role in the differentiation among different cultivars.
Because of the differences in the rootstocks traits and the molecular genetic diversity of these rootstocks, it would be useful to introgress root-knot nematode resistance genes into elite rootstock germplasm. However, the extended juvenile stage of peach impedes such progress by traditional methods. Therefore, it is recommended to use marker-assisted selection (MAS) in peach rootstocks breeding programs, under biotic stresses such as root-knot nematode (Meloidogyne incognita and M. javanica), as an efficient short cut and cost-effective tool for evaluating peach rootstocks.