Determination of genetic stability of grafted marula trees using AFLP markers

Scientia Horticulturae 111 (2007) 293–299 www.elsevier.com/locate/scihorti

Determination of genetic stability of grafted marula trees using AFLP markers K.L.M. Moganedi a,*, N. Colpaert b, P. Breyne c, M.M. Sibara d, E.M.A. Goyvaerts e a

Department of Biochemistry, Microbiology and Biotechnology, School of Molecular and Life Sciences, University of Limpopo, Private Bag No. 1106, Sovenga 0727, Polokwane, South Africa b UZ Gent, Experimental Cancerology, De Pintelaan 185, 9000 Gent, Belgium c Research Institute for Nature and Forest, Gaverstraat 4, 9500 Geraardsbergen, Belgium d Department of Education, Private Bag No. 603, Pretoria 0001, South Africa e Kitso Biotec Pty Ltd, 23 Fernvilla Pl, Pietermaritzburg 3201, South Africa Received 29 July 2004; received in revised form 24 June 2006; accepted 24 October 2006

Abstract Genetic stability of grafted marula trees within seven lines, some of which exhibiting interline phenotypic variations, was evaluated using the AFLP technique. The study was conducted in two-fold using samples from the DR, LP, MR, OS, PH, SW and TR lines. The genetic analysis using leaf materials indicated varying levels of genetic variations between genotypes within the OS, MR, LP and PH lines. The genotypes of the DR, TR and SW lines showed high intraspecific genetic similarity and formed tight clusters on the UPGMA-based dendrogram. The genetic analysis using a subset of the initial sample set constituting both leaf and bark materials which represented the scion and the rootstock components of grafted trees showed high genetic similarities between the graft partners of some genotypes within the OS and PH lines. These high levels of genetic similarity between scion and rootstock partners of a grafted tree suggested that the rootstock graft developed shoots and grew into a tree following graft failure. The observed results imply that grafting is not always successful even within compatible species, however, it can be reliably monitored using molecular markers. # 2006 Elsevier B.V. All rights reserved. Keywords: Sclerocarya birrea; Marula; Genetic stability; Grafted trees; AFLP markers

1. Introduction Marula, Sclerocarya birrea subsp. caffra, is a wild-growing dioecious tree species indigenous to Africa. The marula species belongs to the mango family, Anacardiaceae. This family is widespread in the warmer regions of the world. It is a large group with over 60 genera and more than 500 species (Palmer and Pitman, 1972). Over 50 species of this family grow in South and Southwest Africa as trees, a few of which produce edible fruits and nuts, such as mango (Mangifera indica), cashew nut (Anarcadium occidentale) and pistachio nut (Pistacia vera). Marula has a multitude of uses—with the fruit being the most common tree component used by village communities in countries, such as South Africa, Botswana, Kenya, Tanzania, Swaziland, Namibia, Zimbabwe, Zambia and Malawi (Palgrave, 1984; Agufa et al., 2000; Eloff, 2000; Wynberg et al., 2002). The

* Corresponding author. Tel.: +27 15 268 2996; fax: +27 15 268 3012. E-mail address: [email protected] (K.L.M. Moganedi). 0304-4238/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2006.10.026

fruit is eaten either fresh or processed and is rich in nutritional minerals and carbohydrates. Each fruit has a stony kernel that contains 2–4 seeds. The seeds are rich in oil (50–60%) and protein (28%) and are eaten either raw or roasted as nuts (ANU forestry, 2001). The fruit is commonly brewed into non-alcoholic and predominantly alcoholic drinks in many rural communities. The brew provides a reasonable income to families who turn it into an enterprise during the season. Amarula cream liqueur is a commercial alcoholic drink made from fermented marula fruit flesh and is traded worldwide. The bark and roots of the marula tree are also used in treating a variety of ailments (Palgrave, 1984; Eloff, 2000; ANU forestry, 2001). Marula has been declared a national tree in the Republic of South Africa (Marula natural products, 2003) because of its potential to be developed into a viable commercial crop. This species grows easily from seeds in spring. However, grafting is generally preferred worldwide for commercial fruit crop production because of its desirable advantages over sexual and other forms of vegetative propagation, i.e., fruiting occurs within a short period of time in addition to achieving uniformity

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in fruit yield, size and quality (Empire chestnut company, 2004). Taylor et al. (2004) affirmed that grafting an adult scion hastened fruiting in marula by showing that grafted marula produced the first fruit after 4 years while the seed-propagated cultivars started fruiting in 8–10 years. Several grafted marula lines have been established at the experimental farm of the University of Limpopo-Turfloop, between 1994 and 1995. Each line consisted of several individuals that were raised from scion materials taken from the same tree. The donor trees for each line come from different areas in the Limpopo and Mpumalanga Provinces and their selection was based on the fruit quality (sugar percentage, citric acid and juice contents) for female trees and on size and biomass for male trees. The grafted trees within some lines exhibited phenotypic variations, such as leaf fall, wind tolerance and flowering time. These variations underlie the study to evaluate the genetic uniformity of the grafted marula trees within the marula lines. Previous work on assessing genetic stability using allozymes showed no genetic variation within and between grafted lines (Mafumo et al., 1998, unpublished data). The AFLP assay was used in this study to assess the genetic stability of the grafted marula trees. The AFLP technique has been widely used for assessing genetic relatedness within and between close species (Angiolillo et al., 1999; Hornero et al., 2001; Coulibaly et al., 2002; Balasaravanan et al., 2003; Sensi et al., 2003) and was shown to be time efficient due of its high multiplex ratio and its reproducibility (Russell et al., 1997; Chavarriaga-Aguirre et al., 1999; Soleimani et al., 2002). Hornero et al. (2001) and Rout et al. (1998) successfully used AFLP and RAPD assays to evaluate genetic stability in oak and ginger. DNA markers are currently used for genetic relatedness studies more than protein markers because of their high multiplex ratio and stability to protein markers, which could be influenced by the environment.

Table 1 Samples (leaf) used in genetic stability analysis of grafted marula trees Line

Cultivar

1. Onder Sabie (OS)

1. 2. 3. 4. 5. 6. 7. 8.

2. Pharulani (PH)

9. OS 37 10. PH 38 11. PH 39 12. PH 42 13. PH 45 14. PH 46 15. PH 47

3. Dorarula (DR)

16. 17. 18. 19. 20. 21.

DR DR DR DR DR DR

2 4 6 8 9 10

4. Leeupan (LP)

22. 23. 24. 25. 26. 27.

LP LP LP LP LP LP

8 11 12 16 17 19

5. Toularula (TR)

28. 29. 30. 31. 32. 33. 34.

TR TR TR TR TR TR TR

58 59 60 61 63 65 66

6. Swarula (SW)

35. 36. 37. 38. 39. 40.

SW SW SW SW SW SW

48 52 54 55 56 57

7. Mhalarula (MR)

41. 42. 43. 44. 45.

MR MR MR MR MR

21 22 23 24 26

8. SBL

46. SBL 1

2. Materials and methods 2.1. Grafted marula samples This study was conducted with two sets of samples. The first set included leaf samples to evaluate genetic stability within seven lines of grafted marula trees (Table 1). One wild-growing marula tree (SBL 1) was included in the study. Based on the results observed, a second set constituting a subset of the trees from the initial study included leaf and inner bark materials both from one grafted tree. Collected samples of leaf and inner bark materials of the grafted marula trees were immediately placed in liquid nitrogen. The bark tissue was collected below the graft scar only on trees with visible graft union. Plant materials were ground to fine powder in liquid nitrogen and stored at 80 8C. Trees sampled are listed in Tables 1 and 2. 2.2. AFLP analysis DNA was isolated using the method of Kleinhofs et al. (1993). AFLP analysis of leaf samples was performed

OS OS OS OS OS OS OS OS

29 30 31 32 33 34 35 36

according to Vos et al. (1995) with the restriction enzymes MseI and EcoRI. Two sets of preselective amplification products were generated with two MseI/EcoRI primer combinations with each primer carrying one selective nucleotide (M-G/E-C and M-A/E-A). Selective amplification was performed with the following primer combinations wherein the EcoRI primer was end-labeled with g–33P ATP; M-GGA/ECTC, M-GGA/E-CCT, M-GGA/E-CGG, M-GGA/E-CAT, M-GCC/E-CCT, M-GCC/E-CTC, M-GAC/E-CCT, M-GAC/ E-CAT, M-GTG/E-CGG, M-GTG/E-CTC, M-AGA/E-ATA, M-ATC/E-AGC, M-ACA/E-ACT, M-AGT/E-AAT.

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Table 2 Samples used during further analysis of genetic stability in grafted marula lines Line

Grafted tree

Line

Grafted tree

Line

Grafted tree

Line

Grafted tree

TR

1. 2. 3. 4. 5. 6.

PH

7. PH 38m 8. PH 38g 9. PH 45m 10. PH 45g 11. PH 47m 12. PH 47g

OS

13. 14. 15. 16. 17. 18.

SW

19. 20. 21. 22. 23. 24.

TR TR TR TR TR TR

63m 63g 65m 65g 66m 66g

OS OS OS OS OS OS

32m 32g 35m 35g 37m 37g

SW SW SW SW SW SW

48m 48g 54m 54g 56m 56g

m, leaf material; g, bark material from below the presumed graft union.

Preselective amplification of leaf and bark samples was performed with the primer combinations M-T/E-G, M-A/E-T and M-A/E-G. Selective amplification products were generated with the primer combinations M-TTG/E-GCA, M-TTG/EGTG, M-TCC/E-GCA, M-TCC/E-GTG, M-ACA/E-TGC, MACA/E-TAT, M-AGT/E-TGC, M-AGT/E-TAT, M-ACA/EGTG, M-ACA/E-GCA, M-AGT/E-GTG and M-AGT/EGCA. The fingerprints were visualized with silver staining following the method in Promega protocols and applications guide (1996). 2.3. Data analysis AFLP fingerprints were manually scored for the presence (1) and the absence (0) of a band. Both distinct monomorphic and polymorphic bands were scored. Genetic resemblance matrices based on the Dice (Nei and Li, 1979 for genetic distance estimation) was generated with the software program Treecon (Version 1.3b, Van der Peer and De Wachter, 1994) and NTSYS-pc (Version 2.02i, Rohlf, 1997). Cluster analysis from the generated similarity matrix was performed with the unweighted pair group method with arithmetic average (UPGMA). The dendrograms were created with Treecon program while the similarity index table was obtained from the NTSYS-pc program. The reliability of the trees generated was evaluated with the bootstrap analysis of 100 replications of sampling with replacements. 3. Results and discussion A total of 410 scorable bands with 42% polymorphism obtained from 14 primer combinations was used to generate similarity matrices of grafted marula genotypes sampled from scion leaf materials. A high amount of genetic similarity was

observed between grafted marula lines and was indicative of low levels of genetic diversity in the wild by virtue of the donor trees for each line being from different localities in the two Provinces of South Africa. The genetic similarities presented by the values on the diagonal in Table 3 revealed a varying degree of genetic variation within the grafted marula lines that were supposedly clonal. This genetic difference was also evident on the dendrogram (Fig. 1). The intraspecific genetic variation was in the order OS > MR > LP > PH > DR > TR > SW according to the genetic similarity indices in Table 3. It was apparent from the genetic similarity analysis (Table 3) and the clustering pattern on the dendrogram (Fig. 1) that there is great similarities between the genotypes belonging to each of the lines TR, SW and DR, except for DR 2 in the DR line. The genetic relationship revealed by the clustering patterns on the dendrogram (Fig. 1) between the genotypes belonging to the lines MR, LP, PH and OS demonstrated different levels of intraspecific genetic variations, which suggested different degrees of graft failure in these lines. Due to the observed genetic differences within these lines, a follow-up study that compared the scion and rootstock materials of grafted trees was conducted. The scion grafts within each line were obtained from one donor tree and were expected to be clonal. The rootstock components of the grafts were rooted seedlings, and therefore not genetically uniform. Analysis of the leaf and bark tissue materials using a subset of grafted trees used in the assessment of genetic stability within different grafted lines confirmed the genetic relationship observed previously between genotypes that were common to both studies (Figs. 1 and 2). The genotypes from the lines TR and SW still clustered tightly together and away from their rootstock components. The OS 35 (OS 35m) and PH 47 (PH 47m) genotypes which clustered away from the other scion genotypes from their own lines in the initial study, showed a

Table 3 Similarity indices of grafted marula lines showing relatedness within and between lines based on Nei and Li (1979) coefficient

OS PH DR LP TR SW MR SBL 1

OS (n = 9)

PH (n = 6)

DR (n = 6)

LP (n = 5)

TR (n = 8)

SW (n = 6)

MR (n = 5)

SBL 1 (n = 1)

0.92 0.92 0.91 0.90 0.89 0.88 0.91 0.91

0.97 0.92 0.89 0.89 0.89 0.90 0.91

0.98 0.90 0.90 0.90 0.90 0.92

0.94 0.90 0.88 0.93 0.91

0.98 0.87 0.90 0.91

0.99 0.89 0.89

0.94 0.91

1.00

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Fig. 1. UPGMA-based dendrogram of the grafted marula based on the distance coefficient of Nei and Li (1979). Numbers at branches and brackets indicate bootstrap support and main clusters, respectively.

high genetic relatedness to their rootstock counterparts OS 35g and PH 47g, respectively, both at a similarity index of 0.96 (Table 4) and the pairs clustered at bootstrap values of 100 (Fig. 2). The amount of genetic similarity between the scion and rootstock genotype pairs of the OS 35 and PH 47 trees inferred graft failure that resulted in shooting of the rootstock grafts. This generally explained the genetic variability observed within the OS, MR, LP and PH lines in the initial genetic stability study, which duly emanated from genetic dissimilarity among

rootstock grafts within and across all grafted marula lines. Furthermore, these intraspecific variations partly underlie the phenotypic differences, such as leaf fall, wind tolerance and flowering time that were observed within these grafted marula lines. The occurrence of graft failure between plant materials that belong to the same species has been reported in oak, cherry (Douglas, 1999) and in chestnut (Empire chestnut company, 2004). From the amount of genetic variation observed within

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297

Fig. 2. Dendrogram of grafted marula genotypes generated with the UPGMA clustering method based on the Nei and Li (1979) distance coefficient. Numbers at branches and brackets indicate bootstrap support and clusters, respectively. Letters m and g represent the leaf and bark samples, respectively.

supposedly clonal marula lines, it is deducible that marula is not easily propagated by common grafting technique similar to mango that belongs to the same family as marula, which is commonly approach-grafted (Hartmann et al., 2002). The advantage of grafting that it brings fruiting forward makes the approach desirable for mass propagation of fruit crops for commercial benefit. However, the possibility of graft failure

necessitates the evaluation of genetic similarity between grafted cultivars and the original donor trees. Genetic fingerprinting technology offers a means to reliably screen for genetic stability within vegetatively propagated materials obtained from the same donor plant. It is important especially for marula as a dioecious tree species to determine the genetic stability of clones because graft failure may lead to propagation

298

Table 4 Genetic similarity indices of grafted marula from the Dice coefficient 2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

1.0 0.90 0.96 0.93 0.97 0.87 0.84 0.92 0.84 0.80 0.87 0.85 0.83 0.88 0.84 0.85 0.85 0.85 0.91 0.85 0.90 0.90 0.91 0.89

1.0 0.87 0.92 0.88 0.85 0.81 0.87 0.81 0.80 0.84 0.82 0.80 0.88 0.84 0.84 0.82 0.85 0.89 0.84 0.88 0.87 0.88 0.88

1.0 0.91 0.99 0.85 0.84 0.91 0.84 0.77 0.86 0.83 0.83 0.85 0.83 0.85 0.85 0.83 0.89 0.84 0.88 0.88 0.89 0.87

1.0 0.92 0.90 0.86 0.88 0.86 0.83 0.87 0.85 0.85 0.91 0.86 0.88 0.87 0.84 0.88 0.86 0.87 0.86 0.88 0.88

1.0 0.86 0.85 0.92 0.85 0.78 0.86 0.84 0.84 0.86 0.84 0.86 0.86 0.84 0.90 0.85 0.90 0.90 0.91 0.88

1.0 0.83 0.84 0.83 0.82 0.82 0.80 0.83 0.89 0.84 0.86 0.84 0.84 0.84 0.84 0.83 0.85 0.84 0.86

1.0 0.83 1.0 0.89 0.92 0.85 1.0 0.88 0.87 0.86 0.98 0.85 0.83 0.87 0.82 0.83 0.84 0.86

1.0 0.83 0.82 0.87 0.86 0.82 0.86 0.83 0.86 0.84 0.87 0.90 0.84 0.90 0.89 0.91 0.89

1.0 0.89 0.86 0.85 1.0 0.88 0.87 0.86 0.98 0.85 0.83 0.87 0.83 0.83 0.85 0.86

1.0 0.83 0.81 0.89 0.87 0.83 0.82 0.88 0.84 0.80 0.85 0.80 0.80 0.81 0.85

1.0 0.96 0.86 0.88 0.82 0.82 0.86 0.83 0.86 0.84 0.85 0.83 0.87 0.87

1.0 0.85 0.87 0.80 0.80 0.85 0.84 0.84 0.83 0.83 0.82 0.85 0.86

1.0 0.88 0.87 0.86 0.98 0.5 0.83 0.87 0.82 0.83 0.84 0.86

1.0 0.88 0.88 0.88 0.87 0.86 0.90 0.86 0.88 0.88 0.91

1.0 0.96 0.88 0.86 0.87 0.86 0.87 0.85 0.87 0.89

1.0 0.88 0.87 0.86 0.85 0.87 0.85 0.87 0.90

1.0 0.86 0.85 0.89 0.85 0.84 0.87 0.88

1.0 0.90 0.88 0.89 0.91 0.89 0.89

1.0 0.88 0.98 0.94 0.99 0.91

1.0 0.86 0.90 0.87 0.93

1.0 0.93 0.98 0.90

1.0 0.93 0.2

1.0 0.91

1.0

m, leaf material; g, bark tissue.

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(1) TR 63m (2) TR 63g (3) TR 65m (4) TR 65g (5) TR 66m (6) TR 66g (7) PH 38m (8) PH 38g (9) PH 45m (10) PH 45g (11) PH 47m (12) PH 47g (13) OS 32m (14) OS 32g (15) OS 35m (16) OS 35g (17) OS 37m (18) OS 37g (19) SW 48m (20) SW 48g (21) SW 54m (22) SW 54g (23) SW 56m (24) SW 56g

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