Conservation and Genetic Diversity of Microsatellite Loci in the Genus ...

Aust. J. Bot., 1996,44,331-341

Conservation and Genetic Diversity of Microsatellite Loci in the Genus Eucalyptus M. ~ ~ r nM.eI. ~ a~r ~, u e z - ~ a r cT.i alJrenA, ~ , D. S. smithA and G. F.

or an^

*CSIRO Division of Forestry and Forest Products, PO Box 4008, Queen Victoria Terrace, Canberra, ACT 2600, Australia. B~orrespondingauthor; email: [email protected]

Abstract Four microsatellite loci have been characterised in Eucalyptus nitens Maiden and in six other eucalypt species. The dinucleotide repeats were identified by screening a Sau3AI genomic DNA library from E. nitens with (CA), and (GA), oligonucleotide probes and sequencing the positive clones. Genetic analysis of 20 unrelated individuals from five populations of E. nitens showed all loci to be highly polymorphic with an average of 9.5 alleles per locus and an average heterozygosity of 0.575. Analysis of four individuals from each of six species from three subgenera showed complete conservation of microsatellite loci between species within the same subgenus, Syinphyomyrtus, and conservation of 50% of loci across species between the two main subgenera, Synzphyonzyrtus and Monocalyptus. None of the primers amplified microsatellite loci in Eucalyptus nlaculata from the subgenus Corymbia. All microsatellite loci that were detected were polymorphic. Highly polymorphic microsatellite loci that are conserved across species will be useful for mapping quantitative traits, fingerprinting breeding lines, and for within-population studies requiring fine-scale analysis of genetic variation.

Introduction Microsatellite loci are regions of DNA consisting of short tandem repeats dispersed throughout eukaryotic genomes. These loci exhibit high levels of variation due to variation in the number of repeat units. Therefore, microsatellite markers are ideal for application in plant breeding programs and in genetic studies of specific parameters in natural populations. In agricultural crop species, microsatellites are being used for genetic mapping and individual identification. Microsatellite loci have been used for genetic mapping in soybean (Morgante et al. 1994), barley (Becker and Huen 1995), Arabidopsis (Bell and Ecker 1994), maize (Senior and Huen 1993), rice (Wu and Tanksley 1993; Zhao and Kochert 1993) and Brassica (Lagercrantz et al. 1993). Fingerprinting of cultivars and clones using microsatellite loci has been achieved in soybean (Rongwen et al. 1995) and grapevines (Thomas and Scott 1993). In an analysis of microsatellite polymorphism in Dioscorea tokoro (Terauchi and Konuma 1994), the level of heterozygosity was sufficient to enable monitoring of pollenmediated gene flow within a population. Microsatellites have been shown to be extremely useful in several animal species where low levels of diversity detected with other types of markers have limited the genetic analysis within the species (Hughes and Queller 1993; Jarne et al. 1994; Paetkau and Strobeck 1994; Taylor et al. 1994), and are likely to be just as useful in plant species with low levels of diversity, for example Acacia mangium (Moran et al. 1989). Eucalyptus is a large genus and microsatellites will have applications both in breeding programs of the commercial species, and conservation and population genetic studies of non-domesticated species. Two advantages of microsatellite loci are that they are generally highly variable codominant loci and that they are PCR (polymerase chain reaction) based markers. However, a disadvantage, at least in plants, has been the considerable time taken in development of

M. Byrne et al.

microsatellite loci, which has perhaps been exacerbated by their lower abundance in plants than in animals (Lagercrantz et al. 1993) and the lower availability of sequences in databases. For these reasons, it is important to determine the extent to which sequences isolated in one species are conserved in related species and across wider taxonomic boundaries. For instance, the genus Eucalyptus has about 700 species (Brooker and Kleinig 1994) containing a number of commercially important forest tree species. Since the time and expense of development of microsatellite loci cannot be justified for many species, the conservation and utility of microsatellites across species is an important practical issue in this genus. Microsatellite sequences isolated in one species have been shown to detect loci in other animal species. In mammals, there can be considerable conservation of loci across genera, for example cattle and sheep (Moore et al. 1991), cats (Menotti-Raymond and O'Brien 1995) and whales (Schlatterer et al. 1991). Similar conservation has been observed between two bee genera (Estoup et al. 1993) and some bird genera (Hanotte et al. 1994). In plants, heterologous PCR primers have been found to work across subspecies of barley (Saghai Maroof et al. 1994) and rice (Wu and Tanksley 1993) and between two species of Glycine (Morgante et al. 1994). Five microsatellite loci from grapevine genome were conserved across seven other species in Vitis (Thomas and Scott 1993). Kijas et al. (1995) showed that two microsatellite loci are present in several species of Citrus and species in two related genera. In this study, microsatellite sequences were isolated from Eucalyptus nitens Maiden. The genetic diversity of several of these loci was characterised within E. nitens and their conservation studied in six other species from the subgenera Symphyomyrtus, Monocalyptus and Corymbia.

Materials and Methods Plant Material

A three-generation outcrossed pedigree of E. rzitens was used to assess the inheritance and segregation of alleles at microsatellite loci. This pedigree consists of 4 first-generation individuals, 2 second-generation individuals and 118 progeny, and has been used to construct a genetic linkage map (Byme et al. 1995). To estimate the number and distribution of alleles, four individuals from each of five natural populations (Table 1) from throughout the range of E. nitens were assessed. Determination of the presence of microsatellite loci in other eucalypt species was made by assessing four individuals from different populations covering the geographic distribution of six species representing three subgenera of the genus (Table 1). Genomic DNA was extracted from leaves as described by Byrne et al. (1993), except that twice the concentration of sarkosyl was required to ensure lysis of the cells for E. sieberi and E. marginata. Library Screening

Size-selected (250-600 bp) genomic DNA of E. nitens digested with SaDAI was ligated into BamHI digested and dephosphorylated pUC18 (Phaimacia) and used to transform competent Escherischia coli DHlOa cells (Stratagene). The library was screened by colony hybridisation using 100 ng each of 3 2 ~ labelled poly(CA1GT) and poly(GA1CT) in 6 x SSPE, 5 x Denhardts, 1% sodium dodecyl sulphate (SDS), with three post-hybridisation washes in 0.5 x SSC, 0.1% SDS. Positive colonies were replated and a second round of hybridisation carried out to confirm their positive status. Sequencing and Printer Design

Positive clones identified from the library screening were sequenced on an Applied Biosystems 373A automated sequencer using the recommended dideoxy cycle-sequencing protocol. Sequences flanking the repeat sequence were analysed and compatible primer sequences identified with the program PRIMER (Lincoln et al. 1991).

Microsatellites in the Genus Eucalyptus

Table 1. Locations of sampled individuals for assessment of microsatellite diversity a n d conservation in six Eucalyptus species NSW, New South Wales; Qld, Queensland; Tas., Tasmania; Vic., Victoria; WA, Western Australia Subgenus

Species

Population

Symphyomyrtus

E. nitens

Barrington Tops, NSW Ebor, NSW Tallaganda, NSW Errinundra, Vic. Macaiisiel-, X';ic. Taranna, Tas. King Island, Tas. Macquarie Harbour, Tas. Otways, Vic. Silverton, NSW Lake Albacutya, Vic. Petford, Qld Rudall River, WA Bulahdelah, NSW Whian Whian, NSW Mount George, NSW Crediton, Qld Fingal, Tas. Bega, NSW Mallacoota, Vic. Katoomba, NSW Dwellingup, WA Yanchep, WA Jarrahdale, WA Bussellton, WA Taree, NSW Coffs Harbour, NSW Tathra, NSW Appin, NSW

E. globulus

E. camaldulensis

E. grandis

Monocalyptus

E. sieberi

E. marginata

Corymbia

E. maculata

No. of individuals 4 4 4 4 4 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

PCR Ampl@cation and Fragment Separation PCR amplification of 50 ng genornic DNA was carried out in a total volume of 25 p,L containing 0.2 PM of each primer, 0.2 mM of each dNTP, 67 PM Tris-HCL (pH 8.8), 16.6 mM (NH,),SO,, 0.2 mg m ~ - 'bovine serum albumin, 1.0-2.0 mM MgClz and 1.25 U Taq polymerase. The optimal MgC1, concentration for each primer pair varied (Table 2). Two touch down PCR protocols were used. Protocol 1 consisted of 30 cycles of 94°C for 30 seconds, 6 5 4 6 ° C for 30 seconds and 72OC for 5 seconds. The annealing temperature started at 65'C and dropped 0.3'C each cycle followed by three cycles with annealing at 56OC. Protocol 2 consisted of 20 cycles of 94°C for 30 seconds, 6940°C for

Table 2.

Details of running conditions for microsatellite loci

Locus

MgCl, concentration

PCR protocol

En 6 En 11 En 14 En I6

1.0 mM 1.25 mM 1.0 mM 1.5 mM

1 1 1 2

M. Byrne et al.

30 seconds and 72°C for 5 seconds. The annealing temperature started at 69'C and dropped OS°C each cycle followed by 10 cycles with annealing at 60°C. A particular primer pair was run under one protocol for all species and individuals (Table 2). The PCR products were separated on 20 cm 8% nondenaturing polyacrylamide gels run in 1 x TBE, with cooling, at 300 V for 2.5-3.5 h, depending on the size of the amplification products, then stained in ethidium bromide (0.5bg m ~ - ' )for 30 min. HpaII-digested pUC19 was used as molecular size markers. Data Analysis The inheritance of alleles at microsatellite loci was confirmed using the three-generation pedigree. Segregation of :l!!&s according to Men&!iafi principles afid tests fcr !ifil..ge of loci was &&ermine&by a chi-square analysis of genotype classes. The number of alleles, frequency of alleles, observed heterozygosity (Ho) and expected heterozygosity (H,) in 20 unrelated individuals were calculated. Amplification of microsatellite loci in other eucalypt species was determined and number of alleles per species calculated.

Results Library Screening Previous screening of an individual of E. nitens with di- and tri-nucleotide repeats showed that, as in other species, dinucleotide repeats are more common than trinucleotide repeats. The Sau3AI library generated more than 14000 colonies of which 94 were positive when screened with a combined C N G A probe. After a second round of screening, 28 of these colonies were still presumed positive; however, 15 of these colonies appeared to contain highly repeated DNA when screened with total genomic DNA and were not pursued. The inserts from the remaining 13 clones were sequenced. All of these inserts contained microsatellite sequences, with 10 containing GA repeats, two containing G A repeats associated with a uninucleotide or tetranucleotide repeat, and one containing a complex sequence involving a tetranucleotide repeat with C T and CA dinucleotide repeats. Of the 13 microsatellite sequences, to date 4 have been characterised in E. nitens and other eucalypt species (Table 3).

Table 3.

Segregation and chi-square goodness of fit of microsatellite loci in a pedigree of

Eucalyptus nitens Ahformation on primer sequences may be obtained from G. F. Moran, Division of Forestry and Forest Products, CSIRO, PO Box 4008, Queen Victoria Terrace, Canberra, ACT 2600, Australia Locus Name

Repeat typeA

En 6 En 11 En 14 En 16

(GAIl5 (GA),9+ (A)26 (GA),, (CAGA),(GA),,

Segregation

Expected

x2

Probability

35 : 32 : 33 : 18 38: 26: 23: 31 25 : 40 : 29 : 24 30 : 34 : 24 : 30

1:l:l:l 1:l:l:l 1:l:l:l 1:1:1:1

6.13 4.37 5.45 1.73

0.11 0.22 0.14 0.63

Inheritance All loci showed Mendelian inheritance of alleles through three generations of E. nitens individuals in a pedigree. Inheritance of alleles through three generations for En 6 is shown in Fig. 1. The segregation of genotype classes for all loci is presented in Table 3; it did not depart significantly from Mendelian expectations. The loci are not linked (data not presented) and are on different linkage groups in the eucalypt map (Byme et al. 1995).


1

2

3

4

6

5

7

8

9

10 1 1 1 2 1 3

14

Fig. 1. Inheritance of alleles through three generations for locus En 6. Lanes 1 and 2, female grandparents; Lanes 3 and 4, male grandparents; Lane 5, female parent; Lane 6, male parent; Lanes 7-14, progeny.

Diversity within E. nitens The number of alleles and heterozygosity values detected in 20 unrelated individuals of E. nitens for all loci is shown in Table 4. The number of alleles detected ranged from 5 to 16, with an average of 9.5. The observed heterozygosity is lower than the expected heterozygosity for all loci. En 16 had the largest number of repeats in the sequence, and is also a compound microsatellite with a tetranucleotide repeat associated with a dinucleotide repeat. This locus also detected the largest number of alleles and the highest heterozygosity. The frequency of alleles for each locus is shown in Table 5. The frequencies of all alleles at all loci were less than 0.35, and all loci showed evenness of allele frequencies as reflected in the high expected heterozygosities (Table 4). No two individuals had the same multilocus genotype. At the most polymorphic locus, En 16, all individuals had a unique genotype, except for two individuals from two different populations. The same 20 individuals of E. nitens have been screened with 40 RFLP loci as part of a survey of RFLP variation in the species (M Byme, T. Parish and G. F. Moran, unpublished data). These loci were used to compare the variability in the microsatellite loci to RFLP loci. The average number of alleles (A) detected with the four microsatellite loci is higher than that detected with the 40 RFLP loci (A = 4.5, Ho = 0.33) for these individuals. Although some RFLP loci detect similar numbers of alleles to those detected by some of the microsatellite loci, the average number of alleles detected with microsatellite loci is nearly twice the average number of alleles detected with the RFLP loci. The level of heterozygosity of individuals with the microsatellite loci is also considerably higher than that with the RFLP loci. Table 4. Diversity parameters in 20 unrelated individuals of Eucalyptus nitens for microsatellite loci A, number of alleles; Ho, observed heterozygosity; He, expected heterozygosity

Locus

A

Ho

He

En 6 En 11 En I4 En 16

9 5 8 16

0.65 0.45 0.40 0.80

0.850 0.722 0.831 0.919

Mean

9.5

0.575

0.828

M. Byrne et al.

Table 5. Allelic frequencies at four microsatellite loci, based on 20 unrelated individuals of Eucalyptus nitens A ~ l l e l e have s not been sized in base pairs and are presented in order of migration in the gel AlleleA

Locus

En 6

En 11

En 14

En I6

Conservation in Other Species All primer pairs amplified microsatellite loci in the other Symphyomyrtus species, E. globulus, E. camaldulensis and E. grandis, and two loci were detected in each of the Monocalyptus species, E. sieberi and E, marginata, although not the same loci in both species (Table 6). None of the loci were amplified in E. maculata. The number of alleles detected in each species for each locus is shown in Table 6. Observed heterozygosity in the species ranged from 0.25 to 1.0 with an average of 0.625 over all species and loci. Visualisation of E n 14 in two individuals of the six species tested is shown in Fig. 2. In general, the sizes of alleles in the species were similar, with no major differences for any locus. The individuals of E. grandis were the least variable compared to those of the other species.

Table 6. Detection of microsatellite loci in six Eucalyptus species Number of alleles detected in four individuals of each species; - no amplification products detected Subgenus

Locus Species

Symphyomyrtus Monocalyptus Corymbia

E. globulus E. grandis E. camaldulensis E. sieberi E. marginata E. maculata

En6

En11

En14

En16

5 5

4 2 4

6 2 6 5 5

6 5 6 4

3

-

-

3

-

-

-

Microsatellitesin the Genus Eucalyptus

Fig. 2. Amplification of locus En 14 in two individuals from each of six Eucalyptus species. Lanes 1 and 2, E. globulus; Lanes 3 and 4, E. caiizaldulensis; Lanes 5 and 6, E. grandis; Lanes 7 and 8,

E. iarginata; Lanes 9 and 10, E. sieberi; Lanes 11 and 12, E. inaculata.

Discussion Highly variable microsatellite loci have been identified in eucalypts. Microsatellite loci identified in E. nitens were also detected in other eucalypt species. Detection of loci with primers developed in one species is readily achieved for other species within the same subgenus (Symphyomyrtus). This is an important practical result since 90% of eucalypt plantations worldwide are of species belonging to this subgenus. Detection of loci across species from different subgenera is not as efficient; however, two of the loci could be amplified between the two main subgenera in the genus. None of the loci could be detected in E. maculata from the subgenus Corymbia. Species of the subgenus Corymbia are taxonomically most distant from the rest of the eucalypt species and there has been some debate as to whether they should be included in the genus Eucalyptus or recognised as a separate genus (Ladiges et al. 1995). In animals, conservation of microsatellite sequences has been observed between orders (Moore et al. 1991; Stallings et al. 1991) and genera (Schlotterer et al. 1991; Estoup et al. 1993; Menotti-Raymond and O'Brien 1995). In plants, conservation of microsatellite loci has generally been observed between subspecies and between closely related species (Wu and Tanksley 1993; Morgante et al. 1994; Saghai Maroof et al. 1994). However, except for studies of Vitis (Thomas and Scott 1993) and Citrus (Kijas et al. 1995), extensive assessment of conservation of microsatellites across species within genera has not been investigated. Moreover, extensive investigation of the occurrence of microsatellite loci between plant genera has not been undertaken. The lack of detection of microsatellite loci in E. maculata suggests that conservation of the DNA primer sequences or their relative position in the eucalypt genome may not extend beyond the genus level. The same PCR conditions that were optimised for analysis of the loci in E, nitens, the species of origin of the microsatellite sequences, were used to detect loci in the other six eucalypt species. Some species may benefit from further optimisation of the PCR conditions for some loci. The size of amplification products in the species were similar for each locus, with no significant increase or decrease in the sizes between species. All loci were polymorphic in all species with 2-6 alleles present in only four individuals. A strategic question is whether it is more efficient to put resources into further optimisation of PCR conditions for specific species of the second subgenus or to develop microsatellites in species from both subgenera.

M. Byrne et al.

Variability of microsatellites in plant species has generally been found to be high. Although the number of alleles detected in a study is dependent on sample size, it does give some indication of the degree of variability detected with microsatellite loci. In studies of pine, grapevine, wild yam, soybean, rice and oak, the average number of alleles ranged from 6 to 18.5 and average heterozygosity ranged from 0.62 to 0.87 (Thomas and Scott 1993; Wu and Tanksley 1993; Morgante et al. 1994; Smith and Devey 1994; Terauchi and Konuma 1994; Dow et al. 1995; Rongwen et al. 1995). Variability in some species was low, with an average number of alleles of 3.5 in maize (Senior and Heun 1993) and 2.7 in barley (Becker and Heun 1995); however, two highly variable loci have been identified in barley, with 27 and 38 alleles observed in 207 wild and cultivated accessions (Saghai Maroof et al. 1994). The average number of alleles and average heterozygosity observed for microsatellites in E. nitens are similar to the high values obtained in most other species. Analysis of nuclear RFLPs and cpDNA in E. nitens has shown that this species maintains high levels of genetic diversity (Byrne and Moran 1994; M Byrne, T. Parish and G. F. Moran, unpublished data), therefore high levels of variation were also expected in microsatellite loci. The microsatellite loci in E. nitens show higher levels of polymorphism than were detected with RFLP loci. A direct comparison with the same 20 individuals analysed with both microsatellite and RFLP loci showed much higher levels of polymorphism detected with the microsatellite loci than with the RFLP loci. Data for isozyme polymorphism for these 20 individuals are not available, so a direct comparison with isozymes is not possible. However, analysis of RFLP diversity in E. nitens showed nearly twice as much polymorphism as did isozymes (M Byrne, T. Parish and G. F. Moran, unpublished data), thus microsatellite loci are potentially four times more polymorphic than are isozyme loci in this species. Comparisons of variability between microsatellites and RFLPs in soybean also showed that microsatellites detected twice as much variation as did RFLPs (Morgante et al. 1994), and in rice, microsatellites detected significantly more variation than did RFLPs (Wu and Tanksley 1993). However, in maize, the variation detected with microsatellites was similar to that detected with RFLPs (Senior and Huen 1993). Microsatellite loci in Dioscorea tokoro revealed two or three times more variation than did isozyme loci (Terauchi and Konuma 1994), and, in animals, polymorphic microsatellite loci were found in species showing very little isozyme polymorphism (Hughes and Queller 1993; Jarne et al. 1994; Paetkau and Strobeck 1994). The most polymorphic of the microsatellite loci characterised had the largest number of repeats but also had a tetranucleotide repeat associated with the main dinucleotide repeat of the sequence. The higher variability detected with this locus is probably caused by the larger number of dinucleotide repeats rather than the presence of the tetranucleotide repeat. This is because the tetranucleotide repeat was small, and a positive relationship between number of repeats and number of alleles has been shown in humans (Weber 1990), mice (Love et al. 1990), rice (Wu and Tanksley 1993), barley (Becker and Heun 1995) and wild yam (Terauchi and Konuma 1994), although no such correlation was observed in human microsatellites studied by Valdes et al. (1993). The most variable RFLP locus detected 11 alleles in the 20 individuals that have been analysed here, but detected 15 alleles in the survey of the species involving 160 individuals (M Byrne, T. Parish and G. F. Moran, unpublished data). Therefore, the most variable microsatellite locus is likely to detect even more alleles in a larger survey of E. nitens. The majority of the microsatellites identified in eucalypts contained GA repeats even though a combined CA/GA probe was used to screen the library. This suggests that GA repeats may be more common in the eucalypt genome than CA repeats. Similar results have been observed in other plant species (Condit and Hubbell 1991; Wu and Tanksley 1993; Bell and Ecker 1994; Dow et al. 1995; Liu et al. 1995), by contrast to the observations of CA


repeats being more abundant in human and animal genomes (Weissenbach e t al. 1992; Lagercrantz et al. 1993). Highly variable microsatellite loci that are conserved across species will have many applications in the genus Eucalyptus. They will be highly useful for integration of genetic maps generated in different pedigrees within species, and for investigation of synteny of linkage groups between species. Microsatellites will provide an efficient means of fingerprinting and individual identification for the verification of breeding material and breeding programmes in eucalypt species. Although suitable RFLP loci are already available for this purpose in eucalypts (M Byme, T. Parish and G. F. Moran, unpublished data), the ease of typing of microsaieiiiie ioci makes their use preferable to using RFiPs, particularly when assaying a large number of individuals with small numbers of loci. The need for genotype verification of breeding material in breeding programmes has been highlighted by an investigation that found errors in genotype identification in four sets of Eucalyptus germplasm (Keil and Griffin 1994). This study used RAPDs to identify genotypes; however, microsatellite sequences would be more efficient for these types of investigations. Eucalypt species generally have sufficient allozyme variation to allow analysis of genetic parameters at the species level (Moran 1992) and RFLP loci have been shown to be highly variable in E, nitens (M Byrne, T. Parish and G. F. Moran, unpublished data). However, highly polymorphic microsatellite loci will enable sufficient differentiation of individuals within populations to allow investigations of spatial and temporal genetic variation within populations. Highly variable multilocus microsatellite analysis will allow gamete identification which will enable monitoring of pollen flow and identification of paternity in open-pollinated seeds, and assessment of male fertility and contributions to the pollen pool.

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Manuscript received 2 February 1996, accepted 1 May 1996