0879-B4

Genetic Mapping in an Inbred Populus Family Using Amplified Fragment Length Polymorphism (AFLP) Markers

Zhang Deqiang^[1][2] ·Zhang Zhiyi^[3]·Yang Kai^[4] Li Bailian^[5]

Abstract

The AFLP genetic linkage maps for two poplar cultivars have been constructed with the Pseudo-test-cross mapping strategy. The hybrids were derived from an interspecific backcross between the female hybrid clone “TB01” (Populus tomentosa´Populus bolleana) and the male clone “LM50” (P. tomentosa). A total of 782 polymorphic fragments were obtained with a PCR-based strategy using 49 enzyme-nested primer combinations, among which 632 fragments segregated in a 1:1 ratio (P < 0.01), indicating the DNA polymorphisms heterozygous in one parent and null in the other. The linkage analysis was performed using Mapmaker version 3.0 with LOD 5.0 and a maximum recombination fraction (q) of 3.0. Map distances were estimated using the Kosambi mapping function. In the framework map for “LM50” (P. tomentosa), 218 markers were aligned in 19 major linkage groups. The linked loci spanned along approximately 2683 cM of the poplar genome with an average distance of 12.3 cM between the adjacent markers. For “TB01” (P. tomentosa´P. bolleana), the analysis revealed 144 loci, which were mapped to 19 major linkage groups and covered about 1956 cM with an average distance of 13.6 cM between framework loci.

Key words: Genetic linkage map, AFLP, Populus tomentosa Carr.

Introduction

Genetic linkage mapping based on molecular markers now provides a powerful tool for detecting loci controlling a number of traits and for studying genome organization and evolution in many forest trees species (Bradshaw and Stettler 1995; Grattapaglia et al. 1995; Frewen et al. 2000; Sewell et al. 2002). These in turn, are the basis for map-based gene cloning and for marker-assisted selection of important traits related to faster growth, better stress adaptation or better disease resistance. The advent of molecular-marker systems greatly facilitate the construction of genetic linkage maps in various tree species. The four principal types of molecular markers used in mapping include: Restriction Fragment Length Polymorphisms (RFLPs), Random Amplified Polymorphic DNAs (RAPDs), Simple Sequence Repeats (SSRs) and Amplified Fragment Length Polymorphism (AFLPs). The AFLP marker is generated through a PCR-based approach that does not require sequence information or probe preparation, which are required for generating RFLP and SSR markers. It is highly reliable for its good reproducibility comparing to RAPD, and being a dominant marker, it supplies as much information as codominant markers for analysis of backcross population or pseudo-test-cross population. Currently, the AFLP marker has largely been used in tree genetic mapping. (Marques et al. 1998; Remington et al. 1999; Wu et al. 2000; Cervera et al. 2001).

Various genetic maps have been developed for genomic research in poplar to date. The first poplar genetic map was developed with RFLP and allozymes marker (Liu and Furnier 1993), and much more detailed and complete linkage maps have also been established in cooperating, hundreds of RAPD, RFLP, STS (sequence-tagged site), SSR and AFLP markers (Bradshaw et al. 1994; Wu et al. 2000; Cervera et al. 2001; Yin et al. 2001, 2002). However, diverse genetic maps based on distinct populations are still needed for special objectives such as QTL detection and map based cloning.

In this paper, we report an application of AFLP markers in a two-way pseudo-testcross mapping strategy (Grattapaglia et al. 1994), to map the genome of an elite clone of “LM50” (P. tomentos) and its hybrid clone “TB01” (P. tomentosa´P. bolleana) from progeny data.

Materials and methods

The mapping pedigree

A single, superior “TB01” clone (P. tomentosa´P. bolleana) female tree was crossed to an elite male P. tomentosa, clone “LM50”, to produce one inbred population of 696 individuals and among which 120 individuals were randomly selected and used for genetic mapping.

AFLP procedures

The total genomic DNA was extracted from frozen young leaves by the methods described by Murray and Thompson (1980). The AFLP procedure was performed as reported by Vos et al. (1995).

Data analysis and map construction

Heterozygous AFLP markers present in one parent but not in the other were used to construct separate genetic linkage maps for the male clone “LM50” and female clone “TB01” parents using the two-way pseudo-testcross strategy (Grattapaglia and Sedroff 1994). To detect linkages in repulsion phase, the data set was duplicated and added to the original data. Both parents’ genetic linkage maps were constructed using MAPMAKER version 3.0 software for Macintosh (Lincoln et al. 1992). Linkage groups were assigned with a minimum LOD score of 5.0 and a maximum recombination frequency (q) of 0.30. For each linkage group, markers were ordered with a minimum LOD score of 3.0 and a maximum q of 0.40. Map distances in centiMorgans were computed using Kosambi’s mapping function.

Results

AFLP linkage maps

The linkage analysis was based on 632 testcross AFLP markers with 396 in the male “LM50” and 236 in the female “TB01”, which displayed no significant distorted segregation. For “LM50”, 25 linkage groups, 12 triplets, 23 doublets and 74 unlinked markers were obtained at a LOD score of 5.0 and q = 0.30 using the MAPMAKER 3.0 linkage program. Nineteen of the twenty-five linkage groups were classified as “major” groups (>50 cM), corresponding to 19 haploid chromosomes in poplar, which constituted the framework map for “LM50”. The male map spanned a total length of 2682.8 cM, including 218 markers (Table1), with an average interval of 12.3 cM between adjacent markers (Table1). Linkage groups ranged in length from 57.1 cM (TLG19) to 326.5 cM (TLG1) while the number of markers mapped in each linkage group varied from 6 (TLG16, TLG17, TLG18 and TLG19) to 24 (TLG2).

The same criteria were used to establish linkage map of the maternal tree, “TB01”. The 236 markers were assigned to 19 major groups (>40 cM), including 1 triplet, 20 doublets, with 49 unlinked markers. These 19 groups were set up for the female map covering 1956.3 cM, and 144 markers were located on the map, with an average distance between framework loci of 13.6 cM (Table1). The length of the groups ranged between 42.2 cM (TBLG19) and 274.8 cM (TBLG1) (Table1).

Owing to the relatively low polymorphism levels between P. tomentosa and P. tomentosa´P. bolleana, a few regions of the linkage maps contained gaps larger than 30.0 cM. There were 6 gaps located on the TBLG1, TBLG2, TBLG3, TBLG6, TBLG7, TBLG11, respectively. Of which the largest gap of 38.1 cM was located on TBLG2 in the linkage map of P. tomentosa´P. bolleana while the largest gap in the P. tomentosa map was located on the group TLG1 between the markers E61M31-124 and E63M46-131 and spanned 30.0 cM.

Discussion

Characteristics of AFLP

Unlike P. deltoides, P. trichocarpa or P. nigra that have been intensively studied on molecular level, very few study has been carried out for P. tomentosa on DNA level. In this study, the AFLP marker has been successfully used in genetic mapping for P. tomentosa revealing the existence of high polymorphisms between the mapping parents. AFLP is a PCR-based method that offers an efficient and reliable means of generating the DNA markers needed for linkage map construction (Vos et al. 1995). Enough primer pairs, spanning the average-frequency restriction sites, could scan the entire genome at the DNA levels. Forty-nine pairs of AFLP primers were used to generate 782 polymorphic fragments, among which 632 segregated in a 1:1 ratio, corresponding to DNA polymorphisms heterozygous in one parent and a null in the other. The highly informative pattern of 4 to 27 polymorphic bands was obtained providing a convenient and reliable tool for the construction of genetic maps based on interspecific backcross population. The AFLP analysis can reveal the size differences in restriction fragments caused by DNA insertions, deletions, or changes in target restriction site sequences. As compared to RFLP and RAPD analysis, the labor required in detecting polymorphisms with AFLPs is considerably reduced. On average, each primer pairs could produce 13 test-cross markers, approximately 5 times more than that needed in RAPD approach in a different interspecific cross population in section Populus (Yin et al. 2001).

Table 1 Total number of AFLP markers generated with 49 EcoRI+3/MseI+3 primer combinations.

Map construction

Grattapaglia and Sederoff (1994) put forward the mapping strategy of pseudo-testcross through testcross configuration in forest trees. The major advantage of this approach in our study was the construction of parent-specific maps for male clone “LM50” and female clone “TB01”, respectively. Genetic linkage maps have been established for two closely related poplar species, P. tomentosa and its’ hybrid (P. tomentosa´P. bolleana) using pseudo-testcross strategy and AFLP markers. The genetic linkage map of clone “LM50” (male parent) now comprises 337 markers. There are 19 major linkage groups of 57.1 cM or more in length with an average of 12.3 cM per interval, 38 minor linkage groups ranging from 0.5 to 30 cM, and 61 unlinked markers. The linkage map of clone “TB01” (female parent) consists of 191 markers. That is composed of 19 large linkage groups of 42.2 cM or more in length with an average distance of 13.6 cM between neighboring markers, 21 smaller linkage groups varying from 0.5 - 25 cM, and 47 unlinked markers. Ideally, a complete genetic linkage map should contain 19 linkage groups, consistent with the 19 haploid chromosomes in Populus. The presence of small groups and unlinked markers in both maps indicates some vacant regions present in this study. Nonequivalence between the number of linkage groups and the number of haploid chromosomes has also been reported in foregoing studies (Liu and Furnier 1993; Bradshaw et al. 1994; Wu et al. 2000; Yin et al. 2001). In our study, this may in part be due to the absence of neighboring markers, innate limit of using single marker technique, or small size of mapping population. Therefore, additional efforts will be required to map RFLPs, SSRs, ESTs, allozymes and other valuable markers or gene in larger mapping population to coalesce some of these linkage groups together. Furthermore, a method of bulk segregant analysis (Michelmore et al. 1991) could be used to bridge the breach existing in these maps by screening more co-segregated markers associating with the traits to coalesce the small groups with a larger linkage group.

Analysis of distributions of informative markers in these maps indicated that certain primer combinations produced more informative polymorphic markers than others for mapping. This was the case for E33M38, E63M36, E60M33 and E34M44, although many of the markers generated by E33M38 mapped to the same linkage group TLG13. Apparently, marker screening experiments and effort in searching for suitable primer combination are needed for detection of AFLP on the entire genome. Markers on most linkage groups are randomly distributed in these maps, with obvious gaps (?30 cM) in linkage groups of TLG1, TBLG1, TBLG2, TBLG3, TBLG6, TBLG7 or TBLG11, which could be eliminated by increasing marker numbers. In the future these skeleton maps will be saturated with additional markers like SSR and EST into a consensus map. Such a map will provide a basis for adding other genetic markers, tagging, and ultimately cloning major genes and QTLs controlling economically important traits in P. tomentosa.

References

Bradshaw, H.D. and Stettler, R.F., 1995. Molecular genetics of growth and development in Populus. IV. Mapping QTLs with large effects on growth, form, and phenology traits in a forest tree. Genetics 139: 963-973

Bradshaw, H.D., Villar, M., Watson, B.D., Otto, K.G., Stewart, S., and Stettler, R.F., 1994. Molecular genetics of growth and development in Populus. III. A genetic linkage map of a hybrid poplar composed of RFLP, STS, and RAPD markers. Theor. Appl. Genet. 89:167-178

Brondani, R.P.V., Brondani, C., Tarchini, R. and Grattapaglia, D., 1998. Development, characterization and mapping of microsatellite markers in Eucalyptus grandis and E. utophylla. Theor. Appl. Genet. 97: 816-827

Cato, S.A. and Richardson, T.E., 1996. Inter-and intraspecific polymorphism at chloroplast SSR loci and the inheritence of plastids in Pinus radiata D. Don. Theor. Appl. Genet. 93: 587-592

Cervera, M.T., Storme, V., Ivens B., Gusmão J., Liu, B.H., Hostyn, V., Slycken, J.V., Montagu, M.V. and Boerjan, Wout., 2001. Dense genetic linkage maps of three Populus species (Populus deltoides, P. nigra and P. trichocarpa) based on AFLP and microsatellite markers. Genetics 158: 787-809

Dayanandan, S., Rajora, O.P. and Bawa, K.S., 1998. Isolation and characterization of microsatellites in trembling aspen (Populus tremuloides). Theor. Appl. Genet. 96: 950-956

Devey, M.E., Delfino-Mix, A., Kinloch, B.B. and Neale, D.B., 1995. Random amplified polymorphic DNA markers tightly linked to a gene for resistance to white pine blister rust in sugar pine. Proc. Natl. Acad. Sci. USA 92: 2066-2070

Grattapaglia, D. and Sederoff, R., 1994. Genetic linkage maps of Eucalyptus grandis and Eucalyptus urophylla using a pseudo-test-cross: mapping strategy and RAPD markers. Genetics 137: 1121-1137

Grattapaglia, D., Bertolucci, F.L.G. and Sederoff, R.R., 1995. Genetic mapping of QTL controlling vegetative propagation in Eucalyptus grandis and E. urophylla using a pseudo-testcross map-ping strategy and RAPD markers. Theor. Appl. Genet. 144: 1205-1214

Lincoln, S., Daly, M. and Lander, E., 1992. Constructing genetic maps with MAPMAKER/EXP 3.0. Whitehead Institute Technical Report, 3rd edn. Whitehead Institute, Cambridge, Mass.

Liu, Z. and Furnier, G.R., 1993. Inheritance and linkage of allozymes and restriction fragment length polymorphisms in trembling aspen. J. Hered. 84: 419-424

Marques, C.M., Araujo, J.A., Ferreira, Whetten, R., O’Malley, D.M., Liu, B.H. and Sederoff, R., 1998. AFLP genetic maps of Eucalyptus globulus and E. tereticornis. Theor. Appl. Genet. 96: 727-737

Michelmore, R.W., Paran, I. and Kesseli, R.V., 1991. Identification of markers linked to disease resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating populations. Proc. Natl. Acad. Sci. USA 88: 9828-9832

Murray, M.G. and Thompson, W.F., 1980. Rapid isolation of high-molecular-weight plant DNA. Nucleic Acids Res. 8: 4321 -4325

Remington, D.L., Whetten, R.W., Liu, B.H. and O’Malley, D.M., 1999. Construction of an AFLP genetic map with nearly complete genome coverage in Pinus taeda. Theor. Appl. Genet. 98: 1279-1292

Sewell, M.M., Davis, M.F., Tuskan, G.A., Wheeler NC, Elam CC, Bassoni DL, Neale DB (2002) Identification of QTLs influencing wood property traits in loblolly pine (Pinus taeda L.). II. Chemical wood properties. Theor. Appl. Genet. 104: 214-222

Wu, R.L., Han, Y.F., Hu, J.J., Fang, J.J., Li, L., Li, M.L., Zeng, Z.B., 2000. An integrated genetic map of Populus deltoids based on amplified fragment length polymorphisms. Theor. Appl. Genet. 100: 1249-1256

Vos, P., Hogers, R., Bleeker, M., Reijans, M., Van, L.T., Hornes, M., Frijters, A., Pot, J., Peleman, J., Kuiper, M. and Zabeau, M., 1995. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res. 23: 4407-4414

Yin, T.M., Huang, M.R., Wang, M.X., Zhu, L.H., Zeng, Z.B., Wu, R.L., 2001. Preliminary interspecific genetic maps of the Populus genome constructed from RAPD markers. Genome 44: 602-609

Yin, T.M., Zhang, X.X., Huang, M.R., Wang, M.X., Zhuge, Q., Tu, SM, Zhu, L.H., Zeng, Z.B. and Wu, R.L., 2002. Molecular linkage maps of the Populus genome. Genome 45: 541-555