Linkage Map Construction And Growth QTL Detection In Eucalyptus Urophylla And E. Tereticornis Based On STS Markers

Eucalyptus urophylla and E. tereticornis are two economically important plantation tree species in the world. With the development of morden biology technology, linkage map construction and QTL detection based on molecular markers have been a focus on the research field of woody plant genome in recent years. In this dissertation, we optimised the protocol for fluorescent-dUTP (F-dUTP) based SSR genotyping, and set up a strategy of SNP genotyping by using high resolution melting curve analysis (HRM) for genetic map construction. Combined with the previously mapped EST-CAPS and EST-SSR markers, genetic maps of E.urophylla and E. tereticornis were constructed using an F1 cross of the two species and STS markers, and growth-related quantitative trait loci (QTLs) were detected in the linkage maps by means of composite interval mapping (CIM) method. The results show as follows:(1) An PCR protocol for F-dUTP based SSR genotyping were optimized. In genotyping of SSRs using an automated or semi-automated sequence analyser, there are several methods for fluorescence labelling of PCR amplicons, and integration of F-dUTP in polymerase chain reaction (PCR) appears to be a powerful, fast and cheap option. However, the method has not been explored in terms of performance optimisation and cost control. Here we optimised the protocol for F-dUTP based SSR genotyping in a case study with the genus Eucalyptus. A combination of low dNTP concentration (25μM each) in PCR reaction and a touchdown PCR programme contributed to increase dramatically the fluorescence intensity of SSR amplicons when detected on an automated sequence analyser, thereby facilitating accurate and multiplexed scoring of SSR alleles. At the same time, we found a more cheap and good quality F-dUTP than those of the previous reports. The protocol optimised here provides a reliable and economic high-throughput alternative for SSR genotyping and would have broad applications as the number of available SSR markers and their utility continue to expand for multiple species.(2) The feasibility of map construction based on SNPs genotyped by HRM in Eucalyptus species were explored. By EST re-sequencing for the two parents of a mapping population, E. urophylla and E. tereticornis, we found two within-individual-SNP containing ESTs for the male parent E. tereticornis (A/A:A/G and T/T:C/T). The two EST markers were genotyped using HRM method over the mapping popupation and revealed to segregate in a Mendelian mode. This proved that HRM could be a powerful, cheap and fast procedure for many applications, including linkage map construction.(3) Genetic maps of E. urophylla and E. tereticornis were constructed using a F1 cross between the two species and STS markers (EST-CAPs, gSSR, EST-SSR and EST-SNP). For the E. urophylla map, a total of 273 markers (58 EST-CAPSs, 3 floral genes, 144 gSSRs, 1 new developed gSSR and 64 new developed EST-SSRs) were used for linkage analysis, and finally ordered in 22 linkage groups while 40 loci remained unlinked. Twenty-two linkage groups had 4~14 markers with a length size of 54.6 cM~180.9 cM and an average marker distance 14 cM. The total of 22 linkage groups covered 2680.6 cM of total map distance with 121.8 cM of an average group length. Linkage analysis in the paternal tree (E. tereticornis) was based on 296 markers (76 EST-CAPSs, 3 floral genes, 127 gSSRs, 86 new developed EST-SSRs, 2 new developed gSSRs and 2 SNPs), forming 22 linkage groups while 44 loci remained unlinked. Twenty-two linkage groups had 4~14 markers with a length size of 38.8 cM~178.7 cM and an average marker interval 13.6 cM. The total of 22 linkage groups covered 2639.6 cM of total map distance with 120.0 cM of an average linkage group length.(4) A consensus map of E. urophylla and E. tereticornis were constructed. In total, 209 markers of all segregation types were included for construction of the consensus map. The data set comprised of 74 EST-CAPSs, 2 floral genes, 92 gSSRs and 41 new developed EST-SSRs. Thirteen linkage groups were identified, which spanned a total of 1998.3 cM and averaged at 153.7 cM per group (range 79.8 cM~198.8 cM). The number of markers per linkage group amounted from 9 to 23, and the average interval between markers was 9.8 cM. There were 108 of 191 markers (56.5%) being common between the maternal map and the consensus map, and 118 of 193 markers (60.5%) between the paternal map and the consensus map. The map order of markers present in both parental maps and the consensus map were identical for most linkage groups. Fifty-one SSR markers were used for comparative mapping between this consensus map and that of E. grandis and E. urophylla (Brondani et al. BMC Plant Biol, 2006, 6: 20.). Eleven homological linkage groups were aligned, and the orthologous markers showed the synteny between the two consensus maps. The length covered by the homologous markers in this consensus map (UT) was 1268.8 cM, and that of Brondani et al (GU) was 897.8 cM.(5) The present genetic maps were used to identify QTLs affecting growth-related traits. Fourteen QTLs were identified, including six QTLs for height, three QTLs for diameter at breast height (DBH), and five QTLs for volume. The six QTLs for height at three different growth stages were detected in six linkage groups and explained the amount of phenotypic variance ranged from 7% to 16% and the total R2 was 65%; Three QTLs for DBH at two different growth stages were detected in three linkage groups, exlained the phenotypic variance between 7% and 10% and the total R2 was 26%; and five QTLs for volume at two different growth stages were detected in five linkage groups, exlained the phenotypic variance between 6% and 12% and the total R2 was 47%. The estimated magnitude of the QTL effect ranged between 6% and 16% of the total pheotypic variance with an average of 9.9%. The length for significant effect of a single QTL, i.e. 95% confidence intervals for QTL position, varied from 10.1cM to 24.7cM, with an average of 16.9cM.In summary, this dissertation presented moderate density genetic maps of two important Eucalyptus species based on STS markers. It will be helpful for discovering, separating and validating QTLs as well as analyzing the essence of some complex traits in the genus of Eucalyptus. Also, it will provide landmarks for comparative mapping and facilitate searching for some functional genes in Eucalyptus.