Genetic Linkage Map Construction For Kenaf And Localization Of Some Important Traits, Development Of SCAR Markers

Kenaf (Hibiscus cannabinus L.) is one of the most economically important crops for fiber production. To better understand the genetic basis of kenaf for the improvement of kenaf production and lay the foundation for molecular breeding efforts, a primary genetic linkage map of kenaf was constructed using sequence-related amplified polymorphism (SRAP), inter-simple sequence repeat polymorphism (ISSR) and randomly amplified polymorphic DNA (RAPD). Cultivar Alian kenaf (from Egypt) and Fuhong 992 (from Fujian Agriculture and Forestry University) with fine qualities of high quality and disease resistance were used as parents to construct an F2 population consisting of 180 plants. The genetic linkage map here opened a new prospect for marker - assisted selection program, comparative genomics and quantitative trait loci (QTL) gene location and cloning. On the base of the linkage map, we finally mapped five qualitative traits genes and six yield traits. In addition, we tried to develop SCAR (sequence characterized amplified region) markers for the first time. The results obtained in this study were as the follows:(1) RAPD and two-primer RAPD combinations were used to screen and compare for polymorphisms between Alain kenaf and Fuhong 992, providing the foundation for constructing a genetic linkage map of kenaf. The results showed that RAPD was a potential tool for constructing a genetic linkage map of kenaf. PCR reactions with two different primers tended to produce different amplification products than the single primer, so using two different primers was a convenient and economical way of improving polymorphism.(2) An F₂ population consisted of 180 plants, which was developed from the cross between Alian kenaf and Fuhong 992, was genotyped with SRAP, ISSR, RAPD and two-primer RAPD. A total of 494 SRAP, 60 ISSR, 120 RAPD and 300 two-primer RAPD mixtures primers were used to survey the polymorphism between the parents, the result showed that only 78 pairs of SRAP primers (15.8%), 25 ISSR (41.7%), 28 RAPD (23.3%) and 141 (47%) two-primer RAPD primers could amplify polymorphism bands and revealed 396 polymorphic loci in total (105 SRAPs, 38 ISSRs, 38 RAPDs and 215 two-primer RAPD loci).(3) At a logarithm of the odds (LOD) score threshold of 5.0 and at a maximum map distance of 25 cM, these 396 loci were used to construct the genetic linkage map with Mapmaker/Exp 3.0, 307 loci were mapped into 26 linkage groups which spanned a total map length of around 4,924.8 cM with an average marker density of 16.04 cM per marker. These markers distributed randomly in all linkage groups without any clustering.(4) In this investigation, using a population with 162 F_2:3 individuals from the cross between Alian kenaf and Fuhong 992, on the basis of the linkage map we had finished, we finally mapped five qualitative traits genes including leaf petiole color, leaf shape, dimension of corolla, corolla shape, stem color at late stage. Analysis using the Mapmaker/Exp 3.0 software showed that there were close linkage relation between the gene of stem color at late stage and leaf petiole color gene, with a genetic distance of 2.8 cM, which are mapped on the linkage group 5. Also linkage existed between dimension of corolla and corolla shape, with a genetic distance of 14.7 cM, which are mapped on the linkage group 6. The genetic distance between leaf shape and dimension of corolla, corolla shape, were 38.2 cM and 23.5 cM, respectively. They were mapped on the linkage group 6, but their linkage relation still requires further research.(5) Using this population two location field tests were conducted, with a randomized complete block design, to charactererize six yield traits: plant height, stem diameter, number of nodes, fresh bark weight per plant, dry bark weight per plant, 1000-seed weight. By using the data of field tests and MCIM method, six important yield traits were located and their genetic interactions were analyzed. Correlation analysis of field performance indicated significant positive correlation among all the traits except 1000-seed weight. As a result, two QTLs of plant height, two QTLs of stem diameter, two QTLs of number of nodes, one QTL of fresh bark weight per plant, two QTLs of dry bark weight per plant, and two QTLs of 1000-seed weight were mapped in two places. The eleven QTLs identified mainly clustered in linkage groups 6, 11, 14, 9, 13, 17 and 4, and all of these QTL distributed unevenly on chromosomes and tended to cluster together.(6) Alian kenaf and Fuhong 992 were used as PCR DNA template denotes, totally 22 SCAR markers have been obtained from special primers designed by the sequence of RAPD, ISSR and SRAP polymorphic bands between two parents, which would be used to constructing the molecular linkage map in kenaf.