| Wintersweet [Chimonanthus praecox (L.) Link] belongs to the Calycanthaceae family. It is a famous traditional fragrant flower shrub, which has over 1000 years’history of cultivation as an ornamental plant, native to southeastern China. It is a hardy, fast-growing perennial, which blooms in winter from November to March in China. Researches of wintersweet mainly focus on genetic diversity、cultivar classification、propagation、landscaping application and cut flowers. As the development of molecular biology, some related gene about stress resistance and floral scent were cloned. Normally, the period from seed sowing to flowering takes 2.5-3 years, so it is difficult to breeding by general technology.In this study, in vitro propagation of wintersweet were developed firstly, it could not only be applied on rapidly propagation and germplasm conserved, but also set a foundation for wintersweet’ gene engineering. The genetic linkage maps were studied in the part two by AFLP and ISSR marker, which use for reference about forest and fruit trees with "two-way pseudo-testcross" mapping strategy. In the present study, we used a first intraspecific cross pedigree to construct two genetic linkage maps for wintersweet. These maps are the first reported genetic linkage maps for Ch. praecox. They can provide an overview of wintersweet genome structure for the development of high density genetic linkage maps, aiming to mapping quantitative trait loci (QTLs) and marker-assisted breeding.The main results were as follows:1. In vitro propagation of Ch. praecox(1) Growing twigs from flush watershoot were collected and sterilized, then cultured in MS basal medium with 0.6%(w/v) agar and 3%(w/v) sucrose, supplemented with 2.0 mg/L 6-benzylaminopurine(6-BA),0.5 mg/L zeatin (ZT). After an incubation period of 30 days, regenerated shoots cultured in 1/2 MS medium and supplemented with 1.0 mg/L 6-benzylaminopurine(6-BA),0.1 mg/L indole-3-butyric acid(IBA), the proliferation of subculture was 1.41, during a period of 30 days for antibrowning.(2)Rooting of shoots (after subculture) was carried out on 1/2 MS medium supplemented with 0.6%(w/v) agar and 3%(w/v) sucrose and supplemented with 0.1 mg/L indole-3-butyric acid(IBA). Some white and strong roots produced after one week, and can be transplanting after 15 days. It’s not suitable for transplanting while the roots become long and slender. (3) Cotyledon and hypocotyl were suitable explants for cytodifferentiation of wintersweet, and the results indicate that were easily produced callus and rhizogenesis. Leaf, shoot and flower organ were difficult for cytodifferentiation.(4)There are significantly different for multiplication among different gene-types of wintersweet.2. Construction of molecular genetic map in Ch. praecox(1) A segregating mapping population of Ch. praecox was derived from a cross between two elite unidentified cultivars H4 and H29. The mapping population consisted of 64 F1 test-cross (TC progenies) individuals.(2) Twelve AFLP primer combinations were polymorphism and highly reproducible and were used to assess the mapping population. Of the 312 polymorphism marker,141 markers segregating at 1:1 (α=0.01). Of the 38 ISSR primers, the 9 primers produced a total of 103 amplification products, of which 58 were polymorphic, and 25 markers segregating at 1:1 (α=0.01).The segregation-ration of mendelian segregation, distorted segregation and abnormal segregation was 44.86%、32.16%、22.97%(α=0.01). In total, there are 135 markers showed 1:1 segregation(81.33%), and 31 markers show 3:1 segregation(18.67%).(3) Linkage groups and map construction were accomplished using Mapmaker/Exp Version 3.0 with the data type "F2 backcross" suited for the pseudo-testcross configuration. Distances between adjacent marker loci were calculated from recombination fractions using Kosambi’s mapping function. Finally, we used MapDraw V2.1 to draw genetic linkage maps based on given genetic linkage data. The female parent H4 map had a total of 80 markers distributed in 12 linkage groups. This maternal framework map covers 2471.8 cM of total map distance. The linkage groups had an average length of 205.9 cM and the average spacing of 30.6 cM. In contrast, the male parent map involves 47 markers distributed in 8 linkage groups. This paternal framework map covered 1184.2 cM of total map distance with 2 to 23 markers in each linkage group we estimated that the total genome size of 2974.5 cM and 1581.9 cM for the female and male, respectively. Therefore, the coverage of female and male maps, based on these estimates genome length, was 83.1% and 74.9%, respectively.The results indicated that the "two-way pseudo-testcross" mapping strategy is useful for wintersweet; AFLP and ISSR were suitable molecular marker for constraction of genetic map in wintersweet; this first generation intraspecific pedigree was feasible to mapping research. |
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