| Hippeastrum spp. which belongs to Amaryllidaceae and Hippeastrum, is originated in Central and South American. Its flower is large and brightly colored and its leaf form is beautiful, so there is a high ornamental value in Hippeastrum. Hippeastrum hybrids are higher ornamental valued and adaptable to environment than original species, so the latter are losed in the long-term breeding process. The phylogenetic relationship of most cultivars isn’t found now. At present the yielding of Hippeastrum in our country is in the process of rapid development, and the yielding cultivars are mostly introduced from Holland. It is in the initial process that the new Hippeastrum cultivars are bred independently. There are some problems such as confusion of name and unknown genetic background in the introduced cultivars. It is very difficult to research on the resource survey of Hippeastrum cultivars, cultivars classification and identification, diversity analysis, which seriously limits the process of crossbreeding, production and application. In this paper, genetic diversity analysis and cultivars identification of62Hippeastrum cultivars were studied with the methods of morphological characters and ISSR markers. The main results are as follows:1. Principal component analysis of morphological charactersIn this study,15morphological characters of Hippeastrum cultivars were studied by the method of principal component analysis. Among them, the first principal component includes three characters which are single or multiple petals, pistil length and stamen length; the second principal component includes five characters which are corolla diameter, scape length, scape diameter, maximum leaf length and width in bloom; the third principal component includes four characters which are leaf germination, flower bud germination, period when the first small flower is open and the first scape is wilting; the forth principal component includes three characters which are flower color, number of small flowers and leaves in bloom. The cumulative contribution rate of four principal components reaches to71.517%.2. Cluster analysis based on morphological charactersThe average connection between-method was selected to cluster the Hippeastrum cultivars based on15morphological characters. The results show that a majority of cultivars are divided into5groups at the15point of euclidean distance in order of principal components. Some cultivars whose principal component from one to three are different are clustered together because of similar inflorescence color, which is considered quite important in the classification. The partly clustered together cultivars whose morphological characters are very similar could not be identified.3. The reaction system optimization of ISSR-PCR and primer selectingThe reaction system optimization of ISSR-PCR fit for Hippeastrum was confirmed. The total reaction system is20μg with Taq DNA polymerase1.0U, template DNA40ng,10×PCR Buffer2μl, Mg2+1.5mmol·L-1, dNTP0.15mmol·L-1and primer0.50μmol·L-1. The amplication was1cycle after initial denaturation at94℃for5min, follwed by38cycle of40seconds at94℃,45seconds annealing at55℃(depending on different primers),90seconds at72℃,and a final8min extension at72℃,after which the samples were held at4℃.11primers of high polymorphism and clear bands were selected from100ISSR primers for genetic diversity analysis of Hippeastrum cultivars.4. Cluster analysis based on ISSR markersThe genetic similarity coefficient during62tested materials is0.3714～0.8429which changes largely in amplitude, and it is suggested that there is rich genetic diversity among different cultivars. The results of cluster analysis show that the62cultivars are divided into7groups at the0.63point of genetic similarity coefficient, and the clustered together cultivars are probably originated from the same parents. Some cultivars whose morphology is similar are clustered together such as’Local1’’Local2’,’Picotee’’Picotee Red Lining’’President Johnson’,’Athene’’Charismas Gift’’Matterhorn’’Ludwig Dazzler’, and so on. These are consistent with cluster result based on morphological characters, which shows that genetic diversity analysis for Hippeastrum cultivars based on ISSR markers is exact and feasible.5. Colligate the results of two cluster analysis methodsThere are differences and consistency as well between cluster result of ISSR marekers and morphological characters. Cluster analysis of morphological characters shows that Hippeastrum cultivars are classified according to principal component one to four, however results of classification with clustering of ISSR markers are classified according to genetic similarity coefficients. The two cluster results are similar in the aspect that some cultivars with similar inflorescence shape and color are clustered together. As a whole, inflorescence shape from principal component one, inflorescence color from principal component four and genetic similarity coefficient among cultivars based on ISSR markers should be considered integrating with each other as the first stage classification with Hippeastrum cultivars.6. ISSR fingerprint construction and cultivars identificationTwo primers of high polymorphism and repeatability were selected from11ISSR primers for Hippeastrum cultivars identification. There are two specific bands at450bp of UBC873amplification of ’Xiaohongxing’ and3000bp of UBC835amplification of’Elvas’ respectively.50cultivars could be identified by UBC835, and44cultivars could be identified by UBC873, however all of62cultivars could be identified efficiently by fingerprint based on ISSR primer UBC835and UBC873. |
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