Study On Construction Of Primary Core Collection In Pomegranate Germplasm Resources In China And Its ISSR Marker

Pomegranate (Punica granatum L) is famous economic forest and ornamental plant cultivated for its long florescence and nutritional fruit, its peel and root phloem can be used as material of some Chinese medicines and its leaf can be made as health care tea. there are 35 research organizations in the world which have established one or more core collection related to 63 species of crops and horticultural plants, but there is no work reported about research of core collection in pomegranate till now. It is important for preservation, study and application of pomegranate germplasm resources to establish its primary core collection. ISSR (Inter-Simple Sequence Repeat) is a newly developed molecular marker techniques and is a powerful and convenient method of studying biodiversity in plant at the difference of genomic DNA level. Based on 14 botanic and agronomic traits of 135 pomegranate biotypes cultivated in Chinese eight original areas the genetic diversity of total and primary core collection was analyzed by using of software Popgen version 1.31. Primary core collection was constructed according to systematic and random sample and cluster analysis by SPSS 11.0 for Windows software using squared Euclidean distance coefficient and linkage between groups clustering method. The difference of Shannon's information index and Nei's genetic diversity of traits of primary core collection and total collection in different original areas were analyzed by T-test. According to different sampling rate of 30%, 20%, 15% and 10% the primary core collections with 41, 27 ,20and 14 biotypes were constructed by hierarchical and random sample respectively. The coincidence rate of traits between primary core collection and total collection was calculated and the efficiency of constructed primary core collection was analyzed by F-test. The result showed that the Number of polymorphic loci, Effective locus number, Nei's gene diversity and Shannon's information index was 2-6, 1.211-4.084, 0.174-0.755 and 0.317-1.511 respectively accounting for the constructed primary core collection could represent genetic diversity of the total collection. There was no positive difference between Nei's genetic diversity of primary core collection and total collection. At the sampling rate of 20% the average coincidence rate of mean, range, standard difference and variance coefficient of 14 traits in primary core collection and total collection was 0.956, 0.944, 0.920, and 0.910 respectively. There was no positive difference between total collection and primary core collection at the sampling rate of 10% or 15%. The sampling rate of 20% was optimal sampling rate for construction of primary core collection in pomegranate germplasm resources.In addition the relative relationship of 45 typical biotypes of pomegranate was studied by ISSR molecular marker techniques. The 17specific primers, that could produce repeatable and clear bands in the amplification, were screened out from 45 ones for the ISSR-PCR in the 45 typical biotypes. The polymorphic loci number and the percentage of polymorphic loci were calculated by Popgen Version 1.31 software. The cluster analysis was done by using squared Euclidean distance coefficient and linkage between groups cluster method in SPSS 11.0 for Windows software. The result showed that the 186 fragments were obtained by amplification with 17 primers in the genomic DNA of 45 typical biotypes, eleven fragments had been produced per primer, and the 161 fragments of which were polymorphic loci and the percentage of polymorphic loci was 86.11%. In the phylogenetic dendrogram the 45 typical biotypes would be classified into four clusters, the biotypes in first cluster were mainly native to Shandong or Anhui province, the biotypes in third cluster could be divided into two subdivision according to fruit flavor, the color of petal, the color of pericarp, the color of the aril and the hardness of seed accounting for complex background in genomic DNA of pomegranate in China. The classification result based on ISSR markers in this paper was different greatly with that on botanic characters.