| Diospyros Linn. is the biggest genus including the most species distributed most widely and of the largest economic value in Ebenaceae. China is one of the originating and distributing centers of Diospyros Linn. with abundant genetic resources. D. kaki Thunb. is the representative species cultivated as fruit and classified into PCNA, PVNA, PVA and PCA types. Most of the cultivars in China are PCA types. Chinese non-atringent persimmon and staminate germplasm are only distributed in Dabieshan Mountain around the junction of three provinces, Hubei, Henan and Anhui in central China. They play important roles in the research and industry of the oriental persimmon in the world because that their trait of natural astringency-loss of fruit is dominant to the non-PCNA types whereas Japanese PCNA cultivars’ is recessive. The genetic diversity and phylogenetics of the persimmon germplasm native to Dabieshan region are still unclear now. Here they were analyzed by ISSR, IRAP and cpDNA PCR-RFLP markers, meanwhile DNA barcoding for Diospyros Linn, was researched in the present study, and the main results were shown as follows:1. ISSR and IRAP markers were used to predict the genetic differences among43’Luotian-tianshi’collected from three major producing areas in China. A total number of280amplicons were observed ranging between250～2000bp. Both of them were effective methods because of the high Pi (ISSR=100%; IRAP=97.93%), PIC (ISSR=0.20; IRAP=0.26) and MI (ISSR=1.78; IRAP=1.14). The SM coefficient between ’Luotian-tianshi’were from ranged from0.104to0.985(average0.795) analyzed by ISSR and from0.566to0.903(average0.734) by IRAP, indicating that ISSR is suitable for selecting new germplasm while IRAP for studying the population genetic diversity, origin and evolution of Chinese non-atringent persimmon. On the basis of cluster analysis in the UPGMA dendrogram, two and three clusters were observed by ISSR/ISSR+IRAP and ISSR, indicating that ’Luotian-tianshi’ existed the genetic differences and some could be potential germplasms because of the significant genetic differences, such as Accll, Acc36, Acc40and Acc41. So ’Luotian-tianshi’ was a complex group including different cultivars rather than a single cultivar with the same genetic background. 2. IRAP was used to study the genetic diversity, origin and evolution of non-atringent persimmons native to Dabieshan Mountain. A total number of101amplicons were observed ranging between250-2000bp. IRAP was the effective method because of the high Pi (100%), PIC (0.35) and MI (1.92). The values of H and I of Chinese non-atringent persimmon were0.2992and0.4630in the level of individuals while0.2615and0.4156in the level of population, indicating that the high genetic diversity existed among non-atringent persimmons native to Dabieshan Mountain. Among the seven populations, Badihe population had the highest H (0.3194) and I (0.4864) so that it was supposed to be the origin center of non-atringent persimmons native to Dabieshan Mountain. The values of Hs was88.14%of Ht (0.2967), illustrating that the genetic diversity of non-atringent persimmons native to Dabieshan Mountain is mainly contributed by individuals in the population. The gene flow from Gst was strong (Nm=3.7205) among the populations, suggesting that mutation, crossover or artificial introduction might have happened in the evolution of non-atringent persimmons native to Dabieshan Mountain. Basing on the Nei’s similarity, UPGMA and NJ dendrograms, the probable spreading pathway of non-atringent persimmons native to Dabieshan Mountain was as follows:they origined from Badihe village, then migrated to Tangjiashan village, Zanzishi village, Dongchongfan village and so on in the southwest, to Mashicun village, Dabuhe village and so on in the northeast, to Lingjiabang village and so on in the west, and finally were cultivated widely and evolved in Dabieshan Mountain around the junction of three provinces, Hubei, Henan and Anhui in central China.3. The taxonomy and phylogenetic relationships of Chinese staminate germplasm and the other genotypes in Diospyros Linn. were analyzed by cpDNA PCR-RFLP and IRAP. cpDNA PCR-RFLP was suitable for the identification, phylogenetics and evolution of inter-species or above-species level because of the low values of Pi (75%), PIC (0.13) and MI (0.26), meanwhile IRAP was appropriate for that of intra-species level because of high values (Pi=97.61%; PIC=0.32; MI=2.49). The Jaccard similarity of samples analyzed by cpDNA PCR-RFLP ranged from0.318to1.0(average0.836) and the SM coefficient analyzed by IRAP was from0.394to0.912(average0.671). The UPGMA dendrograms constructed via the combination of the two methods indicated that Chinese staminate germplasm belonged to D. kaki Thunb. indeed and had different genetic background. Male strain No.l, Male strain No.2, Male strain No.3and Male strain No.9were closely related with Chinese PCNA persimmons, such as ’Luotian-tianshi’ and ’Baogai-tianshi’. Interestingly, Male strain No.8was closely related with Chinese PCA persimmons, such as ’Mopanshi’ and ’Tongpanshi’, and Male strain No.10was closely related with Japanese persimmon. In addition, the phylogenetic relationship between D. kaki Thunb. and D. lotus L. was closer than that between D. kaki Thunb. and D. glaucifolia Mete., D. rhombifolia Hemsl., D.virginiana L.4. matK and rbcL sequence fragments were selected to analyze DNA barcode for Diospyros Linn. and its applicability. X+3.2and a_f+724r primers combinations were screened to amplify mat K. and rbcL sequences of17persimmon samples used in the study. The amplification efficiency of mat K and rbcL were the same (84.47%) and the sequencing success rates were82.35%and100%, respectively. Due to the long X+3.2amplicons (1031bp), we recommended2.1a+5and a_f+724r as the universal PCR amplification primers for matK and rbcL sequence in Diospyros Linn, respectively. The length of matK and rbcL sequences, which were deposited in GenBank database (Accession nunbers were from GU471696to GU471727), were1031bp and744bp, respectively. The sequence analysis showed that matK sequence includes gene fragment encoding about343amino acids of mature kinase (matK) superfamily protein, and rbcL sequence is the partial gene fragment encoding about248amino acids of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) superfamily protein, most nucleotide sites in which were highly conserved. matK sequences in all samples had238variable sites (23.92%) and114parsimony informative sites (11.46%), which were approximately twice of those of rbcL sequences, and matK+rbcL sequences between them. The identification of Diospyros Linn, was regarded as successful when the inter-specific genetic distance is greater than0.001because that the largest average intra-specific genetic distance (0.0009) were detected by aligning the matK sequences. When110species in Diospyros Linn.were analyzed by DNA barcoding, the percent of discrimination success were matK+rbcL (92.73%)> matK (91.82%)> rbcL (79.09%), so that matK+rbcL was recommended to be the core DNA barcode for Diospyros Linn.. On the basis of the NJ tree of164samples from110species constructed by matK+rbcL, the taxonomic status and phylogenetic relationships of most species were identified. It was supported that D. kaki Thunb. var. sylvestris Makino and Jinzaoshi would be new species. In addition, D. kaki Thunb. var. sylvestris Makino, D. oleifera Cheng, and D. glandulosa, D. glaucifolia Metc. and D. lotus L. had the close phylogenetic relationships, suggesting that they might have the same ancient. But some unknown species, for example, Diospyros sp. SD-2009voucher Lowry et al.5783(MO), D. sp. K20616and D. sp. K20613, couldn’t be identified so far and DNA barcode for D. spp. needed to be further researched. |
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