Study On Genetic Diversity And Genetic Relationship Of Pomegranate (Punica Granatum L.) Germplasm

Pomegranate(Punica granatum L.)is a small trees or deciduous shrub which belongs to Punicaceae,Punica L.,occurred originally Central Asia and native to Iran,Afghanistan etc.,in which it had already been cultivated more than five thousands of years and more than 2000 years in China.Pomegranate has formed a great deal of cultivars as long-term natural hybridization,gene mutation,seedling stand,grafting etc. in China.According to incomplete statistics, there are 238 cultivars of pomegranate throughout North and South,more than 20 provinces,in China.At present, there is no unified Classification and classification systems for pomegranate Cultivars internationally.The classification of the pomegranate is based mainly on the character of fruit trees and agronomic traits.Researchers who classify and identify it were limited to certain areas and classify it from different angles.These lead to the phenomenon that hybrid Cultivars,synonym and heterogeneous of the same name,which brought difficulties to identify and utilize accurately pomegranate resources.In order to protect and develop pomegranate and provide genetic information for breeding of new pomegranate cultivars and proof for future molecular systematic studies,ISSR markers is used to analyze the genetic diversity and genetic relationship for some pomegranate cultivars in China in this study;AFLP markers is used to analyze genetic diversity and genetic relationship for some pomegranate cultivars in Sichuan and Yunnan.The main results are as follows:(1)Extraction of genomic DNA from leaf of pomegranate:As pomegranate leaves contain lots of polyphenols, polysaccharides and other secondary material characteristics,CTAB method was improved in this study:First, 1.5×10-2 mol.L-1 Na2B4O7.10H2O was choosed as the polyphenol oxidase inhibitors to inhibit phenol oxidation completely and eliminate the negative impact of oxidation phenol on digestion, PCR amplification and other operations;In order to remove polysaccharides, RNA etc.,powder leaves was washed by buffer before the nuclear membrane lysis;In order to remove protein and polysaccharide,NaCl and KAc density are adjusted,extraction time of chloroform/isoamyl alcohol is increased;In order to increase the DNA yield,CTAB density of lysis is increased,centrifugal temperature of suspension is bellow 18℃.The results of agarose gel electrophoresis show that point hole of electrophoresis was clean,no towing,the results of spectrophotometer show that OD260nm/D280nm was 1.7～1.9, OD260nm/OD230nm was greater than 2,the yields ranged is 96.4～127.2μg.g-1.Conclusion:the improved CTAB II is the effective method of high-purity and high-yield extraction of genomic DNA from pomegranate leaves.(2) Optimization of ISSR-PCR amplification system of pomegranate:The orthogonal design was used to optimize the ISSR-PCR amplification system of Pomegranate in five factors (Taq DNA polymerase,Mg2+,DNA template,dNTPs and primer) at four factors respectively.The weighted average method and centesimal grade were used for the results of the orthogonal scoring, the DPS data processing systems is used to data analysis and study the inherent law of the results that different levels of each factor and factors influence on.The results showed that:the order of these five factors is:Mg 2+> dNTPs> Taq DNA polymerase> primer> template DNA,and these five factors among the different levels show a very significant level;The best ISSR-PCR reaction system(25μl)for Pomegranate is:Mg2+1.75 mmol.L-1,dNTPs 0.15 mmol.L-1,Taq DNA polymerase 1 U,DNA template 20 ng, and primer 0.8 mmol.L-1;the best annealing temperature of different primers is different..(3)Genetic analysis of pomegranate resources by ISSR:In this study, these 6 primers with good repetition and high polymorphism have been selected from 100 ISSR primers:UBC 826, UBC 857, UBC 868, UBC 899, UBC 900.The DNA polymorphism of 47 pomegranate cultivars from 7 main production areas in China is analyzed by these 6 primers and 120 bands were amplified,of which 109 were polymorphic loci.The average percentage of polymorphic bands was 90.83%. PopGen32 analysis indicated that the genetic diversity of 7 pomegranate populations as follows:Shandong>Shanxi>Yunnan>Henan>Anhui>Sichuan>Xinjiang.Nei's gene differentiation coefficient of 7 populations (Gst) was 0.191 1,show that genetic variation of among populations is 19.11% and the genetic variation within populations is 81.89%,showing low levels of genetic differentiation among these populations;the gene flow of 7 pomegranate populations(Nm) was 2.116 9,the genetic similarity of these 7 populations (I) ranges from 0.921 8 to 0.975 9,it showed a strong gene flow and high genetic similarity among these populations and the diversity of genetic variation of pomegranates can be maintained.The genetic similarity coefficient (Sg) of 47 pomegranate cultivars ranges from 0.600 0 to 0.925 0,the effective number of alleles (Ne) is 1.2945±0.3094, the gene diversity of Nei (H) is 0.1897±0.1618,the information index of Shannon (I) was 0.3091±0.2198,which show pomegranate germplasm have greater genetic variation and richer genetic diversity among these cultivars.NTSYSpc-2.10 UPGMA method was used to contruct evolutionary trees,47 cultivars were divided into five taxa completely by six primers.As 15 specific bands could be used as referential identification tags for 11 pomegranate cultivars.(4) Optimization of pomegranate AFLP system:In this study, MseⅠwas selected as the high-frequency enzyme, EcoR I was selected as the low-frequency enzyme.The best time of double digestion system of 20μl (MseⅠwas 5 U,Eco RⅠwas 5 U,BSA was 0.2μl,NEBuffer 2 was 2μl,genomic DNA was 300 ng,ddH2O was 14.05μl) was 6～7 h;ligation product was not diluted but for pre-amplification straightly,pre-amplification process was:94℃2 min;94℃30 s,56℃30 s,72℃60 s, Go to 3 29 more times;72℃6 min,10℃hold.The product of pre-amplification is diluted 20-fold for the selective amplification.This AFLP system can be used for genetic analysis of pomegranates.(5) Genetic analysis of pomegranate in Sichuan-yunnan by AFLPIn this study,these 5 pairs of primers with good repetition and high polymorphism are selected from 64 pairs of primers:E-AGG/M-CAA,E-AAG/M-CAA E-ACA/M-CTC,E-AGG/M-CTG,E-AAG/M-CTG.The genetic diversity and the relationship of 42 pomegranate cultivars of Sichuan and Yunnan are analyzed by these 5 pairs of primers,PopGen32 analysis indicated that 362 bands are amplified by 5 pairs of polymorphic AFLP primers,of which 235 were polymorphic loci and the percentage of polymorphic bands is 65.66%.The effective number of alleles per locus (Ne),Nei's gene diversity (H),Shannon information index (I) are 1.260 7,0.160 3,0.253 1 respectively,suggesting there have been greater genetic diversity among pomegranate germplasm in Sichuan and Yunnan cultivars;NTSYSpc-2.10 analysis indicate that the range of genetic similarity coefficient (Sg) is from 0.712 7 to 0.925 4 and with an average of 0.814 2;UPGMA method was used to construct phylogenetic tree,42 cultivars were seperated completely by 5 pairs of AFLP primers with the genetic similarity coefficient (Sg) 0.82 for the threshold value.42 pomegranate cultivars in Sichuan and Yunnan are divided into four taxa,which shows the genetic relationship among 42 pomegranate cultivars in Sichuan and Yunnan at molecular level.18 specific bands are detected,and which can be used as referential identification tags for the 13 pomegranate cultivars.Meng zi'Qing pi'pomegranate and Pan zhihua'Qing pi' pomegranate are homonym.