Population genetic diversity analysis of Chinese jujube branch canker pathogens (Botryosphaeria dothidea) in China using ISSR markers

Abstract: 【Objective】The Chinese jujube branch canker, caused by Ascomycete Botryosphaeria dothidea was a catastrophic branch disease in China and caused significant yield loses. An investigation of the disease incidence showed that the average disease incidence of Lizao grafted by Junzao, Huizao or Suanzao reached 48.65% in Jiaocheng county of Shanxi province. Because of the lack of the researches on the pathogenic fungal population genetic characteristics, there was no commercial disease-resistant variety which could be used in the agricultural production practice. The jujube branch canker pathogen (B. dothidea) was found at the molecular level.【Methods】The DNA of different B. dothidea isolates obtained from different Chinese producing areas in North China were extracted by using the Cetyltrimthylammonium bromide (CTAB) method. The DNA quality and concentration were detected by Nano Drop 1000 ultraviolet and visible spectrophotometer and 1% agarose electrophoresis. The ISSR primers were screened by regular PCR using five B. dothidea isolates from Shandong and Henan provinces, the PCR annealing tem-perature was set according to the primers' Tm temperature. The orthogonal designed experiments wereused to screen the optimal inter simple sequence repeat amplification protocol at three levels of four fac-tors. The four factors included d NTP, primers, Mg2+and Taq enzyme, which could deeply affect the DNAfragment number of ISSR-PCR amplification. The DNA of 161 B. dothidea isolates was used as the tem-plate in the ISSR-PCR amplification. If the particular size DNA fragment appeared, 1 was used to standfor its appearance. If the fragment did not appear, 0 was used to stand for the deficiency. The DNA frag-ments of ISSR-PCR amplification with all screened primers were transformed into a 1 or 0 matrix. Thepopulation genetic diversity and genetic variation were analyzed by using POPGENE Version 1.32. Thefrequency of the same DNA fingerprint was detected by using DCFA Version1.1 software. The analysis ofmolecular variance (AMOVA) program in the Arlequin Version3.11 software was used to detect the popu-lation genetic variation source. The dendrogram relationship of different natural populations was analyzedby using the unweighted pair-group mean average (UPGMA) method in the SHAM module on the basis ofgenetic distance data. The dendrogram of different natural populations was generated by the tree-plotmodule in NYSTS Version2.10.【Results】The agarose electrophoresis results showed that the DNA bandis bright and integral, and there is no significant degradation in the DNA samples. The phenomenon indi-cated that the DNA quality is good enough to be used in the ISSR-PCR amplification reaction. Ten ISSRprimers which can amplify more polymorphic loci were screened from 40 ISSR primers. An optional PCRamplification protocol was established according to the results of the orthogonal designed experiments. Itwas described as 0.30 mmol·L^-1 d NTP, 0.4 μmol·L^-1 ISSR primer, 2.00 mmol·L^-1 Mg2+and 0.25 U Taq enzyme in the volume of the 25.0 μL reaction. The protocol and primers were used to amplify the testedisolates' DNA. Totally, 132 loci were detected of which 129 loci were polymorphic; the polymorphic locipercentage was 97.93%. The POPGENE analysis results showed that the Nei's gene diversity index (H) and Shannon's information index (I) were 0.256 3 and 0.397 8 at the species level. The Shannon's infor-mation index of the Henan geographical population was 0.386 4, it is the maximum value, which indicatedthat there was abundant genetic diversity in this geographical population. There were significant differences among the geographical population on the parameters of the polymorphic loc percentage. The value of Nm (Number of migrants per generation) showed that the gene flow existed between any two geographicalpopulations. The Nm value between Henan and Shandong was 3.605 2, which showed that the gene flowbetween the two geographical populations was the strongest. The AMOVA results revealed that the geneticvariations among geographical groups and within geographical groups were respectively 7.96% and24.16%, and about 67.58% of the total variation located within populations. The dendrogram based onISSR markers showed that the nine natural populations can be clustered into six clusters at the thresholdof the genetic similar coefficient 0.19. The Xinzheng and Liulin natural populations located in cluster Ⅱ, similarly, Lingbao, Zhucheng and Yangling shared the same cluster Ⅲ. This phenomenon showed that thenatural populations from different geographical populations can be clustered into the same cluster.【Conclusion】The ISSR-PCR was a very useful and strong tool to detect the pathogenic fungal population ge-netic diversity. The populations of Chinese jujube branch canker pathogens (B. dothidea) in North Chinapossessed abundant genetic diversity. The intra-populational genetic diversity was much more abundantthan the inter-populational variation. The main genetic variation came from the isolates within the samenatural populations. There was no significant correlation between the genetic relationships and their geo-graphical originals among the natural populations. These finding will pave the way for insight into thepathogenic epidemic rules and establishing an effective disease control strategy in the future.