Development of Simple Sequence Repeat Markers and Genetic Diversity Evaluation of Mycocentrospora acerina in Yunnan Province, China.

Round spot is a destructive disease that limits of Panax notoginseng production in China. However, the genetic diversity of its etiological agent Mycocentrospora acerina has yet to be studied. In this work, firstly, we developed 32 M. acerina polymorphic microsatellite markers using MISA and CERVUS 3.0 and selected 14 for further analysis. Then, we studied the genetic diversity of 187 isolates collected from P. notoginseng round spot using simple sequence repeat markers and polyacrylamide gel electrophoresis. The genetic diversity ranged from 0.813 to 0.946, with 264 alleles detected at the 14 microsatellite loci. The expected average heterozygosity was 0.897.

Development of PCR based SSR markers for Wilsonomyces carpophilus and a PCR based diagnosis protocol for the early detection of shot hole disease in stone fruit crops.

BACKGROUND: The conidial Ascomycota fungus Wilsonomyces carpophilus causing shot hole in stone fruits is a major constraint in the production of stone fruits worldwide. Shothole disease symptoms appear on leaves, fruits, and twigs. Successful isolation of the pathogen from different hosts on synthetic culture medium is a time consuming and tedious procedure for identification of the pathogen based on morpho-cultural characterization. METHODS AND RESULTS: The present research was carried out to develop a successful PCR based early detection protocol for the shot hole disease of stone fruits, viz., peach, plum, apricot, cherry, and almond using the pathogen specific SSR markers developed from the Wilsonomyces carpophilus genome using Genome-wide Microsatellite Analysing Tool package (GMATA) software. Diseased leaf samples of different stone fruits were collected from the SKUAST-K orchard and the pathogen was isolated on potato dextrose agar (PDA) medium and maintained on Asthana and Hawkers' medium with a total of 50 pathogen isolates comprised of 10 isolates each from peach, plum, apricot, cherry and almond. The DNA was extracted from both healthy and infected leaf samples of different stone fruits. The DNA was also extracted from the isolated pathogen cultures (50 isolates). Out of 2851 SSR markers developed, 30 SSRs were used for the successful amplification of DNA extracted from all the 50 pathogen isolates. These SSRs were used for the amplification DNA from shot hole infected leaf samples of different stone fruits, but the amplification was not observed in the control samples (DNA from healthy leaves), thus confirming the detection of this disease directly from the shot hole infected samples using PCR based SSR markers. To our knowledge, this forms the first report of SSR development for the Wilsonomyces carpophilus and their validation for the detection of shot hole disease directly from infected leaves. CONCLUSION: PCR based SSR makers were successfully developed and used for the detection of Wilsonomyces carpophilus causing shot hole disease in stone fruits including almond in nuts for the first time. These SSR markers could successfully detect the pathogen directly from the infected leaves of stone fruits namely peach, plum, apricot and cherry including almond from the nuts.

Genome survey and genetic characterization of Acacia pachyceras O. Schwartz.

Acacia pachyceras O. Schwartz (Leguminoseae), a woody tree growing in Kuwait is critically endangered. High throughput genomic research is immediately needed to formulate effective conservation strategies for its rehabilitation. We therefore, performed a genome survey analysis of the species. Whole genome sequencing generated ~97 Gb of raw reads (92x coverage) with a per base quality score above Q30. The k-mer analysis (17 mer) revealed its genome to be 720Mb in size with an average guanine-cytosine (GC) ratio of 35%. The assembled genome was analyzed for repeat regions (45.4%-interspersed repeats; 9%-retroelements; 2%-DNA transposons). BUSCO assessment of completeness of genome identified 93% of assembly to be complete. Gene alignments in BRAKER2 yielded 34,374 transcripts corresponding to 33,650 genes. Average length of coding sequences and protein sequences were recorded as 1,027nts and 342aa, respectively. GMATA software filtered a total of 901,755 simple sequence repeats (SSRs) regions against which 11,181 unique primers were designed. A subset of 110 SSR primers were PCR validated and demonstrated for its application in genetic diversity analysis of Acacia. The SSR primers successfully amplified A. gerrardii seedlings DNA depicting cross transferability among species. The principal coordinate analysis and the split decomposition tree (bootstrapping runs of 1000 replicates) distributed the Acacia genotypes into two clusters. The flow cytometry analysis revealed the A. pachyceras genome to be polyploid (6x). The DNA content was predicted as 2.46 pg, 1.23 pg, and 0.41 pg corresponding to 2C DNA, 1C DNA and 1Cx DNA, respectively. The results provide a base for further high throughput genomic studies and molecular breeding for its conservation.

Estimation of genetic diversity and relationship in sugar beet pollinators based on SSR markers

Background: Genetic diversity studies are important for the selection of parents with a greater combination capacity that, when crossed, increase the chances of obtaining superior genotypes. Thus, 26 polymorphic simple sequence repeat (SSR) primers were used to assess the genetic diversity of 140 individual samples from 12 diploid sugar beet pollinators (pollen parents) and two cytoplasmic male sterile (cms) lines (seed parents). Eight pollinators originated from three research centers in the United States Department of Agriculture, while four pollinators and cms lines were from the Institute of Field and Vegetable Crops, Novi Sad, Serbia. Results: In total, 129 alleles were obtained, with a mean of 3.2 alleles per SSR marker. The observed heterozygosity ranged from 0.00 to 0.87 (mean = 0.30). Expected heterozygosity and Shannon's information index were the lowest for marker BQ590934 and the highest for markers SB15s and FDSB502s; the same markers were the most informative, with PIC values of 0.70 and 0.69, respectively. Three private alleles were found in pollinator EL0204; two in pollinator C51; and one in pollinators NS1, FC221, and C93035. Molecular variance showed that 77.34% of the total genetic variation was attributed to intrapopulation variability. Cluster and correspondence analysis grouped sugar beet pollinators according to the breeding centers, with few exceptions, which indicate that certain amount of germplasm was shared, although centers had their own breeding programs. Conclusions: The results indicate that this approach can improve the selection of pollinators as suitable parental components and could further be applied in sugar beet breeding programs.

Characterization of SSR genomic abundance and identification of SSR markers for population genetics in Chinese jujube (Ziziphus jujuba Mill.).

Chinese jujube (Ziziphus jujuba Mill. [Rhamnaceae]), native to China, is a major dried fruit crop in Asia. Although many simple sequence repeat (SSR) markers are available for phylogenetic analysis of jujube cultivars, few of these are validated on the level of jujube populations. In this study, we first examined the abundance of jujube SSRs with repeated unit lengths of 1-6 base pairs, and compared their distribution with those in Arabidopsis thaliana. We identified 280,596 SSRs in the assembled genome of jujube. The density of SSRs in jujube was 872.60 loci/Mb, which was much higher than in A. thaliana (221.78 loci/Mb). (A+ T)-rich repeats were dominant in the jujube genome. We then randomly selected 100 SSRs in the jujube genome with long repeats and used them to successfully design 70 primer pairs. After screening using a series of criteria, a set of 20 fluorescently labeled primer pairs was further selected and screened for polymorphisms among three jujube populations. The average number of alleles per locus was 12.8. Among the three populations, mean observed and expected heterozygosities ranged from 0.858 to 0.967 and 0.578 to 0.844, respectively. After testing in three populations, all SSRs loci were in Hardy-Weinberg equilibrium (HWE) in at least one population. Finally, removing high null allele frequency loci and linked loci, a set of 17 unlinked loci was in HWE. These markers will facilitate the study of jujube genetic structure and help elucidate the evolutionary history of this important fruit crop.