Identification and validation of SSR markers for Xanthomonas axonopodis pv. punicae an incitant of bacterial blight of pomegranate

Abstract

This study reports genome wide characterization and development of first set of microsatellite markers through in silico analysis of eight sequenced Xanthomonas axonopodis pv. punicae strains available in the public database. SSR survey resulted in identification of ~ 4638 perfect SSRs, with mean marker frequency 901 SSRs/Mb and densitiy of 11,006 bp/Mb aross the eight genomes. Frequency distribution graphs revealed hexa-nucleotide repeats were more prominent fowllowed by tri-, tetra-, di- and penta-nucleotides in the analysed genomes. We desinged 2927 SSR primers that are specific to the strain LMG 859 and ePCR confirmed on seven other Xap genomes. This resulted in identification of 542 informative SSRs that are producing single amplicons, from which 66 primers were successfully validated through wet lab experiments on eight Xap isolates of pomegranate. Furthermore, utility of these SSRs were demostrated by analysing molecular diversity among 22 Xap isolates using 20 Xap_SSR primers. SSRs revealed moderate genetic diversity among Xap isolates (61%) and grouped 11 isolates that are repersenting six different states into one cluster. This proved the earlier evidence of wider spread of ST3 type Xap acoss India using Multi locus Sequence Typing (MLST) technique. In summary, Xap_SSR will serve as powerful genomics tools that would helps in monitoring of population dynamics, taxonomy, epidomology and quarantine aspects in bacterial blight pathogen through development of microsatellite based Multilocus Variable number of Tandem repeat analysis (MLVA) in future.

Supplementary information: The online version contains supplementary material available at 10.1007/s13205-022-03209-z.

Keywords: Bacterial blight; Microsatellite; Molecular diversity; Pomegranate; Xanthomonas axonopodis; ePCR.

Fig. 1

Circos graph depicting physical locations of 66 Xap_SSRs on the chromosome of reference strain (LMG859)

Fig. 2

Gel image showing a initial PCR validation of SSRs on eight Xap isolates, and b screening on 22 Xap isolates (where, L-100 bp DNA ladder for lanes 1–22 Xap isolates as listed in Table 1)

Fig. 3

Dendrogram (a) and PCA plot (b) showing genetic relationships between 22 Xap isolates based on SSR marker data