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Identification and Control of Latent Bacteria in in vitro Cultures of Sweetpotato [Ipomoea batatas (L.) Lam].

ABSTRACT: Bacterial microorganisms which are latent in in vitro cultures can limit the efficiency of in vitro methods for the conservation of genetic resources. In this study we screened 2,373 accessions from the in vitro sweetpotato germplasm collection of the International Potato Center in Lima, Peru for bacteria associated with plantlets in tissue culture through a combination of morphological methods and partial 16S rDNA sequencing. Bacteria were detected in 240 accessions (10% of the accessions screened) and we were able to isolate 184 different bacterial isolates from 177 different accessions. These corresponded to at least nineteen Operational Taxonomic Units (OTUs) of bacteria, belonging to the genera Sphingomonas, Bacillus, Paenibacillus, Methylobacterium, Brevibacterium, Acinetobacter, Microbacterium, Streptomyces, Staphylococcus, and Janibacter. Specific primers were developed for PCR based diagnostic tests that were able to rapidly detect these bacteria directly from tissue culture plants, without the need of microbial sub-culturing. Based on PCR screening the largest bacterial OTUs corresponded to a Paenibacillus sp. closely related to Paenibacillus taichungensis (41.67%), and Bacillus sp. closely related to Bacillus cereus (22.22%), and Bacillus pumilus (16.67%). Since in vitro plant genetic resources must be microbe-free for international distribution and use, any microbial presence is considered a contamination and therefore it is critical to clean all cultures of these latent-appearing bacteria. To accomplish this, plantlets from in vitro were transferred to soil, watered with Dimanin® (2 ml/l) weekly and then reintroduced into in vitro. Of the 191 accessions processed for bacterial elimination, 100% tested bacteria-free after treatment. It is suspected that these bacteria may be endosymbionts and some may be beneficial for the plants.

SUBMITTER: Izarra ML

PROVIDER: S-EPMC7350948 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Identification and Control of Latent Bacteria in in vitro Cultures of Sweetpotato [Ipomoea batatas (L.) Lam].

Izarra Myriam Lorena ML Panta Ana Luz AL Maza Carmen Rosa CR Zea Brenda Carina BC Cruzado Juan J Gutarra Liliam Rosario LR Rivera Cristina R CR Ellis David D Kreuze Jan Frederik JF

Frontiers in plant science 20200703

Bacterial microorganisms which are latent in in vitro cultures can limit the efficiency of in vitro methods for the conservation of genetic resources. In this study we screened 2,373 accessions from the in vitro sweetpotato germplasm collection of the International Potato Center in Lima, Peru for bacteria associated with plantlets in tissue culture through a combination of morphological methods and partial 16S rDNA sequencing. Bacteria were detected in 240 accessions (10% of ...[more]

PMID: 32719697

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Project description:BackgroundSweetpotato root rot is a devastating disease caused by Fusarium solani that seriously endangers the yield of sweetpotato in China. Although there is currently no effective method to control the disease, breeding of resistant varieties is the most effective and economic option. Moreover, quantitative trait locus (QTL) associated with resistance to root rot have not yet been reported, and the biological mechanisms of resistance remain unclear in sweetpotato. Thus, increasing our knowledge about the mechanism of disease resistance and identifying resistance loci will assist in the development of disease resistance breeding.ResultsIn this study, we constructed genetic linkage maps of sweetpotato using a mapping population consisting of 300 individuals derived from a cross between Jizishu 1 and Longshu 9 by simple sequence repeat (SSR) markers, and mapped seven QTLs for resistance to root rot. In total, 484 and 573 polymorphic SSR markers were grouped into 90 linkage groups for Jizishu 1 and Longshu 9, respectively. The total map distance for Jizishu 1 was 3974.24?cM, with an average marker distance of 8.23?cM. The total map distance for Longshu 9 was 5163.35?cM, with an average marker distance of 9.01?cM. Five QTLs (qRRM_1, qRRM_2, qRRM_3, qRRM_4, and qRRM_5) were located in five linkage groups of Jizishu 1 map explaining 52.6-57.0% of the variation. Two QTLs (qRRF_1 and qRRF_2) were mapped on two linkage groups of Longshu 9 explaining 57.6 and 53.6% of the variation, respectively. Furthermore, 71.4% of the QTLs positively affected the variation. Three of the seven QTLs, qRRM_3, qRRF_1, and qRRF_2, were colocalized with markers IES43-5mt, IES68-6?fs**, and IES108-1?fs, respectively.ConclusionsTo our knowledge, this is the first report on the construction of a genetic linkage map for purple sweetpotato (Jizishu 1) and the identification of QTLs associated with resistance to root rot in sweetpotato using SSR markers. These QTLs will have practical significance for the fine mapping of root rot resistance genes and play an important role in sweetpotato marker-assisted breeding.

Project description:To better define the sweetpotato polyploidy, we sought to reconstruct phylogenies of its subgenomes based on hybridization networks that could trace reticulate lineages of differentiated homoeolog triplets of multiple single-copy genes. In search of such homoeolog triplets, we distinguished cDNA variants of 811 single-copy Conserved Ortholog Set II (COSII) genes from two sweetpotato clones into variation partitions specified by corresponding homologs from two I. trifida lines, I. tenuissima and I. littoralis using a phylogenetic partition method, and amplicon variants of the COSII-marker regions from 729 of these genes from two sweetpotato clones into putative homoeoallele groups using haplotype tree and the partition methods referenced by corresponding homologs from I. tenuissima. These analyses revealed partly or completely differentiated expressed-homoeologs and homoeologs from a majority of these genes with three important features. 1. Two variation types: the predominant interspecific variations (homoeoalleles), which are non-randomly clustered, differentially interspecifically conserved or sweetpotato-specific, and the minor intraspecific ones (alleles), which are randomly distributed mostly at non-interspecifically variable sites, and usually sweetpotato-specific. 2. A clear differentiation of cDNA variants of many COSII genes into the variation partition specified by I. tenuissima or I. littoralis from that by I. trifida. 3. Three species-homolog-specified and one sweetpotato-specific variation partitions among 293 different COSII cDNAs, and two or three out of the four partitions among cDNA variants of 306 COSII genes. We then constructed hybridization networks from two concatenations of 16 and 4 alignments of 8 homologous COSII cDNA regions each, which included three taxa of expressed homoeologs in a triple-partition combination from the 16 or 4 sweetpotato COSII genes and 5 taxa each of respective cDNA homologs from the three sweetpotato relatives and I. nil, and inferred a species tree embodying both networks. The species tree predicted close-relative origins of three partly differentiated sweetpotato subgenomes.

Project description:BackgroundSweetpotato, Ipomoea batatas (L.) Lam., is an important food crop widely grown in the world. However, little is known about the genome of this species because it is a highly heterozygous hexaploid. Gaining a more in-depth knowledge of sweetpotato genome is therefore necessary and imperative. In this study, the first bacterial artificial chromosome (BAC) library of sweetpotato was constructed. Clones from the BAC library were end-sequenced and analyzed to provide genome-wide information about this species.ResultsThe BAC library contained 240,384 clones with an average insert size of 101 kb and had a 7.93-10.82 × coverage of the genome, and the probability of isolating any single-copy DNA sequence from the library was more than 99%. Both ends of 8310 BAC clones randomly selected from the library were sequenced to generate 11,542 high-quality BAC-end sequences (BESs), with an accumulative length of 7,595,261 bp and an average length of 658 bp. Analysis of the BESs revealed that 12.17% of the sweetpotato genome were known repetitive DNA, including 7.37% long terminal repeat (LTR) retrotransposons, 1.15% Non-LTR retrotransposons and 1.42% Class II DNA transposons etc., 18.31% of the genome were identified as sweetpotato-unique repetitive DNA and 10.00% of the genome were predicted to be coding regions. In total, 3,846 simple sequences repeats (SSRs) were identified, with a density of one SSR per 1.93 kb, from which 288 SSRs primers were designed and tested for length polymorphism using 20 sweetpotato accessions, 173 (60.07%) of them produced polymorphic bands. Sweetpotato BESs had significant hits to the genome sequences of I. trifida and more matches to the whole-genome sequences of Solanum lycopersicum than those of Vitis vinifera, Theobroma cacao and Arabidopsis thaliana.ConclusionsThe first BAC library for sweetpotato has been successfully constructed. The high quality BESs provide first insights into sweetpotato genome composition, and have significant hits to the genome sequences of I. trifida and more matches to the whole-genome sequences of Solanum lycopersicum. These resources as a robust platform will be used in high-resolution mapping, gene cloning, assembly of genome sequences, comparative genomics and evolution for sweetpotato.

Dataset Information

Identification and Control of Latent Bacteria in in vitro Cultures of Sweetpotato [Ipomoea batatas (L.) Lam].

Publications

Identification and Control of Latent Bacteria in <i>in vitro</i> Cultures of Sweetpotato [<i>Ipomoea batatas</i> (L.) Lam].

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