Sequence-guided approach to genotyping plant clones and species using polymorphic NB-ARC-related genes

Rapid and economical genotyping tools that can reliably distinguish species and intraspecific variations in plants can be powerful tools for biogeographical and ecological studies. Clones of the cosmopolitan duckweed species, Spirodela polyrhiza, are difficult to distinguish morphologically due to their highly abbreviated architecture and inherently low levels of sequence variation. The use of plastidic markers and generic Amplification Fragment Length Polymorphism approaches have met with limited success in resolving clones of S. polyrhiza from diverse geographical locales. Using whole genome sequencing data from nine S. polyrhiza clones as a training set, we created an informatic pipeline to identify and rank polymorphic regions from nuclear-encoded NB-ARC-related genes to design markers for PCR, Sanger sequencing (barcoding), and fragment length analysis. With seven primer sets, we found 21 unique fingerprints from a set of 23 S. polyrhiza clones. However, three of these clones share the same fingerprint and are indistinguishable by these markers. These primer sets can also be used as interspecific barcoding tools to rapidly resolve S. polyrhiza from the closely related S. intermedia species without the need for DNA sequencing. Our work demonstrates a general approach of using hyper-polymorphic loci within genomes as a resource to produce facile tools that can have high resolving power for genotyping applications

Additional file 5: of Population structure, genetic diversity and downy mildew resistance among Ocimum species germplasm

Genotype data. Binary matrix of accessions (rows) and alleles (columns). (XLSX 186 kb

Additional file 1: of Population structure, genetic diversity and downy mildew resistance among Ocimum species germplasm

Description of 180-accession panel of Ocimum spp., cluster membership and response to downy mildew (Peronospora belbahrii) reported as disease severity. (PDF 115 kb

Additional file 2: of Population structure, genetic diversity and downy mildew resistance among Ocimum species germplasm

Primer sequences and melting temperatures (Tm) for the EST-SSRs used in this study. (PDF 52 kb

A first linkage map and downy mildew resistance QTL discovery for sweet basil (<i>Ocimum basilicum</i>) facilitated by double digestion restriction site associated DNA sequencing (ddRADseq)

<div><p>Limited understanding of sweet basil (<i>Ocimum basilicum</i> L.) genetics and genome structure has reduced efficiency of breeding strategies. This is evidenced by the rapid, worldwide dissemination of basil downy mildew (<i>Peronospora belbahrii</i>) in the absence of resistant cultivars. In an effort to improve available genetic resources, expressed sequence tag simple sequence repeat (EST-SSR) and single nucleotide polymorphism (SNP) markers were developed and used to genotype the MRI x SB22 F₂ mapping population, which segregates for response to downy mildew. SNP markers were generated from genomic sequences derived from double digestion restriction site associated DNA sequencing (ddRADseq). Disomic segregation was observed in both SNP and EST-SSR markers providing evidence of an <i>O</i>. <i>basilicum</i> allotetraploid genome structure and allowing for subsequent analysis of the mapping population as a diploid intercross. A dense linkage map was constructed using 42 EST-SSR and 1,847 SNP markers spanning 3,030.9 cM. Multiple quantitative trait loci (QTL) model (MQM) analysis identified three QTL that explained 37–55% of phenotypic variance associated with downy mildew response across three environments. A single major QTL, <i>dm11</i>.<i>1</i> explained 21–28% of phenotypic variance and demonstrated dominant gene action. Two minor QTL <i>dm9</i>.<i>1</i> and <i>dm14</i>.<i>1</i> explained 5–16% and 4–18% of phenotypic variance, respectively. Evidence is provided for an additive effect between the two minor QTL and the major QTL <i>dm11</i>.<i>1</i> increasing downy mildew susceptibility. Results indicate that ddRADseq-facilitated SNP and SSR marker genotyping is an effective approach for mapping the sweet basil genome.</p></div

Effect and interaction plots for three QTL detected in environment NJSN14.

<p>MRI x SB22 F₂ genotype means (circles) ± 1 SE (error bars) for (A) minor QTL <i>dm9</i>.1, (B) major QTL <i>dm11</i>.<i>1</i> and (C) minor <i>dm14</i>.<i>1</i>. Two-QTL genotype by genotype means ± 1 SE for (D) <i>dm11</i>.<i>1</i> by <i>dm9</i>.<i>1</i> and (E) <i>dm11</i>.<i>1</i> by <i>dm14</i>.<i>1</i>. Allele ‘a’ is inherited from downy mildew resistant grandparent MRI and allele ‘b’ is inherited from susceptible grandparent SB22. Error bars represent ±1 SE.</p

Detection of major downy mildew resistance QTL <i>dm11</i>.<i>1</i> across three environments.

<p>LOD scores for genome-wide scan using square-root transformed phenotype data from three environments: NJSN14 (northern New Jersey; 2014), NJRA14 (southern New Jersey; 2014) and NJRA15 (southern New Jersey; 2015). Significant LOD thresholds (α = 0.05) were calculated by permutation tests with 1,000 iterations and are shown with red, dashed horizontal lines.</p

Frequency distribution of disease severity in the MRI x SB22 F₂ mapping population across three environments.

<p>Codes for each environment are shown on the x-axis and correspond to data recorded in 2014 in southern New Jersey (NJRA14), 2015 in southern New Jersey (NJRA15) and northern New Jersey in 2014 (NJSN14). Disease severity measured on a scale in which 0 = lowest possible severity score and 1 = highest possible severity score.</p

F₂ means for downy mildew (disease) response in environment NJSN14 according to two-QTL genotype by genotype combinations.

<p>F₂ means for downy mildew (disease) response in environment NJSN14 according to two-QTL genotype by genotype combinations.</p