Association study of SNP locus for color related traits in herbaceous peony (Paeonia lactiflora Pall.) using SLAF-seq

Paeonia lactiflora Pall. (P. lactiflora) is a famous ornamental plant with showy and colorful flowers that has been domesticated in China for 4,000 years. However, the genetic basis of phenotypic variation and genealogical relationships in P. lactiflora population is poorly understood due to limited genetic information, which brings about bottlenecks in the application of effective and efficient breeding strategies. Understanding the genetic basis of color-related traits is essential for improving flower color by marker-assisted selection (MAS). In this study, a high throughput sequencing of 99 diploid P. lactiflora accessions via specific-locus amplified fragment sequencing (SLAF-seq) technology was performed. In total, 4,383,645 SLAF tags were developed from 99 P. lactiflora accessions with an average sequencing depth of 20.81 for each SLAF tag. A total of 2,954,574 single nucleotide polymorphisms (SNPs) were identified from all SLAF tags. The population structure and phylogenetic analysis showed that P. lactiflora population used in this study could be divided into six divergent groups. Through association study using Mixed linear model (MLM), we further identified 40 SNPs that were significantly positively associated with petal color. Moreover, a derived cleaved amplified polymorphism (dCAPS) marker that was designed based on the SLAF tag 270512F co-segregated with flower colors in P. lactiflora population. Taken together, our results provide valuable insights into the application of MAS in P. lactiflora breeding programs

Genetic Dissection Of Negative Regulation On Disease Resistance Genes In Arabidopsis

Plant defense responses are repressed under non-pathogenic conditions to ensure optimal plant growth and development. R (Disease Resistance) genes are central regulators mediating robust disease resistance. An R gene SNC1 is negatively regulated by an evolutionarily conserved copine gene BON1 in Arabidopsis. The loss of BON1 function leads to enhanced disease resistance but a growth defect in a SNC1 dependent manner. To understand how the R gene SNC1 is regulated, I analyzed enhancers and suppressors of bon1 mutants. The study of bon1 enhancer ebo30 reveals an effect on R gene expression by cell cycle progression. The ebo30 mutant is an overexpression allele of OSD1 (omission of second division 1). Both OSD1 gene and its homolog UVI4 are negative regulators of anaphase-promoting complex/cyclosome (APC/C), a multisubunit ubiquitin E3 ligase that regulates the progression of cell cycles. Overexpression of OSD1 or UVI4 as well as down regulation of APC10 confers enhanced resistance to a bacterial pathogen. Further, enhanced immune response induced by OSD1 overexpression is dependent on CYCB1;1 and the R gene SNC1. Together, this study suggests that mis-regulated cell cycle progression has an impact on R gene expression and plant immunity. This notion is reinforced by the study of interaction of UVI4 and OSD1 with CPR5, a gene involved in both defense and cell cycle regulation. The cpr5 mutant was reported to have reduced endoreduplication and enhanced disease resistance. These cell cycle defects of osd1 and uvi4 single and double mutants can be suppressed by cpr5 mutation. Therefore, the CPR5 gene may have a direct role in cell cycle regulation and subsequently affect plant immunity. The study of mos1, a suppressor of bon1 reveals a new transcriptional regulator for plant immunity, flowering time and endoreduplication. The mos1 mutant has compromised defense responses, is late flowering, and has enhanced endoreduplication. These phenotypes are due to the change of expression of SNC1, FLC, and potentially CYCD3;1 respectively. The function of MOS1 in modulating flowering time and cell cycle progression is dependent on SUF4, a previously known transcription factor for flowering time control. MOS1 is found to physically interact with SUF4, and may thus repress its function. The interaction of MOS1 and SUF4 might be influenced by MAD2, a component in the spindle assembly checkpoint complex. The interactions among MOS1, MAD2 with SUF4 suggest an intriguingly possibility that checkpoint machinery might have a direct impact on flowering time control. In sum, this study provides insights into complex regulation on R genes in plants, and discusses potential connections among regulations of defense, cell cycle, and flowering time

Interaction of <i>CPR5</i> with Cell Cycle Regulators <i>UVI4</i> and <i>OSD1</i> in Arabidopsis

<div><p>The impact of cell cycle on plant immunity was indicated by the enhancement of disease resistance with overexpressing <i>OSD1</i> and <i>UVI4</i> genes that are negative regulators of cell cycle controller APC (anaphase promoting complex). <i>CPR5</i> is another gene that is implicated in cell cycle regulation and plant immunity, but its mode of action is not known. Here we report the analysis of genetic requirement for the function of <i>UVI4</i> and <i>OSD1</i> in cell cycle progression control and in particular the involvement of <i>CPR5</i> in this regulation. We show that the APC activator CCS52A1 partially mediates the function of <i>OSD1</i> and <i>UVI4</i> in female gametophyte development. We found that the <i>cpr5</i> mutation suppresses the endoreduplication defect in the <i>uvi4</i> single mutant and partially rescued the gametophyte development defect in the <i>osd1 uvi4</i> double mutant while the <i>uvi4</i> mutation enhances the <i>cpr5</i> defects in trichome branching and plant disease resistance. In addition, cyclin B1 genes <i>CYCB1;1</i>, <i>CYCB1;2</i>, and <i>CYCB1;4</i> are upregulated in <i>cpr5</i>. Therefore, <i>CPR5</i> has a large role in cell cycle regulation and this role has a complex interaction with that of <i>UVI4</i> and <i>OSD1</i>. This study further indicates an intrinsic link between plant defense responses and cell cycle progression.</p></div

Both <i>uvi4</i> and <i>osd1</i> mutations enhance defense responses in <i>cpr5</i>.

<p>Bacterial growth assay in Col-0, <i>uvi4</i>, <i>osd1</i>, <i>cpr5</i>, <i>cpr5 uvi4</i> and <i>cpr5 osd1</i> (all diploid plants) inoculated by spray inoculation of <i>Pst</i> DC3000. Error bars indicate standard deviations. Letters a, b and c indicate the statistical significance determined by student <i>t</i>-test.</p

Overexpression of <i>OSD1</i> and <i>UVI4</i> affect endoreduplication in leaves.

<p>(A) Ploidy levels in the first pair of leaves from the wild-type L<i>er</i>, <i>uvi4-2</i>, and <i>osd1-2</i> grown under 12 hour light/day for 4 weeks. (B) Ploidy levels in the first pair of leaves from Ws, <i>bon1-2</i>, <i>osd1-4</i> and <i>bon1 osd1-4</i> grown under 12 hour light/day for 4 weeks. Error bars indicate standard deviations. The number above the column indicates the ploidy index. Averages of three replicas for each sample were shown in (A) and (B). Ploidy indices were used for statistical analysis, and the letter a indicates a statistical significance determined by student <i>t</i>-test. The representative data were shown from two independent measurements. (C) Frequencies of each class of trichome numbers in Col-0, <i>uvi4</i> and <i>UVI4-OE</i> transgenic lines 5 and 17 in Col-0 and lines 3 and 5 in <i>uvi4</i>. Approximately 100 to 150 cells were examined for each genotype. Error bars indicate standard deviations. The difference between overexpression lines and wild-type Col-0 or <i>uvi4</i>was determined by chi-square test (<i>UVI4-OE/</i>Col-0 compared to Col-0, L5: P = 0.01<0.05, L17: P = 0.00<0.05; <i>UVI4-OE/uvi4</i> compared to Col-0, L3: P = 0.02<0.05, L5: P = 0.21>0.05; <i>UVI4-OE/uvi4</i> compared to <i>uvi4</i>, L3: P = 0.00<0.05, L5: P = 0.00<0.05).</p

Genetic interactions of <i>uvi4</i> and <i>osd1</i> with <i>cpr5</i> in the regulation of endoreduplication and meiosis.

<p>(A) Frequency of cells with different branching numbers in Col-0, <i>cpr5</i>, <i>uvi4</i>, and <i>uvi4 cpr5</i> plants on the forth leaves. Approximately 100 to 150 cells were examined for each genotype. (B) DNA ploidy levels of Col-0, <i>cpr5</i>, <i>uvi4</i>, and <i>uvi4 cpr5</i> shown as percentage of cells with 2C to 32C. (C) DNA ploidy levels of Col-0, <i>cpr5</i>, <i>osd1</i> BC7F3, and <i>cpr5 osd1</i> F3 plants shown as percentage of cells with 2C to 32C. Error bars indicate standard deviations. The number above the column indicates the ploidy index. Numbers in (B) and (C) are averages of three replicas. Ploidy indices were used for statistical analysis, and the letter a indicates a statistical significance determined by student <i>t</i>-test. The representative data were shown from two independent measurements.</p

Expression of cell cycle genes in <i>cpr5</i> mutant.

<p>Expression levels of <i>AtCYCB1;1</i> in both the first pair of leaves and whole seedlings of 2-week-old plants, and <i>AtCYCB1;2</i> and <i>AtCYCB1;4</i> in whole seedlings analyzed by quantitative real time RT-PCR. Error bars indicate standard deviations.</p

The <i>cpr5</i> mutation partially suppresses the lethality of <i>osd1 uvi4</i> double mutant.

<p>Shown are numbers of plants of each genotype in an analyzed population. The two numbers separated by a semicolon in parentheses are the observed ratios of that genotype relative to the top left genotype (left) and the expected ratios when there is no reduced transmission of the gametes or zygotes (right). (A) Analysis of gamete transmission inferred from of reciprocal crosses between <i>CPR5/cpr5</i> and Col-0. The <i>cpr5</i> had a higher transmission rate as male gamete (shaded) but not female gamete. The significance was determined by chi-square test (male, P = 0.011<0.05; female, P = 0.78>0.05). (B) Analysis of progenies from <i>osd1/OSD1 uvi4/UVI4 cpr5/cpr5</i>. Notice the presence of <i>osd1/osd1 uvi4/uvi4 cpr5/cpr5</i> (shaded) while there was no <i>osd1/osd1 uvi4/uvi4</i> in progenies of <i>osd1/OSD1 UVI4/uvi4</i> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100347#pone-0100347-g002" target="_blank">Figure 2A</a>). Also notice that plants with <i>osd1/OSD1 cpr5/cpr5</i> genotypes irrespective of the <i>uvi4</i> genotypes (darker shaded) were fewer than expected, suggesting a lower transmission of <i>osd1 cpr5</i> than <i>OSD1 cpr5</i>. The difference between observed segregation ratio and normal segregation ratio was determined by chi-square test (P = 1.07E-14<0.05). (C) Analysis of progenies from <i>osd1</i>/<i>OSD1 uvi4/uvi4 cpr5/cpr5</i>. (D) Morphology of Col-0, <i>uvi4</i>, <i>osd1</i>, <i>cpr5</i>, <i>cpr5 uvi4</i>, <i>cpr5 osd1</i>and <i>cpr5 uvi4 osd1</i> grown for 6 weeks under 12 h light/12 h dark condition. (E) Analysis of gamete transmission inferred from of reciprocal crosses between <i>osd1/OSD1 uvi4/uvi4 CPR5/cpr5</i> and Col-0 or <i>uvi4</i>. Notice a lower transmission (20 versus 50 with Col-0 and 15 versus 33 with <i>uvi4</i>) of <i>osd1uvi4CPR5</i> (shaded) compared to <i>OSD1uvi4CPR5</i> as female gametes but an increased transmission rate (29 versus 40 with Col-0 and 41 versus 38 with <i>uvi4</i>) of <i>osd1uvi4cpr5</i> (darker shaded) compared to that of <i>osd1uvi4CPR5</i>. The difference of gamete transmission between <i>osd1/OSD1 uvi4/uvi4</i> and <i>osd1/OSD1 uvi4/uvi4 CPR5/cpr5</i> was determined by chi-square test (reciprocal cross with Col-0, P = 1.71E-36<0.05; reciprocal cross with <i>uvi4</i>, P = 4.20E-37<0.05).</p

<i>CCS52A1</i> partially mediates the lethality of <i>osd1 uvi4</i>.

<p>Shown are numbers of plants of each genotype in an analyzed population. The two numbers separated by a semicolon in parentheses are the observed ratios of that genotype relative to the top left genotype (left) and the expected ratios when there is no reduced transmission of the gametes or zygotes (right). (A) Analysis of progenies of <i>osd1/OSD1 UVI4/uvi4</i>. No <i>osd1/osd1 uvi4/uvi4</i> plants (shaded) were found. The difference between observed and expected segregation ratio was determined by chi-square test (P = 0.004<0.05). (B) Analysis of progenies of <i>osd1</i>/<i>OSD1 uvi4/uvi4</i>. Notice that both <i>OSD1/OSD1 uvi4/uvi4</i> and <i>osd1/OSD1 uvi4/uvi4</i> (shaded) were about half of expected and there were no <i>osd1/osd1uvi4/uvi4</i> (shaded) progenies produced. The significance was determined by chi-square test (P = 6.24E-12<0.05). (C) Analysis of gamete transmission inferred from reciprocal crosses between <i>osd1/OSD1 uvi4/uvi4</i> and Col-0. The <i>osd1 uvi4</i> genotype had a lower transmission rate through female gametes (shaded) but not male gametes. The significance was determined by chi-square test (male, P = 0.13>0.05; female, P = 2.53E-10<0.05). (D) Opened siliques from wild-type Col-0 (upper) and <i>osd1/OSD1 uvi4/uvi4</i> (lower) plants. Aborted ovules and embryos can be seen in the mutant silique. (E) Analysis of progenies of <i>osd1/OSD1 uvi4/uvi4 ccs52a1/ccs52a1</i>. Notice that two <i>osd1/osd1 uvi4/uvi4 ccs52a1/ccs52a1</i> plants (shaded) were found. (F) Morphology of the <i>osd1/osd1 uvi4/uvi4 ccs52a1/ccs52a1</i> triple mutant plant.</p

Two Different Copy Number Variations of the SOX5 and SOX8 Genes in Yak and Their Association with Growth Traits

Copy number variation (CNV) is a structural variant with significant impact on genetic diversity. CNV has been widely used in breeding for growth traits, meat production or quality, and coat color. SRY-like box genes (SOXs) are a class of transcription factors that play a regulatory role in cell fate specification and differentiation. SOX5 and SOX8 belong to subgroups D and E of the SOXs, respectively. Previous studies have shown that SOX5 and SOX8 are essential in the development of bones. In this study, we explored the association between the growth traits and CNVs of SOX5 and SOX8 in 326 Ashidan yaks and detected mRNA expression levels in different tissues. Our results illustrated that CNVs of SOX5 and SOX8 were significantly associated with withers height at 18 months of age and chest girth at 30 months of age (p p p p < 0.05). Our results provide evidence that the CNVs of SOX5 and SOX8 genes could be used as new markers for the selection of yak growth traits