A new method to uncover signatures of divergent and stabilizing selection in quantitative traits

While it is well understood that the pace of evolution depends on the interplay between natural selection, random genetic drift, mutation, and gene flow, it is not always easy to disentangle the relative roles of these factors with data from natural populations. One popular approach to infer whether the observed degree of population differentiation has been influenced by local adaptation is the comparison of neutral marker gene differentiation (as reflected in FST) and quantitative trait divergence (as reflected in QST). However, this method may lead to compromised statistical power, because FST and QST are summary statistics which neglect information on specific pairs of populations, and because current multivariate tests of neutrality involve an averaging procedure over the traits. Further, most FST-QST comparisons actually replace QST by its expectation over the evolutionary process and are thus theoretically flawed. To overcome these caveats, we derived the statistical distribution of population means generated by random genetic drift and used the probability density of this distribution to test whether the observed pattern could be generated by drift alone. We show that our method can differentiate between genetic drift and selection as a cause of population differentiation even in cases with FST=QST and demonstrate with simulated data that it disentangles drift from selection more accurately than conventional FST-QST tests especially when data sets are small.     

Identification, fine mapping and characterisation of a dwarf mutant (bnaC.dwf) in Brassica napus

In the present study, we have obtained one dwarf mutant (bnaC.dwf) from the Brassica napus inbred line T6 through chemical mutagen ethyl methanesulfonate (EMS). We have determined the phenotypic effects and genetic characteristics of dwarf mutant (bnaC.dwf). The dwarf mutant was insensitive to exogenous GA(3) for plant height, suggesting that it is significantly playing a crucial role in the gibberellins response pathway. Genetic analysis revealed that one recessive gene is responsible for controlling the phenotypic expression of dwarf mutant. Amplified Fragment Length Polymorphism (AFLP) technique was applied for selecting markers linked to the BnaC.DWF gene which assisted in screening of dwarf and normal individuals in the BC(4) population. We have screened 1,024 primer combinations and then identified nine AFLP markers linked to the BnaC.DWF gene. Identification and linkage of the markers were carried out by analysing 2,000 individuals from a larger population of the BC(4). Two markers EA10MC09 and EA12MC02 were located on the flanking region of the BnaC.DWF gene at a distance of 0.2 and 0.05 cM, respectively. Four AFLP markers EA09MG05, EA02MC07, EA01MC01 and EC04MC07 were successfully converted into Sequence Characterised Amplified Region markers namely SCA9G5, SCA2C7, SCA1C1 and SCC4C7. We further integrated BnaC.DWF linked Simple Sequence Repeat markers into two populations (Piquemal et al. Theor Appl Genet 111:1514-1523, 2005; Cheng et al. Theor Appl Genet 118:1121-1131, 2009). BnaC.DWF was mapped to the linkage region N18. The molecular markers developed from these investigations will greatly accelerate the selection process for developing dwarf varieties in B. napus by Marker Assisted Selection and genetic engineering.     

Extracts of Coleus forskohlii relieves cough and asthma symptoms via modulating inflammation and the extracellular matrix

Asthma is characterized by airway inflammatory infiltration, which leads to airway remodeling and airway hyperreactivity. Coleus forskohlii (CFK) has been used to treat asthma, however, the mechanism involved is not clear. To explore the antiasthma mechanism of extracts of Coleus forskohlii (ECFK), guinea pigs were administered with a spray of phosphoric acid histamine, and rats were sensitized with ovalbumin (OVA). Hematoxylin and eosin staining (H&E) were used to evaluate pathological changes in lung tissue. Enzyme-linked immunosorbent assay (ELISA) was used to determine cytokine levels in serum and bronchoalveolar lavage fluid (BALF). Immunohistochemistry and Western blot analysis were used to assess the expression of intercellular cell adhesion molecule-1 (ICAM-1), phosphorylation of p65 (p-p65), matrix metallopeptidase 9 (MMP-9), and tissue inhibitor of metalloproteinase 1 (TIMP-1). After ECFK treatment, the asthma incubation period of guinea pigs was significantly prolonged. The H&E results showed that the number of eosinophils in the 12.8 g/kg ECFK group was significantly lower when compared with the control group. Moreover, ELISA results demonstrated that interleukin (IL)-4, IL-5, and IL-17 in serum and BALF were significantly decreased, and interferon-γ (IFN-γ) and IL-10 were increased after ECFK treatment. In addition, ECFK treatment resulted in downregulation of ICAM-1, p-p65, MMP-9, and TIMP-1 in lung tissue after being sensitized by OVA. In conclusion, our findings indicated that ECFK significantly alleviated OVA-induced inflammatory infiltration and airway remodeling in asthma. This study laid a theoretical foundation for the clinical use of ECFK.     

Neofunctionalization of Duplicated Tic40 Genes Caused a Gain-of-Function Variation Related to Male Fertility in Brassica oleracea Lineages

Gene duplication followed by functional divergence in the event of polyploidization is a major contributor to evolutionary novelties. The Brassica genus evolved from a common ancestor after whole-genome triplication. Here, we studied the evolutionary and functional features of Brassica spp. homologs to Tic40 (for translocon at the inner membrane of chloroplasts with 40 kDa). Four Tic40 loci were identified in allotetraploid Brassica napus and two loci in each of three basic diploid Brassica spp. Although these Tic40 homologs share high sequence identities and similar expression patterns, they exhibit altered functional features. Complementation assays conducted on Arabidopsis thaliana tic40 and the B. napus male-sterile line 7365A suggested that all Brassica spp. Tic40 homologs retain an ancestral function similar to that of AtTic40, whereas BolC9.Tic40 in Brassica oleracea and its ortholog in B. napus, BnaC9.Tic40, in addition, evolved a novel function that can rescue the fertility of 7365A. A homologous chromosomal rearrangement placed bnac9.tic40 originating from the A genome (BraA10.Tic40) as an allele of BnaC9.Tic40 in the C genome, resulting in phenotypic variation for male sterility in the B. napus near-isogenic two-type line 7365AB. Assessment of the complementation activity of chimeric B. napus Tic40 domain-swapping constructs in 7365A suggested that amino acid replacements in the carboxyl terminus of BnaC9.Tic40 cause this functional divergence. The distribution of these amino acid replacements in 59 diverse Brassica spp. accessions demonstrated that the neofunctionalization of Tic40 is restricted to B. oleracea and its derivatives and thus occurred after the divergence of the Brassica spp. A, B, and C genomes.Polyploidy or whole-genome duplication is thought to be a prominent evolutionary force in eukaryotes (Wolfe, 2001; Udall and Wendel, 2006), especially for flowering plants (Blanc and Wolfe, 2004; Van de Peer et al., 2009). Almost 95% of angiosperms show evidence of having undergone at least one round of whole-genome duplication in their evolutionary history, suggesting that most extant diploid flowering plants have evolved from ancient polyploids (Cui et al., 2006; Soltis et al., 2009). Gene duplications in the event of polyploidization provide sources for evolutionary novelties that could benefit plants (Lukens et al., 2004; Chen, 2007). Divergence after gene duplication could result in three primary evolutionary fates of duplicated genes: pseudogenization, neofunctionalization, and subfunctionalization (Force et al., 1999; Conant and Wolfe, 2008; Liu and Adams, 2010). Pseudogenization implies that duplicated genes with redundant functions lose their function by accumulating negative mutations; neofunctionalization denotes that the redundant gene evolves a new adaptive function; while subfunctionalization causes the duplicated genes to adopt a different part of the function of an ancestral gene (Rodríguez-Trelles et al., 2003; Flagel and Wendel, 2009; Liu and Adams, 2010).The Brassica genus consists of three basic diploid species: Brassica rapa (AA; n = 10), Brassica nigra (BB; n = 8), and Brassica oleracea (CC; n = 9), and their derivative allotetraploid species: Brassica juncea (AABB; n = 18), Brassica napus (AACC; n = 19), and Brassica carinata (BBCC; n = 17; Beilstein et al., 2006). Comparative genetic mapping demonstrated that these Brassica spp., which diverged from Arabidopsis thaliana approximately 20 to 40 million years ago (Lagercrantz and Lydiate, 1996; Blanc et al., 2003; Town et al., 2006), descended from a common ancestor after whole-genome triplication (Parkin et al., 2002). Collinear comparison showed that for each of 24 ancestral genomic blocks defined in the ancestral karyotype in A. thaliana, three syntenic copies were identified in each of the diploid Brassica spp. genomes, with only one exception (Schranz et al., 2006; Wang et al., 2011; Cheng et al., 2013). Fractionation (gene loss from homologous genomic regions) and chromosomal rearrangements were prevalent in the diploidization process of the hexaploid Brassica spp. common ancestor (Lagercrantz, 1998; Town et al., 2006; Ziolkowski et al., 2006; Mun et al., 2009). Based on gene density differences caused by varying gene loss rates in the three collinear genomic block copies, these genomic blocks were classified into three subgenomes in the diploid Brassica spp. genomes: the least fractionated (LF), the medium fractionated (MF1), and the most fractionated (MF2) subgenomes (Wang et al., 2011; Tang and Lyons, 2012; Cheng et al., 2013). The different rates of gene loss of the three subgenomes support a two-step origin of the Brassiceae ancestral genome involving a tetraploidization process followed by substantial fractionation of the subgenomes MF1 and MF2 and more recently hybridization with the third subgenome LF to form a hexaploid (Wang et al., 2011; Tang et al., 2012). Although collinearity and changes in genomic structure, including duplications, deletions, and rearrangements of the Brassica spp. and A. thaliana are well studied, there is limited knowledge of the molecular and functional divergence of duplicated or homologous genes in the Brassica spp.To utilize heterosis in B. napus breeding, hybrid production is based mainly on male sterility. Currently, the recessive epistatic genic male-sterile three-type line system 7365ABC is widely used for oilseed heterosis due to its advantages, producing 100% sterile offspring for realizing the triple-cross hybrid (Huang et al., 2007; Xia et al., 2012). The male sterility in this system is controlled by two genes, a recessive male sterile gene Bnms3 and a epistatic gene BnRf (Zhou et al., 2012). Bnms3 was recently reported to be a homolog to A. thaliana Tic40 (Dun et al., 2011). Tic40 was identified as a member of the TIC (for translocon at the inner envelope membrane of chloroplasts) complex that functions as a cochaperone to coordinate Tic110 and the stromal chaperone heat shock protein93 (Hsp93) (Chou et al., 2003, 2006). The A. thaliana tic40 mutant displayed a chlorotic phenotype throughout development (Chou et al., 2003) but a male-fertile phenotype with mature pollen grains (Dun et al., 2011). Interestingly, one allele of Bnms3, BnaC.Tic40, can rescue the fertility of the B. napus male-sterile line 7365A (Dun et al., 2011).In this study, we identified and characterized Tic40 homologs in B. napus and three basic diploid Brassica spp. We suggested that neofunctionalization of BnaC9.Tic40 after the divergence of the Brassica spp. A, B, and C genomes was caused by amino acid replacements in the C terminus of BnaC9.Tic40. In addition, we validated that the allelic genes BnaC9.Tic40 (equivalent to BnaC.Tic40 as described by Dun et al. [2011]) and bnac9.tic40 originate from the C and A genomes, respectively, and became allelic due to a homologous chromosomal rearrangement in B. napus. These results provide further knowledge for the effective utilization of the restoring gene of 7365A and a better insight into the functional divergence of homologous duplicated genes in paleoploid Brassica spp.     

Exploring the role of a stigma-expressed plant U-box gene in the pollination responses of transgenic self-incompatible Arabidopsis thaliana

Recognition of “self” pollen in the self-incompatibility (SI) response of the Brassicaceae is determined by allele-specific interaction between the S-locus receptor kinase (SRK), a transmembrane protein of the stigma epidermis, and its ligand, the pollen coat-localized S-locus cysteine-rich (SCR) protein. The current model for SRK-mediated signaling proposes a central role for the plant U-box (PUB) Armadillo repeat-containing protein ARC1, an E3 ligase that interacts with, and is phosphorylated by, the kinase domain of SRK. According to the model, activated ARC1 causes the degradation of factors required for successful pollen tube growth. However, Arabidopsis thaliana plants transformed with functional SRK and SCR genes isolated from self-incompatible A. lyrata can express an intense SI response despite lacking a functional ARC1 gene. Here, we tested the possibility that a different member of the A. thaliana PUB protein family might have assumed the role of ARC1 in SI. Toward this end, we analyzed the AtPUB2 gene, which is annotated as being highly expressed in stigmas. Our functional analysis of a T-DNA insertion pub2 allele, together with yeast two-hybrid interaction assays and reporter analysis of AtPUB2 promoter activity, demonstrates that AtPUB2 does not function in SI. The results leave open the question of whether the proposed model of ARC1-mediated signaling applies to transgenic SRK–SCR self-incompatible A. thaliana plants.     

Mapping of BnMs4 and BnRf to a common microsyntenic region of Arabidopsis thaliana chromosome 3 using intron polymorphism markers

A recessive epistatic genic male sterile two-type line, 7365AB (Bnms3ms3ms4msRrfRf/BnMs3ms3ms4ms4RfRf), combined with the fertile interim-maintainer 7365C (Bnms3ms3ms4ms4rfrf) is an effective pollination control system in hybrid rapeseed production. We report an effective strategy used to fine map BnMs4 and BnRf. The two genes were both defined to a common microsyntenic region with Arabidopsis chromosome 3 using intron polymorphism (IP) markers developed according to Arabidopsis genome information and published genome organization of the A genome. The near-isogenic lines 7365AC (Bnms3ms3ms4ms4Rfrf/Bnms3ms3ms4ms4rfrf) of BnRf and 736512AB (Bnms3ms3Ms4ms4RfRf/Bnms3ms3ms4ms4RfRf) of BnMs4 were constructed to screen developed markers and create genetic linkage maps. Nine polymorphic IP markers (P1-P9) were identified. Of these, P2, P3, P4, and P6 were linked to both BnMs4 and BnRf with genetic distances <0.6 cM. Three simple sequence repeat markers, SR2, SR3, and SR5, were also identified by using public information. Subsequently, all markers linked to the two genes were used to compare the micro-collinearity of the regions flanking the two genes with Brassica rapa and Arabidopsis. The flanking regions showed rearrangements and inversion with fragments of different Arabidopsis chromosomes, but a high collinearity with B. rapa. This collinearity provided extremely valuable reference for map-based cloning in polyploid Brassica species. These IP markers could be exploited for comparative genomic studies within and between Brassica species, providing an economically feasible approach for molecular marker-assisted selection breeding, accelerating the process of gene cloning, and providing more direct evidence for the presence of multiple alleles between BnMs4 and BnRf.     

Characterization of interploid hybrids from crosses between Brassica juncea and B. oleracea and the production of yellow-seeded B. napus

Yellow-seeded Brassica napus was for the first time developed from interspecific crosses using yellow-seeded B. juncea (AABB), yellow-seeded B. oleracea (CC), and black-seeded artificial B. napus (AACC). Three different mating approaches were undertaken to eliminate B-genome chromosomes after trigenomic hexaploids (AABBCC) were generated. Hybrids (AABCC, ABCC) from crosses AABBCC?×?AACC, AABBCC?×?CC and ABCC?×?AACC were advanced by continuous selfing in approach 1, 2 and 3, respectively. To provide more insight into Brassica genome evolution and the cytological basis for B. napus resynthesis in each approach, B-genome chromosome pairing and segregation were intensively analyzed in AABCC and ABCC plants using genomic in situ hybridization methods. The frequencies at which B-genome chromosomes underwent autosyndesis and allosyndesis were generally higher in ABCC than in AABCC plants. The difference was statistically significant for allosyndesis but not autosyndesis. Abnormal distributions of B-genome chromosomes were encountered at anaphase I, including chromosome lagging and precocious sister centromere separation of univalents. These abnormalities were observed at a significantly higher frequency in AABCC than in ABCC plants, which resulted in more rapid B-genome chromosome elimination in the AABCC derivatives. Yellow or yellow-brown seeds were obtained in all approaches, although true-breeding yellow-seeded B. napus was developed only in approaches 2 and 3. The efficiency of the B. napus construction approaches was in the order 1?>?3?>?2 whereas this order was 3?>?2?>?1 with respect to the construction of yellow-seeded B. napus. The results are discussed in relation to Brassica genome evolution and the development and utilization of the yellow-seeded B. napus obtained here.     

Cloning, sequencing and salt induced expression of PEAMT and BADH in oilseed rape (Brassica napus).

Agriculture productivity is severely hampered by soil salinity, drought and other environmental stresses. Studies on stress-resistant plants (halophytes, xerophytes, accumulating plants for specific toxic ions) have illuminated some mechanisms of stress tolerance in plants at metabolic or molecular levels, which gave some clues on how to genetically engineer stress-tolerant crops. With the isolation of more stress-responsive genes, genetic engineering with modified expression of stress responsive genes may be an effective way to produce stress-tolerant crops. In the present report, two genes (PEAMT and BADH) encoding the corresponding key enzymes for choline and glycine betaine (an important osmoprotectant) biosynthesis in plants were isolated in oilseed rape, an important oil crop in the world. Effects of salt stress on their expression were studied with quantitative PCR and their potential use in the genetic engineering of oilseed rape was discussed.