Map-based cloning and characterization of a gene controlling hairiness and seed coat color traits in <Emphasis Type="Italic">Brassica rapa</Emphasis>

A glabrous, yellow-seeded doubled haploid (DH) line and a hairy, black-seeded DH line in Chinese cabbage (B. rapa) were used as parents to develop a DH line population that segregated for both hairiness and seed coat color traits. The data showed that both traits completely co-segregated each other, suggesting that one Mendelian locus controlled both hairiness and seed coat color in this population. A fine genetic map was constructed and a SNP marker that was located inside a Brassica ortholog of TRANSPARENT TESTA GLABRA 1 (TTG1) in Arabidopsis showed complete linkage to both the hairiness and seed coat color gene, suggesting that the Brassica TTG1 ortholog shared the same gene function as its Arabidopsis counterpart. Further sequence analysis of the alleles from hairless, yellow-seeded and hairy, black-seeded DH lines in B. rapa showed that a 94-base deletion was found in the hairless, yellow-seeded DH lines. A nonfunctional truncated protein in the hairless, yellow-seeded DH lines in B. rapa was suggested by the coding sequence of the TTG1 ortholog. Both of the TTG1 homologs from the black and yellow seeded B. rapa lines were used to transform an Arabidopsis ttg1 mutant and the results showed that the TTG1 homolog from the black seeded B. rapa recovered the Arabidopsis ttg1 mutant, while the yellow seeded homolog did not, suggesting that the deletion in the Brassica TTG1 homolog had led to the yellow seeded natural mutant. This was the first identified gene in Brassica species that simultaneously controlled both hairiness and seed coat color traits.     

A Brassica rapa Linkage Map of EST-based SNP Markers for Identification of Candidate Genes Controlling Flowering Time and Leaf Morphological Traits

For identification of genes responsible for varietal differences in flowering time and leaf morphological traits, we constructed a linkage map of Brassica rapa DNA markers including 170 EST-based markers, 12 SSR markers, and 59 BAC sequence-based markers, of which 151 are single nucleotide polymorphism (SNP) markers. By BLASTN, 223 markers were shown to have homologous regions in Arabidopsis thaliana, and these homologous loci covered nearly the whole genome of A. thaliana. Synteny analysis between B. rapa and A. thaliana revealed 33 large syntenic regions. Three quantitative trait loci (QTLs) for flowering time were detected. BrFLC1 and BrFLC2 were linked to the QTLs for bolting time, budding time, and flowering time. Three SNPs in the promoter, which may be the cause of low expression of BrFLC2 in the early-flowering parental line, were identified. For leaf lobe depth and leaf hairiness, one major QTL corresponding to a syntenic region containing GIBBERELLIN 20 OXIDASE 3 and one major QTL containing BrGL1, respectively, were detected. Analysis of nucleotide sequences and expression of these genes suggested possible involvement of these genes in leaf morphological traits.     

Root hairiness: effect on fluid flow and oxygen transfer in hairy root cultures

The effect of root hairiness on fluid flow and oxygen transfer in hairy root cultures was investigated using wild-type, transgenic and root-hair mutants of Arabidopsis thaliana. The root hair morphologies of the A. thaliana lines were hairless, short hairs, moderately hairy (wild-type) and excessively hairy, and these morphologies were maintained after transformation of seedlings with Agrobacterium rhizogenes. Filtration experiments were used to determine the permeability of packed beds of roots; permeability declined significantly with increasing root hairiness as well as with increasing biomass density. Hairy roots of wild-type A. thaliana grew fastest with a doubling time of 6.9 days, but the hairless roots exhibited the highest specific oxygen uptake rate. In experiments using a gradientless packed bed reactor with medium recirculation, the liquid velocity required to eliminate external mass transfer boundary layer effects increased with increasing root hairiness, reflecting the greater tendency towards liquid stagnation near the surface of roots covered with hairs. External critical oxygen tensions also increased with increasing root hairiness, ranging from 50% air saturation for hairless roots to ca. 150% air saturation for roots with excessive root hairs. These results are consistent with root hairs providing a significant additional resistance to oxygen transfer to the roots, indicating that very hairy roots are more likely than hairless roots to become oxygen-limited in culture. This investigation demonstrates that root hairiness is an important biological parameter affecting the performance of root cultures and suggests that control over root hair formation, either by use of genetically modified plant lines or manipulation of culture conditions, is desirable in large-scale hairy root systems.     

Mapping of genetic locus for leaf trichome in <Emphasis Type="Italic">Brassica oleracea</Emphasis>

Key message

The genetic locus for leaf trichome was identified via marker-based mapping and SNP microarray assay, and a functional marker was developed to facilitate the breeding for hairiness in Brassica oleracea.

Abstract

Plant trichomes are involved in various functions particularly in protecting plants against some biotic and abiotic damages. In the present study, an F2 segregating population was developed from the cross between a glabrous cultivated B. oleracea (CC, 2n = 18) and a hairy wild relative, B. incana (CC, 2n = 18). A 1:3 segregation pattern between glabrous and hairy plants was detected among 1063 F2 genotypes, and the locus for hairiness was mapped in a 4.3-cM genetic region using 267 SSR markers among 149 F2 genotypes, corresponding to a 17.6-Mb genomic region on chromosome C01. To narrow the genetic region for hairiness, the Brassica 60 K SNP Bead Chip Arrays were applied to genotype 64 glabrous and 30 hairy F2 plants, resulting in a 1.04-Mb single peak region located in the 17.6-Mb interval. A candidate gene, BoTRY, was identified by qRT-PCR which revealed significant higher expression in glabrous F2 genotypes as compared with that in hairy plants. A cleaved amplified polymorphic site marker was successfully developed to distinguish the sequence variations of BoTRY between hairy and glabrous plants. Our study will be helpful for molecular breeding for hairiness in B. oleracea.

     

Magnesium and calcium overaccumulate in the leaves of a schengen3 mutant of Brassica rapa

Magnesium (Mg) and calcium (Ca) are essential mineral nutrients poorly supplied in many human food systems. In grazing livestock, Mg and Ca deficiencies are costly welfare issues. Here, we report a Brassica rapa loss-of-function schengen3 (sgn3) mutant, braA.sgn3.a-1, which accumulates twice as much Mg and a third more Ca in its leaves. We mapped braA.sgn3.a to a single recessive locus using a forward ionomic screen of chemically mutagenized lines with subsequent backcrossing and linked-read sequencing of second back-crossed, second filial generation (BC₂F₂) segregants. Confocal imaging revealed a disrupted root endodermal diffusion barrier, consistent with SGN3 encoding a receptor-like kinase required for normal formation of Casparian strips, as reported in thale cress (Arabidopsis thaliana). Analysis of the spatial distribution of elements showed elevated extracellular Mg concentrations in leaves of braA.sgn3.a-1, hypothesized to result from preferential export of excessive Mg from cells to ensure suitable cellular concentrations. This work confirms a conserved role of SGN3 in controlling nutrient homeostasis in B. rapa, and reveals mechanisms by which plants are able to deal with perturbed shoot element concentrations resulting from a “leaky” root endodermal barrier. Characterization of variation in leaf Mg and Ca accumulation across a mutagenized population of B. rapa shows promise for using such populations in breeding programs to increase edible concentrations of essential human and animal nutrients.

“Leaky” root Casparian strips lead to accumulation of two-fold greater magnesium and ∼35% greater calcium concentrations in leaves of a Brassica rapa mutant.     

Characterization of natural variation for zinc, iron and manganese accumulation and zinc exposure response in Brassica rapa L.

Brassica rapa L. is an important vegetable crop in eastern Asia. The objective of this study was to investigate the genetic variation in leaf Zn, Fe and Mn accumulation, Zn toxicity tolerance and Zn efficiency in B. rapa. In total 188 accessions were screened for their Zn-related characteristics in hydroponic culture. In experiment 1, mineral assays on 111 accessions grown under sufficient Zn supply (2 μM ZnSO₄) revealed a variation range of 23.2–155.9 μg g⁻¹ dry weight (d. wt.) for Zn, 60.3–350.1 μg g⁻¹ d. wt. for Fe and 20.9–53.3 μg g⁻¹ d. wt. for the Mn concentration in shoot. The investigation of tolerance to excessive Zn (800 μM ZnSO₄) on 158 accessions, by using visual toxicity symptom parameters (TSPs), identified different levels of tolerance in B. rapa. In experiment 2, a selected sub-set of accessions from experiment 1 was characterized in more detail for their mineral accumulation and tolerance to excessive Zn supply (100 μM and 300 μM ZnSO₄). In this experiment Zn tolerance (ZT) determined by relative root or shoot dry biomass varied about 2-fold. The same six accessions were also examined for Zn efficiency, determined as relative growth under 0 μM ZnSO₄ compared to 2 μM ZnSO₄. Zn efficiency varied 1.8-fold based on shoot dry biomass and 2.6-fold variation based on root dry biomass. Zn accumulation was strongly correlated with Mn and Fe accumulation both under sufficient and deficient Zn supply. In conclusion, there is substantial variation for Zn accumulation, Zn toxicity tolerance and Zn efficiency in Brassica rapa L., which would allow selective breeding for these traits.     

Differential siring success of Pgi genotypes in Clarkia Unguiculata (Onagraceae)

Competition among pollen grains for the fertilization of ovules can play an important role in determining the male and female reproductive success of flowering plants. To examine the influence of pollen-donor genotype on male reproductive success, hand-pollinations were conducted on Clarkia unguiculata and the siring success of pollen-donor plants was compared between donors homozygous for different allelomorphs of the allozyme PGI (phosphoglucoisomerase). Donors homozygous for the B allele sired more seeds than C-allele donors. Single-donor crosses indicated that C-donor-sired seeds are aborted more often than are B-donor-sired seeds, suggesting that the B-allele donor's advantage in mixed pollinations was a result of differential abortion. A negative relationship between pollen load and the siring success of B-allele donors implies that pollen from B-allele donors has reduced performance relative to C-allele donors when pollen loads are high. These data demonstrate consistent differences in siring success between individuals homozygous for different alleles at a single locus and suggest that variation at the Pgi locus may be maintained by a post-pollination trade-off.     

Effect of gametocidal chromosome 4S' on the phenotype segregation ratio in genetic analysis of common wheat lines

Using experimental data on genetic analysis of introgressive lines for the character "hairy leaf sheath" controlled by the "cuckoo" chromosome 4S1, the algorithm for calculation of the theoretical segregation ratio in F2 was developed. Segregation distortion is caused by non-viability of the majority of gametes lacking the chromosome 4S1. The frequency of functioning gametes without the chromosome 4S1 is determined by the probability p versus the theoretically expected ratio 7 nonviable: 9 viable ones. Since segregation involves two characters, gamete viability and hairiness, the ratio 15 hairy: 1 hairless was used as a basis for search of the frequency p by maximum-likelihood method using 16 populations F2 from crossing the lines differing in the character studied.     

Beyond genomic variation - comparison and functional annotation of three Brassica rapa genomes: a turnip,a rapid cycling and a Chinese cabbage

Background

Brassica rapa is an economically important crop species. During its long breeding history, a large number of morphotypes have been generated, including leafy vegetables such as Chinese cabbage and pakchoi, turnip tuber crops and oil crops.

Results

To investigate the genetic variation underlying this morphological variation, we re-sequenced, assembled and annotated the genomes of two B. rapa subspecies, turnip crops (turnip) and a rapid cycling. We then analysed the two resulting genomes together with the Chinese cabbage Chiifu reference genome to obtain an impression of the B. rapa pan-genome. The number of genes with protein-coding changes between the three genotypes was lower than that among different accessions of Arabidopsis thaliana, which can be explained by the smaller effective population size of B. rapa due to its domestication. Based on orthology to a number of non-brassica species, we estimated the date of divergence among the three B. rapa morphotypes at approximately 250,000 YA, far predating Brassica domestication (5,000-10,000 YA).

Conclusions

By analysing genes unique to turnip we found evidence for copy number differences in peroxidases, pointing to a role for the phenylpropanoid biosynthesis pathway in the generation of morphological variation. The estimated date of divergence among three B. rapa morphotypes implies that prior to domestication there was already considerably divergence among B. rapa genotypes. Our study thus provides two new B. rapa reference genomes, delivers a set of computer tools to analyse the resulting pan-genome and uses these to shed light on genetic drivers behind the rich morphological variation found in B. rapa.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-250) contains supplementary material, which is available to authorized users.     

Tryptophan decarboxylase plays an important role in ajmalicine biosynthesis in Rauvolfia verticillata

Tryptophan decarboxylase (TDC) converts tryptophan into tryptamine that is the indole moiety of ajmalicine. The full-length cDNA of Rauvolfia verticillata (RvTDC) was 1,772 bps that contained a 1,500-bp ORF encoding a 499-amino-acid polypeptide. Recombinant 55.5 kDa RvTDC converted tryptophan into tryptamine. The K _m of RvTDC for tryptophan was 2.89 mM, higher than those reported in other TIAs-producing plants. It demonstrated that RvTDC had lower affinity to tryptophan than other plant TDCs. The K _m of RvTDC was also much higher than that of strictosidine synthase and strictosidine glucosidase in Rauvolfia. This suggested that TDC might be the committed-step enzyme involved in ajmalicine biosynthesis in R. verticillata. The expression of RvTDC was slightly upregulated by MeJA; the five MEP pathway genes and SGD showed no positive response to MeJA; and STR was sharply downregulated by MeJA. MeJA-treated hairy roots produced higher level of ajmalicine (0.270 mg g^?1 DW) than the EtOH control (0.183 mg g^?1 DW). Highest RvTDC expression level was detected in hairy root, about respectively 11, 19, 65, and 109-fold higher than in bark, young leaf, old leaf, and root. Highest ajmalicine content was also found in hairy root (0.249 mg g^?1 DW) followed by in bark (0.161 mg g^?1 DW) and young leaf (0.130 mg g^?1 DW), and least in root (0.014 mg g^?1 DW). Generally, the expression level of RvTDC was positively consistent with the accumulation of ajmalicine. Therefore, it could be deduced that TDC might be the key enzyme involved in ajmalicine biosynthesis in Rauvolfia.     

Intake levels of dietary long-chain PUFAs modify the association between genetic variation in FADS and LDL-C

Polymorphisms of the FA desaturase (FADS) gene cluster have been associated with LDL, HDL, and triglyceride concentrations. Because FADS converts α-linolenic acid (ALA) and linoleic acid into PUFAs, we investigated the interaction between different PUFA intakes and the FADS polymorphism rs174547 (T>C) on fasting blood lipid and lipoprotein concentrations. We included 4,635 individuals (60% females, 45–68 years) from the Swedish population-based Malmö Diet and Cancer cohort. Dietary intakes were assessed by a modified diet history method including 7-day registration of cooked meals. The C-allele of rs174547 was associated with lower LDL concentration (P = 0.03). We observed significant interaction between rs174547 and long-chain ω-3 PUFA intakes on LDL (P = 0.01); the C-allele was only associated with lower LDL among individuals in the lowest tertile of long-chain ω-3 PUFA intakes (P < 0.001). In addition, significant interaction was observed between rs174547 and the ratio of ALA and linoleic FA intakes on HDL (P = 0.03). However, no significant associations between the C-allele and HDL were detected within the intake tertiles of the ratio. Our findings suggest that dietary intake levels of different PUFAs modify the associated effect of genetic variation in FADS on LDL and HDL     

Individual variation in thermal performance curves: swimming burst speed and jumping endurance in wild-caught tropical clawed frogs

The importance of studying individual variation in locomotor performance has long been recognized as it may determine the ability of an organism to escape from predators, catch prey or disperse. In ectotherms, locomotor performance is highly influenced by ambient temperature (T _a), yet several studies have showed that individual differences are usually retained across a T _a gradient. Less is known, however, about individual differences in thermal sensitivity of performance, despite the fact that it could represent adaptive sources of phenotypic variation and/or additional substrate for selection to act upon. We quantified swimming and jumping performance in 18 wild-caught tropical clawed frogs (Xenopus tropicalis) across a T _a gradient. Maximum swimming velocity and acceleration were not repeatable and individuals did not differ in how their swimming performance varied across T _a. By contrast, time and distance jumped until exhaustion were repeatable across the T _a gradient, indicating that individuals that perform best at a given T _a also perform best at another T _a. Moreover, thermal sensitivity of jumping endurance significantly differed among individuals, with individuals of high performance at low T _a displaying the highest sensitivity to T _a. Individual differences in terrestrial performance increased with decreasing T _a, which is opposite to results obtained in lizards at the inter-specific and among-individual levels. To verify the generality of these patterns, we need more studies on individual variation in thermal reaction norms for locomotor performance in lizards and frogs.     

Regulation of Respiration in the Leaves and Roots of Two Lolium perenne Populations with Contrasting Mature Leaf Respiration Rates and Crop Yields

Measurements of O₂ uptake were made on leaves and roots of two populations of Lolium perenne L. cv S23 (GL66 and GL72), previously shown to have contrasting rates of CO₂ evolution and yields of dry matter. O₂ uptake was faster in the mature leaves of GL66 than those of GL72, but no difference was observed in the respiratory rates of meristematic leaf bases or mature roots. The growth rate of GL72 was faster than that of GL66. Cyanide resistance was substantial in mature leaves but the alternative path did not contribute to O₂ uptake in the dark. In both populations, adding malate and glycine stimulated O₂ uptake, but exogenous sucrose only stimulated when uncoupler was also present. The difference between the respiratory rates of the two populations was maintained under all investigated conditions. We conclude that the rate of mature leaf respiration in the dark in L. perenne is limited by adenylate control of glycolysis. The difference between the fast (GL66) and slow (GL72) respiring populations reflected a greater respiratory capacity and higher turnover of ATP in GL66. Alternative path capacity was also high in the roots of both and contributed substantially to O₂ uptake, as indicated by inhibition by salicylhydroxamic acid in the absence of KCN. The alternative path capacity of meristematic leaf bases was considerably less than that in mature leaves.

Transverse and cross-sections were made of mature leaves of both populations to study anatomical features which might explain the differences in ATP turnover, suggested by the biochemical experiments. Leaves of GL72 were thicker but did not show a different anatomy when compared with GL66. The increased thickness was not due to more or larger cells but entirely to a larger intercellular volume.

     

The influence of seasonality in estimating mangrove leaf chlorophyll-a content from hyperspectral data

Mangrove photosynthetic activity and, consequently, physiological stress can be monitored indirectly using leaf chlorophyll-a (Chla) measurements. Recent studies have demonstrated the feasibility of mangrove leaf Chla content estimation from in situ hyperspectral vegetation indices (VI) but no such research has been conducted using data collected from contrasting seasons (i.e. dry and rainy). In this study, mangrove leaves were collected in a sub-tropical forest of the Mexican Pacific for Chla content determination and in situ hyperspectral measurements (450–1,000 nm). Specifically, we tested 35 VI to estimate Chla content based on a leaf sample of 360 collected from the same trees during both the dry and rainy seasons. The forest examined contained three species of mangrove (Rhizophora mangle, Avicennia germinans and Laguncularia racemosa) exhibiting various conditions of health (dwarf condition, tall and healthy). A principal component analysis, followed by linear regression analyses, were conducted in order to identify those VI that best predict mangrove leaf Chla content during the two seasons. The results indicate that VI derived from hyperspectral measurements collected during the dry season are better at estimating leaf Chla content than those collected during the rainy season. Among the 35 VI, the Vog1 (R₇₄₀/R₇₂₀) index was found to be the best predictor of mangrove leaf Chla content, resulting in R ² values of 0.80 and 0.68 for the dry and rainy season respectively. These results would suggest that for identifying variation in mangrove forest stress (i.e. health) in sub-tropical regions, hyperspectral measurements should be carried out during the dry season.     

Seasonal variation in ammonia compensation point and nitrogen pools in beech leaves (Fagus sylvatica)

Ammonia (NH₃) fluxes between beech leaves (Fagus sylvatica) and the atmosphere were investigated in a 90-year-old forest canopy and related to leaf nitrogen (N) pools and glutamine synthetase (GS) activities. The stomatal ammonia compensation point, ?? _NH3, was measured by both a twig cuvette and bioassay techniques involving measurements of pH and ammonium (NH ₄ ⁺ ) concentration in the leaf apoplastic solution. The ?? _NH3 determined on the basis of the gas exchange measurements followed a seasonal variation with early-season peaks during leaf expansion (9.6 nmol NH₃ mol^?1 air) and late-season peaks during leaf senescence (7.3 nmol NH₃ mol^?1 air). In the mid-season, the ?? _NH3 of mature green leaves was much lower (around 3 nmol NH₃ mol^?1 air) and dropped below the NH₃ concentration in the ambient atmosphere. For comparison, ?? _NH3 obtained by the apoplastic bioassay were 7.0, 3.7 and 6.4 nmol NH₃ mol^?1 air in early-, mid-, and late -season, thus agreeing reasonably well with ?? _NH3 values derived from the gas exchange measurements. Potential NH₃ emission fluxes during early and late season were 1.31 and 0.51 nmol m^?2 leaf surface area s^?1, respectively, while leaves were a sink for NH₃ during mid-season. During leaf establishment and senescence, both apoplastic and bulk tissue NH ₄ ⁺ concentrations were relatively high coinciding with low activities of glutamine synthetase, which is a key enzyme in leaf N metabolism. In conclusion, the exchange of NH₃ between beech leaves and the atmosphere followed a seasonal variation with NH₃ emission peaks being related to N mobilization during early leaf establishment and remobilization during late leaf senescence.