Combined use of bulked segregant analysis and microarrays reveals SNP markers pinpointing a major QTL for resistance to Phytophthora capsici in pepper

Key message

Bulked segregant analysis (BSA) using Affymetrix GeneChips revealed candidate genes underlying the major QTL for Phytophthora capsici resistance in Capsicum . Using the candidate genes, reliable markers for Phytophthora resistance were developed and validated.

Abstract

Phytophthora capsici L. is one of the most destructive pathogens of pepper (Capsicum spp.). Resistance of pepper against P. capsici is controlled by quantitative trait loci (QTL), including a major QTL on chromosome 5 that is the predominant contributor to resistance. Here, to maximize the effect of this QTL and study its underlying genes, an F₂ population and recombinant inbred lines were inoculated with P. capsici strain JHAI1-7 zoospores at a low concentration (3 × 10³/mL). Resistance phenotype segregation ratios for the populations fit a 3:1 and 1:1 (resistant:susceptible) segregation model, respectively, consistent with a single dominant gene model. Bulked segregant analysis (BSA) using Affymetrix GeneChips revealed a single position polymorphism (SPP) marker mapping to the major QTL. When this SPP marker (Phyto5SAR) together with other SNP markers located on chromosome 5 was used to confirm the position of the major QTL, Phyto5SAR showed the highest LOD value at the QTL. A scaffold sequence (scaffold194) containing Phyto5SAR was identified from the C. annuum genome database. The scaffold contained two putative NBS-LRR genes and one SAR 8.2A gene as candidates for contributing to P. capsici resistance. Markers linked to these genes were developed and validated by testing 100 F₁ commercial cultivars. Among the markers, Phyto5NBS1 showed about 90 % accuracy in predicting resistance phenotypes to a low-virulence P. capsici isolate. These results suggest that Phyto5NBS1 is a reliable marker for P. capsici resistance and can be used for identification of a gene(s) underlying the major QTL on chromosome 5.     

Use of synteny to identify candidate genes underlying QTL controlling stomatal traits in faba bean (Vicia faba L.)

Key message

We have identified QTLs for stomatal characteristics on chromosome II of faba bean by applying SNPs derived from M. truncatula , and have identified candidate genes within these QTLs using synteny between the two species.

Abstract

Faba bean (Vicia faba L.) is a valuable food and feed crop worldwide, but drought often limits its production, and its genome is large and poorly mapped. No information is available on the effects of genomic regions and genes on drought adaptation characters such as stomatal characteristics in this species, but the synteny between the sequenced model legume, Medicago truncatula, and faba bean can be used to identify candidate genes. A mapping population of 211 F₅ recombinant inbred lines (Mélodie/2 × ILB 938/2) were phenotyped to identify quantitative trait loci (QTL) affecting stomatal morphology and function, along with seed weight, under well-watered conditions in a climate-controlled glasshouse in 2013 and 2014. Canopy temperature (CT) was evaluated in 2013 under water-deficit (CTd). In total, 188 polymorphic single nucleotide polymorphisms (SNPs), developed from M. truncatula genome data, were assigned to nine linkage groups that covered ~928 cM of the faba bean genome with an average inter-marker distance of 5.8 cM. 15 putative QTLs were detected, of which eight (affecting stomatal density, length and conductance and CT) co-located on chromosome II, in the vicinity of a possible candidate gene—a receptor-like protein kinase found in the syntenic interval of M. truncatula chromosome IV. A ribose-phosphate pyrophosphokinase from M. truncatula chromosome V, postulated as a possible candidate gene for the QTL for CTd, was found some distance away in the same chromosome. These results demonstrate that genomic information from M. truncatula can successfully be translated to the faba bean genome.     

High-throughput root phenotyping screens identify genetic loci associated with root architectural traits in Brassica napus under contrasting phosphate availabilities

Background and Aims

Phosphate (Pi) deficiency in soils is a major limiting factor for crop growth worldwide. Plant growth under low Pi conditions correlates with root architectural traits and it may therefore be possible to select these traits for crop improvement. The aim of this study was to characterize root architectural traits, and to test quantitative trait loci (QTL) associated with these traits, under low Pi (LP) and high Pi (HP) availability in Brassica napus.

Methods

Root architectural traits were characterized in seedlings of a double haploid (DH) mapping population (n = 190) of B. napus [‘Tapidor’ × ‘Ningyou 7’ (TNDH)] using high-throughput phenotyping methods. Primary root length (PRL), lateral root length (LRL), lateral root number (LRN), lateral root density (LRD) and biomass traits were measured 12 d post-germination in agar at LP and HP.

Key Results

In general, root and biomass traits were highly correlated under LP and HP conditions. ‘Ningyou 7’ had greater LRL, LRN and LRD than ‘Tapidor’, at both LP and HP availability, but smaller PRL. A cluster of highly significant QTL for LRN, LRD and biomass traits at LP availability were identified on chromosome A03; QTL for PRL were identified on chromosomes A07 and C06.

Conclusions

High-throughput phenotyping of Brassica can be used to identify root architectural traits which correlate with shoot biomass. It is feasible that these traits could be used in crop improvement strategies. The identification of QTL linked to root traits under LP and HP conditions provides further insights on the genetic basis of plant tolerance to P deficiency, and these QTL warrant further dissection.     

Quantitative trait loci (QTL) mapping of blush skin and flowering time in a European pear (<Emphasis Type="Italic">Pyrus communis</Emphasis>) progeny of ‘Flamingo’ × ‘Abate Fetel’

Blush skin and flowering time are agronomic traits of interest to the Agricultural Research Council (ARC) Infruitec-Nietvoorbij pear breeding programme. The genetic control of these traits was investigated in the pear progeny derived from ‘Flamingo’ (blush cultivar) × ‘Abate Fetel’ (slightly blush) made up of 121 seedlings. Blush skin was scored phenotypically over three seasons and flowering time was scored over two seasons. A total of 160 loci from 137 simple sequence repeat (SSR) markers were scored in the progeny and used to construct parental genetic linkage maps. Quantitative trait loci (QTL) analysis revealed two QTLs for blush skin, a major QTL on linkage group (LG) 5 in ‘Flamingo’, and a major QTL on LG9 in ‘Abate Fetel’. Two SSR markers, NB101a and SAmsCO865954, were closely linked with the major QTL on LG5 in ‘Flamingo’, with alleles 139 bp and 462 bp in coupling, respectively. These markers were present in approximately 90% of the seedlings scored as good blush (class 4) based on the average data set. These two markers were used to genotype other pear accessions to validate the QTL on LG5 with the view of marker-assisted selection. Two candidate genes, MYB86 and UDP-glucosyl transferase, were associated with the QTL on LG5 and MYB21 and MYB39 were associated with the QTL on LG9. QTL analysis for flowering time revealed a major QTL located on LG9 in both parents. Marker GD142 with allele 161 bp from ‘Flamingo’ was present in approximately 88% of the seedlings that flowered earlier than either parent, based on the average data set. The QTLs and linked markers will facilitate marker-assisted selection for the improvement of these complex traits.     

A comparison study of Agrobacterium-mediated transformation methods for root-specific promoter analysis in soybean

Key message

Both in vitro and in vivo hairy root transformation systems could not replace whole plant transformation for promoter analysis of root-specific and low-P induced genes in soybean.

Abstract

An efficient genetic transformation system is crucial for promoter analysis in plants. Agrobacterium-mediated transformation is the most popular method to produce transgenic hairy roots or plants. In the present study, first, we compared the two different Agrobacterium rhizogenes-mediated hairy root transformation methods using either constitutive CaMV35S or the promoters of root-preferential genes, GmEXPB2 and GmPAP21, in soybean, and found the efficiency of in vitro hairy root transformation was significantly higher than that of in vivo transformation. We compared Agrobacterium rhizogenes-mediated hairy root and Agrobacterium tumefaciens-mediated whole plant transformation systems. The results showed that low-phosphorous (P) inducible GmEXPB2 and GmPAP21 promoters could not induce the increased expression of the GUS reporter gene under low P stress in both in vivo and in vitro transgenic hairy roots. Conversely, GUS activity of GmPAP21 promoter was significantly higher at low P than high P in whole plant transformation. Therefore, both in vitro and in vivo hairy root transformation systems could not replace whole plant transformation for promoter analysis of root-specific and low-P induced genes in soybean.     

Construction of a dense SNP map of a highly heterozygous diploid potato population and QTL analysis of tuber shape and eye depth

Key message

Generation of a dense SNP-based linkage map of a diploid potato population and identification of major QTLs for tuber shape and eye depth on chromosomes 2 and 10.

Abstract

This paper reports the construction of a genetic map of a highly heterozygous full-sib diploid potato population (06H1) based on the use of a set of 8,303 single nucleotide polymorphism (SNP) markers. The map contains 1,355 distinct loci and 2,157 SNPs, 802 of which co-segregate with other markers. We find high levels of collinearity between the 12 chromosomal maps with a recently improved version of the potato genome assembly, with the expected genetic clustering in centromeric regions. The linkage maps are used in combination with highly detailed phenotypic assessments conducted over two growing seasons to perform quantitative trait loci analysis of two important potato traits, tuber shape and eye depth. The major loci segregating for tuber shape in 06H1 map to loci on chromosomes 2 and 10, with smaller effects mapping to three other chromosomes. A major locus for tuber eye depth co-locates with the tuber shape locus on chromosome 10. To assess when tuber shape is established in the developing tuber, we have performed staged observations of tuber formation. Our observations suggest that tuber shape is determined very early in tuber development.     

Development of a genetic linkage map for Pinus radiata and detection of pitch canker disease resistance associated QTLs

Key message

The heritability of genetic resistance of radiata pine against Fusarium circinatum was not clear. We demonstrated that there are at least 3 QTLs that could be involved in this resistance/susceptibility.

Abstract

A genetic linkage map was developed for Pinus radiata, using Amplified Fragment Length Polymorphism (AFLP), Inter-Simple Sequence Repeat (ISSR), Selective Amplification of Microsatellite Polymorphic Loci (SAMPL), and Simple Sequence Repeat (SSR) molecular markers, based on a two-way pseudo-testcross strategy, using 86 individuals of a F1 full-sib family and 787 molecular markers for genotyping. Linkage analysis generated a map of medium to high density for each parent, with 1,060 and 1,258 cM for parents XO and XP, respectively. A total of 458 markers were mapped on 12 linkage groups (LG) in XO and XP, which equals the number of haploid chromosomes present in P. radiata. Analysis of quantitative trait loci (QTL) for resistance against pitch canker disease caused by Fusarium circinatum was made using Bayesian Information Criterion (BIC). In the XO parental map, two groups (LG-1 and LG-9) showed high probabilities for one or more QTLs. Only one group (LG-9) in the XP parental map showed probability for one or more QTLs. The results indicate that resistance to pitch canker is inherited from both parents. These results provide the basis for further studies focused on structure, evolution, and function of the P. radiata genome.     

Evaluation of root reinforcement models using numerical modelling approaches

Background and aims

The root reinforcement (RR) models commonly used in slope stability modelling can be simply explained as a single soil additional cohesion parameter estimated with simple analytical functions of root traits. We have simulated 3D direct shear tests using the standard implicit Finite Element Method (FEM) and the Discrete Element Method (DEM), aiming to (i) evaluate the RR models and (ii) compare the two numerical approaches.

Methods

In homogeneous soil with low cohesion, 36 straight, non-branched and thin root models were implanted in three parallel lines. Root traits, including orientation relative to the shear direction (45°, 90° and ?45°), longitudinal modulus of elasticity (10 MPa and 100 MPa), and bending and compressive behaviours (beam, truss and cable) were investigated.

Results

Compared to the FEM, the DEM achieved consistent results and avoided convergence problems, but required longer computation time and used parameters potentially difficult to identify. Root reinforcement did not occur until significant plastic deformation of soil. The RR values estimated by the shear tests were much lower than those estimated by the usual RR models and were significantly dependent upon root traits.

Conclusions

Ignoring the effect of root traits in RR models might lead to an important bias when using slope stability models.     

Use of single nucleotide polymorphisms and haplotypes to identify genomic regions associated with protein content and water-soluble protein content in soybean

Key message

Four major SPC-specific loci were identified, and these accounted for 8.5–15.1 % of the phenotypic variation, thus explaining why certain soybean varieties have a high PC but a low SPC.

Abstract

Water-soluble protein content (SPC) is a critical factor in both food quality and the production of isolated soybean proteins. However, few data are available regarding the genetic control and the mechanisms contributing to elevated SPC. In this study, a soybean collection of 192 accessions from a wide geographic range was used to identify genomic regions associated with soybean protein content (PC) and SPC using an association mapping approach employing 1,536 SNP makers and 232 haplotypes. The diverse panel revealed a large genetic variation in PC and SPC. Association mapping was performed using three methods to minimize false-positive associations. This resulted in 4/8 SNPs and 3/6 haplotypes that were significantly associated with soybean PC/SPC in two or more environments based on the mixed model. An SNP that was highly significantly associated with PC, BARC-021267-04016, was localized 0.28 cM away from a published glycinin gene, G7, and was detected across all four environments. Four major SPC-specific loci, BARC-029149-06088, BARC-018023-02499, BARC-041663-08059 and haplotype 15 (hp15), were stably identified on chromosomes five and eight and explained 8.5–15.1 % of the phenotypic variation. Moreover, a glutelin type-B 2-like gene was identified on chromosome eight and may be related to soybean protein solubility. These markers, which are located in previously reported QTL, reconfirmed previous findings and may be important targets for the identification of protein-related genes. These novel SNPs and haplotypes are important for further understanding the genetic basis of PC and SPC. In addition, by comparing the correlation and genetic loci between PC and SPC, we provide new insights into why certain soybean varieties have a high protein content but a low SPC.     

A high-resolution linkage map of the Rfd1, a restorer-of-fertility locus for cytoplasmic male sterility in radish (Raphanus sativus L.) produced by a combination of bulked segregant analysis and RNA-Seq

Key message

We utilized a combination of BSA and RNA-Seq to identify SNPs linked to the Rfd1 locus, a restorer-of-fertility gene in radish. A high-density linkage map was constructed using this approach.

Abstract

Male fertility of cytoplasmic male sterility conditioned by the Dongbu cytoplasmic and genic male-sterility cytoplasm can be restored by a restorer-of-fertility locus, Rfd1, in radish. To construct a high-density linkage map and to identify a candidate gene for the Rfd1 locus, bulked segregant analysis and RNA-seq approaches were combined. A total of 26 and 28 million reads produced from male-fertile and male-sterile bulked RNA were mapped to the radish reference unigenes. After stringent screening of SNPs, 327 reliable SNPs of 109 unigenes were selected. Arabidopsis homologs for 101 of the 109 genes were clustered around the 4,000 kb region of Arabidopsis chromosome 3, which was syntenic to the Rfd1 flanking region. Since the reference unigene set was incomplete, the contigs were de novo assembled to identify 134 contigs harboring SNPs. Most of SNP-containing contigs were also clustered on the same syntenic region in Arabidopsis chromosome. A total of 21 molecular markers positioned within a 2.1 cM interval including the Rfd1 locus were developed, based on the selected unigenes and contigs. A segregating population consisting of 10,459 individuals was analyzed to identify recombinants containing crossovers within this interval. A total of 284 identified recombinants were then used to construct a high-density map, which delimited the Rfd1 locus into an 83-kb syntenic interval of Arabidopsis chromosome 3. Since no candidate gene, such as a pentatricopeptide repeat (PPR)-coding gene, was found in this interval, 231 unigenes and 491 contigs containing putative PPR motifs were analyzed further, but no PPR gene in linkage disequilibrium with the Rfd1 locus could be found.     

Genome-wide QTL and bulked transcriptomic analysis reveals new candidate genes for the control of tuber carotenoid content in potato (Solanum tuberosum L.)

Key message

Genome-wide QTL analysis of potato tuber carotenoid content was investigated in populations of Solanum tuberosum Group Phureja that segregate for flesh colour, revealing a novel major QTL on chromosome 9.

Abstract

The carotenoid content of edible plant storage organs is a key nutritional and quality trait. Although the structural genes that encode the biosynthetic enzymes are well characterised, much less is known about the factors that determine overall storage organ content. In this study, genome-wide QTL mapping, in concert with an efficient ‘genetical genomics’ analysis using bulked samples, has been employed to investigate the genetic architecture of potato tuber carotenoid content. Two diploid populations of Solanum tuberosum Group Phureja were genotyped (AFLP, SSR and DArT markers) and analysed for their tuber carotenoid content over two growing seasons. Common to both populations were QTL that explained relatively small proportions of the variation in constituent carotenoids and a major QTL on chromosome 3 explaining up to 71 % of the variation in carotenoid content. In one of the populations (01H15), a second major carotenoid QTL was identified on chromosome 9, explaining up to 20 % of the phenotypic variation. Whereas the major chromosome 3 QTL was likely to be due to an allele of a gene encoding β-carotene hydroxylase, no known carotenoid biosynthetic genes are located in the vicinity of the chromosome 9 QTL. A unique expression profiling strategy using phenotypically distinct bulks comprised individuals with similar carotenoid content provided further support for the QTL mapping to chromosome 9. This study shows the potential of using the potato genome sequence to link genetic maps to data arising from eQTL approaches to enhance the discovery of candidate genes underlying QTLs.     

Development of an STS map of an interspecific progeny of <Emphasis Type="Italic">Malus</Emphasis>

Simple sequence repeat (SSR) markers developed from Malus, as well as Prunus, Pyrus and Sorbus, and some other sequence-tagged site (STS) loci were analysed in an interspecific F₁ apple progeny from the cross ‘Fiesta’ × ‘Totem’ that segregated for several agronomic characters. A linkage map was constructed using 259 STS loci (247 SSRs, four SCARs and eight known-function genes) and five genes for agronomic traits—scab resistance (Vf), mildew resistance (Pl-2), columnar growth habit (Co), red tissues (Rt) and green flesh background colour (Gfc). Ninety SSR loci and three genes (ETR1, Rt and Gfc) were mapped for the first time in apple. The transferability of markers from other Maloideae to Malus was found to be around 44%. The loci are spread across 17 linkage groups, corresponding to the basic chromosome number of Malus and cover 1,208 cM, approximately 85% of the estimated length of the apple genome. Interestingly, we have extended the top of LG15 with eight markers covering 25 cM. The average map density is 4.7 cM per marker; however, marker density varies greatly between linkage groups, from 2.5 in LG14 to 8.9 in LG7, with some areas of the genome still in need of further STS markers for saturation.     

Chromosome numbers in selectedHieracium species in the Krkonoše Mts. (the West Sudeten)

Chromosome numbers are given of 15 species of the genusHieracium L. s. str., representing seven species groups (in the sense of Flora Europaea, roughly corresponding to Zahn's “species principales”) from the Krkono?e Mts., N. Bohemia and SW Poland. For the first time chromosome numbers are reported forH. melanocephalum Tausch (2n=27),H. tubulosum Tausch (2n=36),H. schustleri Zlatník (2n=36),H. fritzei F. Schultz (2n=27),H. rohlenae Zlatník (2n=27),H. nigrescens Willd. (2n=36),H. decipiens Tausch (2n=36),H. atrellum Juxip inSchischkin etBobrov (2n=27),H. subnigrescens (Fries exNorrlin)Dahlst. (2n=36),H. sudeticum Sternberg (2n=36),H. pedunculare Tausch (2n=36),H. glandulosodentatum Uechtr. (2n=36),H. wimmeri Uechtr. (2n=27). InHieracium alpinum L. s. str. the number 2n=27 has been confirmed. The results show a high proportion of tetraploid taxa; no diploids have been found.     

Comparative QTL analysis of root lesion nematode resistance in barley

Key message

This study demonstrates for the first time that resistance to different root lesion nematodes ( P. neglectus and P. penetrans ) is controlled by a common QTL. A major resistance QTL ( Rlnnp6H ) has been mapped to chromosome 6H using two independent barley populations.

Abstract

Root lesion nematodes (Pratylenchus spp.) are important pests in cereal production worldwide. We selected two doubled haploid populations of barley (Igri × Franka and Uschi × HHOR 3073) and infected them with Pratylenchus penetrans and Pratylenchus neglectus. Nematode multiplication rates were measured 7 or 10 weeks after infection. In both populations, continuous phenotypic variations for nematode multiplication rates were detected indicating a quantitative inheritance of resistance. In the Igri × Franka population, four P. penetrans resistance QTLs were mapped with 857 molecular markers on four linkage groups (2H, 5H, 6H and 7H). In the Uschi × HHOR 3073 population, eleven resistance QTLs (P. penetrans and P. neglectus) were mapped with 646 molecular markers on linkage groups 1H, 3H, 4H, 5H, 6H and 7H. A major resistance QTL named Rlnnp6H (LOD score 6.42–11.19) with a large phenotypic effect (27.5–36.6 %) for both pests was mapped in both populations to chromosome 6H. Another resistance QTL for both pests was mapped on linkage group 5H (Igri × Franka population). These data provide first evidence for common resistance mechanisms against different root lesion nematode species. The molecular markers are a powerful tool for the selection of resistant barley lines among segregating populations because resistance tests are time consuming and laborious.     

Ultrastructural changes associated with the induction of premature chromosome condensation in Vicia faba root meristem cells

Key message

PCC induction is regulated by several signaling pathways, and all observed effects associated with PCC induction are strongly dependent on the mechanism of action of each PCC inducer used.

Abstract

Electron microscopic observations of cells with symptoms of premature chromosome condensation (PCC) showed that the interphase chromatin and mitotic chromosomes differed with respect to a chemical compound inducing PCC. Induction of this process under the influence of hydroxyurea and caffeine as well as hydroxyurea and sodium metavanadate led to a slight decrease in interphase chromatin condensation and the formation of chromosomes with a considerably loosened structure in comparison with the control. Incubation in the mixture of hydroxyurea and 2-aminopurine brought about clear chromatin dispersion in interphase and very strong mitotic chromosome condensation. Electron microscopic examinations also revealed the characteristic features of the structural organization of cytoplasm of Vicia faba root meristems, which seemed to be dependent on the type of the PCC inducer used. The presence of the following was observed: (i) large plastids filled with starch grains (caffeine), (ii) mitochondria and plastids of electron dense matrix with dilated invaginations of their internal membranes (2-aminopurine), and (iii) large mitochondria of electron clear matrix and plastids containing protein crystals in their interior (sodium metavanadate). Moreover, since caffeine causes either the most effective loosening of chromatin fibrils (within the prematurely condensed chromosomes) or induction of starch formation (in the plastids surrounding the nuclei), this may be a proof that demonstrates the existence of a link between physical accessibility to chromatin and the effectiveness of cellular signaling (e.g., phosphothreonine-connected).     

Influence of agricultural practices and seasons on the abundance and community structure of culturable pseudomonads in soils under no-till management in Argentina

Background and aims

Members of the genus Pseudomonas are common inhabitants of rhizospheres and soils, and it is known that soil types and crop species influence their population density and structure. 20?×?10⁶ ha are cultivated under no-tillage in Argentina and there is a need to find new biologically-based soil quality indexes to distinguish between sustainable and non-sustainable agricultural practices. Pseudomonads abundance and community structure were analyzed in no-till soils with different agricultural practices, in productive fields along 400 km of Argentinean Pampas.

Methods

We sampled soils and root systems from agricultural plots in which sustainable or non-sustainable agricultural practices have been applied. Samples were collected in summer and winter during 2010 and 2011. Culturable fluorescent and total pseudomonads were enumerated by plating on Gould’s selective medium S1. Colonies from these plates served as DNA source to carry out PCR-RFLP community structure analysis of the pseudomonads-specific marker genes oprF and gacA.

Results

Abundance of total and fluorescent culturable pseudomonads in bulk soils was influenced by seasonal changes and agricultural practices. Rhizospheric counts from the same crop were affected by agricultural treatments. Also, crop species influenced pseudomonads density in the rhizosphere. Combined PCR-RFLP profile of both genes showed a seasonal grouping of samples.

Conclusions

Sustainable soil management seems to promote pseudomonads development in soils, favoring root colonization of crops from those plots. Crop species influence total pseudomonads load of rhizospheres and its community structure. Total or relative pseudomonads load could function as soil quality indicator of good agricultural practices.