Candidate gene association mapping of Sclerotinia stalk rot resistance in sunflower (Helianthus annuus L.) uncovers the importance of COI1 homologs

Key message

Functional markers for Sclerotinia basal stalk rot resistance in sunflower were obtained using gene-level information from the model species Arabidopsis thaliana.

Abstract

Sclerotinia stalk rot, caused by Sclerotinia sclerotiorum, is one of the most destructive diseases of sunflower (Helianthus annuus L.) worldwide. Markers for genes controlling resistance to S. sclerotiorum will enable efficient marker-assisted selection (MAS). We sequenced eight candidate genes homologous to Arabidopsis thaliana defense genes known to be associated with Sclerotinia disease resistance in a sunflower association mapping population evaluated for Sclerotinia stalk rot resistance. The total candidate gene sequence regions covered a concatenated length of 3,791 bp per individual. A total of 187 polymorphic sites were detected for all candidate gene sequences, 149 of which were single nucleotide polymorphisms (SNPs) and 38 were insertions/deletions. Eight SNPs in the coding regions led to changes in amino acid codons. Linkage disequilibrium decay throughout the candidate gene regions declined on average to an r ² = 0.2 for genetic intervals of 120 bp, but extended up to 350 bp with r ² = 0.1. A general linear model with modification to account for population structure was found the best fitting model for this population and was used for association mapping. Both HaCOI1-1 and HaCOI1-2 were found to be strongly associated with Sclerotinia stalk rot resistance and explained 7.4 % of phenotypic variation in this population. These SNP markers associated with Sclerotinia stalk rot resistance can potentially be applied to the selection of favorable genotypes, which will significantly improve the efficiency of MAS during the development of stalk rot resistant cultivars.     

Multiple sclerosis and pregnancy: what does the patient think? a questionnaire study

Background

The first objective of a DNA microarray experiment is typically to generate a list of genes or probes that are found to be differentially expressed or represented (in the case of comparative genomic hybridizations and/or copy number variation) between two conditions or strains. Rank Products analysis comprises a robust algorithm for deriving such lists from microarray experiments that comprise small numbers of replicates, for example, less than the number required for the commonly used t-test. Currently, users wishing to apply Rank Products analysis to their own microarray data sets have been restricted to the use of command line-based software which can limit its usage within the biological community.

Findings

Here we have developed a web interface to existing Rank Products analysis tools allowing users to quickly process their data in an intuitive and step-wise manner to obtain the respective Rank Product or Rank Sum, probability of false prediction and p-values in a downloadable file.

Conclusions

The online interactive Rank Products analysis tool RankProdIt, for analysis of any data set containing measurements for multiple replicated conditions, is available at: http://strep-microarray.sbs.surrey.ac.uk/RankProducts     

Association mapping, patterns of linkage disequilibrium and selection in the vicinity of the PHYTOCHROME C gene in pearl millet

Key message

Linkage analysis confirmed the association in the region of PHYC in pearl millet. The comparison of genes found in this region suggests that PHYC is the best candidate.

Abstract

Major efforts are currently underway to dissect the phenotype–genotype relationship in plants and animals using existing populations. This method exploits historical recombinations accumulated in these populations. However, linkage disequilibrium sometimes extends over a relatively long distance, particularly in genomic regions containing polymorphisms that have been targets for selection. In this case, many genes in the region could be statistically associated with the trait shaped by the selected polymorphism. Statistical analyses could help in identifying the best candidate genes into such a region where an association is found. In a previous study, we proposed that a fragment of the PHYTOCHROME C gene (PHYC) is associated with flowering time and morphological variations in pearl millet. In the present study, we first performed linkage analyses using three pearl millet F₂ families to confirm the presence of a QTL in the vicinity of PHYC. We then analyzed a wider genomic region of ~100 kb around PHYC to pinpoint the gene that best explains the association with the trait in this region. A panel of 90 pearl millet inbred lines was used to assess the association. We used a Markov chain Monte Carlo approach to compare 75 markers distributed along this 100-kb region. We found the best candidate markers on the PHYC gene. Signatures of selection in this region were assessed in an independent data set and pointed to the same gene. These results foster confidence in the likely role of PHYC in phenotypic variation and encourage the development of functional studies.     

Mapping and identification of a Cicer arietinum NSP2 gene involved in nodulation pathway

Key message

For the first time the putative NSP2 gene in chickpea has been identified using pairs of NILs differing for the Rn1 / rn1 nodulation gene that was located in LG5 of chickpea genetic map.

Abstract

An intraspecific cross between the mutant non-nodulating genotype PM233, carrying the recessive gene rn1, and the wild-type CA2139 was used to develop two pairs of near-isogenic lines (NILs) for nodulation in chickpea. These pairs of NILs were characterized using sequence tagged microsatellite site (STMS) markers distributed across different linkage groups (LGs) of the chickpea genetic map leading to the detection of polymorphic markers located in LG5. Using this information, together with the genome annotation in Medicago truncatula, a candidate gene (NSP2) known to be involved in nodulation pathway was selected for mapping in chickpea. The full length sequence obtained in chickpea wild-type (CaNSP2) was 1,503 bp. Linkage analysis in an F₃ population of 118 plants derived from the cross between the pair of NILS NIL7-2A (nod) × NIL7-2B (non-nod) revealed a co-localization between CaNSP2 and Rn1 gene. These data implicate the CaNSP2 gene as a candidate for identity to Rn1, and suggest that it could act in the nodulation signaling transduction pathway similarly to that in other legumes species.     

Jasmonate-dependent plant defense restricts thrips performance and preference

Background

Worldwide, diseases are important reducers of peanut (Arachis hypogaea) yield. Sources of resistance against many diseases are available in cultivated peanut genotypes, although often not in farmer preferred varieties. Wild species generally harbor greater levels of resistance and even apparent immunity, although the linkage of agronomically un-adapted wild alleles with wild disease resistance genes is inevitable. Marker-assisted selection has the potential to facilitate the combination of both cultivated and wild resistance loci with agronomically adapted alleles. However, in peanut there is an almost complete lack of knowledge of the regions of the Arachis genome that control disease resistance.

Results

In this work we identified candidate genome regions that control disease resistance. For this we placed candidate disease resistance genes and QTLs against late leaf spot disease on the genetic map of the A-genome of Arachis, which is based on microsatellite markers and legume anchor markers. These marker types are transferable within the genus Arachis and to other legumes respectively, enabling this map to be aligned to other Arachis maps and to maps of other legume crops including those with sequenced genomes. In total, 34 sequence-confirmed candidate disease resistance genes and five QTLs were mapped.

Conclusion

Candidate genes and QTLs were distributed on all linkage groups except for the smallest, but the distribution was not even. Groupings of candidate genes and QTLs for late leaf spot resistance were apparent on the upper region of linkage group 4 and the lower region of linkage group 2, indicating that these regions are likely to control disease resistance.     

High-resolution mapping of zym, a recessive gene for Zucchini yellow mosaic virus resistance in cucumber

Key message

Using a high-resolution mapping approach, we identified a candidate gene for ZYMV resistance in cucumber. Our findings should assist the development of high-versatility molecular markers for MAS for ZYMV resistance.

Abstract

Zucchini yellow mosaic virus (ZYMV) causes significant disease, which leads to fruit yield loss in cucurbit crops. Since ZYMV resistance is often inherited recessively in cucumber, marker-assisted selection (MAS) is a useful tool for the development of resistant cucumber cultivars. Using 128 families of an F_2:3 population derived from a cross between susceptible ‘CS-PMR1’ and resistant ‘A192-18’ cucumber inbred lines, we confirmed that ZYMV resistance is conferred by a single recessive locus: zym ^A192-18 . We constructed a cucumber genetic linkage map that included 125 simple sequence repeat (SSR) markers segregating into 7 linkage groups (chromosomes). The zym ^A192-18 locus was mapped to chromosome 6, at genetic distances of 0.9 and 1.3 cM from two closely linked SSR markers. For high-resolution genetic mapping, we identified new molecular markers cosegregating with the zym ^A192-18 locus; using cucumber genomic and molecular marker resources and screening an F₂ population of 2,429 plants, we narrowed down the zym ^A192-18 locus to a <50-kb genomic region flanked by two SSR markers, which included six candidate genes. Sequence analysis of the candidate genes’ coding regions revealed that the vacuolar protein sorting-associated protein 4-like (VPS4-like) gene had two SNPs between the parental lines. Based on SNPs of the VPS-4-like gene, we developed zym ^A192-18 -linked DNA markers and found that genotypes associated with these markers were correlated with the ZYMV resistance phenotype in 48 cucumber inbred lines. According to our data, the gene encoding VPS4-like protein is a candidate for the zym ^A192-18 locus. These results may be valuable for MAS for ZYMV resistance in cucumber.     

Fine mapping and candidate gene analysis of the novel thermo-sensitive genic male sterility tms9-1 gene in rice

Key message

Fine mapping of the novel thermo-sensitive genic male sterility locus tms9 - 1 in the traditional TGMS line HengnongS-1 revealed that the MALE STERILITY1 homolog OsMS1 is the candidate gene.

Abstract

Photoperiod-thermo-sensitive genic male sterility (P/TGMS) has been widely used in the two-line hybrid rice breeding system. HengnongS-1 is one of the oldest TGMS lines and is often used in indica two-line breeding programs in China. In this study, our genetic analysis showed that the TGMS gene in HengnongS-1 was controlled by a single recessive gene that was non-allelic with the other TGMS loci identified, including C815S, Zhu1S and Y58S. Using SSR markers and bulked segregant analysis, we located the TGMS locus on chromosome 9 and named the gene tms9-1. Fine mapping further narrowed the tms9-1 loci to a 162 kb interval between two dCAPS markers. Sequence analysis revealed that a T to C substitution results in an amino acid change in the tms9-1 candidate gene (Os09g27620) in HengnongS-1 as compared to Minghui63. Sequencing of other rice accessions, including six P/TGMS lines, seven indica varieties and nine japonica varieties, showed that this SNP was exclusive to HengnongS-1. With multiple sequence alignment and expression pattern analyses, the rice MALE STERILITY1 homolog OsMS1 gene was identified as the candidate gene for tms9-1. Therefore, our study identified a novel TGMS locus and will facilitate the functional identification of the tms9-1 gene. Moreover, the markers linked to the tms9-1 gene will provide useful tools for the development of new TGMS lines by marker-assisted selection in two-line hybrid rice breeding programs.     

MulSatDB: a first online database for mulberry microsatellites

Key message

Simple sequence repeat motifs were mined from the genome and EST sequences of Morus notabilis and archived in MulSatDB. Bioinformatics tools were integrated with the database for the analysis of genomic datasets.

Abstract

Mulberry is a crop of economic importance in sericulture, which shapes the lives of millions of rural people among different Eurasian and Latin American countries. Limited availability of genomic resources has constrained the molecular breeding efforts in mulberry, a poorly studied crop. Microsatellite or simple sequence repeat (SSR) has revolutionized the plant breeding and is used in linkage mapping, association studies, diversity, and parentage analysis, etc. Recent availability of mulberry whole genome assembly provided an opportunity for the development of mulberry-specific DNA markers. In this study, we mined a total of 217,312 microsatellites from whole genome and 961 microsatellites from EST sequences of Morus notabilis. Mono-repeats were predominant among both whole genome and EST sequences. The SSR containing EST sequences were functionally annotated, and SSRs mined from whole genome were mapped on chromosomes of the phylogenetically related genus—Fragaria vesca, to aid the selection of markers based on the function and location. All the mined markers were archived in the mulberry microsatellite database (MulSatDB), and the markers can be retrieved based on different criteria like marker location, repeat kind, motif type and size. Primer3plus and CMap tools are integrated with the database to design primers for PCR amplification and to visualize markers on F. vesca chromosomes, respectively. A blast tool is also integrated to collate new markers with the database. MulSatDB is the first and complete destination for mulberry researchers to browse SSR markers, design primers, and locate markers on strawberry chromosomes. MulSatDB is freely accessible at http://btismysore.in/mulsatdb.     

Regulation of Arabidopsis defense responses against Spodoptera littoralis by CPK-mediated calcium signaling

Background

Genetic markers and linkage mapping are basic prerequisites for marker-assisted selection and map-based cloning. In the case of the key grassland species Lolium spp., numerous mapping populations have been developed and characterised for various traits. Although some genetic linkage maps of these populations have been aligned with each other using publicly available DNA markers, the number of common markers among genetic maps is still low, limiting the ability to compare candidate gene and QTL locations across germplasm.

Results

A set of 204 expressed sequence tag (EST)-derived simple sequence repeat (SSR) markers has been assigned to map positions using eight different ryegrass mapping populations. Marker properties of a subset of 64 EST-SSRs were assessed in six to eight individuals of each mapping population and revealed 83% of the markers to be polymorphic in at least one population and an average number of alleles of 4.88. EST-SSR markers polymorphic in multiple populations served as anchor markers and allowed the construction of the first comprehensive consensus map for ryegrass. The integrated map was complemented with 97 SSRs from previously published linkage maps and finally contained 284 EST-derived and genomic SSR markers. The total map length was 742 centiMorgan (cM), ranging for individual chromosomes from 70 cM of linkage group (LG) 6 to 171 cM of LG 2.

Conclusions

The consensus linkage map for ryegrass based on eight mapping populations and constructed using a large set of publicly available Lolium EST-SSRs mapped for the first time together with previously mapped SSR markers will allow for consolidating existing mapping and QTL information in ryegrass. Map and markers presented here will prove to be an asset in the development for both molecular breeding of ryegrass as well as comparative genetics and genomics within grass species.     

Minor differences in haplotype frequency estimates can produce very large differences in heterogeneity test statistics

Background

Genome wide association (GWA) studies provide the opportunity to develop new kinds of analysis. Analysing pairs of markers from separate regions might lead to the detection of allelic association which might indicate an interaction between nearby genes.

Methods

396,591 markers typed in 541 subjects were studied. 7.8*10¹⁰ pairs of markers were screened and those showing initial evidence for allelic association were subjected to more thorough investigation along with 10 flanking markers on either side.

Results

No evidence was detected for interaction. However 6 markers appeared to have an incorrect map position according to NCBI Build 35. One of these was corrected in Build 36 and 2 were dropped. The remaining 3 were left with map positions inconsistent with their allelic association relationships.

Discussion

Although no interaction effects were detected the method was successful in identifying markers with probably incorrect map positions.

Conclusion

The study of allelic association can supplement other methods for assigning markers to particular map positions. Analyses of this type may usefully be applied to data from future GWA studies.     

Identification of quantitative trait loci and candidate genes for specific cellular resistance responses against Didymella pinodes in pea

Key message

Phenotyping of specific cellular resistance responses and improvement of previous genetic map allowed the identification of novel genomic regions controlling cellular mechanisms involved in pea resistance to ascochyta blight and provided candidate genes suitable for MAS.

Abstract

Didymella pinodes, causing ascochyta blight, is a major pathogen of the pea crop and is responsible for serious damage and yield losses. Resistance is inherited polygenically and several quantitative trait loci (QTLs) have been already identified. However, the position of these QTLs should be further refined to identify molecular markers more closely linked to the resistance genes. In previous works, resistance was scored visually estimating the final disease symptoms; in this study, we have conducted a more precise phenotyping of resistance evaluating specific cellular resistance responses at the histological level to perform a more accurate QTL analysis. In addition, P665 × Messire genetic map used to identify the QTLs was improved by adding 117 SNP markers located in genes. This combined approach has allowed the identification, for the first time, of genomic regions controlling cellular mechanisms directly involved in pea resistance to ascochyta blight. Furthermore, the inclusion of the gene-based SNP markers has allowed the identification of candidate genes co-located with QTLs and has provided robust markers for marker-assisted selection.     

Distinct gene expression profiles of human type 1 and type 2 T helper cells

Background

The development and activation of CD4⁺ helper T cell (Th) subsets with distinct patterns of unbalanced production of cytokines play an important part in infectious, allergic and autoimmune diseases. Human neonatal cord blood CD4⁺ Th cells can be polarized into type 1 or type 2-like effector cells in vitro by culturing them in the presence of interleukin (IL)-12 or IL-4, respectively. We have exploited this experimental system to identify marker genes that are differentially expressed by polarized Th1 and Th2 cells. An oligonucleotide microarray specifically designed to screen for inflammation-related candidate genes was used and the differential expression was further validated with a quantitative real-time RT-PCR method.

Results

In addition to the previously described marker genes of Th cells, we report subtle changes in the expression of several other genes that represent growth factors, receptors and other signaling molecules in polarized Th1 and Th2 cell subsets. Additionally, we describe a novel set of genes as Th1/Th2 differentiation markers for cells activated by anti-CD3 and anti-CD28 antibodies.

Conclusions

This study demonstrates the power of the targeted use of microarrays in combination with quantitative real-time RT-PCR in identifying and validating new marker genes for gene expression studies.     

Autosomal recessive cerebellar ataxia caused by mutations in the PEX2 gene

Objective

To expand the spectrum of genetic causes of autosomal recessive cerebellar ataxia (ARCA).

Case report

Two brothers are described who developed progressive cerebellar ataxia at 3 1/2 and 18 years, respectively. After ruling out known common genetic causes of ARCA, analysis of blood peroxisomal markers strongly suggested a peroxisomal biogenesis disorder. Sequencing of candidate PEX genes revealed a homozygous c.865_866insA mutation in the PEX2 gene leading to a frameshift 17 codons upstream of the stop codon. PEX gene mutations usually result in a severe neurological phenotype (Zellweger spectrum disorders).

Conclusions

Genetic screening of PEX2 and other PEX genes involved in peroxisomal biogenesis is warranted in children and adults with ARCA.     

Genetic diversity and genetic relationships of Chukrasia spp. (Meliaceae) as revealed by inter simple sequence repeat (ISSR) markers

Key message

ISSR characterization of Chukrasia populations from the natural range revealed two distinct groups of populations consonant with morphological differentiation. Results suggest the current taxonomic classification of the genus should be reviewed.

Abstract

There are different views as to whether the genus Chukrasia (Meliaceae) consists of one species, C. tabularis, or two species C. tabularis and C. velutina. Despite a clear pattern of variation in many morphological characteristics such as leaves and bark, some authors regard the latter merely an ecotype of the former in seasonal forest. In the present study, we used ISSR markers to determine the genetic diversity and population structure among 23 Chukrasia subpopulations from across the natural range in Asia. Molecular analysis clearly differentiated two distinct groups of subpopulations, corresponding to the putative species, as well as well-defined subpopulations corresponding to geographic regions within the two groups. The molecular results are in concordance with morphological differentiation and corresponded to the two recognized taxa. The present study suggests that current taxonomic classification of the genus Chukrasia should be reviewed.     

A novel allele of the P-starvation tolerance gene OsPSTOL1 from African rice (Oryza glaberrima Steud) and its distribution in the genus Oryza

Key message

We have developed allele-specific markers for molecular breeding to transfer the PSTOL1 gene from Kasalath to African mega-varieties, including NERICAs, to improve their tolerance to P-deficient soil.

Abstract

The deficiency of phosphorus (P) in soil is a major problem in Sub-Saharan Africa due to general nutrient depletion and the presence of P-fixing soils. Developing rice cultivars with enhanced P efficiency would, therefore, represent a sustainable strategy to improve the livelihood of resource-poor farmers. Recently the Pup1 locus, a major QTL for tolerance to P deficiency in soil, was successfully narrowed-down to a major gene, the protein kinase OsPSTOL1 (P-starvation tolerance), which was found to be generally absent from modern irrigated rice varieties. Our target is to improve the tolerance of African mega-varieties to P deficiency through marker-assisted introgression of PSTOL1. As a first step, we have determined the Pup1 haplotype and surveyed the presence or absence of PSTOL1 and other genes of the Pup1 locus in African mega-varieties, NERICAs (New Rice for Africa) and their Oryza glaberrima parents. Here, we report the presence of a novel PSTOL1 allele in upland NERICAs that was inherited from the O. glaberrima parent CG14. This allele showed a 35 base-pair substitution when aligned to the Kasalath allele, but maintained a fully conserved kinase domain, and is present in most O. glaberrima accessions evaluated. In-silico and marker analysis indicated that many other genes of the Kasalath Pup1 locus were missing in the O. glaberrima genome, including the dirigent-like gene OsPupK20-2, which was shown to be downstream of PSTOL1. We have developed several allele-specific markers for the use for molecular breeding to transfer the PSTOL1 gene from Kasalath to African mega-varieties, including NERICAs.     

Fine mapping of LrSV2, a race-specific adult plant leaf rust resistance gene on wheat chromosome 3BS

Key message

Fine mapping permits the precise positioning of genes within chromosomes, prerequisite for positional cloning that will allow its rational use and the study of the underlying molecular action mechanism.

Abstract

Three leaf rust resistance genes were identified in the durable leaf rust resistant Argentinean wheat variety Sinvalocho MA: the seedling resistance gene Lr3 on distal 6BL and two adult plant resistance genes, LrSV1 and LrSV2, on chromosomes 2DS and 3BS, respectively. To develop a high-resolution genetic map for LrSV2, 10 markers were genotyped on 343 F2 individuals from a cross between Sinvalocho MA and Gama6. The closest co-dominant markers on both sides of the gene (3 microsatellites and 2 STMs) were analyzed on 965 additional F2s from the same cross. Microsatellite marker cfb5010 cosegregated with LrSV2 whereas flanking markers were found at 1 cM distal and at 0.3 cM proximal to the gene. SSR markers designed from the sequences of cv Chinese Spring BAC clones spanning the LrSV2 genetic interval were tested on the recombinants, allowing the identification of microsatellite swm13 at 0.15 cM distal to LrSV2. This delimited an interval of 0.45 cM around the gene flanked by the SSR markers swm13 and gwm533 at the subtelomeric end of chromosome 3BS.     

Characterization and mapping of <Emphasis Type="Italic">Dt1</Emphasis> locus which co-segregates with <Emphasis Type="Italic">CcTFL1</Emphasis> for growth habit in pigeonpea

Key message

We report growth habit profiling following SEM, genetic mapping and QTL analysis. Highlighted CcTFL1 , a candidate for determinacy in pigeonpea, since an Indel marker derived from this gene co-segregated with Dt1 locus.

Abstract

Pigeonpea (Cajanus cajan) is one of the most important legume crops grown in arid and semi-arid regions of the world. It is characterized with few unique features compared with other legume species, such as Lotus, Medicago, and Glycine. One of them is growth habit, an important agronomic trait. In the present study, identification of mutations affecting growth habit accompanied by a precise analysis of phenotype has been done which will shed more light upon developmental regulation in pigeonpea. A genetic study was conducted to examine the inheritance of growth habit and a genotyping by sequencing (GBS)-based genetic map constructed using F₂ mapping population derived from crossing parents ICP 5529 and ICP 11605. Inheritance studies clearly demonstrated the dominance of indeterminate (IDT) growth habit over determinate (DT) growth habit in F₂ and F_2:3 progenies. A total of 787 SNP markers were mapped in the genetic map of 1454 cM map length. Growth habit locus (Dt1) was mapped on the CcLG03 contributing more than 61% of total phenotypic variations. Subsequently, QTL analysis highlighted one gene, CcTFL1, as a candidate for determinacy in pigeonpea, since an Indel marker derived from this gene co-segregated with the Dt1 locus. Ability of this Indel-derived marker to differentiate DT/IDT lines was also validated on 262 pigeonpea lines. This study clearly demonstrated that CcTFL1 is a candidate gene for growth habit in pigeonpea and a user-friendly marker was developed in the present study which will allow low-cost genotyping without need of automation.