Heritable differences in abundance of bacterial rhizosphere taxa are correlated with fungal necrotrophic pathogen resistance.

Host-microbe interactions are increasingly recognized as important drivers of organismal health, growth, longevity and community-scale ecological processes. However, less is known about how genetic variation affects hosts' associated microbiomes and downstream phenotypes. We demonstrate that sunflower (Helianthus annuus) harbours substantial, heritable variation in microbial communities under field conditions. We show that microbial communities co-vary with heritable variation in resistance to root infection caused by the necrotrophic pathogen Sclerotinia sclerotiorum and that plants grown in autoclaved soil showed almost complete elimination of pathogen resistance. Association mapping suggests at least 59 genetic locations with effects on both microbial relative abundance and Sclerotinia resistance. Although the genetic architecture appears quantitative, we have elucidated previously unexplained genetic variation for resistance to this pathogen. We identify new targets for plant breeding and demonstrate the potential for heritable microbial associations to play important roles in defence in natural and human-altered environments.

Genetic analysis of basal stalk rot resistance introgressed from wild Helianthus petiolaris into cultivated sunflower (Helianthus annuus L.) using an advanced backcross population.

Introduction: Sclerotinia sclerotiorum is a serious pathogen causing severe basal stalk rot (BSR) disease on cultivated sunflower (Helianthus annuus L.) that leads to significant yield losses due to insufficient resistance. The wild annual sunflower species H. petiolaris, commonly known as prairie sunflower is known for its resistance against this pathogen. Sunflower resistance to BSR is quantitative and determined by many genes with small effects on the resistance phenotype. The objective of this study was to identify loci governing BSR resistance derived from H. petiolaris using a quantitative trait loci (QTL) mapping approach. Methods: BSR resistance among lines of an advanced backcross population (AB-QTL) with 174 lines developed from a cross of inbred line HA 89 with H. petiolaris PI 435843 was determined in the field during 2017-2019, and in the greenhouse in 2019. AB-QTL lines and the HA 89 parent were genotyped using genotyping-by-sequencing and a genetic linkage map was developed spanning 997.51 cM and using 1,150 SNP markers mapped on 17 sunflower chromosomes. Results and discussion: Highly significant differences (p<0.001) for BSR response among AB-QTL lines were observed disease incidence (DI) in all field seasons, as well as disease rating (DR) and area under the disease progress curve (AUDPC) in the greenhouse with a moderately high broad-sense heritability (H 2) of 0.61 for the tested resistance parameters. A total of 14 QTL associated with BSR resistance were identified on nine chromosomes, each explaining a proportion of the phenotypic variation ranging from 3.5% to 28.1%. Of the 14 QTL, eight were detected for BSR resistance in the field and six were detected under greenhouse conditions. Alleles conferring increased BSR resistance were contributed by the H. petiolaris parent at 11 of the 14 QTL.

A Quantitative Genetic Study of Sclerotinia Head Rot Resistance Introgressed from the Wild Perennial Helianthus maximiliani into Cultivated Sunflower (Helianthus annuus L.).

Sclerotinia head rot (HR), caused by Sclerotinia sclerotiorum, is an economically important disease of sunflower with known detrimental effects on yield and quality in humid climates worldwide. The objective of this study was to gain insight into the genetic architecture of HR resistance from a sunflower line HR21 harboring HR resistance introgressed from the wild perennial Helianthus maximiliani. An F2 population derived from the cross of HA 234 (susceptible-line)/HR21 (resistant-line) was evaluated for HR resistance at two locations during 2019−2020. Highly significant genetic variations (p < 0.001) were observed for HR disease incidence (DI) and disease severity (DS) in both individual and combined analyses. Broad sense heritability (H2) estimates across environments for DI and DS were 0.51 and 0.62, respectively. A high-density genetic map of 1420.287 cM was constructed with 6315 SNP/InDel markers developed using genotype-by-sequencing technology. A total of 16 genomic regions on eight sunflower chromosomes, 1, 2, 10, 12, 13, 14, 16 and 17 were associated with HR resistance, each explaining between 3.97 to 16.67% of the phenotypic variance for HR resistance. Eleven of these QTL had resistance alleles from the HR21 parent. Molecular markers flanking the QTL will facilitate marker-assisted selection breeding for HR resistance in sunflower.

Genomic Insights Into Sclerotinia Basal Stalk Rot Resistance Introgressed From Wild Helianthus praecox Into Cultivated Sunflower (Helianthus annuus L.).

Crop wild relatives of the cultivated sunflower (Helianthus annuus L.) are a valuable resource for its sustainable production. Helianthus praecox ssp. runyonii is a wild sunflower known for its resistance against diseases caused by the fungus, Sclerotinia sclerotiorum (Lib.) de Bary, which infects over 400 broadleaf hosts including many important food crops. The objective of this research was to dissect the Sclerotinia basal stalk rot (BSR) resistance introgressed from H. praecox ssp. runyonii into cultivated sunflower. An advanced backcross quantitative trait loci (AB-QTL) mapping population was developed from the cross of a H. praecox accession with cultivated sunflower lines. The AB-QTL population was evaluated for BSR resistance in the field during the summers of 2017-2018 and in the greenhouse in the spring of 2018. Highly significant genetic variations (p < 0.001) were observed for the BSR disease in the field and greenhouse with a moderately high broad-sense heritability (H 2) ranging from 0.66 to 0.73. Genotyping-by-sequencing approach was used to genotype the parents and the progeny lines of the AB-QTL population. A genetic linkage map spanning 1,802.95 cM was constructed using 1,755 single nucleotide polymorphism (SNP) markers mapped on 17 sunflower chromosomes. A total of 19 BSR resistance QTL were detected on nine sunflower chromosomes, each explaining 2.21%-16.99% of the phenotypic variance for resistance in the AB-QTL population. Sixteen of the 19 QTL had alleles conferring increased BSR resistance derived from the H. praecox parent. SNP markers flanking the identified QTL will facilitate marker-assisted breeding to combat the disease in sunflower.

Map and sequence-based chromosome walking towards cloning of the male fertility restoration gene Rf5 linked to R11 in sunflower.

The nuclear fertility restorer gene Rf5 in HA-R9, originating from the wild sunflower species Helianthus annuus, is able to restore the widely used PET1 cytoplasmic male sterility in sunflowers. Previous mapping placed Rf5 at an interval of 5.8 cM on sunflower chromosome 13, distal to a rust resistance gene R11 at a 1.6 cM genetic distance in an SSR map. In the present study, publicly available SNP markers were further mapped around Rf5 and R11 using 192 F2 individuals, reducing the Rf5 interval from 5.8 to 0.8 cM. Additional SNP markers were developed in the target region of the two genes from the whole-genome resequencing of HA-R9, a donor line carrying Rf5 and R11. Fine mapping using 3517 F3 individuals placed Rf5 at a 0.00071 cM interval and the gene co-segregated with SNP marker S13_216392091. Similarly, fine mapping performed using 8795 F3 individuals mapped R11 at an interval of 0.00210 cM, co-segregating with two SNP markers, S13_225290789 and C13_181790141. Sequence analysis identified Rf5 as a pentatricopeptide repeat-encoding gene. The high-density map and diagnostic SNP markers developed in this study will accelerate the use of Rf5 and R11 in sunflower breeding.

Genetic loci underlying quantitative resistance to necrotrophic pathogens Sclerotinia and Diaporthe (Phomopsis), and correlated resistance to both pathogens.

KEY MESSAGE: We provide results rooted in quantitative genetics, which combined with knowledge of candidate gene function, helps us to better understand the resistance to two major necrotrophic pathogens of sunflower. Necrotrophic pathogens can avoid or even benefit from plant defenses used against biotrophic pathogens, and thus represent a distinct challenge to plant populations in natural and agricultural systems. Sclerotinia and Phomopsis/Diaporthe are detrimental pathogens for many dicotyledonous plants, including many economically important plants. With no well-established methods to prevent infection in susceptible plants, host-plant resistance is currently the most effective strategy. Despite knowledge of a moderate, positive correlation in resistance to the two diseases in sunflower, detailed analysis of the genetics, in the same populations, has not been conducted. We present results of genome-wide analysis of resistance to both pathogens in a diversity panel of 218 domesticated sunflower genotypes of worldwide origin. We identified 14 Sclerotinia head rot and 7 Phomopsis stem canker unique QTLs, plus 1 co-located QTL for both traits, and observed extensive patterns of linkage disequilibrium between sites for both traits. Most QTLs contained one credible candidate gene, and gene families were common for the two disease resistance traits. These results suggest there has been strong, simultaneous selection for resistance to these two diseases and that a generalized mechanism for defense against these necrotrophic pathogens exists.

Genetic Dissection of Phomopsis Stem Canker Resistance in Cultivated Sunflower Using High Density SNP Linkage Map.

Phomopsis stem canker (PSC) caused by Diaporthe helianthi is increasingly becoming a global threat for sunflower production. In this study, the genetic basis of PSC resistance was investigated in a recombinant inbred line (RIL) population developed from a cross between HA 89 (susceptible) and HA-R3 (resistant). The RIL population was evaluated for PSC disease incidence (DI) in seven screening trials at multiple locations during 2016-2018. The distribution of PSC DI in the RIL population was continuous, confirming a polygenic inheritance of the trait. A moderately high broad-sense heritability (H2, 0.76) was estimated for the trait across environments. In the combined analysis, both the genotype and the genotype × environment interactions were highly significant. A linkage map spanning 1505.33 cM was constructed using genotyping-by-sequencing derived markers. Marker-trait association analysis identified a total of 15 quantitative trait loci (QTL) associated with PSC resistance on 11 sunflower chromosomes, each explaining between 5.24 and 17.39% of the phenotypic variation. PSC resistance QTL were detected in two genomic regions each on chromosomes 3, 5, 13, and 17, while one QTL each was detected in the remaining seven chromosomes. Tightly linked single nucleotide polymorphism (SNP) markers flanking the PSC resistance QTL will facilitate marker-assisted selection in PSC resistance sunflower breeding.

Unraveling the Sclerotinia Basal Stalk Rot Resistance Derived From Wild Helianthus argophyllus Using a High-Density Single Nucleotide Polymorphism Linkage Map.

Basal stalk rot (BSR), caused by the fungus Sclerotinia sclerotiorum, is a serious disease of sunflower (Helianthus annuus L.) in the humid temperate growing areas of the world. BSR resistance is quantitative and conditioned by multiple genes. Our objective was to dissect the BSR resistance introduced from the wild annual species Helianthus argophyllus using a quantitative trait loci (QTL) mapping approach. An advanced backcross population (AB-QTL) with 134 lines derived from the cross of HA 89 with a H. argophyllus Torr. and Gray accession, PI 494573, was evaluated for BSR resistance in three field and one greenhouse growing seasons of 2017-2019. Highly significant genetic variations (p < 0.001) were observed for BSR disease incidence (DI) in all field screening tests and disease rating and area under the disease progress curve in the greenhouse. The AB-QTL population and its parental lines were genotyped using the genotyping-by-sequencing method. A genetic linkage map spanning 2,045.14 cM was constructed using 3,110 SNP markers mapped on 17 sunflower chromosomes. A total of 21 QTL associated with BSR resistance were detected on 11 chromosomes, each explaining a phenotypic variation ranging from 4.5 to 22.6%. Of the 21 QTL, eight were detected for BSR DI measured in the field, seven were detected for traits measured in the greenhouse, and six were detected from both field and greenhouse tests. Thirteen of the 21 QTL had favorable alleles from the H. argophyllus parent conferring increased BSR resistance.

Introgression and monitoring of wild Helianthus praecox alien segments associated with Sclerotinia basal stalk rot resistance in sunflower using genotyping-by-sequencing.

Sclerotinia basal stalk rot (BSR) and downy mildew are major diseases of sunflowers worldwide. Breeding for BSR resistance traditionally relies upon cultivated sunflower germplasm that has only partial resistance thus lacking an effective resistance against the pathogen. In this study, we report the transfer of BSR resistance from sunflower wild species, Helianthus praecox, into cultivated sunflower and molecular assessment of the introgressed segments potentially associated with BSR resistance using the genotyping-by-sequencing (GBS) approach. Eight highly BSR-resistant H. praecox introgression lines (ILs), H.pra 1 to H.pra 8, were developed. The mean BSR disease incidence (DI) for H.pra 1 to H.pra 8 across environments for four years ranged from 1.2 to 11.1%, while DI of Cargill 270 (susceptible check), HA 89 (recurrent parent), HA 441 and Croplan 305 (resistant checks) was 36.1, 31.0, 19.5, and 11.6%, respectively. Molecular assessment using GBS detected the presence of H. praecox chromosome segments in chromosomes 1, 8, 10, 11, and 14 of the ILs. Both shared and unique polymorphic SNP loci were detected throughout the entire genomes of the ILs, suggesting the successful transfer of common and novel introgression regions that are potentially associated with BSR resistance. Downy mildew (DM) disease screening and molecular tests revealed that a DM resistance gene, Pl17, derived from one of the inbred parent HA 458 was present in four ILs. Introgression germplasms possessing resistance to both Sclerotinia BSR and DM will extend the useful diversity of the primary gene pool in the fight against two destructive sunflower diseases.

Linkage Mapping and Genome-Wide Association Studies of the Rf Gene Cluster in Sunflower (Helianthus annuus L.) and Their Distribution in World Sunflower Collections.

Commercial hybrid seed production in sunflower currently relies on a single cytoplasmic male sterility (CMS) source, PET1 and the major fertility restoration gene, Rf1, leaving the crop highly vulnerable to issues with genetic bottlenecks. Therefore, having multiple CMS/Rf systems is important for sustainable sunflower production. Here, we report the identification of a new fertility restoration gene, Rf7, which is tightly linked to a new downy mildew (DM) resistance gene, Pl34 , in the USDA sunflower inbred line, RHA 428. The Rf7 gene was genetically mapped to an interval of 0.6 cM on the lower end of linkage group (LG) 13, while Pl34 was mapped 2.1 cM proximal to the Rf7. Both the genes are located in a cluster of Rf and Pl genes. To gain further insights into the distribution of Rf genes in the sunflower breeding lines, we used a genome-wide association study (GWAS) approach to identify markers associated with the fertility restoration trait in a panel of 333 sunflower lines genotyped with 8,723 single nucleotide polymorphism (SNP) markers. Twenty-four SNP markers on the lower end of LG13 spanning a genomic region of 2.47 cM were significantly associated with the trait. The significant markers were surveyed in a world collection panel of 548 sunflower lines and validated to be associated with the Rf1 gene. The SNP haplotypes for the Rf1 gene are different from Rf5 and the Rf7gene located in the Rf gene cluster on LG13. The SNP and SSR markers tightly flanking the Rf7 gene and the Pl34 gene would benefit the sunflower breeders in facilitating marker assisted selection (MAS) of Rf and Pl genes.

SNP Discovery and QTL Mapping of Sclerotinia Basal Stalk Rot Resistance in Sunflower using Genotyping-by-Sequencing.

Basal stalk rot (BSR), caused by the ascomycete fungus (Lib.) de Bary, is a serious disease of sunflower ( L.) in the cool and humid production areas of the world. Quantitative trait loci (QTL) for BSR resistance were identified in a sunflower recombinant inbred line (RIL) population derived from the cross HA 441 × RHA 439. A genotyping-by-sequencing (GBS) approach was adapted to discover single nucleotide polymorphism (SNP) markers. A genetic linkage map was developed comprised of 1053 SNP markers on 17 linkage groups (LGs) spanning 1401.36 cM. The RILs were tested in five environments (locations and years) for resistance to BSR. Quantitative trait loci were identified in each environment separately and also with integrated data across environments. A total of six QTL were identified in all five environments: one of each on LGs 4, 9, 10, 11, 16, and 17. The most significant QTL, and , were identified at multiple environments on LGs 10 and 17, explaining 31.6 and 20.2% of the observed phenotypic variance, respectively. The remaining four QTL, , , , and , were detected in only one environment on LGs 4, 9, 11, and 16, respectively. Each of these QTL explains between 6.4 and 10.5% of the observed phenotypic variation in the RIL population. Alleles conferring increased resistance were contributed by both parents. The potential of the and in marker-assisted selection (MAS) breeding are discussed.

Genotyping-by-Sequencing Uncovers the Introgression Alien Segments Associated with Sclerotinia Basal Stalk Rot Resistance from Wild Species-I. Helianthus argophyllus and H. petiolaris.

Basal stalk rot (BSR), caused by Sclerotinia sclerotiorum, is a devastating disease in sunflower worldwide. The progress of breeding for Sclerotinia BSR resistance has been hampered due to the lack of effective sources of resistance for cultivated sunflower. Our objective was to transfer BSR resistance from wild annual Helianthus species into cultivated sunflower and identify the introgressed alien segments associated with BSR resistance using a genotyping-by-sequencing (GBS) approach. The initial crosses were made between the nuclear male sterile HA 89 with the BSR resistant plants selected from wild Helianthus argophyllus and H. petiolaris populations in 2009. The selected resistant F1 plants were backcrossed to HA 458 and HA 89, respectively. Early generation evaluations of BSR resistance were conducted in the greenhouse, while the BC2F3 and subsequent generations were evaluated in the inoculated field nurseries. Eight introgression lines; six from H. argophyllus (H.arg 1 to H.arg 6), and two from H. petiolaris (H.pet 1 and H.pet 2), were selected. These lines consistently showed high levels of BSR resistance across seven environments from 2012 to 2015 in North Dakota and Minnesota, USA. The mean BSR disease incidence (DI) for H.arg 1 to H.arg 6, H.pet 1, and H.pet 2 was 3.0, 3.2, 0.8, 7.2, 7.7, 1.9, 2.5, and 4.4%, compared to a mean DI of 36.1% for Cargill 270 (susceptible hybrid), 31.0% for HA 89 (recurrent parent), 19.5% for HA 441 (resistant inbred), and 11.6% for Croplan 305 (resistant hybrid). Genotyping of the highly BSR resistant introgression lines using GBS revealed the presence of the H. argophyllus segments in linkage groups (LGs) 3, 8, 9, 10, and 11 of the sunflower genome, and the H. petiolaris segments only in LG8. The shared polymorphic SNP loci in the introgression lines were detected in LGs 8, 9, 10, and 11, indicating the common introgression regions potentially associated with BSR resistance. Additionally, a downy mildew resistance gene, Pl17 , derived from one of the parents, HA 458, was integrated into five introgression lines. Germplasms combining resistance to Sclerotinia BSR and downy mildew represent a valuable genetic source for sunflower breeding to combat these two destructive diseases.

A high-density SNP Map of sunflower derived from RAD-sequencing facilitating fine-mapping of the rust resistance gene R12.

A high-resolution genetic map of sunflower was constructed by integrating SNP data from three F2 mapping populations (HA 89/RHA 464, B-line/RHA 464, and CR 29/RHA 468). The consensus map spanned a total length of 1443.84 cM, and consisted of 5,019 SNP markers derived from RAD tag sequencing and 118 publicly available SSR markers distributed in 17 linkage groups, corresponding to the haploid chromosome number of sunflower. The maximum interval between markers in the consensus map is 12.37 cM and the average distance is 0.28 cM between adjacent markers. Despite a few short-distance inversions in marker order, the consensus map showed high levels of collinearity among individual maps with an average Spearman's rank correlation coefficient of 0.972 across the genome. The order of the SSR markers on the consensus map was also in agreement with the order of the individual map and with previously published sunflower maps. Three individual and one consensus maps revealed the uneven distribution of markers across the genome. Additionally, we performed fine mapping and marker validation of the rust resistance gene R12, providing closely linked SNP markers for marker-assisted selection of this gene in sunflower breeding programs. This high resolution consensus map will serve as a valuable tool to the sunflower community for studying marker-trait association of important agronomic traits, marker assisted breeding, map-based gene cloning, and comparative mapping.

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. 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.

Minerals (Zn, Fe, Ca and Mg) and Antinutrient (Phytic Acid) Constituents in Common Bean.

In this study, the variation of zinc (Zn), iron (Fe), calcium (Ca) and magnesium (Mg) and the interference of phytic acid (PA) on their availability was investigated in 29 US grown and CIAT breeding genotypes of common bean. Fe levels showed the highest variation (8.9-112.9 mg kg-1) followed by Ca (58.67-122.98 mg kg-1) and Zn (30.90-64.60 mg kg-1) while variability of Mg concentration (6.47-11.05 mg kg-1) is the least among the mineral components. PA showed a wide range of variability (12.52-316.42 m kg-1) and inversely correlated with Fe, Ca and Mg concentrations. The results of the minerals and PA concentration can be interpreted in terms of expected bio-availability of minerals and the correlation study indicated that the presence of high concentration of PA inhibit the availability of most minerals under study in common beans. We suggest that the genotypes, MIB466, MIB465, MIB152 and JaloEEP 558 could be considered as sources of high Zn and Vista and NUA56-1770 for high seed Fe. We also identified G122 for high Ca and JaloEEP558 genotype for high Mg. We conclude that there is scope for the enhancement of mineral contents of common bean by selecting suitable genotype and bean products require processing for dephytinization for the improvement of mineral availability.

Loci controlling partial resistance to rice blast do not show marked QTL x environment interaction when plant nitrogen status alters disease severity.

Plant disease susceptibility is often increased by nitrogen (N) application. Therefore, it is important to know if resistance loci are effective in different plant N environments. One-hundred lines of the Bala x Azucena rice (Oryza sativa) mapping population were grown in two N treatments and tested for partial resistance to blast (Magnaporthe grisea) isolate CD100. Disease severity (DS), the number and size of lesions and plant N and C concentrations were measured and the results subject to quantitative trait loci (QTL) and QTL x environment analysis. There was a 66% higher plant N concentration in the high N treatment and DS increased significantly, mostly as a result of increased numbers of lesions. Nine regions contained QTL for disease traits but only one showed evidence of statistically significant QTL x treatment interaction. This was a large effect quantitative trait locus at marker R1933 on chromosome 12 which was less effective at high N. Apparently, blast disease is increased by higher plant N, but the efficacy of partial resistance genes is not greatly affected by N application.