Whole-genome sequencing enables molecular dissection and candidate gene identification of the rust resistance gene R12 in sunflower (Helianthus annuus L.).

KEY MESSAGE: We finely mapped the rust resistance gene R12 to a 0.1248-cM region, identified a potential R12 candidate gene in the XRQ reference genome, and developed three diagnostic SNP markers for R12. Rust is a devastating disease in sunflower that is damaging to the sunflower production globally. Identification and utilization of host-plant resistance are proven to be preferable means for disease control. The rust resistance gene R12 with broad-spectrum specificity to rust was previously localized to a 2.4 Mb region on sunflower chromosome 11. To understand the molecular mechanism of resistance, we conducted whole-genome sequencing of RHA 464 (R12 donor line) and reference genome-based fine mapping of the gene R12. Overall, the 213 markers including 186 SNPs and 27 SSRs' were identified from RHA 464 sequences and used to survey polymorphisms between the parents HA 89 and RHA 464. Saturation mapping identified 26 new markers positioned in the R12 region, and fine mapping with a large population of 2004 individuals positioned R12 at a genetic distance of 0.1248 cM flanked by SNP markers C11_150451336 and S11_189205190. One gene, HanXRQChr11g0348661, with a defense-related NB-ARC-LRR domain, was identified in the XRQr1.0 genome assembly in the R12 region; it is predicted to be a potential R12 candidate gene. Comparative analysis clearly distinguished R12 from the rust R14 gene located in the vicinity of the R12 gene on chromosome 11. Three diagnostic SNP markers, C11_147181749, C11_147312085, and C11_149085167, specific for R12 were developed in the current study, facilitating more accurate and efficient selection in sunflower rust resistance breeding. The current study provides a new genetic resource and starting point for cloning R12 in the future.

Introgression and targeting of the Pl37 and Pl38 genes for downy mildew resistance from wild Helianthus annuus and H. praecox into cultivated sunflower (Helianthus annuus L.).

KEY MESSAGE: Two new downy mildew resistance genes, Pl37 and Pl38, were introgressed from wild sunflower species into cultivated sunflower and mapped to sunflower chromosomes 4 and 2, respectively Downy mildew (DM), caused by the oomycete pathogen Plasmopara halstedii (Farl.) Berl. & de Toni, is known as the most prevalent disease occurring in global sunflower production areas, especially in North America and Europe. In this study, we report the introgression and molecular mapping of two new DM resistance genes from wild sunflower species, Helianthus annuus and H. praecox, into cultivated sunflower. Two mapping populations were developed from the crosses of HA 89/H. annuus PI 435417 (Pop1) and CMS HA 89/H. praecox PRA-417 (Pop2). The phenotypic evaluation of DM resistance/susceptibility was conducted in the BC1F2-derived BC1F3 populations using P. halstedii race 734. The BC1F2 segregating Pop1 was genotyped using an Optimal GBS AgriSeq™ Panel consisting of 768 mapped SNP markers, while the BC1F2 segregating Pop2 was genotyped using a genotyping-by-sequencing approach. Linkage analysis and subsequent saturation mapping placed the DM resistance gene, designated Pl37, derived from H. annuus PI 435417 in a 1.6 cM genetic interval on sunflower chromosome 4. Pl37 co-segregated with SNP markers SPB0003 and C4_5738736. Similarly, linkage analysis and subsequent saturation mapping placed the DM resistance gene, designated Pl38, derived from H. praecox PRA-417 in a 0.8 cM genetic interval on sunflower chromosome 2. Pl38 co-segregated with seven SNP markers. Multi-pathotype tests revealed that lines with Pl37 or Pl38 are immune to the most prevalent and virulent P. halstedii races tested. Two germplasm lines, HA-DM15 with Pl37 and HA-DM16 with Pl38, were developed for use in sunflower DM-resistance breeding.

Discovery and mapping of two new rust resistance genes, R17 and R18, in sunflower using genotyping by sequencing.

KEY MESSAGE: Discovery of two rust resistance genes, R17 and R18, from the sunflower lines introduced from South Africa and genetic mapping of them to sunflower chromosome 13. Rust, caused by the fungus Puccinia helianthi Schw., is one of the most serious diseases of sunflower in the world. The rapid changes that occur in the virulence characteristics of pathogen populations present a continuous threat to the effectiveness of existing rust-resistant hybrids. Thus, there is a continued need for the characterization of genetically diverse sources of rust resistance. In this study, we report to identify two new rust resistance genes, R17 and R18, from the sunflower lines, KP193 and KP199, introduced from South Africa. The inheritance of rust resistance was investigated in both lines using two mapping populations developed by crossing the resistant plants selected from KP193 and KP199 with a common susceptible parent HA 89. The F2 populations were first genotyped using genotyping by sequencing for mapping of the rust genes and further saturated with markers in the target region. Molecular mapping positioned the two genes at the lower end of sunflower chromosome 13 within a large gene cluster. Two co-segregating SNP markers, SFW01497 and SFW08875, were distal to R17 at a 1.9 cM genetic distance, and a cluster of five co-segregating SNPs was proximal to R17 at 0.7 cM. R18 co-segregated with the SNP marker SFW04317 and was proximal to two cosegregating SNPs, SFW01497 and SFW05453, at 1.9 cM. These maps provide markers for stacking R17 or R18 with other broadly effective rust resistance genes to extend the durability of rust resistance. The relationship of the six rust resistance genes in the cluster was discussed.

Development and dissection of diagnostic SNP markers for the downy mildew resistance genes Pl Arg and Pl 8 and maker-assisted gene pyramiding in sunflower (Helianthus annuus L.).

Diagnostic DNA markers are an invaluable resource in breeding programs for successful introgression and pyramiding of disease resistance genes. Resistance to downy mildew (DM) disease in sunflower is mediated by Pl genes which are known to be effective against the causal fungus, Plasmopara halstedii. Two DM resistance genes, Pl Arg and Pl 8 , are highly effective against P. halstedii races in the USA, and have been previously mapped to the sunflower linkage groups (LGs) 1 and 13, respectively, using simple sequence repeat (SSR) markers. In this study, we developed high-density single nucleotide polymorphism (SNP) maps encompassing the Pl arg and Pl 8 genes and identified diagnostic SNP markers closely linked to these genes. The specificity of the diagnostic markers was validated in a highly diverse panel of 548 sunflower lines. Dissection of a large marker cluster co-segregated with Pl Arg revealed that the closest SNP markers NSA_007595 and NSA_001835 delimited Pl Arg to an interval of 2.83 Mb on the LG1 physical map. The SNP markers SFW01497 and SFW06597 delimited Pl 8 to an interval of 2.85 Mb on the LG13 physical map. We also developed sunflower lines with homozygous, three gene pyramids carrying Pl Arg , Pl 8 , and the sunflower rust resistance gene R 12 using the linked SNP markers from a segregating F2 population of RHA 340 (carrying Pl 8 )/RHA 464 (carrying Pl Arg and R 12 ). The high-throughput diagnostic SNP markers developed in this study will facilitate marker-assisted selection breeding, and the pyramided sunflower lines will provide durable resistance to downy mildew and rust diseases.

Genetic diversity and selection of genotypes to enhance Zn and Fe content in common bean.

Common bean (Phaseolus vulgaris L.) is an important source of dietary protein and minerals worldwide. Genes conditioning variability for mineral contents are not clearly understood. Our ultimate goal is to identify genes conditioning genetic variation for Zn and Fe content. To establish mapping populations for this objective, we tested mineral content of 29 common bean genotypes. Chemical analyses revealed significant genetic variability for seed Zn and Fe contents among the genotypes. Genetic diversity was evaluated with 49 primer pairs, of which 23 were simple sequence repeats (SSR), 16 were developed from tentative consensus (TC) sequences, and 10 were generated from common bean NBS-LRR gene sequences. The discriminatory ability of molecular markers for identifying allelic variation among genotypes was estimated by polymorphism information content (PIC) and the genetic diversity was measured from genetic similarities between genotypes. Primers developed from NBS-LRR gene sequences were highly polymorphic in both PIC values and number of alleles (0.82 and 5.3), followed by SSRs (0.56 and 3.0), and markers developed from TC (0.39 and 2.0). genetic similarity values between genotypes ranged from 14.0 (JaloEEP558 and DOR364) to 91.4 (MIB152 and MIB465). Cluster analysis clearly discriminated the genotypes into Mesoamerican and Andean gene pools. Common bean genotypes were selected to include in crossing to enhance seed Zn and Fe content based on genetic diversity and seed mineral contents of the genotypes.