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.

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.

Characterization and mapping of a downy mildew resistance gene, Pl36, in sunflower (Helianthus annuus L.).

Downy mildew (DM) is one of the most serious diseases in sunflower-growing regions worldwide, often significantly reducing sunflower yields. The causal agent of sunflower DM, the oomycete pathogen Plasmopara halstedii, is highly virulent and aggressive. Studying regional disease spread and virulence evolution in the DM pathogen population is important for the development of new sunflower inbred lines with resistance to the existing DM pathogen. The sunflower line 803-1, as one of nine international differential hosts, has been used in the identification of P. halstedii virulent pathotypes in sunflower since 2000. The DM resistance gene in 803-1 was temporally designated Pl5 + based on allelic analysis but has not been molecularly characterized. In the present study, bulked segregant analysis and genetic mapping confirmed the presence of the Pl gene within a large gene cluster on sunflower chromosome 13 in 803-1, as previously reported. Subsequent saturation mapping in the gene target region with single nucleotide polymorphism (SNP) markers placed this gene at an interval of 3.4 Mb in the XRQ reference genome assembly, a location different from that of Pl5. Therefore, the Pl gene in 803-1 was re-designated Pl36 because it is not allelic with Pl5. Four SNP markers co-segregated with Pl36, and SNP SFW05743 was 1.1 cM proximal to Pl36. The relationship of eight Pl genes in the cluster is discussed based on their origin, map position, and specificity of resistance/susceptibility to DM infection. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01280-1.

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.

[Risk factors for lung cancer based on low-dose computed tomography screening].

Objective: To analyze the risk factors related to lung cancer in participants with low-dose computed tomography (LDCT) screening, to provide data support for identifying high-risk groups of lung cancer and to improve the effectiveness of LDCT lung cancer screening. Methods: A total of 5 366 asymptomatic subjects (2 762 males and 2 604 females) who underwent LDCT lung cancer screening were recruited at Cancer Hospital, Chinese Academy of Medical Sciences from 2014 to 2017. The result of LDCT and the risk factors of participants were analyzed. The LDCT positive results were defined as solid or part-solid nodules≥5 mm and non-solid nodule≥8 mm. A total of 12 factors were included and multivariate logistic regression was used to analyze the risk factors associated with lung cancer in the study. Results: Of the 5 366 asymptomatic subjects, 389 were positive and 4 977 were negative for LDCT screening. Among them, 26 of 389 positive cases were confirmed as lung cancers pathologically, and the detection rate of stage I lung cancer was 92.3% (24/26). Multivariate logistic regression showed that age, smoking, low level of education were the relevant risk factors for lung cancer and positive nodules. A stratified analysis of age showed that no risk factors were detected in the 40-49 years old group, while age, smoking, low level of education (primary school and below) were recognized as risk factors in the ≥50 years old group. No statistically significant risk factor was detected between the lung cancer group and the positive nodules group. Conclusions: Age, smoking, and low level of education (primary school and below) are related risk factors for lung cancer and positive nodules. People aged 50 years or older, smoking, and low level of education may be a high risk group for lung cancer. LDCT can effectively detect early lung cancer.

Diversification of the downy mildew resistance gene pool by introgression of a new gene, Pl35, from wild Helianthus argophyllus into oilseed and confection sunflowers (Helianthus annuus L.).

KEY MESSAGE: We have mapped a new downy mildew resistance gene, Pl35, derived from wild Helianthus argophyllus to sunflower linkage group 1. New germplasms incorporating the Pl35 gene were developed for both oilseed and confection sunflower Sunflower downy mildew (DM), caused by the oomycete pathogen Plasmopara halstedii, is an economically important and widespread sunflower disease worldwide. Non-race-specific resistance is not available in sunflower, and breeding for DM resistance relies on race-specific resistance to control this disease. The discovery of the novel DM resistance genes is a long-term task due to the highly virulent and aggressive nature of the P. halstedii pathogen, which reduces the effectiveness of resistance genes. The objectives of this study were to: (1) transfer DM resistance from a wild sunflower species Helianthus argophyllus (PI 494576) into cultivated sunflowers; (2) map the resistance gene; and (3) develop diagnostic single-nucleotide polymorphism (SNP) markers for efficient targeting of the gene in breeding programs. The H. argophyllus accession PI 494576 previously identified with resistance to the most virulent P. halstedii race 777 was crossed with oilseed and confection sunflower in 2012. Molecular mapping using the BC2F2 and BC2F3 populations derived from the cross CONFSCLB1/PI 494576 located a new resistance gene Pl35 on linkage group 1 of the sunflower genome. The new gene Pl35 was successfully transferred from PI 494576 into cultivated sunflowers. SNP markers flanking Pl35 were surveyed in a validation panel of 548 diversified sunflower lines collected globally. Eleven SNP markers were found to be diagnostic for Pl35 SNP alleles, with four co-segregating with Pl35. The developed oilseed and confection germplasms with diagnostic SNP markers for Pl35 will be very useful resources for breeding of DM resistance in sunflower.

High-throughput genotyping-by-sequencing facilitates molecular tagging of a novel rust resistance gene, R 15 , in sunflower (Helianthus annuus L.).

KEY MESSAGE: A novel rust resistance gene, R 15 , derived from the cultivated sunflower HA-R8 was assigned to linkage group 8 of the sunflower genome using a genotyping-by-sequencing approach. SNP markers closely linked to R 15 were identified, facilitating marker-assisted selection of resistance genes. The rust virulence gene is co-evolving with the resistance gene in sunflower, leading to the emergence of new physiologic pathotypes. This presents a continuous threat to the sunflower crop necessitating the development of resistant sunflower hybrids providing a more efficient, durable, and environmentally friendly host plant resistance. The inbred line HA-R8 carries a gene conferring resistance to all known races of the rust pathogen in North America and can be used as a broad-spectrum resistance resource. Based on phenotypic assessments of 140 F2 individuals derived from a cross of HA 89 with HA-R8, rust resistance in the population was found to be conferred by a single dominant gene (R 15 ) originating from HA-R8. Genotypic analysis with the currently available SSR markers failed to find any association between rust resistance and any markers. Therefore, we used genotyping-by-sequencing (GBS) analysis to achieve better genomic coverage. The GBS data showed that R 15 was located at the top end of linkage group (LG) 8. Saturation with 71 previously mapped SNP markers selected within this region further showed that it was located in a resistance gene cluster on LG8, and mapped to a 1.0-cM region between three co-segregating SNP makers SFW01920, SFW00128, and SFW05824 as well as the NSA_008457 SNP marker. These closely linked markers will facilitate marker-assisted selection and breeding in sunflower.

[Comparison of parameters for diffusion-weighted intravoxel incoherent motion imaging in lung cancer patients with different histopathological subtypes].

Objective: To explore the intravoxel incoherent motion diffusion-weighted imaging (IVIM-DWI) model in lung cancer patients with different histopathological subtypes. Methods: A total of 105 patients were recruited, including 68 cases of adenocarcinoma, 22 cases of squamous carcinoma and 15 cases of small cell carcinoma. All patients underwent magnetic resonance examination consisting of axial IVIM-DWI sequence on a 3.0 T whole body scanner, then the standard ADC (sADC), diffusion coefficient (D), pseudo-diffusion coefficient(D(*)), perfusion fraction (f), distributed diffusion coefficient (DDC) and water diffusion heterogeneity index (α) were calculated for each lesion within the IVIM-DWI model. Results: Mean sADC values were (1.45±0.26) ×10(-3)mm(2)/s, (1.36±0.48) ×10(-3)mm(2)/s and (1.35±0.40) ×10(-3)mm(2)/s for adenocarcinoma, squamous carcinoma and small cell carcinoma, respectively. Mean f values were (59.75±16.37) %, (47.41±18.69) % and (48.96±19.88) % for adenocarcinoma, squamous carcinoma and small cell carcinoma, respectively. Mean α values were 0.72±0.13 for adenocarcinoma, 0.62±0.12 for squamous carcinoma, and 0.63±0.11 for small cell carcinoma, respectively. Statistical analyses indicated that the sADC, f and α values among different histopathological subtypes were significantly different (P<0.05), while there was no significant difference in D, D(*) and DDC values (P>0.05). Furthermore, the comparison showed that the sADC, f and α values of patients with adenocarcinoma were significantly higher than those with squamous carcinoma or small cell carcinoma (P<0.05), whereas there was no significant difference between squamous carcinoma group and small cell carcinoma group (P>0.05). Conclusions: The sADC, f and α values derived from the IVIM-DWI model can be used for comprehensive non-invasive evaluation of diffusion, perfusion and heterogeneity of microenvironment in lung cancer patients. And the IVIM-DWI model may be a promising tool for predicting histopathological subtypes of lung cancer.

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.

Genotyping-by-sequencing targeting of a novel downy mildew resistance gene Pl 20 from wild Helianthus argophyllus for sunflower (Helianthus annuus L.).

KEY MESSAGE: Genotyping-by-sequencing revealed a new downy mildew resistance gene, Pl 20 , from wild Helianthus argophyllus located on linkage group 8 of the sunflower genome and closely linked to SNP markers that facilitate the marker-assisted selection of resistance genes. Downy mildew (DM), caused by Plasmopara halstedii, is one of the most devastating and yield-limiting diseases of sunflower. Downy mildew resistance identified in wild Helianthus argophyllus accession PI 494578 was determined to be effective against the predominant and virulent races of P. halstedii occurring in the United States. The evaluation of 114 BC1F2:3 families derived from the cross between HA 89 and PI 494578 against P. halstedii race 734 revealed that single dominant gene controls downy mildew resistance in the population. Genotyping-by-sequencing analysis conducted in the BC1F2 population indicated that the DM resistance gene derived from wild H. argophyllus PI 494578 is located on the upper end of the linkage group (LG) 8 of the sunflower genome, as was determined single nucleotide polymorphism (SNP) markers associated with DM resistance. Analysis of 11 additional SNP markers previously mapped to this region revealed that the resistance gene, named Pl 20 , co-segregated with four markers, SFW02745, SFW09076, S8_11272025, and S8_11272046, and is flanked by SFW04358 and S8_100385559 at an interval of 1.8 cM. The newly discovered P. halstedii resistance gene has been introgressed from wild species into cultivated sunflower to provide a novel gene with DM resistance. The homozygous resistant individuals were selected from BC2F2 progenies with the use of markers linked to the Pl 20 gene, and these lines should benefit the sunflower community for Helianthus improvement.

Discovery and introgression of the wild sunflower-derived novel downy mildew resistance gene Pl 19 in confection sunflower (Helianthus annuus L.).

KEY MESSAGE: A new downy mildew resistance gene, Pl 19 , was identified from wild Helianthus annuus accession PI 435414, introduced to confection sunflower, and genetically mapped to linkage group 4 of the sunflower genome. Wild Helianthus annuus accession PI 435414 exhibited resistance to downy mildew, which is one of the most destructive diseases to sunflower production globally. Evaluation of the 140 BC1F2:3 families derived from the cross of CMS CONFSCLB1 and PI 435414 against Plasmopara halstedii race 734 revealed that a single dominant gene controls downy mildew resistance in the population. Bulked segregant analysis conducted in the BC1F2 population with 860 simple sequence repeat (SSR) markers indicated that the resistance derived from wild H. annuus was associated with SSR markers located on linkage group (LG) 4 of the sunflower genome. To map and tag this resistance locus, designated Pl 19 , 140 BC1F2 individuals were used to construct a linkage map of the gene region. Two SSR markers, ORS963 and HT298, were linked to Pl 19 within a distance of 4.7 cM. After screening 27 additional single nucleotide polymorphism (SNP) markers previously mapped to this region, two flanking SNP markers, NSA_003564 and NSA_006089, were identified as surrounding the Pl 19 gene at a distance of 0.6 cM from each side. Genetic analysis indicated that Pl 19 is different from Pl 17 , which had previously been mapped to LG4, but is closely linked to Pl 17 . This new gene is highly effective against the most predominant and virulent races of P. halstedii currently identified in North America and is the first downy mildew resistance gene that has been transferred to confection sunflower. The selected resistant germplasm derived from homozygous BC2F3 progeny provides a novel gene for use in confection sunflower breeding programs.

An innovative SNP genotyping method adapting to multiple platforms and throughputs.

KEY MESSAGE: An innovative genotyping method designated as semi-thermal asymmetric reverse PCR (STARP) was developed for genotyping individual SNPs with improved accuracy, flexible throughputs, low operational costs, and high platform compatibility. Multiplex chip-based technology for genome-scale genotyping of single nucleotide polymorphisms (SNPs) has made great progress in the past two decades. However, PCR-based genotyping of individual SNPs still remains problematic in accuracy, throughput, simplicity, and/or operational costs as well as the compatibility with multiple platforms. Here, we report a novel SNP genotyping method designated semi-thermal asymmetric reverse PCR (STARP). In this method, genotyping assay was performed under unique PCR conditions using two universal priming element-adjustable primers (PEA-primers) and one group of three locus-specific primers: two asymmetrically modified allele-specific primers (AMAS-primers) and their common reverse primer. The two AMAS-primers each were substituted one base in different positions at their 3' regions to significantly increase the amplification specificity of the two alleles and tailed at 5' ends to provide priming sites for PEA-primers. The two PEA-primers were developed for common use in all genotyping assays to stringently target the PCR fragments generated by the two AMAS-primers with similar PCR efficiencies and for flexible detection using either gel-free fluorescence signals or gel-based size separation. The state-of-the-art primer design and unique PCR conditions endowed STARP with all the major advantages of high accuracy, flexible throughputs, simple assay design, low operational costs, and platform compatibility. In addition to SNPs, STARP can also be employed in genotyping of indels (insertion-deletion polymorphisms). As vast variations in DNA sequences are being unearthed by many genome sequencing projects and genotyping by sequencing, STARP will have wide applications across all biological organisms in agriculture, medicine, and forensics.

Genetics and mapping of a novel downy mildew resistance gene, Pl(18), introgressed from wild Helianthus argophyllus into cultivated sunflower (Helianthus annuus L.).

KEY MESSAGE: A novel downy mildew resistance gene, Pl(18), was introgressed from wild Helianthus argophyllus into cultivated sunflower and genetically mapped to linkage group 2 of the sunflower genome. The new germplasm, HA-DM1, carrying Pl(18) has been released to the public. Sunflower downy mildew (DM) is considered to be the most destructive foliar disease that has spread to every major sunflower-growing country of the world, except Australia. A new dominant downy mildew resistance gene (Pl 18) transferred from wild Helianthus argophyllus (PI 494573) into cultivated sunflower was mapped to linkage group (LG) 2 of the sunflower genome using bulked segregant analysis with 869 simple sequence repeat (SSR) markers. Phenotyping 142 BC1F2:3 families derived from the cross of HA 89 and H. argophyllus confirmed the single gene inheritance of resistance. Since no other Pl gene has been mapped to LG2, this gene was novel and designated as Pl (18). SSR markers CRT214 and ORS203 flanked Pl(18) at a genetic distance of 1.1 and 0.4 cM, respectively. Forty-six single nucleotide polymorphism (SNP) markers that cover the Pl(18) region were surveyed for saturation mapping of the region. Six co-segregating SNP markers were 1.2 cM distal to Pl(18), and another four co-segregating SNP markers were 0.9 cM proximal to Pl(18). The new BC2F4-derived germplasm, HA-DM1, carrying Pl(18) has been released to the public. This new line is highly resistant to all Plasmopara halstedii races identified in the USA providing breeders with an effective new source of resistance against downy mildew in sunflower. The molecular markers that were developed will be especially useful in marker-assisted selection and pyramiding of Pl resistance genes because of their close proximity to the gene and the availability of high-throughput SNP detection assays.

Relocation of a rust resistance gene R 2 and its marker-assisted gene pyramiding in confection sunflower (Helianthus annuus L.).

KEY MESSAGE: The rust resistance gene R 2 was reassigned to linkage group 14 of the sunflower genome. DNA markers linked to R 2 were identified and used for marker-assisted gene pyramiding in a confection type genetic background. Due to the frequent evolution of new pathogen races, sunflower rust is a recurring threat to sunflower production worldwide. The inbred line Morden Cross 29 (MC29) carries the rust resistance gene, R 2 , conferring resistance to numerous races of rust fungus in the US, Canada, and Australia, and can be used as a broad-spectrum resistance resource. Based on phenotypic assessments and SSR marker analyses on the 117 F2 individuals derived from a cross of HA 89 with MC29 (USDA), R 2 was mapped to linkage group (LG) 14 of the sunflower, and not to the previously reported location on LG9. The closest SSR marker HT567 was located at 4.3 cM distal to R 2 . Furthermore, 36 selected SNP markers from LG14 were used to saturate the R 2 region. Two SNP markers, NSA_002316 and SFW01272, flanked R 2 at a genetic distance of 2.8 and 1.8 cM, respectively. Of the three closely linked markers, SFW00211 amplified an allele specific for the presence of R 2 in a marker validation set of 46 breeding lines, and SFW01272 was also shown to be diagnostic for R 2 . These newly developed markers, together with the previously identified markers linked to the gene R 13a , were used to screen 524 F2 individuals from a cross of a confection R 2 line and HA-R6 carrying R 13a . Eleven homozygous double-resistant F2 plants with the gene combination of R 2 and R 13a were obtained. This double-resistant line will be extremely useful in confection sunflower, where few rust R genes are available, risking evolution of new virulence phenotypes and further disease epidemics.

Pl(17) is a novel gene independent of known downy mildew resistance genes in the cultivated sunflower (Helianthus annuus L.).

KEY MESSAGE: Pl 17, a novel downy mildew resistance gene independent of known downy mildew resistance genes in sunflowers, was genetically mapped to linkage group 4 of the sunflower genome. Downy mildew (DM), caused by Plasmopara halstedii (Farl.). Berl. et de Toni, is one of the serious sunflower diseases in the world due to its high virulence and the variability of the pathogen. DM resistance in the USDA inbred line, HA 458, has been shown to be effective against all virulent races of P. halstedii currently identified in the USA. To determine the chromosomal location of this resistance, 186 F 2:3 families derived from a cross of HA 458 with HA 234 were phenotyped for their resistance to race 734 of P. halstedii. The segregation ratio of the population supported that the resistance was controlled by a single dominant gene, Pl 17. Simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) primers were used to identify molecular markers linked to Pl 17. Bulked segregant analysis using 849 SSR markers located Pl 17 to linkage group (LG) 4, which is the first DM gene discovered in this linkage group. An F2 population of 186 individuals was screened with polymorphic SSR and SNP primers from LG4. Two flanking markers, SNP SFW04052 and SSR ORS963, delineated Pl 17 in an interval of 3.0 cM. The markers linked to Pl 17 were validated in a BC3 population. A search for the physical location of flanking markers in sunflower genome sequences revealed that the Pl 17 region had a recombination frequency of 0.59 Mb/cM, which was a fourfold higher recombination rate relative to the genomic average. This region can be considered amenable to molecular manipulation for further map-based cloning of Pl 17.

Sequence organization and evolutionary dynamics of Brachypodium-specific centromere retrotransposons.

Brachypodium distachyon is a wild annual grass belonging to the Pooideae, more closely related to wheat, barley, and forage grasses than rice and maize. As an experimental model, the completed genome sequence of B. distachyon provides a unique opportunity to study centromere evolution during the speciation of grasses. Centromeric satellite sequences have been identified in B. distachyon, but little is known about centromeric retrotransposons in this species. In the present study, bacterial artificial chromosome (BAC)-fluorescence in situ hybridization was conducted in maize, rice, barley, wheat, and rye using B. distachyon (Bd) centromere-specific BAC clones. Eight Bd centromeric BAC clones gave no detectable fluorescence in situ hybridization (FISH) signals on the chromosomes of rice and maize, and three of them also did not yield any FISH signals in barley, wheat, and rye. In addition, four of five Triticeae centromeric BAC clones did not hybridize to the B. distachyon centromeres, implying certain unique features of Brachypodium centromeres. Analysis of Brachypodium centromeric BAC sequences identified a long terminal repeat (LTR)-centromere retrotransposon of B. distachyon (CRBd1). This element was found in high copy number accounting for 1.6 % of the B. distachyon genome, and is enriched in Brachypodium centromeric regions. CRBd1 accumulated in active centromeres, but was lost from inactive ones. The LTR of CRBd1 appears to be specific to B. distachyon centromeres. These results reveal different evolutionary events of this retrotransposon family across grass species.

Molecular tagging of a novel rust resistance gene R(12) in sunflower (Helianthus annuus L.).

Sunflower production in North America has recently suffered economic losses in yield and seed quality from sunflower rust (Puccinia helianthi Schwein.) because of the increasing incidence and lack of resistance to new rust races. RHA 464, a newly released sunflower male fertility restorer line, is resistant to both of the most predominant and most virulent rust races identified in the Northern Great Plains of the USA. The gene conditioning rust resistance in RHA 464 originated from wild Helianthus annuus L., but has not been molecularly marked or determined to be independent from other rust loci. The objectives of this study are to identify molecular markers linked to the rust resistance gene and to investigate the allelism of this gene with the unmapped rust resistance genes present in HA-R6, HA-R8 and RHA 397. Virulence phenotypes of seedlings for the F(2) population and F(2:3) families suggested that a single dominant gene confers rust resistance in RHA 464, and this gene was designated as R(12). Bulked segregant analysis identified ten markers polymorphic between resistant and susceptible bulks. In subsequent genetic mapping, the ten markers covered 33.4 cM of genetic distance on linkage group 11 of sunflower. A co-dominant marker CRT275-11 is the closest marker distal to R(12) with a genetic distance of 1.0 cM, while ZVG53, a dominant marker linked in the repulsion phase, is proximal to R(12) with a genetic distance of 9.6 cM. The allelism test demonstrated that R(12) is not allelic to the rust resistance genes in HA-R6, HA-R8 and RHA 397, and it is also not linked to any previously mapped rust resistance genes. Discovery of the R(12) novel rust resistance locus in sunflower and associated markers will potentially support the molecular marker-assisted introgression and pyramiding of R(12) into sunflower breeding lines.

Genetic mapping of rust resistance genes in confection sunflower line HA-R6 and oilseed line RHA 397.

Few widely effective resistance sources to sunflower rust, incited by Puccinia helianthi Schwein., have been identified in confection sunflower (Helianthus annuus L.). The USDA inbred line HA-R6 is one of the few confection sunflower lines resistant to rust. A previous allelism test indicated that rust resistance genes in HA-R6 and RHA 397, an oilseed-type restorer line, are either allelic or closely linked; however, neither have been characterized nor molecularly mapped. The objectives of this study are (1) to locate the rust resistance genes in HA-R6 and RHA 397 on a molecular map, (2) to develop closely linked molecular markers for rust resistance diagnostics, and (3) to determine the resistance spectrum of two lines when compared with other rust-resistant lines. Two populations of 140 F2:3 families each from the crosses of HA 89, as susceptible parent, with HA-R6 and RHA 397 were inoculated with race 336 of P. helianthi in the greenhouse. The resistance genes (R-genes) in HA-R6 and RHA 397 were molecularly mapped to the lower end of linkage group 13, which encompasses a large R-gene cluster, and were designated as R 13a and R 13b, respectively. In the initial maps, SSR (simple sequence repeat) and InDel (insertion and deletion) markers revealed 2.8 and 8.2 cM flanking regions for R 13a and R 13b, respectively, linked with a common marker set of four co-segregating markers, ORS191, ORS316, ORS581, and ZVG61, in the distal side and one marker ORS464 in the proximal side. To identify new markers closer to the genes, sunflower RGC (resistance gene candidate) markers linked to the downy mildew R-gene Pl 8 and located at the same region as R 13a and R 13b were selected to screen the two F2 populations. The RGC markers RGC15/16 and a newly developed marker SUN14 designed from a BAC contig anchored by RGC251 further narrowed down the region flanking R 13a and R 13b to 1.1 and 0.1 cM, respectively. Both R 13a and R 13b are highly effective against all rust races tested so far. Our newly developed molecular markers will facilitate breeding efforts to pyramid the R 13 genes with other rust R-genes and accelerate the development of rust-resistant sunflower hybrids in both confection and oilseed sunflowers.

Genetics and mapping of the R11 gene conferring resistance to recently emerged rust races, tightly linked to male fertility restoration, in sunflower (Helianthus annuus L.).

Sunflower oil is one of the major sources of edible oil. As the second largest hybrid crop in the world, hybrid sunflowers are developed by using the PET1 cytoplasmic male sterility system that contributes to a 20 % yield advantage over the open-pollinated varieties. However, sunflower production in North America has recently been threatened by the evolution of new virulent pathotypes of sunflower rust caused by the fungus Puccinia helianthi Schwein. Rf ANN-1742, an 'HA 89' backcross restorer line derived from wild annual sunflower (Helianthus annuus L.), was identified as resistant to the newly emerged rust races. The aim of this study was to elucidate the inheritance of rust resistance and male fertility restoration and identify the chromosome location of the underlying genes in Rf ANN-1742. Chi-squared analysis of the segregation of rust response and male fertility in F(2) and F(3) populations revealed that both traits are controlled by single dominant genes, and that the rust resistance gene is closely linked to the restorer gene in the coupling phase. The two genes were designated as R ( 11 ) and Rf5, respectively. A set of 723 mapped SSR markers of sunflower was used to screen the polymorphism between HA 89 and the resistant plant. Bulked segregant analysis subsequently located R ( 11 ) on linkage group (LG) 13 of sunflower. Based on the SSR analyses of 192 F(2) individuals, R ( 11 ) and Rf5 both mapped to the lower end of LG13 at a genetic distance of 1.6 cM, and shared a common marker, ORS728, which was mapped 1.3 cM proximal to Rf5 and 0.3 cM distal to R ( 11 ) (Rf5/ORS728/R ( 11 )). Two additional SSRs were linked to Rf5 and R ( 11 ): ORS995 was 4.5 cM distal to Rf5 and ORS45 was 1.0 cM proximal to R ( 11 ). The advantage of such an introduced alien segment harboring two genes is its large phenotypic effect and simple inheritance, thereby facilitating their rapid deployment in sunflower breeding programs. Suppressed recombination was observed in LGs 2, 9, and 11 as it was evident that no recombination occurred in the introgressed regions of LGs 2, 9, and 11 detected by 5, 9, and 22 SSR markers, respectively. R ( 11 ) is genetically independent from the rust R-genes R ( 1 ), R ( 2 ), and R ( 5 ), but may be closely linked to the rust R-gene R ( adv ) derived from wild Helianthus argophyllus, forming a large rust R-gene cluster of R ( adv )/R ( 11 )/R ( 4 ) in the lower end of LG13. The relationship of Rf5 with Rf1 is discussed based on the marker association analysis.

Genetic compensation abilities of Aegilops speltoides chromosomes for homoeologous B-genome chromosomes of polyploid wheat in disomic S(B) chromosome substitution lines.

The S genome of Aegilops speltoides is closely related to the B and G genomes of polyploid wheats. However, little work has been reported on the genetic relationships between the S-genome and B-genome chromosomes of polyploid wheat. Here, we report the isolation of a set of disomic substitutions (DS) of S-genome chromosomes for the B-genome chromosomes and their effects on gametophytic and sporophytic development. Ae. speltoides chromosomes were identified by their distinct C-banding and fluorescence in situ hybridization patterns with the Ae. speltoides-derived clone pGc1R-1. Although no large structural differences between S-genome and B-genome chromosomes exist, significant differences in gametophytic compensation were observed for chromosomes 1S, 3S, 5S and 6S. Similarly, chromosomes 1S, 2S, 4S, 5S and 6S affected certain aspects of sporophytic development in relation to spike morphology, fertility and meiotic pairing. The DS5S(5B) had disturbed meiosis with univalents/multivalents and suffered chromosome elimination in the germ tissues leading to haploid spikes in 50% of the plants. The effect of the Ph1 gene on meiosis is well known, and these results provide evidence for the role of Ph1 in the maintenance of polyploid genome integrity. These and other data are discussed in relation to the structural and functional differentiation of S- and B-genome chromosomes and the practical utility of the stocks in wheat improvement.