Characterization of a new greenbug resistance gene Gb9 in a synthetic hexaploid wheat.

Greenbug [Schizaphis graminum (Rondani)] is a serious insect pest that not only damages cereal crops, but also transmits several destructive viruses. The emergence of new greenbug biotypes in the field makes it urgent to identify novel greenbug resistance genes in wheat. CWI 76364 (PI 703397), a synthetic hexaploid wheat (SHW) line, exhibits greenbug resistance. Evaluation of an F2:3 population from cross OK 14319 × CWI 76364 indicated that a dominant gene, designated Gb9, conditions greenbug resistance in CWI 76364. Selective genotyping of a subset of F2 plants with contrasting phenotypes by genotyping-by-sequencing identified 25 SNPs closely linked to Gb9 on chromosome arm 7DL. Ten of these SNPs were converted to Kompetitive allele-specific polymerase chain reaction (KASP) markers for genotyping the entire F2 population. Genetic analysis delimited Gb9 to a 0.6-Mb interval flanked by KASP markers located at 599,835,668 bp (Stars-KASP872) and 600,471,081 bp (Stars-KASP881) on 7DL. Gb9 was 0.5 cM distal to Stars-KASP872 and 0.5 cM proximal to Stars-KASP881. Allelism tests indicated that Gb9 is a new greenbug resistance gene which confers resistance to greenbug biotypes C, E, H, I, and TX1. TX1 is one of the most widely virulent biotypes and has overcome most known wheat greenbug resistance genes. The introgression of Gb9 into locally adapted wheat cultivars is of economic importance, and the KASP markers developed in this study can be used to tag Gb9 in cultivar development.

Characterization of Quantitative Trait Loci for Leaf Rust Resistance in the Uzbekistani Wheat Landrace Teremai Bugdai.

Leaf rust, caused by Puccinia triticina, is a major cause of wheat yield losses globally, and novel leaf rust resistance genes are needed to enhance wheat leaf rust resistance. Teremai Bugdai is a landrace from Uzebekistan that is highly resistant to many races of P. triticina in the United States. To unravel leaf rust resistance loci in Teremai Bugdai, a recombinant inbred line (RIL) population of Teremai Bugdai × TAM 110 was evaluated for response to P. triticina race Pt54-1 (TNBGJ) and genotyped using single nucleotide polymorphism (SNP) markers generated by genotyping-by-sequencing (GBS). Quantitative trait loci (QTL) analysis using 5,130 high-quality GBS-SNPs revealed three QTLs, QLr-Stars-2DS, QLr-Stars-6BL, and QLr.Stars-7BL, for leaf rust resistance in two experiments. QLr-Stars-2DS, which is either a new Lr2 allele or a new resistance locus, was delimited to an ∼19.47-Mb interval between 46.4 and 65.9 Mb on 2DS and explained 31.3 and 33.2% of the phenotypic variance in the two experiments. QLr-Stars-6BL was mapped in an ∼84.0-kb interval between 719.48 and 719.56 Mb on 6BL, accounting for 33 to 36.8% of the phenotypic variance in two experiments. QLr.Stars-7BL was placed in a 350-kb interval between 762.41 and 762.76 Mb on 7BL and explained 4.4 to 5.3% of the phenotypic variance. Nine GBS-SNPs flanking these QTLs were converted to kompetitive allele specific PCR (KASP) markers, and these markers can be used to facilitate their introgression into locally adapted wheat lines.

Identification of a new Rsg1 allele conferring resistance to multiple greenbug biotypes from barley accessions PI 499276 and PI 566459.

Greenbug [Schizaphis graminum (Rondani)] is a major insect pest that significantly affects barley production worldwide. The identification of novel greenbug resistance genes is crucial for sustainable barley production and global food security. To identify greenbug resistance genes from a US breeding line PI 499276 and a Chinese cultivar PI 566459, two F6:7 recombinant inbred line (RIL) populations developed from crosses Weskan × PI 499276 and Weskan × PI 566459 were phenotyped for responses to greenbug biotype E and genotyped using genotyping-by-sequencing (GBS). Linkage analysis using single nucleotide polymorphism and kompetitive allele-specific polymorphism (KASP) markers delimited the greenbug resistance genes from PI 499276 and PI 566459 to a 1.2 Mb genomic region between 666.5 and 667.7 Mb on the long arm of chromosome 3H in the Morex Hordeum vulgare r1 reference sequence. Allelism tests based on responses of four F2 populations to greenbug biotype E indicated that the greenbug resistance gene in PI 499276 and PI 566459 is either allelic or very close to Rsg1. Given that PI 499276 and PI 566459 shared the same unique resistance pattern to a set of 14 greenbug biotypes, which is different from those of other Rsg1 alleles, they carry a new Rsg1 allele. The greenbug resistance genes in Post 90, PI 499276/PI 566459, and WBDC 336 were designated as Rsg1.a1, Rsg1.a2, and Rsg1.a3, respectively. KASP markers KASP-Rsg1a3-1, KASP-Rsg1a3-2, and KASP160 can be used to tag Rsg1.a2 in barley breeding.

Identification of a Novel Pm65 Allele Conferring a Wide Spectrum of Resistance to Powdery Mildew in Wheat Accession PI 351817.

Powdery mildew is caused by the highly adaptive biotrophic fungus Blumeria graminis f. sp. tritici infecting wheat worldwide. Novel powdery mildew resistance genes are urgently needed that can be used rapidly in wheat cultivar development with minimal disruption of trait advances elsewhere. PI 351817 is a German cultivar exhibiting a wide spectrum of resistance to B. graminis f. sp. tritici isolates collected from different wheat-growing regions of the United States. Evaluation of an F2 population and 237 F2:3 lines derived from OK1059060-2C14 × PI 351817 for responses to B. graminis f. sp. tritici isolate OKS(14)-B-3-1 identified a single dominant gene, designated Pm351817, for powdery mildew resistance in PI 351817. Using bulked segregant analysis (BSA) and simple sequence repeat (SSR) markers, Pm351817 was mapped in the terminal region of the long arm of chromosome 2A. Deep sequencing of the genotyping-by-sequencing libraries of the two parental lines identified a set of single-nucleotide polymorphism (SNP) markers in the 2AL candidate gene region. Those SNP markers was subsequently converted to Kompetitive allele-specific PCR (KASP) markers for genotyping the mapping population. Linkage analysis delimited Pm351817 to a 634-kb interval between Stars-KASP656 (771,207,512 bp) and Stars-KASP662 (771,841,609 bp) on 2AL, based on the Chinese Spring reference sequence IWGSC RefSeq v 2.1. Tests of allelism indicated that Pm351817 is located at the Pm65 locus. Pm351817 shows resistance to all B. graminis f. sp. tritici isolates used in this study and can be used to enhance powdery mildew resistance in the United States. KASP markers flanking Pm351817 can be used to select Pm351817 in wheat breeding programs after further tests for polymorphism.

Quantitative trait loci for rolled leaf in a wheat EMS mutant from Jagger.

KEY MESSAGE: Two QTLs with major effects on rolled leaf trait were consistently detected on chromosomes 1A (QRl.hwwg-1AS) and 5A (QRl.hwwg-5AL) in the field experiments. Rolled leaf (RL) is a morphological strategy to protect plants from dehydration under stressed field conditions. Identification of quantitative trait loci (QTLs) underlining RL is essential to breed drought-tolerant wheat cultivars. A mapping population of 154 recombinant inbred lines was developed from the cross between JagMut1095, a mutant of Jagger, and Jagger to identify quantitative trait loci (QTLs) for the RL trait. A linkage map of 3106 cM was constructed with 1003 unique SNPs from 21 wheat chromosomes. Two consistent QTLs were identified for RL on chromosomes 1A (QRl.hwwg-1AS) and 5A (QRl.hwwg-5AL) in all field experiments. QRl.hwwg-1AS explained 24-56% of the phenotypic variation and QRl.hwwg-5AL explained up to 20% of the phenotypic variation. The combined percent phenotypic variation associated with the two QTLs was up to 61%. Analyses of phenotypic and genotypic data of recombinants generated from heterogeneous inbred families of JagMut1095 × Jagger delimited QRl.hwwg-1AS to a 6.04 Mb physical interval. This work lays solid foundation for further fine mapping and map-based cloning of QRl.hwwg-1AS.

Characterization of Rsg3, a novel greenbug resistance gene from the Chinese barley landrace PI 565676.

Greenbug (Schizaphis graminum Rondani) is a pest that poses a serious threat to cereal production worldwide. Yield losses caused by greenbug are predicted to increase because of global warming. To date, only a few barley (Hordeum vulgare L.) greenbug resistance genes have been reported and new genes are urgently needed because of the continuous occurrence of novel greenbug biotypes. PI 565676, a landrace collected from Henan province of China, exhibits high resistance to several predominant greenbug biotypes. An F6:7 recombinant inbred line (RIL) population derived from the cross PI 565676 × 'Weskan' was evaluated for response to greenbug biotypes E and F using a standard aphid assay protocol, and a randomized complete block design with two replicates was adopted. The RIL population was genotyped using single-nucleotide polymorphisms (SNPs) markers generated by genotyping-by-sequencing (GBS). Gene mapping placed the greenbug resistance gene in PI 565676, designated Rsg3, to an interval of 93,140 bp between 667,558,306 and 667,651,446 bp on the long arm of chromosome 3H. Four high-confidence genes were annotated in this region with one encoding a leucine-rich repeat-containing protein. An allelism test indicated that Rsg3 is independent of the Rsg1 locus, with estimated recombination frequency of 12.85 ± 0.20% and genetic distance of 13.14 ± 0.21 cM between the two loci. Therefore, Rsg3 represents a new locus for greenbug resistance. Two SNPs flanking Rsg3 were converted to Kompetitive Allele Specific PCR (KASP) markers, which can be used to tag Rsg3 in barley breeding.

Identification and characterization of the novel leaf rust resistance gene Lr81 in wheat.

KEY MESSAGE: The novel, leaf rust seedling resistance gene, Lr81, was identified in a Croatian breeding line and mapped to a genomic region of less than 100 Kb on chromosome 2AS. Leaf rust, caused by Puccinia triticina, is the most common and widespread rust disease in wheat. Races of Puccinia triticina evolve rapidly in the southern Great Plains of the USA, and leaf rust resistance genes often lose effectiveness shortly after deployment in wheat production. PI 470121, a wheat breeding line developed by the University of Zagreb in Croatia, showed high resistance to Puccinia triticina races collected from Oklahoma, suggesting that PI 470121 could be a leaf rust resistance source for the southern Great Plains of the USA. Genetic analysis based on an F2 population and F2:3 families derived from the cross PI 470121 × Stardust indicated that PI 470121 carries a dominant seedling resistance gene, designated as Lr81. Linkage mapping delimited Lr81 to a genomic region of 96,148 bp flanked by newly developed KASP markers Xstars-KASP320 and Xstars-KASP323 on the short arm of chromosome 2A, spanning 67,030,206-67,132,354 bp in the Chinese Spring reference assembly (IWGSC RefSeq v1.0). Deletion bin mapping assigned Lr81 to the terminal bin 2AS-0.78-1.00. Allelism tests indicated that Lr81 is a distinctive leaf rust resistance locus with the physical order Lr65-Lr17-Lr81. Marker-assisted selection based on a set of markers closely linked to leaf rust resistance genes in PI 470121 and Stardust enabled identification of a recombinant inbred line RIL92 carrying Lr81 only. Lr81 is a valuable leaf rust resistance source that can be rapidly introgressed into locally adapted cultivars using KASP markers Xstars-KASP320 and Xstars-KASP323.

A natural variation in Ribonuclease H-like gene underlies Rht8 to confer "Green Revolution" trait in wheat.

Rht8 is a gibberellin-sensitive Reduced height (Rht) locus that has been widely used in crop wheat semi-dwarfing breeding. In this study, the authors reported the map-based cloning of Rht8 candidate gene, and confirmed that loss of Ribonuclease H-Like 1 (RNHL-D1) is responsible for Rht8 semi-dwarfing effect.

Characterization of an Incomplete Leaf Rust Resistance Gene on Chromosome 1RS and Development of KASP Markers for Lr47 in Wheat.

Leaf rust, caused by Puccinia triticina, is one of the most common wheat (Triticum aestivum) diseases in the Great Plains of the United States. A population of recombinant inbred lines from CI 17884 × 'Bainong 418' was evaluated for responses to leaf rust race Pt52-2 and genotyped using single nucleotide polymorphism (SNP) markers. Quantitative trait locus analysis identified a minor gene for resistance to leaf rust, designated QLr.stars-1RS, on the 1BL.1RS translocation segment in 'Bainong 418', and another leaf rust resistance gene, Lr47, on chromosome 7A of CI 17884. Lr47, originally identified in CI 17884 and located in a wheat-T. speltoides translocation segment 7S#1S, remains one of only a few race-specific resistance genes still effective in the Great Plains. A set of 7A-specific simple sequence repeat markers were developed and used to genotype CI 17884 and a pair of near-isogenic lines differing in the presence or absence of 7S#1S, PI 603918, and 'Pavon F76'. Haplotype analysis indicated that the estimated length of 7S#1S was 157.23 to 174.42 Megabases, accounting for ∼23% of the 7A chromosome. Two SNPs on 7S#1S and four SNPs on the 1RS chromosome arm were converted to Kompetitive allele-specific PCR (KASP) markers, which were subsequently validated in a panel of cultivars and elite breeding lines released within the last decade. Of these, one- and two-KASP markers are specific to the 1RS chromosome arm and 7S#1S, respectively, indicating that they can facilitate the introgression of Lr47 and QLr.stars-1RS into locally adapted wheat cultivars and breeding lines.

Pleiotropic QTL influencing spikelet number and heading date in common wheat (Triticum aestivum L.).

KEY MESSAGE: Three pleiotropic QTL regions associated with spikelet number and heading date were identified, with FT-A1 considered the candidate gene for QTspn/Hd.cau-7A. Spikelet number traits and heading date (HD) play key roles in yield improvement of wheat and its wide adaptation to different environments. Here, we used a Recombinant Inbred Lines population derived from a cross between Yi5029 (5029) and Nongda4332 (4332) to construct a high-density genetic linkage map and identify quantitative trait loci (QTL) associated with total spikelet number per spike (TSPN), fertile spikelet number per spike (FSPN), sterile spikelet number per spike (SSPN) and HD. A total of 22 environmentally stable QTL for TSPN, FSPN, SSPN and HD were identified. Notably, three pleiotropic QTL regions for TSPN and HD were detected on chromosomes 2A, 7A and 7D. The QTL associated with TSPN and HD on chromosome 7AS was designated QTspn/Hd.cau-7A. Furthermore, the candidate gene FT-A1 located in the region of QTspn/Hd.cau-7A had a single-nucleotide polymorphism (T-G) within the third exon, which might be the cause of diversity in spikelet number and HD between the two parents. Additionally, we developed a semi-thermal asymmetric reverse PCR (STARP) marker to analyze the geographical distribution and evolution of FT-A1 (T or G) alleles. This study contributes to our understanding of the molecular mechanisms of the four traits (TSPN, FSPN, SSPN and HD) and provides further insights into the genetic relationship between spikelet number traits and HD in wheat.

Dissection of genetic factors underlying grain size and fine mapping of QTgw.cau-7D in common wheat (Triticum aestivum L.).

KEY MESSAGE: Thirty environmentally stable QTL controlling grain size and/or plant height were identified, among which QTgw.cau-7D was delimited into the physical interval of approximately 4.4 Mb. Grain size and plant height (PHT) are important agronomic traits in wheat breeding. To dissect the genetic basis of these traits, we conducted a quantitative trait locus (QTL) analysis using recombinant inbred lines (RILs). In total, 30 environmentally stable QTL for thousand grain weight (TGW), grain length (GL), grain width (GW) and PHT were detected. Notably, one major pleiotropic QTL on chromosome arm 3DS explained the highest phenotypic variance for TGW, GL and PHT, and two stable QTL (QGw.cau-4B and QGw.cau-7D) on chromosome arms 4BS and 7DS contributed greater effects for GW. Furthermore, the stable QTL controlling grain size (QTgw.cau-7D and QGw.cau-7D) were delimited into the physical interval of approximately 4.4 Mb harboring 56 annotated genes. The elite NILs of QTgw.cau-7D increased TGW by 12.79-21.75% and GW by 4.10-8.47% across all three environments. Collectively, these results provide further insight into the genetic basis of TGW, GL, GW and PHT, and the fine-mapped QTgw.cau-7D will be an attractive target for positional cloning and marker-assisted selection in wheat breeding programs.

Correction to: Dissection of two quantitative trait loci with pleiotropic effects on plant height and spike length linked in coupling phase on the short arm of chromosome 2D of common wheat (Triticum aestivum L.).

Theoretical and Applied Genetics.

Dissection of two quantitative trait loci with pleiotropic effects on plant height and spike length linked in coupling phase on the short arm of chromosome 2D of common wheat (Triticum aestivum L.).

KEY MESSAGE: Two QTL with pleiotropic effects on plant height and spike length linked in coupling phase on chromosome 2DS were dissected, and diagnostic marker for each QTL was developed. Plant height (PHT) is a crucial trait related to plant architecture and yield potential, and dissection of its underlying genetic basis would help to improve the efficiency of designed breeding in wheat. Here, two quantitative trait loci (QTL) linked in coupling phase on the short arm of chromosome 2D with pleiotropic effects on PHT and spike length, QPht/Sl.cau-2D.1 and QPht/Sl.cau-2D.2, were separated and characterized. QPht/Sl.cau-2D.1 is a novel QTL located between SNP makers BS00022234_51 and BobWhite_rep_c63957_1472. QPht/Sl.cau-2D.2 is mapped between two SSR markers, SSR-2062 and Xgwm484, which are located on the same genomic interval as Rht8. Moreover, the diagnostic marker tightly linked with each QTL was developed for the haplotype analysis using diverse panels of wheat accessions. The frequency of the height-reduced allele of QPht/Sl.cau-2D.1 is much lower than that of QPht/Sl.cau-2D.2, suggesting that this novel QTL may be an attractive target for genetic improvement. Consistent with a previous study of Rht8, a significant difference in cell length was observed between the NILs of QPht/Sl.cau-2D.2. By contrast, there was no difference in cell length between NILs of QPht/Sl.cau-2D.1, indicating that the underlying molecular mechanism for these two QTL may be different. Collectively, these data provide a new example of QTL dissection, and the developed diagnostic markers will be useful in marker-assisted pyramiding of QPht/Sl.cau-2D.1 and/or QPht/Sl.cau-2D.2 with the other genes in wheat breeding.

Dissection of two quantitative trait loci with pleiotropic effects on plant height and spike length linked in coupling phase on the short arm of chromosome 2D of common wheat (Triticum aestivum L.).

KEY MESSAGE: Two QTL with pleiotropic effects on plant height and spike length linked in coupling phase on chromosome 2DS were dissected, and diagnostic marker for each QTL was developed. Plant height (PHT) is a crucial trait related to plant architecture and yield potential, and dissection of its underlying genetic basis would help to improve the efficiency of designed breeding in wheat. Here, two quantitative trait loci (QTL) linked in coupling phase on the short arm of chromosome 2D with pleiotropic effects on PHT and spike length, QPht/Sl.cau-2D.1 and QPht/Sl.cau-2D.2, were separated and characterized. QPht/Sl.cau-2D.1 is a novel QTL located between SNP makers BS00022234_51 and BobWhite_rep_c63957_1472. QPht/Sl.cau-2D.2 is mapped between two SSR markers, SSR-2062 and Xgwm484, which are located on the same genomic interval as Rht8. Moreover, the diagnostic marker tightly linked with each QTL was developed for the haplotype analysis using diverse panels of wheat accessions. The frequency of the height-reduced allele of QPht/Sl.cau-2D.1 is much lower than that of QPht/Sl.cau-2D.2, suggesting that this novel QTL may be an attractive target for genetic improvement. Consistent with a previous study of Rht8, a significant difference in cell length was observed between the NILs of QPht/Sl.cau-2D.2. By contrast, there was no difference in cell length between NILs of QPht/Sl.cau-2D.1, indicating that the underlying molecular mechanism for these two QTL may be different. Collectively, these data provide a new example of QTL dissection, and the developed diagnostic markers will be useful in marker-assisted pyramiding of QPht/Sl.cau-2D.1 and/or QPht/Sl.cau-2D.2 with the other genes in wheat breeding.