Whole-genome sequencing of allotetraploid bermudagrass reveals the origin of Cynodon and candidate genes for salt tolerance.

Bermudagrass (Cynodon dactylon) is a globally distributed, extensively used warm-season turf and forage grass with high tolerance to salinity and drought stress in alkaline environments. However, the origin of the species and genetic mechanisms for salinity tolerance in the species are basically unknown. Accordingly, we set out to study evolution divergence events in the Cynodon genome and to identify genes for salinity tolerance. We developed a 604.0 Mb chromosome-level polyploid genome sequence for bermudagrass 'A12359' (n = 18). The C. dactylon genome comprises 2 complete sets of homoeologous chromosomes, each with approximately 30 000 genes, and most genes are conserved as syntenic pairs. Phylogenetic study showed that the initial Cynodon species diverged from Oropetium thomaeum approximately 19.7-25.4 million years ago (Mya), the A and B subgenomes of C. dactylon diverged approximately 6.3-9.1 Mya, and the bermudagrass polyploidization event occurred 1.5 Mya on the African continent. Moreover, we identified 82 candidate genes associated with seven agronomic traits using a genome-wide association study, and three single-nucleotide polymorphisms were strongly associated with three salt resistance genes: RAP2-2, CNG channels, and F14D7.1. These genes may be associated with enhanced bermudagrass salt tolerance. These bermudagrass genomic resources, when integrated, may provide fundamental insights into evolution of diploid and tetraploid genomes and enhance the efficacy of comparative genomics in studying salt tolerance in Cynodon.

Comparative analysis reveals chromosome number reductions in the evolution of African bermudagrass (Cynodon transvaalensis Burtt-Davy).

African bermudagrass (Cynodon transvaalensis Burtt-Davy) (2n = 2x = 18) belongs to the genus Cynodon, tribe Cynodonteae, subfamily Chloridoideae in the grass family Poaceae. The species is frequently crossed with common bermudagrass (Cynodon dactylon Pers.) in developing high-quality hybrid turf cultivars. Molecular resources for C. transvaalensis are scarce; thus, its genomic evolution is unknown. Recently, a linkage map consisting of 1278 markers provided a powerful tool for African bermudagrass genomic research. The objective of this study was to investigate chromosome number reduction events that resulted in the nine haploid chromosomes in this species. Tag sequences of mapped single nucleotide polymorphism markers in C. transvaalensis were compared against genome sequences of Oropetium thomaeum (L.f.) Trin. (2n = 2x = 20), a genomic model in the Cynodonteae tribe. The comparative genomic analyses revealed broad collinearity between the genomes of these two species. The analyses further revealed that two major interchromosomal rearrangements of the paleochromosome ρ12 (ρ1-ρ12-ρ1 and ρ6-ρ12-ρ6) resulted in nine chromosomes in the genome of C. transvaalensis. The findings provide novel information regarding the formation of the initial diploid species in the Cynodon genus.

Development and deployment of KASP markers for multiple alleles of Lr34 in wheat.

KEY MESSAGE: Heterogeneous Lr34 genes for leaf rust in winter wheat cultivar 'Duster' and KASP markers for allelic variation in exon 11 and exon 22 of Lr34. Wheat, Triticum aestivum (2n = 6x = 42, AABBDD), is a hexaploid species, and each of three homoeologous genomes A, B, and D should have one copy for a gene in its ancestral form if the gene has no duplication. Previously reported leaf rust resistance gene Lr34 has one copy on the short arm of chromosome 7D in hexaploid wheat, and allelic variation in Lr34 is in intron 4, exon 11, exon 12, or exon 22. In this study, we discovered that Oklahoma hard red winter wheat cultivar 'Duster' (PI 644,016) has two copies of the Lr34 gene, the resistance allele Lr34a and the susceptibility allele Lr34b. Both Lr34a and Lr34b were mapped in the same linkage group on chromosome 7D in a doubled-haploid population generated from a cross between Duster and a winter wheat cultivar 'Billings' which carries the susceptibility allele Lr34c. A chromosomal fragment including Lr34 and at least two neighboring genes on its proximal side but excluding genes on its distal side was duplicated in Duster. The Duster Lr34ab allele was associated with tip necrosis and increased resistance against leaf rust at adult plants in the Duster × Billings DH population tested in the field, demonstrating the function of the Duster Lr34ab allele in wheat. We have developed KASP markers for allelic variation in exon 11 and exon 22 of Lr34 in wheat. These markers can be utilized to accelerate the selection of Lr34 in wheat.

Nitrogen use efficiency is regulated by interacting proteins relevant to development in wheat.

Wheat (Triticum aestivum) has low nitrogen use efficiency (NUE). The genetic mechanisms controlling NUE are unknown. Positional cloning of a major quantitative trait locus for N-related agronomic traits showed that the vernalization gene TaVRN-A1 was tightly linked with TaNUE1, the gene shown to influence NUE in wheat. Because of an Ala180 /Val180 substitution, TaVRN-A1a and TaVRN-A1b proteins interact differentially with TaANR1, a protein encoded by a wheat orthologue of Arabidopsis nitrate regulated 1 (ANR1). The transcripts of both TaVRN-A1 and TaANR1 were down-regulated by nitrogen. TaANR1 was functionally characterized in TaANR1::RNAi transgenic wheat, and in a natural mutant with a 23-bp deletion including 10-bp at the 5' end of intron 5 and 13-bp of exon 6 in gDNA sequence in its gDNA sequence, which produced transcript that lacked the full 84-bp exon 6. Both TaANR1 and TaHOX1 bound to the Ala180 /Val180 position of TaVRN-A1. Genetically incorporating favourable alleles from TaVRN-A1, TaANR1 and TaHOX1 increased grain yield from 9.84% to 11.58% in the field. Molecular markers for allelic variation of the genes that regulate nitrogen can be used in breeding programmes aimed at improving NUE and yield in novel wheat cultivars.

Vernalization requirement duration in winter wheat is controlled by TaVRN-A1 at the protein level.

Winter wheat requires a period of low temperatures to accelerate flowering (vernalization). This requirement could make winter wheat more vulnerable to elevated global temperature via insufficient vernalization. All known vernalization genes are cloned according to qualitative variation in vernalization requirement between spring and winter wheat, but the genes controlling quantitative variation for more or less vernalization requirement among winter wheat cultivars remain unknown. We report here that the gene for the vernalization requirement duration in winter wheat was cloned using a BC(1)F(2:3) population that segregated in a 3:1 ratio of early-flowering plants and late-flowering plants after vernalization for 3 weeks. The positional cloning of the gene for vernalization requirement duration demonstrated that this trait is controlled by TaVRN-A1 at the protein level. The Ala(180) in vrn-A1a, encoded by the dominant allele for 3-week vernalization, was mutated to Val(180) in vrn-A1b, encoded by the recessive allele for 6-week vernalization. Further studies with in vitro protein pull-down assays and immunoprecipitation analyses indicated that the mutated Val(180) in vrn-A1b protein decreased the ability to bind with TaHOX1 (the first homeobox protein in Triticum aestivum). The direct binding of TaVRN-A1 and TaHOX1 proteins was confirmed in the nucleus of living plant cells by bimolecular fluorescence complementation (BiFC) analyses. The TaHOX1 gene was found to be upregulated by low temperatures, and to have a significant genetic effect on heading date, suggesting that TaHOX1 functions in the flowering pathway in winter wheat.

Genetic and QTL mapping in African bermudagrass.

Cynodon transvaalensis Burtt-Davy is frequently used to cross with C. dactylon Pers. in the creation of F1 hybrid cultivars that are some of the most widely used in the worldwide turf industry. However, molecular resource development in this species is limited. Accordingly, the objectives of this study were to construct a high-density genetic map, and to identify genomic regions associated with establishment rate. In this study, we constructed the first high-density linkage map for African bermudagrass using a genotyping by sequencing approach based on 109 S1 progenies. A total of 1,246 single nucleotide polymorphisms and 32 simple sequence repeat markers were integrated in the linkage map. The total length of nine linkage groups was 882.3 cM, with an average distance of 0.69 cM per interval. Four genomic regions were identified to be associated with sod establishment rate. The results provide important genetic resources towards understanding the genome as well as marker-assisted selection for improving the establishment rate in bermudagrass breeding.

A single-nucleotide polymorphism that accounts for allelic variation in the Lr34 gene and leaf rust reaction in hard winter wheat.

Leaf rust, caused by Puccinia triticina Eriks, is one of the most common and persistent wheat diseases in the US Great Plains. We report that the Lr34 gene was mapped in the center of a QTL for leaf rust reaction and explained 18-35% of the total phenotypic variation in disease severity of adult plants in a Jagger x 2174 population of recombinant inbred lines (RILs) field-tested for 3 years. The sequence of the complete Lr34 gene was determined for the susceptible Jagger allele and for the resistant 2174 allele. The two alleles had exactly the same sequence as the resistant allele reported previously in Chinese Spring at three polymorphic sites in intron 4, exon 11, and exon 12. A G/T polymorphism was found in exon 22, where a premature stop codon was found in the susceptible Jagger allele (Lr34E22s), confirming a previous report, due to a point mutation compared with the resistant 2174 allele (Lr34E22r). We have experimentally demonstrated a tight association between the point mutation at exon 22 of Lr34 and leaf rust susceptibility in a segregating biparental population. A PCR marker was developed to distinguish between the Lr34E22r and Lr34E22s alleles. A survey of 33 local hard winter wheat cultivars indicated that 7 cultivars carry the Lr34E22s allele and 26 cultivars carry the Lr34E22r allele. This study significantly improves our genetic understanding of allelic variation in the Lr34 gene and provides a functional molecular tool to improve leaf rust resistance in a major US wheat gene pool.

Sequence-based genetic mapping of Cynodon dactylon Pers. reveals new insights into genome evolution in Poaceae.

Bermudagrass (Cynodon dactylon Pers.) is an important warm-season perennial used extensively for turf, forage, soil conservation and remediation worldwide. However, limited genomic information has hindered the application of molecular tools towards understanding genome evolution and in breeding new cultivars. We genotype a first-generation selfed population derived from the tetraploid (4x = 36) 'A12359' using genotyping-by-sequencing. A high-density genetic map of 18 linkage groups (LGs) is constructed with 3,544 markers. Comparative genomic analyses reveal that each of nine homeologous LG pairs of C. dactylon corresponds to one of the first nine chromosomes of Oropetium thomaeum. Two nested paleo-ancestor chromosome fusions (ρ6-ρ9-ρ6, ρ2-ρ10-ρ2) may have resulted in a 12-to-10 chromosome reduction. A segmental dissemination of the paleo-chromosome ρ12 (ρ1-ρ12-ρ1, ρ6-ρ12-ρ6) leads to the 10-to-9 chromosome reduction in C. dactylon genome. The genetic map will assist in an ongoing whole genome sequence assembly and facilitate marker-assisted selection (MAS) in developing new cultivars.

Molecular identification of a new powdery mildew resistance gene Pm41 on chromosome 3BL derived from wild emmer (Triticum turgidum var. dicoccoides).

Powdery mildew caused by Blumeria graminis f. sp. tritici is an important wheat disease in China and other parts of the world. Wild emmer (Triticum turgidum var. dicoccoides) is the immediate progenitor of cultivated tetraploid and hexaploid wheats and thus an important resource for wheat improvement. Wild emmer accession IW2 collected from Mount Hermon, Israel, is highly resistant to powdery mildew at the seedling and adult plant stages. Genetic analysis using an F(2) segregating population and F(2:3) families, derived from a cross between susceptible durum cultivar Langdon and wild emmer accession IW2, indicated that a single dominant gene was responsible for the resistance of IW2. Bulked segregant and molecular marker analyses detected that six polymorphic SSR, one ISBP, and three EST-STS markers on chromosome 3BL bin 0.63-1.00 were linked to the resistance gene. Allelic variations of resistance-linked EST-STS marker BE489472 revealed that the allele was present only in wild emmer but absent in common wheat. Segregation distortion was observed for the powdery mildew resistance allele and its linked SSR markers with preferential transmission of Langdon alleles over IW2 alleles. The resistance gene was introgressed into common wheat by backcrossing and marker-assisted selection. Since no designated powdery mildew resistance gene has been found on chromosome 3BL, the resistance gene derived from wild emmer accession IW2 appears to be new one and was consequently designated Pm41.

Mis-Spliced Lr34 Transcript Events in Winter Wheat.

Lr34 in wheat is a non-race-specific gene that confers resistance against multiple fungal pathogens. The resistant allele Lr34 and the susceptible allele Lr34s can be distinguished by three polymorphisms that cause alternation of deduced amino acid sequences of Lr34 at the protein level. In seedlings of a cultivar carrying the resistant Lr34r allele, only a portion (35%) of its transcripts was correctly spliced and the majority (65%) of its transcripts were incorrectly spliced due to multiple mis-splicing events. Lr34 mis-splicing events were also observed at adult plant age when this gene exerts its function. All of the mis-spliced Lr34r cDNA transcripts observed in this study resulted in a premature stop codon due to a shift of the open reading frame; hence, the mis-spliced Lr34r cDNAs were deduced to encode incomplete proteins. Even if a cultivar has a functional Lr34 gene, its transcripts might not completely splice in a correct pattern. These findings suggested that the partial resistance conferred by a quantitative gene might be due to mis-splicing events in its transcripts; hence, the resistance of the gene could be increased by eliminating or mutating regulators that cause mis-splicing events in wheat.

Quantitative Trait Loci Mapping for Tillering-Related Traits in Two Switchgrass Populations.

Plant tillering and related traits are morphologically important components contributing to switchgrass ( L.) biomass yield. The objectives of this study were to estimate broad-sense heritabilities for tillering-related traits, to analyze correlations between biomass yield and the traits, and to identify quantitative trait loci (QTL) for them. A first-generation selfed population of NL94 plant and a hybrid population between NL94 and SL93 plants were field established in a randomized complete block design with three replications in Stillwater and Perkins, OK. Phenotypic data were collected in 2 yr and genotypic data were obtained by genotyping simple-sequence repeat (SSR) markers in the two populations on the basis of two preexisting genetic maps. Plant base size (PBS), plant girth (PG), tillering ability (TA), tiller diameter (TD), and tiller dry weight (TDW) were positively correlated with biomass yield in both populations. Consistently, PBS had the largest correlation coefficients for biomass yield, suggesting its value as an indirect selection criterion for biomass yield. Twenty and 26 QTL for six tillering-related traits were detected in the hybrid and selfed population, respectively. Among the QTL, one on linkage group (LG) 5a between sww-2387/PVCAG-2197/2198 and PVGA-1649/1650 for PBS, PG, and TA and another on LG 2a between sww-2640/sww-2545 and PVCA-765/766 for TD and TDW were stably detected in multiple environments in the two populations. The findings add to the knowledge base regarding the genetics of tillering-related traits that could be used in accelerating the development of high-yielding cultivars through marker-assisted selection.