Publications by Colleges and Departments (MSU - Bozeman)
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Item Dosage response to reduced height‐1 (Rht‐1) loss‐of‐function mutations and characterization of slender phenotype in hexaploid wheat(Wiley, 2023-10) Ugrin, Josey M.; Hogg, Andrew C.; Tracy, Emma M.; Tillet, Brandon J.; Cook, Jason P.; Martin, John M.; Giroux, Michael J.The reduced height (Rht-1) genes in wheat (Triticum aestivum L.) are integral in controlling plant height. Previous studies in other plant species have demonstrated that loss-of-function mutations in their orthologous Rht-1 genes results in plants with a slender phenotype illustrated by increased plant heights, sterility, and a constitutive gibberellic acid (GA3) response; however, this phenotype has not been described in wheat. In this study, nonsense alleles occurring in the GRAS domain of Rht-A1, B1, and D1 were combined to create single, double, and triple Rht-1 mutants. Homozygous lines possessing none, one, two, or three Rht-1 stop mutations were grown in replicated field trials in three environments to assess agronomic traits. Germination tests to measure GA3 responsiveness and gene expression analysis via RNA-seq were also performed. Rht-1 triple mutants exhibited a slender phenotype characterized by rapid growth, elongated coleoptiles and internodes, elongated spikes, decreased tiller and spikelet number, and sterile heads. The presence of a single functional Rht-1 gene resulted in a normal phenotype. Differences in plant height among the Rht-1 double mutants, Rht-1 single mutants, and Rht-1 all wild-type dosages trended toward increased plant height with increased Rht-1 stop mutation dosage. Differences in Rht-1 homeolog gene expression did not equate to differences in plant height between the different Rht-1 stop mutations.Item Missense alleles of the HMW glutenin subunits Dx5 and Dy10 have small changes in function relative to missense changes in Puroindoline a and b(Wiley, 2022-12) Martin, John M.; Zhang, Jinrui; Hogg, Andrew C.; Giroux, Michael J.Background and Objectives. The high molecular weight glutenin subunit genes (HMW-GS) in wheat (Triticum aestivum L.) play a key role in determining dough functionality. More specifically allelic variation for the Glu-D1 subunit loci is consistently associated with bread making quality. Since much of the hard wheat germplasm is fixed for the more favorable 1Dx5 + 1Dy10 haplotype, our goal was to identify allelic variation in 1Dx5 and 1Dy10 HMW-GS genes using an ethyl methanesulfonate (EMS) mutagenized population in the soft white spring wheat cultivar “Alpowa.” The same source population was previously used in screening for creating novel alleles in Puroindoline a (Pina) and Puroindoline b (Pinb) which are responsible for grain hardness variation. Direct sequencing of 384 M3 families identified 135 point mutations equally dispersed across 1Dx5 and 1Dy10. The mutation discovery rate was 1/12.7 kb of DNA, equal to that found in the Pin loci. Mutation carrying plants were crossed to the nonmutagenized Alpowa parent to create F2 populations segregating for the induced EMS HMW Glutenin mutations. Swelling index of gluten (SIG) was measured from seed from field grown plants to assess the impact of each EMS mutation upon Glutenin function. Findings. Nonsense mutations in both 1Dx5 and 1Dy10 reduced SIG values, indicating both 1Dx5 and 1Dy10 are needed for gluten functionality. Missense mutations did not alter SIG values compared to their wild type counterparts. In contrast missense mutations within the tryptophan-rich region of both PINS increased grain hardness pointing to the importance of this protein region. No such region in 1Dx5 or 1Dy10 was found indicating these proteins are structurally stable and tolerant of amino acid substitution. Conclusions. Results from the Glu-D1 subunit and Pin loci show that useful phenotypic variation can be created using EMS mutagenesis for genes where naturally occurring mutations occur from amino substitutions as is the case for the Pin loci. Significance and Novelty. EMS induced mutations are useful for creating new alleles in storage proteins with much greater impacts in genes that have a defined active site as for the Pin loci than in genes such as the HMW Glu-1 genes which have a long repetitive region.Item TAMFT‐3A and TAMFT‐3B2 homeologs are associated with wheat preharvest sprouting(Wiley, 2022-08) Vetch, Justin Michael; Tillett, Brandon J.; Bruckner, Phil L.; Martin, John M.; Marlowe, Karol; Hooker, Marcus Alan; See, Deven Robert; Giroux, Michael J.The phenomenon of preharvest sprouting (PHS), caused by rain after physiological maturity and prior to harvest, negatively affects wheat (Triticum aestivum L.) production and end use. Investigating the genetics that control PHS resistance may result in increased control of seed dormancy. Multiple genes involved in the development of seed dormancy are associated with PHS. In this study, the TaMFT (3A, 3B1, 3B2, 3D), TaMKK3-4A, and TaVP1-3B genes were assessed for association with PHS in a double-haploid line (DHL) hard red winter wheat population derived from a BC1 cross between the cultivars Loma and Warhorse, where Loma was the recurrent and PHS susceptible parent. The 162 BC1 DHL lines were grown over two field seasons and PHS susceptibility was assessed by measuring PHS resistance in physiologically mature heads. The PHS variation was associated with the TaMFT-A and the B2 homeolog with Loma carrying mutant forms of each gene. No sequence variation between Loma and Warhorse was detected in the exons of the TaMFT-B1 and D homeologs. No association between PHS resistance and TaMKK3-4A or TaVp1-3B variation was observed, though Loma and Warhorse vary for TaMKK3-4A and TaVp1-3B mutations reported to be PHS associated. Previous research has shown TaMFT-3A as having a large impact on PHS resistance. In the current study, the TaMFT-3A and TaMFT-3B2 alleles each explained 14% of observed PHS variation. Markers for both TaMFT-3A and TaMFT-3B2 should be used in selecting for increased wheat dormancy and PHS resistance.Item Evaluating the impact of Rht hypomorphic mutations in durum wheat(Wiley, 2021-12) Brown, McKenna M.; Martin, John M.; Jobson, Emma M.; Hogg, Andrew C.; Carr, Patrick M.; Giroux, Michael J.Increasing the yield of wheat (Triticum spp.) requires identifying new allelic combinations by crossing or by creating useful variation in yield limiting genes. Wheat yield is impacted by many factors, including tiller number and seeds per tiller, both of which are impacted by the Reduced height (Rht) gene. Durum wheat [T. turgidum L. subsp. durum (Desf.) van Slageren] varieties are either standard height, wild type for Rht (Rht-B1a), or are semidwarf and carry the Rht-B1b allele. Rht-B1b increases productive tillers but can result in plants too short for easy harvest in the northern United States and shorter coleoptiles that reduce dry soil germination. In this study, durum plants varying for Rht alleles created by ethyl methanesulfonate (EMS) mutagenesis were studied to determine the impact of each allele upon agronomic and seed traits. The projects’ goal is to increase durum wheat yield through the development of a plant with height intermediate between current full-height and semidwarf varieties. Experiments included field trials, coleoptile length and gibberellic acid (GA) responsiveness assays, and an in vitro test to determine the impact of each Rht mutation upon binding to Gibberellin Insensitive Dwarf1 (GID1). It was found that the Rht-B1b-E529K allele conferred plant height and coleoptile length intermediate between Rht-B1b and Rht-B1a containing plants, while two Rht-A1 alleles had lesser impacts with trends toward intermediate-height plants. The results of this research demonstrate that hypomorphic Rht alleles that alter Rht binding to GID1 may prove useful in optimizing durum wheat height to increase yield across different growing conditions.Item Rht‐1 semi‐dwarfing alleles increase the abundance of high molecular weight glutenin subunits(Wiley, 2020-11) Jobson, Emma M.; Ohm, Jae-Bom; Martin, John M.; Giroux, Michael J.Background and Objectives Grain protein and starch abundance and composition are quantitative traits that play key roles in wheat quality. The semi-dwarfing alleles of the Reduced height (Rht-1) gene increase tillers and yield but also reduce seed size and protein content. Despite their negative impact on grain protein content, the semi-dwarfing alleles increase dough mixing time and tolerance. This study used near isogenic lines that were either tall or semi-dwarf lines that carried Rht-B1b, Rht-D1b, or Rht-8 to investigate how each semi-dwarfing allele impacts gluten composition and flour pasting properties. Findings None of the semi-dwarfing alleles impacted starch properties. Each reduced flour protein content compared to the tall variety with the largest decreases in Rht-B1b (1.8%) and Rht-D1b (1.5%). The semi-dwarfing lines increased the gluten index (21.5%) compared to Rht-1a. Using SE-HPLC, we determined that the semi-dwarfing lines had an increased relative abundance of high molecular weight glutenins compared to the tall variety. Conclusions This study indicates that the Rht-1 semi-dwarfing alleles increase dough mixing time and tolerance by increasing the relative abundance of high molecular weight glutenins yielding stronger dough. Significance and Novelty The semi-dwarfing alleles developed primarily for agronomic purposes have significant impacts on gluten index and starch swelling power.Item Allelic Impacts of TaPHS1, TaMKK3, and Vp1B3 on Preharvest Sprouting of Northern Great Plains Winter Wheats(2018-12) Vetch, Justin M.; Stougaard, Robert N.; Martin, John M.; Giroux, Michael J.Preharvest sprouting (PHS) of bread wheat (Triticum aestivum L.) is a common problem that can lead to negative economic impacts arising from yield loss and undesirable end-use quality. Twenty-one winter wheats adapted to northwestern Montana were grown over two field seasons and used to assess three loci observed in previous studies to have moderate to large impacts on PHS. The main goal was to validate the usefulness of TaPHS1-3A (a Mother of Flowering Time-like gene), TaMKK3-4A (a mitogen-activated protein kinase kinase 3), and Vp1-1B (Viviparous 1) in breeding for modified dormancy before harvest, as well as to determine their potential relationships to agronomic and seed traits, specifically, falling number and α-amylase concentrations. Variation in PHS susceptibility across entries ranged from 0% sprout (fully dormant) to 95% sprout (fully nondormant) after 7 d of wetting. Most entries showed an intermediate level of sprouting susceptibility ranging between 10 and 50% sprouted. Alleles previously reported to impact dormancy were found for all three genes but TaPHS1 was the only locus found to be significantly associated with PHS. It is unclear whether variation caused by TaPHS1 may be masking the effects of the other loci, but it is evident that TaPHS1 could be used in a breeding program to modify the level of seed dormancy in winter wheat before harvest.Item The Impact of the Wheat Rht-B1b Semi-Dwarfing Allele on Photosynthesis and Seed Development Under Field Conditions(2019-02) Jobson, Emma M.; Johnston, Rachel E.; Oiestad, Alanna J.; Martin, John M.; Giroux, Michael J.The Reduced Height (Rht) genes formed the basis for the green revolution in wheat by decreasing plant height and increasing productive tillers. There are two current widely used Rht mutant alleles, Rht-B1b and Rht-D1b. Both reduce plant height by 20% and increase seed yield by 5-10%. They are also associated with decreased seed size and protein content. Here, we tested the degree to which Rht-B1b impacts flag leaf photosynthetic rates and carbon and nitrogen partitioning to the flag leaf and grain during grain fill under field conditions using near isogenic lines (NILs) that were either standard height (Rht-B1a) or semi-dwarf (Rht-B1b). The results demonstrate that at anthesis, Rht-B1b reduces flag leaf photosynthetic rate per unit area by 18% and chlorophyll A content by 23%. Rht-B1b significantly reduced grain protein beginning at 14 days post anthesis (DPA) with the greatest difference seen at 21 DPA (12%). Rht-B1b also significantly decreased individual seed weight beginning at 21 DPA and by 15.2% at 28 DPA. Global expression analysis using RNA extracted from developing leaves and stems demonstrated that genes associated with carbon and nitrogen metabolism are not substantially altered by Rht-B1b. From this study, we conclude that Rht-B1b reduces flag leaf photosynthetic rate at flowering while changes in grain composition begin shortly after anthesis.Item Creation and Characterization of a Double Null Puroindoline Genotype in Spring Wheat(2017-09) Martin, John M.; Hogg, Andrew C.; Webster, Richard W.; Giroux, Michael J.Wheat (Triticum aestivum L.) grain hardness is controlled by the Ha locus, which is composed of two closely linked genes, Puroindoline a (Pina) and Puroindoline b (Pinb). Hard grain results from mutations in either of the Pin genes. Previous results have shown that the Pina-D1b (Pina null) allele has harder grain than other naturally occurring Pin alleles. Our goal was to create, identify, and characterize a double null Pin genotype by identifying a Pinb null mutation in a variety carrying the Pina-D1l null allele. Seeds of Fortuna, which has a premature stop codon in Pina, were treated with ethyl methanesulfonate. Two premature stop codon mutations were identified in Pinb using direct sequencing. The double null Pin haplotype was characterized after backcrossing to the parent variety Fortuna to create Pina null populations segregating for the presence of Pinb. The double null group was 6 units harder than the single null with no difference in other kernel characteristics. The milling characteristics differed between the two classes; the double null class had less break flour with a greater fraction of large and a smaller fraction of small flour particles compared with the single null class. Neither water absorption nor loaf volume was impacted by the change in grain hardness; however, Na2CO3 tests indicated greater starch damage in the double nulls. The double null Pin genotype may find a niche in hard wheat products for which flours with larger particle size are desired.Item Identification of Candidate Genes Responsible for Stem Pith Production Using Expression Analysis in Solid-Stemmed Wheat(2017-07) Oiestad, Alanna J.; Martin, John M.; Cook, Jason P.; Varella, Andrea C.; Giroux, Michael J.The wheat stem sawfly (WSS) is an economically important pest of wheat in the Northern Great Plains. The primary means of WSS control is resistance associated with the single quantitative trait locus (QTL) Qss. msub. 3BL, which controls most stem solidness variation. The goal of this study was to identify stem solidness candidate genes via RNA-seq. This study made use of 28 single nucleotide polymorphism (SNP) makers derived from expressed sequence tags (ESTs) linked to Qss. msub. 3BL contained within a 5.13 cM region. Allele specific expression of EST markers was examined in stem tissue for solid and hollow-stemmed pairs of two spring wheat near isogenic lines (NILs) differing for the Qss. msub-3BL QTL. Of the 28 ESTs, 13 were located within annotated genes and 10 had detectable stem expression. Annotated genes corresponding to four of the ESTs were differentially expressed between solid and hollow-stemmed NILs and represent possible stem solidness gene candidates. Further examination of the 5.13 cM region containing the 28 EST markers identified 260 annotated genes. Twenty of the 260 Qss. msub. 3BL linked genes were up-regulated in hollow NIL stems, while only seven genes were up-regulated in solid NIL stems. An O-methyltransferase within the region of interest was identified as a candidate based on differential expression between solid and hollow-stemmed NILs and putative function. Further study of these candidate genes may lead to the identification of the gene(s) controlling stem solidness and an increased ability to select for wheat stem solidness and manage WSS.Item Genetic characterization and expression analysis of wheat (Triticum aestivum) line 07OR1074 exhibiting very low polyphenol oxidase (PPO) activity(2015-08) Hystad, Steven M.; Martin, John M.; Graybosch, R.A.; Giroux, Michael J.Wheat (Triticum aestivum) polyphenol oxidase (PPO) contributes to the time-dependent discoloration of Asian noodles. Wheat contains multiple paralogous and orthologous Ppo genes, Ppo-A1, Ppo-D1, Ppo-A2, Ppo-D2, and Ppo-B2, expressed in wheat kernels. To date, wheat noodle color improvement efforts have focused on breeding cultivars containing Ppo-D1 and Ppo-A1 alleles conferring reduced PPO activity. A major impediment to wheat quality improvement is a lack of additional Ppo alleles conferring reduced kernel PPO. In this study, a previously reported very low PPO line, 07OR1074, was found to contain a novel allele at Ppo-A2 and null alleles at the Ppo-A1 and Ppo-D1 loci. To examine the impact of each mutation upon kernel PPO, populations were generated from crosses between 07OR1074 and the hard white spring wheat cultivars Choteau and Vida. Expression analysis using RNA-seq demonstrated no detectable Ppo-A1 transcripts in 07OR1074 while Ppo-D1 transcripts were present at less than 10 % of that seen in Choteau and Vida. Novel markers specific for the Ppo-D1 and Ppo-A2 mutations discovered in 07OR1074, along with the Ppo-A1 STS marker, were used to screen segregating populations. Evaluation of lines indicated a substantial genotypic effect on PPO with Ppo-A1 and Ppo-D1 alleles contributing significantly to total PPO in both populations. These results show that the novel mutations in Ppo-A1 and Ppo-D1 present in 07OR1074 are both important to lowering overall wheat seed PPO activity and may be useful to produce more desirable and marketable wheat-based products.