Development and Characterization of SSR Markers in Proso Millet Based on Switchgrass Genomics

Proso millet (Panicummiliaceum) has highwater use efficiency (WUE), a short growing-season, and is highly adapted to a semi-arid climate. Genomic resources for proso millet are very limited. Large numbers of DNA markers and other genomic tools in proso millet can readily be developed by using genomic resources in related grasses. The objectives of the present report were to 1) test and characterize switchgrass SSR markers for use in proso millet, and 2) elucidate repeat-motifs in proso millet based on new SSR marker analysis. A total of 548 SSR markers were tested on 8 proso millet genotypes. Out of these, 339 amplified SSR markers in proso millet. This showed that 62% of the switchgrass SSR markers were transferable to proso millet. Of these 339 markers, 254 were highly polymorphic among the 8 proso genotypes. The resolving power of these 254 polymorphic SSR markers ranged from 0.25-14.75 with an average of 2.71. The 254 polymorphic SSR markers amplified 984 alleles in the ranges of 50 bp to 1300 bp. The majority of the SSR markers (221 of 254) amplified dinucleotide repeats. Based on SSR marker analysis, AG/GA was the most abundant repeat-motifs in proso millet. Switchgrass genomic information seems to be the most useful for developing DNA markers in proso millet. Markers developed in this study will be helpful for linkage map construction, mapping agronomic traits and future molecular breeding efforts in proso millet.

Bioassay of Winter Wheat for Gibberellic Acid Sensitivity

Increasing winter wheat seedling growth would make it a better winter cover crop. Gibberellic acid (GA3) seed treatment may accomplish this by stimulating stem growth. A bioassay, mimicking field conditions, could determine the relative sensitivity of conventional and semi-dwarf cultivars. In growth chambers set for cool (10℃/4℃) and warm (21℃/4℃) conditions, wheat seeds were treated with 0 and 125 to 16,000 ppm GA3. The cultivars Goodstreak (tall or conventional) and Wesley (semi-dwarf) were compared as standards. Emergence and plant height were measured. “Goodstreak” showed a significant growth promotion at 500 ppm GA3 when seeds were dipped and 2000 ppm when GA3 was applied in-furrow under both temperature regimes. “Wesley” in general required the same or a higher dose of GA3. Separately, the seeds of nine other cultivars were treated with GA3 as the standards. Based on maximum height promotion, the most sensitive cultivars under cool conditions were Goodstreak, Harry, Millenium, and Wahoo;under warm conditions, the most sensitive cultivars were Alliance, Goodstreak, Jagalene, and Millenium. In general, the least GA3 sensitive cultivars were Arrowsmith, Scout66, and Wesley. “Buckskin” and “InfinityCL” were intermediate. The rye cultivar Rymin also was tested and showed less sensitivity to GA3 than “Goodstreak”. When 6 benzyladenine (6BA) with GA3 was applied to “Goodstreak” and “Wesley” seed, emergence, plant height and weight, and tiller formation were reduced. Wheat cultivars will respond to GA3 and differ in the amount of GA3 needed. The results of this growth chamber study will guide subsequent field trials.

Gibberellic Acid Promotes Early Growth of Winter Wheat and Rye

Winter wheat (Triticum aestivum) planting in Nebraska is recommended for mid Sep but summer crops are often harvested around Oct 1. Also, weather may delay planting. Could gibberellic acid (GA3), a growth stimulant, overcome the delayed seedling growth from late planting? Irrigated field trials were planted from 2005 to 2010 applying GA3 to wheat seed of cvs. Goodstreak and Wesley. In 2005, dip, spray and furrow GA3 applications to seed were tested. Dip and spray gave similar results. Furrow application was calculated too costly. Further tests used seed dips. In 2006 to 2008 planting, wheat was planted about 15 Sep, 1 Oct and 15 Oct. Trials planted in 2007 and 2008 included winter rye (Secale cereale) cv. Rymin. Heights in mid Nov showed a reduction with later planting. When planted about 1 Oct and 15 Oct, the delayed growth of Goodstreak, compared to planting two weeks earlier, was fully overcome by GA3 at 250 ppm. For Wesley, 1000 ppm GA3 was needed to nearly overcome later planting. Rymin rye gave an intermediate response between the two wheat cultivars. Spring and summer heights were not affected by GA3. Spring biomass, yield and harvested seed germination showed no GA3 effect. In later trials (planted in 2009 and 2010), the cytokinin 6-benzyl adenine (6BA) was added to GA3 to stimulate tiller formation. In the 1st year, 6BA at 2000 ppm depressed height, weight and yield without tiller promotion. Repeating the trial with lower rates (31 - 125 ppm 6BA) did not offer an additional advantage to GA3. The results indicate that seed application of GA3 can overcome delayed growth resulting from delayed planting of winter wheat and rye under irrigation.

Effect of Cold-Mediated Pretreatment on Microspore Culture in Winter and Spring Wheat

Microspore culture of wheat generates completely homozygous (doubled haploid) plants in a single generation thereby reducing the time required for wheat variety development. Success of microspore culture in spring wheat is relatively higher than that in winter wheat. Cold mediated pretreatment was reported to improve response of microspore culture in wheat. The objective of the study was to determine and compare the influence of cold pretreatment on microspore culture in spring and winter wheat. Three spring (“Chris”, “Express”, and “Macon”) and three winter (“Anton”, “Antelope”, and “Camelot”) wheat cultivars were used. In cold pretreatment, excised anthers were incubated in solution B at 25°C-28°C for 4-5 days followed by cold treatment at 4°C for 5 days and were compared with the no-cold pretreatment at 25°C-28°C for 4-5 days. Isolated microspores were cultured in induction medium (MMS4) at 27°C-28°C for 25-30 days in the dark. Embryos (1-2 mm size) were transferred to regeneration medium (MMS5). Numbers of multicellular structures, transferable embryos and green plants were counted and data were used for analysis of variance using a generalized linear model. It was observed that cold pretreatment increased multicellular structures, transferable embryos and green plants in both spring and winter wheat. However, the degree of improvement was higher in spring wheat compared to winter wheat. The cultivars within spring and winter wheat responded differently. Development of embryos from pro-embryos was 4-5 folds lower in winter wheat than that in spring wheat, indicating requirement of a possibly different hormonal composition in induction medium for improving embryo induction in winter wheat. This report may provide future direction of research to improve microspore culture response in winter wheat.