Development of Root Phenotyping Platforms for Identification of Root Architecture Mutations in EMS-Induced and Low-Path-Sequenced Sorghum Mutant Population

Sorghum’s natural adaptation to a wide range of abiotic stresses provides diverse genetic reserves for potential improvement in crop stress tolerance. Growing interest in sorghum research has led to the expansion of genetic resources though establishment of the sorghum association panel (SAP), generation of mutagenized populations, and recombinant inbred line (RIL) populations, etc. Despite rapid improvement in biotechnological tools, lack of efficient phenotyping platforms remains one of the major obstacles in utilizing these genetic resources. Scarcity of efforts in root system phenotyping hinders identification and integration of the superior root traits advantageous to stress tolerance. Here, we explored multiple approaches in root phenotyping of an ethyl methanesulfonate (EMS)-mutagenized sorghum population. Paper-based growth pouches (PGP) and hydroponics were employed to analyze root system architecture (RSA) variations induced by mutations and to test root development flexibility in response to phosphorus deficiency in early growing stages. PGP method had improved capabilities compared to hydroponics providing inexpensive, space-saving, and high-throughput phenotyping of sorghum roots. Preliminary observation revealed distinct phenotypic variations which were qualitatively and quantitatively systemized for association analysis. Phenotypes/ideotypes with root architecture variations potentially correlated with Pi acquisition were selected to evaluate their contribution to P-efficiency (PE). Sand mixed with P-loaded activated alumina substrate (SAS) provided closely to natural but still controlled single-variable conditions with regulated Pi availability. Due to higher labor and cost input we propose SAS to be used for evaluating selected sorghum candidates for PE. The ability of rapidly screening root phenotypes holds great potential for discovering genes responsible for relevant root traits and utilizing mutations to improve nutrient efficiency and crop productivity.

Canopy Light Interception of a Conventional and an Erect Leafed Sorghum

Two sorghum lines, an erect leafed mutant sorghum and the wild type from which the mutant was generated, were field grown in rectilinear arrays at low (10 plants m-2) and high (23 plants m-2) population densities. Canopy light interception, biomass accretion and yield were measured. Photosynthetically active radiation under the canopy at ground level and midway through the canopy were higher in the erect leafed line, as compared to the normal leafed line. Planting density had less effect on mean grain yield and biomass in the erect leaf line than in the wild type. Though not conclusive, when taken together, the results suggested that optimal planting densities are higher for the erect leaf line and that the erect leaf trait could be useful for incorporation into breeding programs.

Evaluation of Sweet Sorghum as a Feedstock by Multiple Harvests for Sustainable Bioenergy Production

Sweet sorghum has become an important feedstock for bioethanol production. Total sugar yield and multiple harvests can directly affect ethanol production cost. Little is known about stem traits and multiple harvests that contribute to sugar yield in sweet sorghum. Stem traits were evaluated from 25 sweet and grain sorghum accessions. Stems were harvested twice at the soft-dough stage and the stems were pressed with a hydraulic press. Sugars in the stem juice were quantified by high performance liquid chromatography. Sweet sorghum produced five times more fresh stem weight and dry stem mass (830 gand164 g) than grain sorghum (150 gand27g). Sweet sorghum produced a much higher volume of juice and higher yield of sugars (366 ml and42 g) per stem than grain sorghum (70 ml and4 g). Significant variability in fresh stem weight (72 - 1837 g), juice volume (31 - 753 ml), sugar yield (3 - 81 g), dry stem mass (14 - 383 g), and sugar yield/dry stem mass ratio (0.11 - 0.53) per stem was detected among sweet sorghum accessions. Stem sugar yield was significantly correlated with stem fresh weight and juice volume. Sorghum was harvested twice within one growing season resulting in some sweet sorghum accessions producing double amount of sugars. Sweet sorghum produced three times more dry mass weight (bagasse) than fermentable sugar weight. To reduce feedstock cost, methods have to be developed for efficiently utilizing bagasse. Our results showed high fresh stem weight, high ratio of sugar yield to dry stem mass, and double harvests are prime traits to boost sugar yield. Sweet sorghum may be suitable for multiple harvests in certain regions of theU.S.TheU.S.sweet sorghum collection needs to be screened for acces- sions that can be harvested twice with an extended feedstock-production season and used as a feedstock for sustainable and renewable bioenergy production.

Short Communication: Analysis of Grain Size Distribution through Image Analysis

Herein we describe an approach to measure the volume and to characterize the volume distribution of large numbers of individual small regularly shaped seeds. The results of a preliminary investigation into the distribution of seed size of a multi-seeded sorghum mutant, as compared to the wild type from which it was developed are also reported, and are used as an example of the method’s utility.

Genetic Diversity in a Collection of Chinese Sorghum Landraces Assessed by Microsatellites

Genetic diversity was characterized in a collection of 159 sorghum (Sorghum bicolor L. Moench) landraces gathered from the colder region (primarily the northeastern region) of China. A set of 41 microsatellites or simple sequence repeat (SSR) markers distributed throughout the 10 chromosomes of sorghum was utilized in the analysis. Majority (40 out of 41 SSRs, 98%) of the SSR markers were polymorphic and highly informative with polymorphism information content (PIC) value ranging from 0.05 to 0.92. An estimation of genetic similarity coefficients (GSC, with values ranging from 0.42 to 0.96) revealed a range of variability in this collection of sorghum landraces. Eight clusters of accessions were identified at a cut off at 0.50 GSC and these groupings were supported by the analysis of subpopulations using Structure. This study provides evidence that Chinese landraces of sorghum exhibit moderate genetic diversity and results from cluster analysis may well aid in identification of diverse accessions that can serve as parental lines for efficient utilization and application of these germplasm into sorghum breeding programs.

Systematic Errors Introduced into Sorghum Grain Yield Data: Does the Multiseed (<i>msd</i>) Trait Increase Sorghum Seed Yield?

Multiseed (msd) mutant sorghum [Sorghum bicolor (L.) Moench] lines with greatly increased seed numbers were developed. It was originally thought that the msd trait could increase grain yield several times in comparison with the wild type from which the mutant was derived. However, in a small plot trial, msd seed yield decreased when compared to the parent line. Herein we report results that msd seed yield remained either unchanged or slightly increased in comparison to the parent line. We suggest that attempts to measure msd sorghum seed yield were complicated due to systematic errors associated with the post-harvest processing methods, including threshing and pneumatic winnowing equipment that was used for harvest. That is, seed recovery and seed loss from individual panicles were affected by the post-harvest processing. When evaluating sorghum grain yield of types with different seed sizes, threshing and seed cleaning harvesting methods should be optimized for each sorghum line.

Description of a Novel Allelic “Thick Leafed” Mutant of Sorghum

An allelic sorghum [Sorghum bicolor (L.) Moench] mutant with thick and narrow erect leaves (Thl) and reduced adaxial stomatal density was isolated from the Annotated Individually pedigreed Mutagenized Sorghum mutant library developed at the Plant Stress and Germplasm Development Unit at Lubbock TX. The mutant, Thl, was isolated from a pedigreed M3 family generated by ethyl methanesulfonate mutagenization from an elite inbred sorghum line, BTx623, which had been used to sequence the sorghum genome. The mutant has been backcrossed to the wild-type BTx623 confirming that the trait results derive from a stable recessive nuclear gene mutation. Herein, we briefly described morphological and selected physiological characteristics of this mutant sorghum.

The Microenvironment within and Pollen Transmission through Polyethylene Sorghum Pollination Bags

Bird damage is a problem in sorghum breeding and germplasm maintenance operations. Paper pollination bags are damaged by rain and provide minimal deterrent to birds. Earlier we reported upon bird resistance of spun polyethylene pollination bags. Herein, we report the potential for pollen transmission through, and the microenvironment within, hard form (HfT) and soft form (SfT) spun polyethylene pollination bags as compared to traditional Paper pollination bags. Within Paper pollination bags morning temperatures were 10°C - 15°C above ambient and high temperature excursions as high as 45°C were measured. Heating in Sft and HfT was 25% and 50% that of Paper, respectively. Temperature differences between bags were attributed to differences in albedo and air permeability of the bag materials. No difference in pollen transmission through Paper and HfT was found. Although SfT allowed 35% - 40% wind borne pollen through the pores as compared to controls, male sterile plants covered with SfT produced only 30 seeds/panicle, about 1% of a self-pollinating fertile plant. Our results suggested that SfT could adequately reduce or eliminate cross-pollination in self-pollinating plants while maintaining near ambient environmental conditions.

Research Note: Bird-Resistant Pollination Bags for Sorghum Breeding and Germplasm Maintenance

Bird damage is a problem in sorghum breeding and germplasm maintenance operations. Paper pollination bags are damaged by rain and provide only a minimal deterrent to birds. To overcome these limitations we fabricated pollination bags from spun polyethylene fiber sheeting. No seed yield difference was found between plants bagged with either spun polyethylene or paper. Seed loss by bird damage was nearly eliminated under the polyethylene bags. In areas where bird damage is problematic bird resistant pollination bags can allow for a reduction in the plot size required for breeding and germplasm maintenance operations, increase the productivity of such operations as genetic diversity per unit land area, and make direct measurement of seed yield possible in agronomic field experiments.

Comparison of Stem Damage and Carbohydrate Composition in the Stem Juice between Sugarcane and Sweet Sorghum Harvested before and after Late Fall Frost

A late fall frost may significantly affect sugar crops’ stem sugar composition, yield and juice quality for biofuel and bioproduct manufacture. Research on the effects of late fall frost in sugarcane is well documented, but information is lacking for sweet sorghum. Three and six commercial cultivars of sugarcane and sweet sorghum, respectively, were selected and evaluated for exposure to a late fall frost (-2.8&degC) in Griffin, Georgia, USA. Under the same controlled environmental conditions in a screen house, the late fall frost induced more damage to sugarcane than sweet sorghum stems. The frost caused damage to sugarcane tissue and for juice to exude from stems, whereas similar behavior was not observed for sweet sorghum. In both sugarcane and sweet sorghum, the glucose/fructose ratio was significantly reduced, but this change may not be totally directly related to the frost effect. Overall, these initial results suggest that sweet sorghum may have a better tolerance to fall frost than sugarcane. Two sweet sorghum cultivars, Grassl and M81E, responded well to the late fall frost, and they can possibly be used as feedstocks for biofuel/bioproduct manufacture in areas susceptible to frosts including northern regions of the Southeastern US.

Characterization of novel Brown midrib 6 mutations affecting lignin biosynthesis in sorghum

The presence of lignin reduces the quality of lignocellulosic biomass for forage materials and feedstock for biofuels. In C4 grasses, the brown midrib phenotype has been linked to mutations to genes in the monolignol biosynthesis pathway. For example, the Bmr6 gene in sorghum （Sorghum bicolor） has been previously shown to encode cinnamyl alcohol dehydrogenase （CAD）, which catalyzes the final step of the monolignol biosynthesis pathway. Mutations in this gene have been shown to reduce the abundance of lignin, enhance digestibility, and improve saccharification efficiencies and ethanol yields. Nine sorghum lines harboring five different bmr6 alleles were identified in an EMS-mutagenized TILLING population. DNA sequencing of Bmr6 revealed that the majority of the mutations impacted evolutionarily conserved amino acids while three-dimensional structural modeling predicted that all of these alleles interfered with the enzyme＇s ability to bind with its NAPPH cofactor. All of the new alleles reduced in vitro CAD activity levels and enhanced glucose yields following saccharification. Further, many of these lines were associated with higher reductions in acid detergent lignin compared to lines harboring the previously characterized bmr6-ref allele. These bmr6 lines represent new breeding tools for manipulating biomass composition to enhance forage and feedstock quality.