Thomas Tai, research geneticist, USDA-ARS Crops Pathology and Genetics Research Unit, Dept. of Plant Sciences, UC Davis

The overall goal of this project is to employ trait and DNA-based genetic screens to identify and characterize novel rice germplasm to advance the understanding of agronomic performance and grain quality for incorporation into breeding programs for the California rice industry.

Primary emphasis is on screening rice populations generated by traditional mutagenesis for new traits that improve grain quality and reduce production costs. This is achieved by directly screening plant materials for traits of interest and by identifying changes in the DNA sequence of genes that may result in the expression of these traits. Specific targets currently include reduced uptake and/or localization of arsenic in milled rice grains and resistance or tolerance of rice plants to selected herbicides.

The traditional approach to mutant populations is to conduct screens that identify new characteristics of interest. Examples of this “forward” genetics approach in rice include the identification of the semidwarf trait, conditional male sterility, and herbicide tolerance.

Reverse genetics is a complementary approach for exploiting mutant populations. This strategy requires prior knowledge of the genes that are responsible for the target traits. These genes are used to screen populations to identify mutated versions that may result in the expression of novel characteristics.

One method of reverse genetics is the Targeting of Induced Local Lesions in Genomes (TILLING) strategy. It is based on the detection of mutations in target gene sequences by screening DNA isolated and pooled from hundreds of mutant lines (typically 2,000 lines total). A service to identify mutations of interest with this technique is operated by the TILLING Core Lab at the UC Davis Genome Center. Using this service, researchers have successfully identified mutations in genes involved in arsenic uptake and accumulation and genes that encode protein targets of various herbicides.

The major objectives of research in 2018 were:

Response of rice seedlings to germanium after nine days of treatment, part of a genetic screening experiment on mutant lines.

Arsenic is known to enter rice plants through the same pathway used by silicon. Genetic screening can identify mutants exhibiting altered uptake and accumulation of silicon based on resistance to the element germanium. Because of similar chemical properties, germanium is also taken up the same way as silicon, but it is toxic to rice plants and results in the formation of lesions and eventually death in seedlings. 

Previously, several lines carrying mutations in genes involved in silicon and arsenic uptake or accumulation were identified by TILLING and confirmed using DNA sequencing. Seeds were harvested from mutants harboring fixed mutations (important for trait expression). Genetic screening using germanium also resulted in the identification of several mutant lines that are potentially altered in their ability to take up silicon and arsenic.

In 2018, mutant lines carrying fixed mutations in the genes targeted in this study were grown in a greenhouse and in a field on the UC Davis campus to provide tissue samples for analysis of arsenic (organic and inorganic) and silicon content. These included three lines containing mutations in the Lsi1 gene, nine lines containing mutations in the Lsi2 line and one line containing a mutation in the OsABCC1 gene. Two wild-type lines and one wild-type sibling of an Lsi2 mutant line were also included in the field test as controls. The lines were started in the greenhouse and then transplanted to the field for subsequent harvest of mature plants and elemental analysis of tissue samples.

Greenhouse-based evaluation of these mutants using a germanium assay revealed that two of three mutations detected in the Lsi1 gene showed strong effects on reducing straw silicon content and sensitivity to germanium. This is consistent with the reduced transport of both elements from the environment into the rice plant. All three Lsi1 mutants exhibited increased total arsenic in straw, but one, NM-3403, may actually increase the content of total arsenic in brown rice.

In contrast to the Lsi1 mutants, most Lsi2 mutants showed increased total silicon in shoot tissues but no effect on total arsenic in the straw and grain or in germanium response. Notable exceptions are Lsi2 mutants NM-2902, NM-E2244, and NME-2249, which all showed significant increases in total arsenic in their brown rice samples.

Interestingly, the OsABCC1 mutant, NM-4903, was the only line to exhibit a significant reduction in total arsenic in its brown rice. Unlike the Lsi1and Lsi2, which are involved in the transport of arsenic from the soil into the root and from the root into the rest of the plant, OsABCC1encodes a transporter involved in sequestering arsenic, thus preventing accumulation in rice grains. Identification of a mutation that enhances this activity would prove useful in the development of rice varieties with reduced grain arsenic.

These results must be confirmed by testing materials grown in 2018. Total arsenic analysis does not provide information on the various arsenic species. The toxicity of arsenic depends very heavily on the two main chemical forms, inorganic arsenic and organic arsenic. Detailed analysis of arsenic species needs to be conducted if differences observed in total arsenic are confirmed.

In 2019, additional crosses of low-silicon mutants identified by the TILLING process in 2016 and 2017 will be made as warranted by evaluation of silicon and arsenic content and germanium assays.