1 Summary

Sequencing, phasing and annotating the diploid genome of basin big sagebrush (Artemisia tridentata subsp. tridentata). This taxon as 2n=2x=18 chromosomes and a 2C genome size of 9.5Gbp (see Barron et al., 2020). In addition, preliminary analyses demonstrated that the genome is highly out-bred. For these reasons, we are aiming at not only produce enough biomass of the same individual line to produce the genome, but also maintain individual lines both in vitro and ex vitro to support genotype-by-environment experiments.

2 Organism

For this project, we are focusing on establishing in vitro individual lines for:

More details on the classification of this organism is provided here:

Eukaryota; Viridiplantae; Streptophyta; Streptophytina; Embryophyta; Tracheophyta; Euphyllophyta; Spermatophyta; Magnoliopsida; Mesangiospermae; eudicotyledons; Gunneridae; Pentapetalae; asterids; campanulids; Asterales; Asteraceae; Asteroideae; Anthemideae; Artemisiinae; Artemisia; Artemisia tridentata

3 Geographical origin of source populations

Seeds of the mother plants used to establish the in vitro tissue culture propagation program of sagebrush individual lines were provided by Bryce Richardson (US Forest Service). Table 3.1 and Figure 3.1 provide details on the two source populations referred to as ID3 and UT2. All the seedlings generated from mother plants collected in these locations were identified as Artemisia tridentata subsp. tridentata by Richardson et al. (2012) and these authors also demonstrated that these populations were diploids (2n=2x=18).

Table 3.1: Meta-data information on source populations used to generate sagebrush individual lines.
Population Mother plant State County Elevation (m) Latitude Longitude
ID3 95 Hilltop 4 Idaho Owyhee 1358 43.337 -116.964
UT2 La Sal B18 Utah San Juan 1820 38.306 -109.388

Figure 3.1: Map of locations of the source populations where seeds from mother plants were collected to establish sagebrush individual lines.

4 Overview of tissue culture program

4.1 In vitro

The in vitro tissue culture propagation that we have recently published for Artemisia tridentata subsp. tridentata (Barron et al., 2020) is composed of three phases described in Table 4.1.

Table 4.1: In vitro tissue culture protocol for Artemisia tridentata subsp. tridentata to maintain clonal lines.
Phase Duration (week) Location Vessel Notes
Growth 15 P4/P5 Magenta vessel During the first generation, we started from seeds, but later this step was based on rooted shoot tips cultured on MS growth media in Magenta vessels.
Cutting NA P4/P5 NA Shoot tips are cut from the grown seedlings or plantlets and transferred into rooting media.
Rooting 3 P4/P5 Petri plates Shoot tips are cultivated on a rooting media containing IBA growth regulator (1 ml/l) in plates to initiate rooting. Rooted shoot tips are then transferred into growth media and we are back to step 1.

The Cutting phase is labor intensive and usually takes 1 hour to process 5 plantlets (per person).

More details on the methodology (incl. protocols) and analyses associated to in vitro sagebrush propagation can be found on our companion website:

4.2 Ex planting

The ex planting protocol to produce plantlets of Artemisia tridentata subsp. tridentata for GxE experiments is composed of five phases described in Table 4.2. Plantlets used for this protocol are sourced from the in vitro tissue program after the Growth phase (see Table 4.1.

Table 4.2: Ex planting protocol for Artemisia tridentata subsp. tridentata to produce plantlets for GxE experiments.
Phase Duration (week) Location Vessel Notes
Ex1 2 P4 Magenta vessel (sealed with couplers) Transfer plantlet into peat pots (containing sand, MS, vitamins and PPM)
Ex2 1 Cart Magenta vessel (sealed with couplers) Adjusting to lower light intensity
Ex3 2 Cart Magenta vessel (cracked open) Lower light intersity and gas exchange (plantlet starts changing phenotype)
Ex4 3 Cart Fully open Magenta vessel Soil is drying out, requires watering and fertilizing. At the end of this phase, plantlet is acclimatized.
Ex5 1 P4 Fully open Magenta vessel Adaptation to light and temperature conditions in P4. After this phase, plantlet is ready for GxE experiment.

For the Ex5 phase, the light intensity of the P4 culture chamber will be set identical to the one at the Cart.

5 Individual lines

Information on individual lines in culture at BSU is provided in Table 5.1. The individual line used for biomass production underpinning the genome project is G2_b24_1.

Table 5.1: Meta-data information on sagebrush individual lines.
PopID Type Individual Line Ploidy Genome size Genotype Germination Date Rooting rate Survival rate N shoot tips Biomass gr
ID3 In vitro G1_b2_1 2x NA G1 2020-02-13 0.86 0.8,0.9 14 1.7
ID3 In vitro G1_b13_1 2x NA G1 2020-02-13 0.96 NULL 11 NA
ID3 In vitro G1_b29_1 2x NA G1 2020-02-13 0.9 NULL 11 NA
ID3 In vitro G1_b10_1 2x NA G1 2020-02-13 0.96 NULL 11 NA
UT2 In vitro G2_b27_1 2x NA G2 2020-02-13 0.89,0.89 0.45,0.35 9.5,9.2 NA
UT2 In vitro G2_b24_1 2x NA G2 2020-02-13 0.93,0.87 0.8 9.5, 11.9 0.8
UT2 In vitro G2_b24_2 2x NA G2 2020-02-13 0.96 NULL 12 NA
UT2 Explant G2_b4_1 2x NA G2 2020-02-13 NULL NULL NULL NA

6 Status on in vitro culture program

The status of the propagation of sagebrush individual lines is in Table 6.1.

Table 6.1: Data on propagation for sagebrush individual lines.
Individual Line Current Date Total Stage Generation Phase Chamber Shelf Predicted Actual Next Date Action Needed
G1_b10_1 2021-03-04 18 Growing 2 NULL P5 5 14 NA 2021-03-24 3_wk_Mortality_Check
G1_b13_1 2021-03-04 12 Growing 2 NULL P5 5 9 NA 2021-03-24 3_wk_Mortality_Check
G1_b2_1 2021-03-04 90 Growing 2 NULL P4 1 NULL 90 2021-03-24 10_wk_Mortality
G1_b29_1 2021-03-04 9 Growing 2 NULL P5 5 7 NA 2021-03-24 3_wk_Mortality_Check
G2_b24_1 2021-03-04 279 Growing 3 NULL P4 2,3,4 223 NA 2021-03-22 3_wk_Mortality Check
G2_b24_1 2021-03-04 229 Growing 3 NULL P5 3,4,5 183 NA 2021-03-22 3_wk_Mortality_Check
G2_b24_2 2021-03-04 10 Growing 2 NULL P5 5 8 NA 2021-03-24 3_wk_Mortality_Check
G2_b27_1 2021-03-17 84 Growing 3 1 P5 1 82 84 2021-03-31 9_wk_Mortality Check
G2_b27_1 2021-03-17 21 Growing 3 2 P5 2 49 21 2021-03-31 7_wk_Mortality Check
G2_b4_1 2021-03-10 15 Explant Ex3 NA Cart 1 NA 15 2021-03-23 Fully open boxes

7 Culture chamber

The culture chambers used in this experiment taking place at BSU are described in Table 7.1.

Table 7.1: Carrying capacity of culture chambers.
Type Model Light intensity Temperature (Celsius) Carrying capacity Current occupancy Percent
P4 Percival Culture Chamber CU41L4C8 152 24 396 369 0.93
P5 Percival Culture Chamber CU41L5C8 160 24 495 383 0.77
Cart Seedling Cart GA3P4T8 80 24 288 15 0.05

References

Barron, R., P. Martinez, M. Serpe, and S. Buerki. 2020. Development of an in vitro method of propagation for artemisia tridentata subsp. Tridentata to support genome sequencing and genotype-by-environment research. Plants 9: Available at: https://www.mdpi.com/2223-7747/9/12/1717.
Richardson, B.A., J.T. Page, P. Bajgain, S.C. Sanderson, and J.A. Udall. 2012. Deep sequencing of amplicons reveals widespread intraspecific hybridization and multiple origins of polyploidy in big sagebrush (artemisia tridentata; asteraceae). American Journal of Botany 99: 1962–1975. Available at: https://onlinelibrary.wiley.com/doi/abs/10.3732/ajb.1200373.