Identifying the genetic basis of a particular trait is the building block on which molecular and transgenic breeding strategies build upon. How we connect the dots between a genotype and trait is the basis of a past post “How do we find genes related to traits? A review of bulked sample analysis”.
Despite only being behind rice and wheat as the most important crop worldwide, quantitative trait loci (QTLs) for heat tolerance in potatoes have not, until recently, been identified.
Potatoes are particularly sensitive to heat, with temperatures above the ideal maximum of 22°C resulting in significant decreases in growth and yield. The higher temperatures have a number of effects:
- Strongly suppresses tuberisation;
- Reduces the proportion of carbon assimilated for tuber starch;
- Reduces chlorophyll levels and CO2 fixation rates;
- Halts tuber dormancy resulting in early sprouting and secondary growths.
A recent study in the Plant Biotechnology Journal sought to identify QTLs for heat tolerance by comparing differences in gene expression and tuber yield between progeny of a biparental diploid potato (06H1) when grown at 22°C and 28°C.
Temperature response analysis and gene identification
Single node cuttings were used as a method to quickly induce tuberisation of subject potato plants, allowing a faster assessment of tuber yield (see this article discussing its use – paywalled unfortunately). 170 genotypes from the 06H1 population were analysed using 6 nodal cuttings from each to assess tuber yield under 22°C and another 6 for each genotype assessed at 28°C. On average, tuber yield at the lower temperature (1.017 grams) was greater than for nodes grown at the higher temperature (0.285 grams). The difference in performance was used in combination with single nucleotide polymorphism (SNP) maps of the 06H1 genome to identify genetic markers which may be connected to the genes which alter expression rates at the two temperatures.
Figure 1 from article. Comparison of tuber fresh weight yield when grown at 22°C and 28°C.
Statistical analysis of the differences in expression levels at each marker found 3 markers which may be connected to the difference in phenotype. Using the Potato Genome Browser the researchers browsed the list of genes connected to the markers for suitable gene candidates for further research. Of the three markers, one marker contained a possible gene candidate, that encoding Heat Shock Cognate 70, a gene that has been suggested as involved in the heat tolerance in cabbage and potato.
With a candidate gene identified, the gene was isolated at amplified for analysis of allelic differences within the gene, of which 4 were identified and named A1, A2, A3 and A4. Nodes of potatoes containing each the four alleles were assessed for tuber yield, with allele A2 having the greatest fresh weight at both 22°C and 28°C and combined A2A3 genotypes having the highest yield. Gene transcript levels were assessed in plants grown at both temperatures which showed that the HSc70 gene was upregulated in A2A3 plants but not in the A1A4 plants, while the HSc70 allele up-regulated in plants grown at the warmer temperature were all from the A2 allele.
Analysis of the amino acids of each of the alleles themselves found that the A2 and A3 proteins contained an additional C-terminal sequence of KIEEVD (thought to be involved in interactions with co-chaperones), while no other variations were unique in a single allele.
What was unique to the A2 allele was a difference in the promoter sequence where a run of ten TA repeats was found as opposed to a run of four TA repeats in the promoters of the other repeats. The researchers later hypothesize that the greater number of TA repeats may result in enhanced expression levels, a hypothesis based on previous research in yeast, although the specific effect of the repeat TA sequence is not known.
Confirming the effects of the HSc70 A2 allele
To test the A2 allele and its promoter sequence, the gene and upstream component was transferred into Nicotiana benthamiana plants and subjected to 45°C heat in a growth chamber. After 24 hours the expression of the HSc70 gene was 7 times greater than in the control plants and cell membrane injury in the transgenic plants were significantly lower.
The unique promoter was then targeted for further analysis. The A2 allele was put downstream of a series of promoters with TA repeats varying between four and ten times. After being exposed to the higher temperatures the level of HSc70 transcription was measured. Alleles with eight to ten TA repeats in the promoter showed statistically significant elevated transcript levels compared to alleles with four to six TA repeats in the promoter.
Figure 6 from article. a) HSc70 expression levels for promoters containing 4, 6, 8 and 10 TA repeats and b) cell membrane injury percentage for plants containing the same numbers of TA repeats.
A2 allele-containing plants with significantly increased expression under 28°C heat were selected for analysis under 40°C conditions for 24 hours in comparison to wild-type plants. Extracts of plants after 4 hours at this temperature showed HSc70 expression being enhanced by up to 50 times more than in control plants. However, after 24 hours the transcript levels fell back to that seen prior to being subjected to the extreme heat. The A2 allele-containing lines has significantly less cell membrane damage compared to the wild-type.
Finally, the ability of the A2 allele to protect tuber yield at elevated temperatures was tested. Using the same nodal cutting tuber yield assay as before, A2 allele containing plants were compared with wild Desiree potato plants when grown at 20°C showed little difference in tuber yield. However, at 28°C the Desiree plants reduced yield by 75% compared to yield at 22°C. By comparison, transgenic lines had significantly lower yield reductions, with some lines have twice the fresh and dry weight yield as the wild type.
The identification of the heat shock cognate protein 70 within the potato genome and the quantification of the effects of the specific A2 allele demonstrates the significance of quality genome research, genome annotation and the ability of powerful molecular tools to seek out expression variances that allow us to pinpoint the basis of variances in phenotype. Coupled with well prepared and executed experiments, the development of these tools will continue to enable us to extract information that will assist us to assist crops and food production.
Whether it is through transgenic technology or molecular breeding, the identification of genes such as the one discovered in this study will assist food production meet the challenges of a changing climate.
Identifying the specific allele of the HSc70 protein that contributes to its resistance to the negative effects of elevated temperature is surely a big step into future-proofing the security of this important crop.