An international breeding project using a wild potato relative Solanum commersonii resulted in two new frost-tolerant native potato cultivars for the Andes and the Altiplano.

This breeding project, initiated at the United States Potato Genebank (USPG) in collaboration with Peruvian partners Instituto Nacional de Innovacion Agraria (INIA), International Potato Center, Peru (CIP), and local farmers, sought to enhance cold hardiness and frost tolerance in native potato cultivars in Peru. The Andes and Altiplano are often affected by frost, which causes significant reduction in yield; creating varieties with superior resilience is a critical undertaking. The goal was to transfer outstanding non-acclimated cold tolerance and acclimation capacity found in wild potato species Solanum commersonii (cmm). Breeding families segregating for cold hardiness were created using (a) a somatic hybrid cmm + haploid Solanum tuberosum (tbr) (cv. Superior, US variety from Wisconsin) as male and (b) seven cultivars native to Peru of the species S. tuberosum sbsp. andigenum (adg) as females. All plant materials were part of the USPG germplasm collection. Sexual seeds of each family were sent to Peru for evaluations under the natural conditions of the Andean highlands and Altiplano. The plants were assessed for their response to frost, and genotypes showing exceptional tolerance were selected. Plants were also evaluated for good tuber traits and yield. Initial planting involving ~2,500 seedlings in five locations resulted in selecting 58 genotypes with exceptional frost tolerance, good recovery capacity after frost, and good tuber traits. Over the years, evaluations continued and were expanded to replicated field trials in the harsher conditions of the Altiplano (Puno). All trials confirmed consistency of frost tolerance over time and location, tuber quality, and yield. After 8 years, two advanced clones were considered for cultivar release because of their exceptional frost tolerance and superior field productivity that outyielded many of the established cultivars in the region. In November 2018, a new native cultivar named Wiñay, a Quechua word meaning "to grow" was released in Peru. In 2022, a second cultivar followed with the name Llapanchispaq (meaning "for all of us"). This project evidenced that a multinational and all-encompassing approach to deploy valuable genetic diversity can work and deliver effective results. This is even more significant when outcomes can promote food security and sustainability in very vulnerable regions of the world.

Fractionating of Calcium in Tuber and Leaf Tissues Explains the Calcium Deficiency Symptoms in Potato Plant Overexpressing CAX1.

Consistent with reports on other plants we recently reported that a potato transgenic line (AT010901) overexpressing sCAX1 show classic symptoms of calcium deficiency shoot tip injury, leaf curling, leaf margin necrosis and tuber internal defects such as hollow heart and brown spots. The present study was undertaken to quantify calcium in various fraction of leaf and tuber tissues of this transgenic and wild type potato clones to understand the development of these deficiency symptoms at normal calcium nutrition (1mM) and its mitigation at higher calcium nutrition (10mM). Plants were grown in controlled environment growth chamber and watered with balanced nutrient solution containing either 1 or 10 mM calcium. The plants overexpressing sCAX1 showed calcium deficiency symptoms while sequestering calcium in the vacuole as calcium oxalate crystals. Various fractions of calcium were qualified in the young and mature leaves as well as tuber tissue. A reduced concentration of water soluble fraction of calcium was most important factor related to the development of calcium deficiency symptoms in the line overexpressing sCAX1. Furthermore, an increase in this fraction appear to explain the alleviation of the deficiency symptoms in these transgenic plants.Ours is the first study to document the significance of water-soluble calcium in the development of calcium-deficiency symptoms in the potato transgenic lines overexpressing sCAX1. Furthermore, our result demonstrates that an increase in this fraction plays a significant role in the alleviation of calcium deficiency symptoms when calcium concentration in the nutrient media is increased. These results provide important insight on the role of sCAX1 in the calcium homeostasis.

Genetic Variance Partitioning and Genome-Wide Prediction with Allele Dosage Information in Autotetraploid Potato.

As one of the world's most important food crops, the potato (Solanum tuberosum L.) has spurred innovation in autotetraploid genetics, including in the use of SNP arrays to determine allele dosage at thousands of markers. By combining genotype and pedigree information with phenotype data for economically important traits, the objectives of this study were to (1) partition the genetic variance into additive vs. nonadditive components, and (2) determine the accuracy of genome-wide prediction. Between 2012 and 2017, a training population of 571 clones was evaluated for total yield, specific gravity, and chip fry color. Genomic covariance matrices for additive (G), digenic dominant (D), and additive × additive epistatic (G#G) effects were calculated using 3895 markers, and the numerator relationship matrix (A) was calculated from a 13-generation pedigree. Based on model fit and prediction accuracy, mixed model analysis with G was superior to A for yield and fry color but not specific gravity. The amount of additive genetic variance captured by markers was 20% of the total genetic variance for specific gravity, compared to 45% for yield and fry color. Within the training population, including nonadditive effects improved accuracy and/or bias for all three traits when predicting total genotypic value. When six F1 populations were used for validation, prediction accuracy ranged from 0.06 to 0.63 and was consistently lower (0.13 on average) without allele dosage information. We conclude that genome-wide prediction is feasible in potato and that it will improve selection for breeding value given the substantial amount of nonadditive genetic variance in elite germplasm.

Automated tetraploid genotype calling by hierarchical clustering.

KEY MESSAGE: New software to make tetraploid genotype calls from SNP array data was developed, which uses hierarchical clustering and multiple F1 populations to calibrate the relationship between signal intensity and allele dosage. SNP arrays are transforming breeding and genetics research for autotetraploids. To fully utilize these arrays, the relationship between signal intensity and allele dosage must be calibrated for each marker. We developed an improved computational method to automate this process, which is provided as the R package ClusterCall. In the training phase of the algorithm, hierarchical clustering within an F1 population is used to group samples with similar intensity values, and allele dosages are assigned to clusters based on expected segregation ratios. In the prediction phase, multiple F1 populations and the prediction set are clustered together, and the genotype for each cluster is the mode of the training set samples. A concordance metric, defined as the proportion of training set samples equal to the mode, can be used to eliminate unreliable markers and compare different algorithms. Across three potato families genotyped with an 8K SNP array, ClusterCall scored 5729 markers with at least 0.95 concordance (94.6% of its total), compared to 5325 with the software fitTetra (82.5% of its total). The three families were used to predict genotypes for 5218 SNPs in the SolCAP diversity panel, compared with 3521 SNPs in a previous study in which genotypes were called manually. One of the additional markers produced a significant association for vine maturity near a well-known causal locus on chromosome 5. In conclusion, when multiple F1 populations are available, ClusterCall is an efficient method for accurate, autotetraploid genotype calling that enables the use of SNP data for research and plant breeding.

Changes in freezing tolerance, plasma membrane H+-ATPase activity and fatty acid composition in Pinus resinosa needles during cold acclimation and de-acclimation.

It has previously been suggested that plasma membrane ATPase (PM H+-ATPase, EC 3.6.1.3.) is a site of incipient freezing injury because activity increases following cold acclimation and there are published data indicating that activity of PM H+-ATPase is modulated by changes in lipids associated with the enzyme. To test and extend these findings in a tree species, we analyzed PM H+-ATPase activity and the fatty acid (FA) composition of glycerolipids in purified plasma membranes (PMs) prepared by the two-phase partition method from current-year needles of adult red pine (Pinus resinosa Ait.) trees. Freezing tolerance of the needles decreased from -56 degrees C in March to -9 degrees C in May, and increased from -15 degrees C in September to -148 degrees C in January. Specific activity of vanadate-sensitive PM H+-ATPase increased more than two-fold following cold acclimation, despite a concurrent increase in protein concentration. During de-acclimation, decreases in PM H+-ATPase activity and freezing tolerance were accompanied by decreases in the proportions of oleic (18:1) and linoleic (18:2) acids and increases in the proportions of palmitic (16:0) and linolenic (18:3) acids in total glycerolipids extracted from the plasma membrane fraction. This pattern of changes in PM H+-ATPase activity and the 18:1, 18:2 and 18:3 fatty acids was reversed during cold acclimation. In the PM fractions, changes in FA unsaturation, expressed as the double bond index (1 x 18:1 + 2 x 18:2 + 3 x 18:3), were closely correlated with changes in H+-ATPase specific activity (r2 = 0.995). Changes in freezing tolerance were well correlated with DBI (r2 = 0.877) and ATPase specific activity (r2 = 0.833) in the PM fraction. Total ATPase activity in microsomal fractions also closely followed changes in freezing tolerance (r2 = 0.969). We conclude that, as in herbaceous plants, simultaneous seasonal changes in PM H+-ATPase activity and fatty acid composition occur during cold acclimation and de-acclimation in an extremely winter hardy tree species under natural conditions, lending support to the hypothesis that FA-regulated PM H+-ATPase activity is involved in the cellular response underlying cold acclimation and de-acclimation.

Characterization of a cDNA encoding Arabidopsis secretory phospholipase A2-alpha, an enzyme that generates bioactive lysophospholipids and free fatty acids.

Phospholipase A2s (PLA2s) are enzymes that liberate lysophospholipids and free fatty acids (FFAs) from membrane phospholipids in response to hormones and other external stimuli. This report describes the cloning and functional characterization of a PLA2 cDNA from Arabidopsis thaliana, AtsPLA2-alpha, which represents one of four secretory PLA2 (sPLA2) genes in Arabidopsis. The encoded protein is 148-amino acid polypeptide and is predicted to contain a 20-amino acid signal peptide at its amino terminus. The predicted mature form (Mr=14,169) of AtsPLA2-alpha exhibited approximately 5 times the specific activity of its pre-processed form. Different from animal sPLA2s, AtsPLA2-alpha showed a significant preference for the acyl group linoleic acid over palmitic acid in phospholipid hydrolysis. Like some animal sPLA2s, however, it has a slight preference for phosphatidylethanolamine over phosphatidylcholine as the substrate. The specific activity of AtsPLA2-alpha continuously increased as the Ca2+ concentration was increased to 10 mM, and the optimal pH range was very broad and biphasic between 6 and 11. AtsPLA2-alpha transcript was detected at low levels in roots, stems, leaves, and flowers but not in siliques.

Secretory low molecular weight phospholipase A2 plays important roles in cell elongation and shoot gravitropism in Arabidopsis.

To elucidate the cellular functions of phospholipase A(2) in plants, an Arabidopsis cDNA encoding a secretory low molecular weight phospholipase A(2) (AtsPLA(2)beta) was isolated. Phenotype analyses of transgenic plants showed that overexpression of AtsPLA(2)beta promotes cell elongation, resulting in prolonged leaf petioles and inflorescence stems, whereas RNA interference-mediated silencing of AtsPLA(2)beta expression retards cell elongation, resulting in shortened leaf petioles and stems. AtsPLA(2)beta is expressed in the cortical, vascular, and endodermal cells of the actively growing tissues of inflorescence stems and hypocotyls. AtsPLA(2)beta then is secreted into the extracellular spaces, where signaling for cell wall acidification is thought to occur. AtsPLA(2)beta-overexpressing or -silenced transgenic plants showed altered gravitropism in inflorescence stems and hypocotyls. AtsPLA(2)beta expression is induced rapidly by auxin treatment and in the curving regions of inflorescence stems undergoing the gravitropic response. These results suggest that AtsPLA(2)beta regulates the process of cell elongation and plays important roles in shoot gravitropism by mediating auxin-induced cell elongation.

Root zone calcium modulates the response of potato plants to heat stress.

Potato plant growth and development are known to be severely impacted by heat stress. Here plants grown in a chemically inert medium of 1 : 1 quartzite : perlite (v : v) were subjected to either 35/25 degrees C (stress) or 20/15 degrees C (control) day/night air temperatures and four concentrations of root zone calcium (5, 25, 125 and 600 &mgr;M Ca) for 3 weeks. We report for the first time that potato plant growth under heat stress can persist at specific levels of Ca2+ in the root zone and that the Ca2+ level required for growth under heat stress exceeds that required for growth under normal temperatures. We also provide strong, initial evidence that the ability of high Ca2+ levels to mitigate heat stress effects results from shifts in meristematic activity. Total foliar mass and leaf area were essentially unaffected by Ca2+ level under control temperatures. Under heat stress, leaf area was reduced to about 5% of the control at 5 and 25 &mgr;M Ca but to only 70% of the control at 125 and 600 &mgr;M Ca. Likewise, total foliar mass was reduced under heat stress to about 30% of the control at 5 and 25 &mgr;M Ca but total foliar mass was greater under heat stress than control conditions at 125 and 600 &mgr;M Ca. This increase at higher Ca2+ concentrations was due primarily to axillary shoot growth. Anatomical studies of leaves grown under heat stress show that cell expansion was impaired by heat stress and this impairment was overcome by increasing root zone calcium levels. These results provide insight into the mechanism by which root zone Ca2+ may modulate plant response to heat stress.