Protoplast fusion and plant regeneration
Somatic hybridization between AC142 and MLM266-2 was successfully conducted via protoplast fusion. Among 980 calli, 248 differentiate calli were selected for plant regeneration, and one vigorous shoot of each callus was transferred to MS medium for root development. The brown and dead shoots were removed during subculture, and in total 80 vigorous shoots with strong roots were used for the analysis of their ploidy and genetic constitution (Fig. 1).
Identification and ploidy analysis of somatic hybrids
Two pairs of specific SSR primers (S215 and S165) of MLM266-2 and AC142 were used to identify somatic hybrids from the 80 regenerated plantlets. Amplified by primers S165, we observed the specific bands of 110 bp in AC142 and 150 bp in MLM266-2 (Fig 2A). For primers S215, a specific band (270 bp) was found in AC142 and a 250 bp band was found in MLM266-2, respectively (Fig. 2B). As a result, 51 regenerated plantlets were identified as somatic hybrids among 80 tested plantlets, which led to the successful protoplast fusion rate to 63.75%.
The ploidy of 51 somatic hybrids determined by flow cytometry were diverse, which included 23 octoploids, 20 hexaploids, 7 tetraploids, and a mixoploid. Unexpectedly, somatic hybrids of protoplasts fusion between diploid AC142 and MLM266-2 were mostly hexaploids and octoploids with much fewer tetraploids (Fig. 3A).
After subcultured for 4 years, the ploidy of 44 somatic hybrids out of 51 was analyzed again by flow cytometry, together with chromosome counting. Results showed that 23 had ploidy changes, accounting for 52.27% among 44 hybrids (Fig. 3B). Compared with the ploidy analysis in the years 2016, 16 out of 17 were still hexaploids, while only one hybrid changed to aneuploid, which was the most consistent type. Among 6 tetraploids, one changed to a hexaploid. A chimera of hexaploid and octoploid changed to be a hexaploidy. The most unstable ploidy was octoploidy, in which all the 20 hybrids had ploidy changes, turning out to be a tetraploid, 5 pentaploids, 9 hexaploids, a heptaploid, and 4 aneuploids. In a word, hexaploids and tetraploids were more stable at ploidy level in somatic hybrids (Fig 3, Supplement Table S1).
Analysis of freezing tolerance and agronomic traits in somatic hybrids
Electrolyte leakage was used to evaluate the freezing tolerance of NA and CA in 44 somatic hybrids. The results showed that the mean value of their NA reached -2.85°C and the coefficient of variation (CV) was 47.36% (Table 2). Among them, 88.64% of hybrids (39/44) hold intermediate NA between AC142 (-2.38°C) and MLM266-2 (-5.10°C), which suggested somatic hybrids had improvement of NA compared with the cultivated parent. On the other hand, most hybrids had more obvious enhancement in ACC than AC142 (0.74°C) and greater variation than NA (Table 2).
We analyzed the agronomic traits of fusion parents and 44 somatic hybrids, including plant morphology, flowering habit, and tuber yield, etc. The cultivated parent AC142 was semi-erect, abundant in flowers and had excellent tuber characters. And the wild species MLM266-2 was semi-erect and abundant in flowers, with high pollen viability and low yield (a large number of small tubers per plant). The somatic hybrids grew well on the whole, which exhibited 24 semi-erect plants, 16 decumbent plants, and 4 erect plants. Among the 44 hybrids, 75% of them showed heterobeltiosis in plant height and 30 hybrids (68.18%) produced normal flowers. All the somatic hybrids can produce tuber normally, tuber shape including oval, flat oblong, and oblate, etc., with a wide range of distribution and great differences in tuber weight per plant and tuber number per plant (Table 2). However, like the wild parent, most of them had many small tubers per plant.
Selection of excellent somatic hybrids and analysis of backcross efficiency with tetraploid cultivars
Nine somatic hybrids with strong freezing tolerance and relatively good agronomic traits were selected to backcross with 21 tetraploid cultivars to produce 46 backcross combinations. A total of 2175 seeds within 142 berries were obtained after 344 times of pollination (Supplement Table S2). The berry rates varied greatly for different somatic hybrids, ranging from 20% to 100%, and the average rate was 47.08% of the 9 somatic hybrids. In the backcross, the top three somatic hybrids of berry rate were the M+A74-1, M+A70-1, and M+A76-1. Meanwhile, M+A59-1, with the most backcross combinations, successfully backcrossed with 15 cultivars and obtained 755 seeds within 62 berries. However, a barrier of the post-zygotic development was observed in the backcross between somatic hybrids and cultivars, resulting in very few seeds per berry (Table 3).
Phenotype assessment of BC1 progenies
Three backcross combinations mentioned above were selected for further research and the progenies of M+A17-1 (6x) × Redsen (4x), M+A17-1 (6x) × Adirondack (4x) and M+A2-1 (6x) × 393160-4 (4x) were named FT069, FT070, and FT071 respectively. The ploidy level of the BC1 progenies ranged from 4x to 6x, and the majority were 5x (25/84) and 5x-6x (28/84), in which the proportion of mixoploids reached 50% by the female parent of M+A2-1 (Table 4).
The results of the electrolyte leakage test at -3°C showed that the freezing tolerance of three BC1 progenies largely varied (Table 4). Their progenies with NA (electrolyte leakage values) less than 50% accounted for 80.56%, 84.21%, and 70.37%, respectively; and with CA (electrolyte leakage values) less than 50% accounted for 83.78%, 90.91%, and 96.29%, respectively, which revealed the improved freezing tolerance in the materials compared with S. tuberosum (Table 4, Supplement Table S3).
The assessments of agronomic traits suggested that BC1 progenies had great variations in plant height, tuber number per plant, and tuber yield per plant (Fig. 4). The plant height varied from 30 cm to 110 cm, the number of tubers per plant ranged from zero to 135, and tuber yield per plant weighed from none to 534 g. The average tuber number per plant in FT070 was significantly less than that of the other two backcross combinations, while its tuber yield per plant was significantly higher (Fig. 4). The tuber traits of BC1 progenies were significantly improved compared with fusion parents, and some progenies had higher tuber yields, among which the tuber weight of FT069-16, FT069-32, FT070-1, FT070-22, and FT071-57 was close to that of the tetraploid cultivar (Supplement Table S3).
Correlation analysis between freezing tolerance and multiple traits, including ploidy, NA, CA, agronomic traits (tuber yield per plant and number of tubers per plant), were examined by 74 BC1 progenies of the three backcross combinations mentioned above. The results showed no significant correlation between ploidy with other traits and no significant correlation between NA or CA and agronomic traits (tuber yield per plant and number of tubers per plant). The results suggested that tuberization capacity and freezing tolerance were regulated by independent genetic loci with no interaction, which was more beneficial for the genetic improvement of freezing-resistant breeding materials (Table 5). To evaluate whether the aneuploids are useful for potato breeding, the differences in freezing tolerance and tuberization capacity between euploids and aneuploids were analyzed by two independent T-tests. There were no significant differences observed (Table 6), which proved that aneuploids did not affect freezing tolerance and yield-related traits.