Production of DH lines
During summer sowings in 2014-2016, wheat DHs were produced from four F1s and sterile plants selected from F1 and F2 generations by wheat ´ maize system (Fig.1). A total of 920 DH lines were obtained from all eight crosses (Table 1). In Dec. 2016, a major frost caused damage of plants that were heading, resulting in partial failure to obtain DH seeds. Variance analysis showed that there were significant difference in the rate of immature embryos (P=0.00) and haploid seedling rate (No. of seedlings germinated from every 100 inoculated embryos, P=0.00) among different combinations, suggesting that embryo rate and haploid seedling rate were more susceptible to genotypes. The averages of embryo rate, seedling rate of embryos and chromosome doubling rate of seedlings were 36.76%, 62.65% and 86.42% respectively, exhibiting a good efficiency in DH production as demonstrated in our previous studies [25-29].
Temperate climate at Kunming, especially from May to October, allows planting spring and vernalized winter wheat materials throughout the year under natural condition (Fig. 2 and Supplemental Data 1), which facilitates mass production of wheat DHs by wheat ´ maize crosses because fresh pollens are available from multiple rounds of planting of maize plants from late April to early November [27,29].
Selection of candidate DH sterile lines
During 2016-2018, all 920 DH lines produced from F1, F2 and F3 generations were evaluated independently using a one-year sterility test to screen candidate sterile lines (Fig. 3). A total of 295 DH lines showed normal seed set were excluded from further analysis. These lines were mostly from F1 generations as expected. In the 1st (Oct. 15) and 2nd (Nov. 20) sowings, 210 (33.60%) and 66 (10.65%) lines from the total of 625 DH lines had seed setting rates less than 5%, respectively. Furthermore, 41 (6.56%) DH lines showed seed setting rate less than 5% in both sowings (Table 2, Fig. 4 and Supplemental Data 2). When further considering other desired traits of out-crossing potential, disease resistance (to stripe rust and powdery mildew), plant height (50-60 cm), tillering and spike formation ability, 24 lines were selected from 41 lines as our final set of elite DH lines.
When sterile lines are sown on Oct. 15 (1st sowing) and Nov. 20 (2nd sowing), the critical periods for causing fertility alteration are from middle to late February and from late March to early April, respectively. Consequently, during the critical periods, the 1st sown sterile lines would go through lower temperature and shorter days to fully exhibit sterility, while the 2nd sown lines would have relatively higher temperature and longer days that can cause the early heading spikes sterile and the late heading tillers partially fertile to produce a few seeds for propagation (Fig. 2 and Supplemental Data 1).
Based on our experience, TPSGMS lines that exhibit 100% sterility in the 2nd sowing date are usually stable in sterility but are difficult for propagation, which make them not suitable for practical application. In southwest of China wheat is normally sown from middle Oct. to early Nov., thus a TPSGMS line with seed setting rate <5% in both sowing dates (from Oct. 15 to Nov. 20) would meet the demand for safe production of qualified hybrid seeds.
Stability evaulation of sterility for DH lines derived from F1 generation
To test the stablilty of sterility in different years, ten F1-derived elite DH sterile lines were evaluated in two growing seasons from 2016 to 2018 by planting in two and ten different sowing dates respectively (Fig. 3). In the 2016/2017 growing season, the seed setting rates of all lines were 0 in the 1st sowing date, and ranged from 2.98% to 4.87% in the 2nd sowing date (Table 3). In further tests using ten sowing dates in the 2017/2018 season, the seed setting rates of the ten elite lines were < 1% from the 1st to the 3rd sowings (Oct.22-Nov. 5), < 5% till the 5th sowing (Nov. 19), and ≥ 50% in the 10th sowing (Dec. 24), suggesting sowings before Nov. 5-19 were optimum for hybrid seed production, and sowings after Dec. 24 is suitable for propagation of these sterile lines (Fig. 5 and Supplemental Data 3).
The ten elite TPSGMS lines derived from F1 generation showed nearly 100% sterility in three years when sown from Oct. 15 to Nov. 5, although the average temperatures varied from 12℃ to 15℃ during the critical periods from the second half Feb. to the first half Mar. (Fig.2 and Supplemental Data 1). These lines are stable in sterility in three years’ tests, thus selected for hybrid breeding. Stable sterility is a prerequisite to commercial utilization for a TPSGMS line .
Stability evaulation of sterility for DH sterile lines derived from F2 generation
Ten F2-derived DH lines were selected in 2016/2017 and were evaluated again in the 2017/2018 growing season (Fig 3). The seed setting rates of all lines were 0 in the 1st sowing, and ranged from 1.99% to 4.04% in the 2nd sowing (Table 4). These ten lines showed stable sterility in two years, were selected to plant in ten sowing dates for further evaluation in stability of sterility, as well as determination of suitable sowing times for hybrid seed production and self propagation.
Evaluation of out-crossing potential for elite TPSGMS lines
In winter sowing of 2018/2019, the out-crossing potential of 20 elite TPSGMS lines derived from F1 and F2 generations were evaluated. The out-crossing rates of the 20 lines ranged from 70.46 % to 93.90% with an average of 82.87%. There were 13 lines, including 8 derived from F2 generation, showing out-crossing rate > 80%, 4 lines between 75% and 80%, and 3 lines between 70% and 75% (Table 5). All 20 lines showed high out-crossing potential after only one round of selection after DH production. Thus, doubled haploids showed a great efficiency in fixation of this trait, which confirmed our previous results. More lines derived from F2 generation appeared to have better out-crossing ability (>80%) compared with those from F1 generation, suggesting that one more cycle of selection before DH production is helpful to further enhance the target trait. The results of out-crossing rates here were obtained by pollination with nearly unlimited pollen supply, it needs to be further confirmed in practical hybrid seed production.
Breeding efficiency of different generations
Based on the seed setting rates < 5% in both sowing dates, 41 DH sterile lines, including 13, 15, and 13 lines derived from F1, F2 and F3 generations, respectively, were selected (Table 2), the breeding efficiency (percentage of selected DH lines in total DH lines tested) was 4.14%, 7.35% and 12.15% for F1, F2 and F3, respectively. A total of 24 elite lines were selected based on further evaluation of other desired traits. The breeding efficiency for F1, F2 and F3 was 3.18%, 4.90% and 3.74%, respectively. U-test analyses indicated that there were significant differences (P<0.01) in breeding efficiency of producing DHs from F1, F2 and F3 generations (Table 2). The trend of breeding efficiency for a single trait (sterility) was in the order of F3 > F2 > F1, while for comprehensive traits was F2 > F3 > F1, which suggests that producing DHs from F2 generation is better in overall breeding efficiency.