Production of DH lines
During summer sowings of 2014-2016, we produced DHs with four F1s and sterile plants selected from F1 and F2 segregating populations by wheat ´ maize system (Table 2). A total of 920 DH lines were obtained from all eight crosses (Table 3). In Dec. 2016, a serious frost damaged some doubling treated plants that were heading, resulting in partial failure to obtain DH seeds. Variance analysis showed that there were significant difference in embryo rate (P=0.00) and haploid seedling rate (P=0.00) among different combinations, which indicated that embryo rate and haploid seedling rate were more susceptible to genotypic influence. 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 good efficiency in DH production as showed in our previous study [22].
Temperate climate at Kunming especially from May to October allows planting of spring and vernalized winter wheat materials throughout the year under natural conditions here (Fig. 1 and Supplemental Figure 1), which facilitates mass production of wheat DHs by wheat ´ maize crosses because plenty of fresh pollens are available from naturally and repeatedly planted maize plants from late April to early November [22-26].
Selection of candidate DH sterile lines
All 920 DH lines produced from F1, F2 and F3 generations were separately tested for sterility during 2016-2018. Among them, 295 DH lines showed normal seed set were excluded from further testing. These lines were mainly from F1 generations as expected. In the 1st (Oct. 15) and 2nd (Nov. 20) sowings, 210 lines (33.60%) and 66 lines (10.65%) of 625 DH lines in total tested had seed setting rates less than 5%, respectively. 41 (6.56%) DH lines showed seed setting rate less than 5% in both sowings (Table 4, Fig. 2 and Supplemental Figure 2). When keeping in view other desired traits of out-crossing rate (≥70%), disease resistance (to stripe rust, powdery mildew), plant height (50-60 cm), tillering and spike formation ability, 24 elite lines were finally selected from 41 lines above mentioned.
When sterile lines were sown on Oct. 15 (1st sowing) and Nov. 20 (2nd sowing), their sensitive periods (causing fertility alteration) would be the dates from middle to late February and from late March to early April, respectively. Consequently, during sensitive periods, the 1st sown sterile lines would have low temperature and short-day to fully exhibit sterility, while the 2nd sown lines would have relatively higher temperature and longer day length that cause the early heading spikes sterile and the late heading tillers partially fertile to produce a few seeds for propagation (Fig. 1 and Supplemental Figure 1).
According to our experiences, TPSGMS lines that exhibit 100% sterility in the 2nd sowing date are usually stable in sterility but difficult in 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 be able to meet the demand for safe production of qualified hybrid seeds.
Sterility determination of DH lines derived from F1 generation
Ten elite DH sterile lines derived from F1 were repeatedly tested during 2016-2018. In 2016/2017, 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 5). In further test of ten sowing dates in 2017/2018, 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 and Nov. 19 were separately the optimum and suitable times for hybrid seed production while after Dec. 24 the suitable time for propagation of these sterile lines (Fig. 3 and Supplemental Figure 3).
During 2016-2018, ten elite TPSGMS lines derived from F1 generation showed highly or 100% sterility in three years when sown from Oct. 15 to Nov. 5, although the average temperatures varied from 12℃ to 15℃ during their sensitive periods from the second half Feb. to the first half Mar. (Fig.1 and Supplemental Figure1), will be utilized in hybrid breeding later.
Sterility evaluation of sterile lines derived from F2 generation
Ten selected lines in 2016/2017 derived from F2 were tested again in winter sowing of 2017/2018. 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 6). All elite lines showed stably in sterility during two-year cycles, and will be planted in ten sowing dates for further determination in sterility, suitable times for hybrid seed production and propagation. Meanwhile, preliminary test-crosses will be conducted with these lines.
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 20 lines ranged from 70.46 % to 93.90% with average of 82.87%. There were 13 lines including 8 derived from F2 generation with out-crossing rate > 80%, 4 lines between 75% and 80% and 3 lines between 70% and 75% (Table 7). All 20 lines showed high out-crossing potential even only one round of selection was done after DH production, because doubled haploids had ‘genetically fixed’ the trait, which confirmed our previous results[27]. However, more lines derived from F2 generation appeared to have better out-crossing ability (>80%) compared with that from F1 generation, suggesting one more cycle of selection before DH production would help to further concentrate the target trait. In fact, the results of out-crossing rates here were obtained by pollination with nearly unlimited pollen supply, and need to be further assessed in practical hybrid seed production.
Breeding efficiency of different generations
According to seed setting rates < 5% in both sowing dates, 41 DH sterile lines including 13, 15, and 13 lines separately derived from F1, F2 and F3 generations were selected, with breeding efficiency (percentage of selected DH lines in total DH lines tested) of 4.14%, 7.35% and 12.15% in F1, F2 and F3, respectively. When out-crossing ability, resistance to diseases and other desired traits were further considered, 24 elite lines were left and the overall breeding efficiency in F1, F2 and F3 were 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 4). 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.