Development of thermo-photo sensitive genic male sterile lines in wheat using doubled haploid breeding

Background Two-line hybrid wheat system using thermo-photo sensitive genic male sterility (TPSGMS) is currently the most promising approach for wheat heterosis utilization in China. However, during past 20 years only few TPSGMS lines were developed in hybrid wheat breeding, which has been the main limiting factor to create heterotic hybrids. Application of doubled haploid (DH) breeding provides a useful strategy to efficiently develop practically usable TPSGMS lines. Results F1s and selected F2 and F3 sterile plants of eight crosses made from two commercial TPSGMS lines were used to produce DH lines. We developed a total of 24 elite DH sterile lines with stable sterility, good outcrossing and yield potential, resistance to yellow rust and powdery mildew, as well as desirable plant height (50–60 cm). These DH lines were developed within 4 years through at least 1 year of evaluation. The stability of male sterility was confirmed for most (20/24) of these elite DH sterile lines by multiple tests in two or 3 years. These lines are expected to be used in hybrid wheat breeding. The percentage of elite lines developed from the tested DH lines produced from filial generations was in the order of F2 > F3 > F1. Conclusions We demonstrate that coupling DH techniques with conventional breeding is an efficient strategy for accelerating the development of more practical wheat TPSGMS lines. Generation of DHs from F2 generation appeared to be the better choice considering the balance of shortening breeding time and overall breeding efficiency.


Background
Wheat provides about 20% of the world's nutrition supply [1]. Heterosis utilization in wheat is one of the most promising ways for increasing yield potential and stability, which has importance for increasing the productivity of wheat to meet the growing demand in the world [2][3][4]. However, developing hybrids with high level of heterosis and producing hybrid seeds with low cost remain challenges in hybrid wheat breeding and its commercial application [1,4].
Unlike hybrid wheat systems based on cytoplasmic male sterility (CMS) [5] and photoperiod-sensitive cytoplasmic male sterility (PCMS) [6], the two-line hybrid wheat system using thermo-photo sensitive genic male sterility (TPSGMS) is a new methodology of wheat heterosis utilization in China. The TPSGMS line is characterized as being sterile under low-temperature and short-day for hybrid seed production, and fertile under high-temperature and long-day for self propagation. Therefore, this system does not need a sterility 'maintainer' line and makes hybrid seed production easier [7][8][9]. From 2002 to 2018, 20 hybrid wheat varieties were released in China [10], 14 of them were developed using TPSGMS-based two-line system with yield increase of 10-15%, especially in marginal lands [10][11][12]. Meanwhile, encouraging multi-location evaluations have been conducted and observed in Vietnam, where "Yunza" hybrid varieties performed much better than local inbred cultivars in yield, drought tolerance and fertilizer input [13]. Nevertheless, the 14 TPSGMS-based hybrid varieties accounted only 0.5% of total 2691 wheat varieties released in China from 2002 to 2018 [14]. In addition, few hybrid varieties were applied in main producing areas such as Yellow-Huai River wheat zone of China, where inbred varieties perform well in yield while most hybrid varieties available did not exhibit enough yield advantage. An important cause for this situation is that only eight practically usable TPSGMS lines across China were developed over past 20 years, which greatly restricted the opportunities and efficiency of creating heterotic hybrids although ten thousands of restorers were test-crossed. A practical TPSGMS line is commonly characterized as stable sterile duration for ≥20 days in different years [15,16], outcrossing rate for ≥70% [17], dwarf plant height at 50-70 cm [15], as well as good combining ability and agronomic traits [9]. In addition, the recessive genes controlled sterility only express at a restricted temperature and day-length condition and produces a low ratio of sterile plants in segregating population [15]. These factors result in a low efficiency in developing practical TPSGMS lines by conventional breeding methods. Therefore, the current breeding strategy needs to be improved to develop more practical wheat TPSGMS lines for producing more heterotic hybrids.
The Doubled Haploid (DH) technique allows to homogenize a heterozygous material in one generation. It has been widely used in crop breeding to improve the efficiency of selection and to accelerate the breeding process [18][19][20][21][22]. In wheat, DHs can be produced by intergeneric cross between wheat (Triticum aestivum L.) and maize (Zea mays L.). This methodology has become an integral part of many commercial wheat breeding programs. It has advantages of stable induction and few genotypic restrictions in producing haploids over anther and microspore cultures [23,24]. This study aimed to evaluate the efficiency of developing wheat TPSGMS lines by using DH technique based on wheat × maize in breeding program with sterile materials derived from different filial generations of F 1 , F 2 and F 3 .

Successful generation of DH lines by wheat × maize system
During summer sowings in 2014-2016, wheat DHs were produced from four F 1 s and sterile plants selected from F 1 and F 2 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][26][27][28][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].

Candidates of elite DH sterile lines
During 2016-2018, all 920 DH lines produced from F 1 , F 2 and F 3 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 F 1 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. To test the stability of sterility in different years, ten F 1derived 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 (   The ten elite TPSGMS lines derived from F 1 generation showed nearly 100% sterility in 3 years when sown from Oct. 15 to Nov. 5, although the average temperatures varied from 12°C to 15°C 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 3 years' tests, thus selected for hybrid breeding. Stable sterility is a prerequisite to commercial utilization for a TPSGMS line [16].
Sterility stability of F 2 -derived elite sterile lines was confirmed by repeat tests Ten F 2 -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 2 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.

Elite DH sterile lines showed high out-crossing ability
In winter sowing of 2018/2019, the out-crossing potential of 20 elite TPSGMS lines derived from F 1 and F 2 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 F 2 generation, showing out-crossing rate > 80%, 4 lines between 75 and 80%, and 3 lines between 70 and    (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 [17]. More lines derived from F 2 generation appeared to have better out-crossing ability (> 80%) compared with those from F 1 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.

Comparison of DH breeding efficiency among generations
Based on the seed setting rates < 5% in both sowing dates, 41 DH sterile lines, including 13, 15, and 13 lines derived from F 1 , F 2 and F 3 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 F 1, F 2 and F 3 , respectively. A total of 24 elite lines were selected based on further evaluation of other desired traits. The breeding efficiency for F 1, F 2 and F 3 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 F 1 , F 2 and F 3 generations ( Table 2). The trend of breeding efficiency for a single trait (sterility) was in the order of F 3 > F 2 > F 1 , while for comprehensive traits was F 2 > F 3 > F 1 , which suggests that producing DHs from F 2 generation is better in overall breeding efficiency.

Discussion
Discovery and application of male sterility is the foundation of commercial production of hybrid wheat. The chance of creating elite heterotic hybrids is correlated with the number of sterile lines and restorers available  in breeding programs. Although the TPSGMS-based two-line hybrid wheat system was established in 1990s [7,30], less than ten TPSGMS lines capable for commercial usage have been developed in north and south wheat zones of China up to now. Pedigree method is commonly used in developing TPSGMS lines [7,15,30,31], however, several difficulties have hindered the breeding efficiency. The sterility of TPSGMS line is controlled by two or three recessive major genes plus several minor genes [7,16,[32][33][34], causing a very low proportion of highly sterile plants in segregating populations, especially in F 2 s derived from crosses between sterile lines and normal fertile lines. When other desired traits are considered together during selection, the breeding efficiency would become extremely low. Theoretically, the probability of homozygous recessive individuals in F 2 population is 1/4 n , the probability would be 1/2 n if DH lines are produced from F 1 , suggesting DH breeding is more efficient for selection of traits controlled by recessive genes, such as the sterility here. Crosses between semisterile materials and sterile lines can further increase the proportion of highly sterile plants in segregating populations of this study, which is similar in effectiveness to backcrossing with sterile lines [31]. Few effective molecular markers are currently available for marker-assisted selection in sterility of wheat TPSGMS lines [35]. Consequently, it is time-consuming to develop a genetically stable TPSGMS line because the expression of sterility needs strict temperature and light condition which is only available one season per year [15]. In our previous breeding program, only two practical TPSGMS lines (K78S and K456S) were developed by pedigree method from 1996 to 2010, while in this study we developed 24 elite TPSGMS lines with complete homozygosity and other desired traits within 4 years by introducing DH techniques.
Another issue addressed in this study is to identify the ideal generation for producing DHs. Most breeders prefer to produce DHs from F 1 generation to shorten the breeding cycles, but this approach may limit the chances for recombination [18]. Therefore, producing DHs with selected individuals from F 2 generation of single crosses or F 1 generation of pyramiding crosses seems to be better than that from F 1 generation of single crosses [36]. Similarly, Snape and Simpson (1981) inclined to produce DHs from F 2 generation in barley by comparing the gain in genetic variation for six agronomic traits with DH lines derived from F 1 , F 2 , F 3 and intermated F 2 (S3) generations [37]. In contrast, Iyamabo and Hayes (1995) did not find more favorable genotypes in DH lines produced from F 2 generation than that from F 1 generation in barley, therefore, they preferred to use F 1 generation for producing DHs [38]. In the present study, the overall breeding efficiency of producing DHs from filial generations was in the order of F 2 > F 3 > F 1 , indicating that F 2 generation is better for producing DHs in breeding efficiency. However, it still needs to be further investigated by comparing the breeding efficiency of producing DHs with F 1 and selected plants of F 2 and F 3 derived from the same cross.  Producing DHs from F 1 generation had less breeding efficiency because only one round of recombination occurred and no selection was applied. As a result, a high frequency of agronomically undesirable lines were produced [37], which was confirmed in this study as most fertile lines were discarded from F 1 generation. However, as showed in this study, using F 1 generation for DH breeding has the edge in saving time; it could be useful for crosses with better predictability and coping with urgent needs for developing varieties with resistance to diseases, such as yellow rust for its frequently varying pathogenic races.

Conclusion
In this study we developed 20 practical TPSGMS lines of wheat. These lines showed stable sterility in replicated evaluation in multiple years, as well as good outcrossing potential and other desirable traits. We demonstrated that introducing DH technique is an efficient strategy in accelerating development of TPSGMS lines of wheat. Producing DHs from F 2 generation appeared to be the better choice with balance of breeding efficiency and shortening of breeding cycle. Nevertheless, it will be necessary to conduct further investigations by using diverse genetic materials of different filial generations derived from the same combinations. More practically usable TPSGMS lines would further increase the opportunity of creating heterotic hybrids in hybrid wheat breeding.

Plant materials
Two TPSGMS lines and five semi-sterile advanced lines of wheat were used in the study (Table 6). A maize variety "Baitiannuo" was used as pollen donor in DH production. All wheat and maize materials were bred by Institute of Food Crops, Yunnan Academy of Agricultural Sciences, Kunming, China.

Crossing and DH production
Wheat materials were late sown in Jan. 2014 to make semi-sterile materials fertile for crossing with sterile lines K78S and K456S in Mar. 2014 at Kunming, Yunnan province, China (25°02′N, 102°42′E, altitude 1960 m). For DH production, maize sowing (in April) began 2 months before wheat sowing (in June) to synchronize their flowering dates. Maize was sown in three dates with an interval of 14 days.
Before producing doubled haploids, pedigree methods were adopted to select sterile plants from segregating population of F 1 and F 2 generations according to performances in sterility, out-crossing potential including glume opening and stigma exsertion [17], plant height (50-60 cm), resistance to yellow rust and powdery mildew, tillering ability and the yield potential. Seeds of sterile plants were harvested from regenerated tillers by cutting all spikes of sterile plants followed by intensive management in irrigation and fertilization. The crossings and subsequent breeding work and DH production are summarized in Fig. 1.

Method of producing DHs
For DH production, we adopted an improved protocol from previous reports [18,23,24]. Wheat spikes were pollinated with fresh maize pollen 24 to 48 h after emasculation. Pollinated tillers were cut 24 h after pollination and sprayed with 100 ppm 2,4-D, then were cultured in growth chambers for 14 days with nutrition solution containing 100 mg L − 1 2,4-D, 40 g L − 1 sucrose, 10 mg L − 1 silver nitrate, 3 g L − 1 potassium dihydrogen phosphate and 3 g L − 1 urea. The nutrient solution was replaced every 3 days. During culture, the growth chamber keeps a regime of 14 h darkness / 10 h light with light intensity of 6000 Lux, constant temperature of 25 ± 1°C and relative humidity of 80 ± 5%. Embryos were aseptically dissected from 14-day caryopses and cultured on half-strength MS medium [39] under darkness at 24 ± 1°C until germination, then moved to the growth house at a regime of 14 h darkness /10 h light (at 3000 Lux), keeping constant temperature of 25 ± 1°C and humidity of 75 ± 5%. When seedlings developed two to three tillers, the plantlets were taken out and immersed in 0.05% colchicine solution for 8 h at 25°C to induce doubling of chromosomes. Treated seedlings were transplanted into pots to grow until booting stage, then moved into greenhouse for 15 days, keeping temperature > 20°C to ensure fertility of DH plants. All plants were bagged before flowering and harvested one by one.

DH sterile line selection and its stability evaluation in sterility
The sterility of DH lines obtained during 2015-2017 was independently evaluated by sowing at two dates on Oct.15 and Nov. 20, respectively (Fig. 3). At least 10 spikes per line in each sowing were randomly bagged before flowering to measure the seed setting rate. The out-crossing potential (glume opening, stigma exsertion) [17], and other important traits such as disease resistances and yield potential were also recorded. Lines with sterility higher than 95% in both sowings were kept for stability evaluation of sterility next year.
Where gn means the number of grains from bagged spikes, sn the number of spikelets. A TPSGMS line with SSR < 5% was recognized as highly sterile and qualified for hybrid seed production [16].
Temperatures during 2016-2018 were collected from a data-logger 'HUATO S100-TH' in thermometer screen near the field, and daylengths from the meteorological station of Kunming.
Out-crossing potential assessment of elite sterile lines Twenty elite DH sterile lines derived from F 1 and F 2 generations were separately planted as 10 rows in a plot of 1 m × 2.5 m surrounded by about 600 restorers on Oct. 15, 2018. Open pollination was aided by natural wind of grade 3-6, which is usual in Yunnan throughout wheat growing seasons. Twenty spikes of each line were randomly bagged before flowering. Twenty open pollinated spikes were randomly harvested from 10 rows of each line with the bagged spikes to measure the outcrossing rate (OR) of sterile lines as follow [16,17]: Where ugn and bgn are un-bagged and bagged grain numbers at two basal florets of each spikelet respectively, sn the number of spikelets per spike. bgn is counted to exclude the possible self-pollination seed setting because TPSGMS lines are not always keeping 100% sterile.

Evaluation of disease resistance
Yellow rust and powdery mildew are two most important and frequently occurred diseases at Kunming. A highly susceptible cultivar was planted close to tested DH sterile lines as the control and spreader. The adult plant resistance of sterile lines was scored in the field according to Han et al. (2010) for yellow rust [40] and  for powdery mildew [41].

Statistical analysis
The embryo rate, haploid seedling rate and chromosome doubling rate were analyzed using one-way analysis of variance (ANOVA) followed by Fisher's least significant difference (LSD) test. U-test of multiple percentage comparison [42] was conducted for evaluating the significant differences in breeding efficiency of producing DHs from F 1 , F 2 and F 3 generations. SPSS and Excel Office were used in statistical analyses.