Heterosis is the phenomenon that the first generation of hybrid is superior to its parents in one or more traits. Using heterosis to create new crop varieties can improve crop yield, quality and resistance (Gupta et al., 2019). Compared with conventional wheat varieties, hybrid wheat tends to increase production by 10%~20%. Therefore, the study of wheat heterosis is of great theoretical and practical significance (Zhao, 2010).

As early as the 1960s, China began to study hybrid wheat, and there were mainly two ways to use heterosis (Zhao et al., 2018): the first was genetic mechanism, mainly including “three-line method” and “two-line method”; the second was the “chemical killing method”. A lot of research has been done on these two ways in the utilization of wheat heterosis. Now, the research mainly focuses on the “two-line method” and “chemical killing method”. The research and application results of the former are remarkable, which makes the utilization of wheat heterosis in China reach the international leading level. “Two-line method” is the research and utilization of light and thermo sensitive male sterile lines, including photosensitive type (BS Series), thermo sensitive type (C49S series, BNS Series) and photo thermo interactive type (ES Series). Many national and provincial varieties have been bred. This method is simple to operate, but the fertility of male sterile lines is unstable due to environmental changes, which limits the wide application of this method. “Chemical-killing method” is to produce male sterile lines through abnormal stamen development caused by the use of chemical reagents, which has the advantages of simple procedure of seed production, quick selection for strong dominant combinations. The varieties such as “Xiza1” and “Yun97CH4” have been selected. However, problems such as the effect of male killing, the purity and yield of hybrid and environmental pollution restrict the large-scale use (Wen et al., 2017). Male sterile lines in the “three-line method” include cytoplasmic controlled male sterile lines, nuclear gene controlled male sterile lines and nuclear-cytoplasmic interaction male sterile lines. Up to now, it has been found that the used male sterile materials include T type, K type, V type, AL type and F type (Wen et al., 2017). A new hybrid wheat variety ‘Xindong43’ was selected by using AL male sterile lines (Nie et al., 2014, Journal of Triticeae Crops, 34 (11): 1450), and a new hybrid wheat variety ‘Xinan 112’ was selected by using recessive genic male sterile lines (Ruan et al., 2016). However, the male sterile line is controlled by multiple genes, and there are also problems such as unstable fertility, which limits the development of this technology.

The male sterile line of K-type wheat (CMS-K) was bred by replacing the nucleus of Chinese lB/IR varieties into the cytoplasm of Aegilops kotschyi (Mukai and Tsunewaki, 1979). The preliminary study showed that the fertility of K-type male sterile line was complete, controlled by main effect and micro effect multiple restoration genes. The restoration source was wide, and the hybrid seeds were full, so it had a broad application prospect in hybrid wheat. The male sterile line of F-type wheat was a new type of cytoplasmic male sterile line of common wheat. It was bred by Lanhong Hybrid Wheat Research Center. At present, a series of research results have been achieved (Zhao et al., 2015; Zhang et al., 2016; Yuan et al., 2018). However, it is necessary to continuously identify male sterile lines in different regions, screen matched restoration sources and strong dominant combinations, so as to select new hybrid wheat varieties. The K-type and F-type male sterile lines are “three-line method” male sterile materials, so it is necessary to screen maintainer lines, restorer lines and strong dominant combinations for relevant male sterile lines, so as to accelerate the theoretical and practical basis of hybrid wheat. In this experiment, the K-type and F-type male sterile lines were used as female parents to be hybridized with 583 common wheat varieties. By investigating the self-seed set ratio on F₁ of combinations, the recurrent parents which were suit for the transfer of new male sterile lines and restorer lines with high recovery degree, were selected. At the same time, the cytological identification of male sterile line materials was carried out, and the pollen abortive characteristics of different male sterile lines were compared, so as to provide reference for the transfer of new male sterile lines and the screening of strong restorer lines, and to accelerate the process of hybrid wheat breeding.

1 Results and Analysis

1.1 Pollen fertility characteristics of K-type and F-type male sterile lines

The self-seed set ratio of male sterile line materials was counted for two consecutive years from 2017 to 2018 and 2018 to 2019. The average self-seed set ratio of K-type male sterile line was 4.61% (domestic algorithm), and the average self-seed set ratio of F-type male sterile line was 1.98% (domestic algorithm).

The pollen of male sterile line wheat was dyed by 1% I₂-KI, and then the type and proportion of abortive pollen were identified. The results (Figure 1) showed that the pollen abortive characteristics of K-type male sterile line were stained aborted tape 68.66%, round aborted tape 23.88% and fertility 3.03%; The pollen abortive characteristics of F-type male sterile lines were typical aborted tape 54.62%, round aborted tape 37.69% and fertility 2.27%. Therefore, the pollen abortion types of K-type and F-type male sterile lines were stained aborted tape and typical aborted tape respectively.

Through the measurement of pollen grain size, the normal pollen radius of K-type male sterile line was 39.0~40.2 µm, and the coefficient of variation was 0.084; The pollen radius of F-type male sterile line was 40.1~43.9 µm, and the coefficient of variation was 0.095; The pollen radius of ‘Jinmai84’ was 40.0~40.3 µm, and the coefficient of variation was 0.039. Compared with ‘Jinmai84’, the normal pollen grains of F-type male sterile line were significantly larger than that of ‘Jinmai84’, while the normal pollen grains of K-type male sterile line and ‘Jinmai84’ didn’t show significant difference. At the same time, the normal pollen grains of F-type male sterile line and K-type male sterile line didn’t show significant difference.

1.2 Determination of recovery capability of K-type and F-type male sterile lines

1.2.1 Effect of different male parents on sterile restorer lines

According to the cross combination of K-type and F-type male sterile lines in 2017-2018 and 2018-2019, the range distribution of self-seed set ratio of F₁ (domestic algorithm) (Figure 2) showed that the high fertility and better combinations of FA accounted for 67.74% in 2017-2018, and the high fertility and better combinations of CMS-K only accounted for 33.82%. From 2018 to 2019, the high fertility and better combinations of FA accounted for 48.6%, and the high fertility and better combinations of K-type male sterile lines accounted for 72.50%. The results of the recovery test showed that the recovery degree of F₁ was mostly concentrated in high fertility areas. Therefore, the male sterile lines of K-type and F-type wheat were easy to recover and had a wide range of restoration sources.

The survey results of 2017-2018 and 2018-2019 (Table 1) showed that the recovery of K-type and F-type male sterile lines varied greatly, and the range distribution of self-seed set ratio of F₁ (international algorithm) was 9.55%~131.67% and 12.35%~134.85% respectively. There were 17 high restoring combinations with self-seed set ratio of F₁ (international algorithm) greater than 100%, and there are 5 K-type male sterile lines and 6 F-type male sterile lines belonging to low restoring combinations with seed setting rate (international algorithm) less than 20%. The high recovery hybrid combinations of K-type male sterile lines included Shandong varieties such as ‘Liangxing805’, ‘Jimai22’ and ‘Yannong999’, Shanxi varieties such as Yunmai, Yunhan and Linmai series, and Shaanxi varieties, so the restoration sources were wide. Besides Henan and Shandong varieties, Shanxi varieties sunch as Linmai or Yunmai varieties (lines) have better recovery among the high recovery hybrid parents of F-type male sterile lines. Among the low recovery hybrid combinations, ‘Bao8696’, ‘Nongda3291’, ‘Ke1201’, and ‘Ji 8906069’ can be used to screen the excellent maintainers of K-type male sterile lines, and ‘15Zhan11’, ‘Wenhang6’, ‘Linyu5’ and ‘Fengyou8’ can be used to screen the excellent maintainers of F-type male sterile lines. Through backcross breeding, F-type agronomic characters can be improved and new male sterile lines can be obtained for further production and application.

1.2.2 Effect of the same male parents on sterile restorer lines

In 2017-2018 and 2018-2019, K-type and F-type male sterile lines were hybridized with 39 same male parents respectively. The survey results (Table 2) showed that there was a great difference in the self-seed set ratio of F₁, and the total proportions of high fertility and full fertility were 64.10% and 87.18% respectively. There were significant (p<0.05) or very significant (p<0.01) differences in 18 combinations. The self-seed set ratio of F₁ generation of CMS-K with ‘Wangfeng369’, ‘Yannong999’, ‘Hengyan56’, ‘15Zhan11’ was significantly higher than that of F₁ generation of FA with same male parents. The first three were high fertility and the last was semi-sterility. The self-seed set ratio of F₁ of F-type male sterile lines with 14 male parents such as ‘15Zhan24’, ‘16Zhan12’, ‘16Zhan16’, ‘15BiA2’, ‘15Guan295’, ‘Fen40929’, ‘Yan1604’, ‘Yu607’ and ‘Bao8696’ was significantly higher than that of F₁ of CMS-K with same male parents. The F₁ hybrids of ‘Wangfeng369’, ‘Shannong88069’ with FA were semi-sterility, and the other F₁ generations were high fertility, which showed FA had more restoration sources. Among the highly sterile materials with significant differences, F₁ of ‘Wangfeng369’, ‘15zhan11’ with FA were high sterility, and F₁ of ‘Shannong88069’, ‘Bao8696’, ‘Ke1201’ with CMS-K were high sterility.

2 Discussion

This study found that the fertile pollen rate of K-type male sterile line and F-type male sterile line were less than 5%, and their self-seed set ratio was also low. It can be seen that the pollen abortion of both lines was complete and stable. CMS-K pollen abortion was mainly stained aborted tape, and FA pollen abortion was mainly and typical aborted tape. Previous studies believe that the abortion of F-type male sterile lines was mainly stained aborted tape (Zhang et al., 2016; Yuan et al., 2018) or the common type of stained aborted tape and typical aborted tape (Yuan et al., 2018). The reason may be related to the nucleocytoplasmic gene interaction or environmental differences of F-type male sterile lines. A large number of facts showed that there were fewer restorer lines in the male sterile lines with early pollen abortion, while there were more restorer lines in the male sterile lines with late pollen abortion (Li and Sun, 1996), CMS-K pollen was stained and aborted in the late mononuclear stage (Meng et al., 2015), and FA pollen mother cells began to degrade in the reproductive nucleus and vegetative nucleus in the binuclear stage (Zhao et al., 2015). The difference between FA normal pollen grains and CMS-K normal pollen grains was not significant, but the pollen abortion of FA was late, and the pollen grains were mostly empty, shriveled or degraded, indicating that FA abortion was complete and had wider restoration sources. In this study, different varieties (lines) were used as male parents to cross with K-type and F-type male sterile lines. The analysis of self-seed set ratio of F₁ showed that K-type and F-type male sterile lines had more restoration sources, but the self-seed set ratio of F₁ of K-type male sterile lines with ‘Bao8696’, ‘Wangfeng369’ and ‘Ke1201’ was different from that of F₁ of F-type male sterile lines with these male parents, which was consistent with the different types of pollen abortion and was mainly related to the different sources of male sterile lines; Using the same varieties (lines) as male parents and K-type and F-type male sterile lines as female parents, the self-seed set ratio of F₁ showed that F-type male sterile lines had stronger recovery and were mostly concentrated in self bred varieties, which may be related to the fact that the breeding process of F-type male sterile lines occurred in Shanxi.

Through pollen fertility identification and recovery degree investigation, the fertility characteristics of the two male sterile lines were preliminarily revealed, which provided a basis for screening restorer lines, maintainer lines and strong dominant combinations (Ba et al., 2015). Through two years of fertility investigation of male sterile lines, it was preliminarily speculated that there was a certain degree of environmental induction effect on the fertility of K-type male sterile lines and F-type male sterile lines (Qi et al., 2015; Qin et al., 2018), but suitable local restorer lines with high recovery and stability can be obtained through screening, so as to form strong dominant hybrid combinations. In view of the common influence of environment and gene on the fertility of male sterile lines, the strong dominant combinations suitable for local K-type and F-type male sterile lines could be used for the breeding of local hybrids, and the low restorer could be used to transfer new types of male sterile lines. By selecting recurrent parents with good agronomic traits and strong disease resistance, new K-type and F-type male sterile lines suitable for local could be transferred (Chen et al., 2016; Wen et al., 2017).

In order to meet the needs of characteristic agricultural development, K-type and F-type male sterile lines can be fully combined with local dryland wheat and characteristic wheat to screen hybrids with stable yield and high quality (Li et al., 2016; Zhang, 2019). For example, it has been proved that the local dryland wheat variety ‘Jinmai47’ was an excellent restorer line of FA and could form strong dominant hybrid combinations (Wen et al., 2017). At present, molecular marker technology (Li et al., 2019) and gene editing technology (Okada et al., 2019) have been applied to hybrid wheat. Combining molecular breeding technology with hybrid wheat production can accelerate the breeding process and quickly select hybrids with advantages such as water saving, stress resistance, stable yield and high yield, so as to meet the needs of production and life and ensure food security.

3 Materials and Methods

3.1 Test materials

K-type wheat male sterile line was provided by the project of “Wheat Heterosis Utilization Technology and Strong Heterosis Hybrid Creation”, and F-type wheat male sterile line was bred and provided by Lanhong Hybrid Wheat Research Center in Yuncheng City of Shanxi. Most of the materials used for hybridization came from germplasm resources in different ecological areas in China, a few were self bred varieties (lines), and the control variety was ‘Jinmai84’, which was bred and preserved by our lab.. All materials were normally sown in autumn in 2017-2018 and 2018-2019 at the Experimental Farm in Institute of Cotton Research, Shanxi Agricultural University.

3.2 Cytological observation

Five spikes were selected from the main stem of K-type and F-type male sterile lines and ‘Jinmai 84’ at the flowering stage, and two anthers from the middle spikelet of the selected spike were picked and placed on the slide. The pollen grains were squeeze out with an anatomical needle and the residue was removed. Stain with 1% I₂-KI solution for 1 min, cover the slide, and observe them under a 200W eyepiece with JNOEC (XSP-16A) biological microscope and take photos under 10×, 40×objectives. The number of abortive and fertile pollen in 10 visual fields was counted. Pollen abortion rate = (number of abortive pollen grains/total number of pollen grains)×100% (Zhang et al., 2016). At the same time, the length of micrographs was calibrated and tested with a 0.01 mm dividing plate, and then the size of normal pollen grains was measured.

3.3 Investigation on seed setting rate and sterility of bagged selfing

In 2017-2018 and 2018-2019, 583 conventional wheat varieties (lines) were used as male parents to hybridize with K-type and F-type male sterile lines. Each combination hybridized 10 spikes and harvested. F₁ was planted in single row in October of that year. 10 spikes were randomly selected for bagging at the heading stage of the next year. The number of grains and spikelets per spike were investigated at the maturity stage to calculate the average self-seed set ratio of F₁; At the same time, the male sterile lines were self bagged, and the self-seed set ratio of male sterile lines was investigated at the maturity stage (Zhang et al., 2016).

Seed setting rate (international algorithm)=(the setting number of available spikelets/total number of florets at the base of available spikelets×2)×100%

Seed setting rate (domestic algorithm)=(the setting number of florets on both sides of the base of available spikelets/total number of florets at the base of available spikelets×2)×100%

The recovery degree of varieties (lines) was expressed by self-seed set ratio of F₁ generation (domestic algorithm). According to the recovery degree, it was divided into four grades: the recovery of 0~20% was high sterility (H_s); The recovery of 20%~50% was semi-sterility (S_s); The recovery of 50%~80% was high fertility (H_f); If the recovery was over 80%, it was complete fertility (C_f).

3.4 Statistical analysis

Data sorting was carried out by Excel 2013; Statistical analysis was carried out by SPSS 24.0 software.

Authors’ Contributions

QYL was the executor of the experimental design and research of this study; XSG was the conceiver and the person in charge of the project, guiding experimental design, data analysis, manuscript writing and revision; RWB and WQ completed field phenotypic investigation, pollen microscopic observation and data statistical analysis; YJZ participated in the experimental design and field material planting and management. All authors read and approved the final manuscript.

Acknowledgments

This study was funded by National Key Research and Development Program “Wheat Heterosis Utilization Technology and Strong Heterosis Hybrid Creation” in China (2016YFD0101600).

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