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Abstract Great achievements have been made in the exploration of male sterile resources， gene mapping and cloning and molecular mechanism revealing， as well as in breeding and application of twoline rice in China. This paper briefly reviewed the discovery， types and cloning of sterile genes in rice and molecular breeding of twoline rice， and summarized the research progress of critical sterility inducing temperature， so as to provide new ideas for the research and breeding of twoline hybrid rice.
Key words Rice; Male sterile gene; Critical sterility inducing temperature; Molecular breeding
Rice is an important model crop， as well as the staple food grain for nearly half of the worlds population. As the contradiction between increasing population and decreasing cultivated land area becomes increasingly prominent， increasing the yield per unit area of rice is of great significance for ensuring China and even global food security. Since Shi Mingsong discovered Nongken 58S in 1973， twoline hybrid rice has become an important part of hybrid rice in China， and has made a huge contribution to increasing grain production in China. The discovery of a number of major male sterile resources provides a material basis for the invention of hybrid rice. Hybrid rice can be divided into two types， namely， threeline hybrid rice and twoline hybrid rice. Twoline hybrid rice has also become an important part of hybrid rice in China. This paper briefly reviewed the discovery， types and cloning of sterile genes in rice and molecular breeding of twoline rice， and summarized the research progress of critical sterility inducing temperature of twoline rice， aiming to provide new ideas for the research of twoline hybrid rice.
Discovery of Twoline Male Sterile Lines
In 1973， Chinas breeder Shi Mingsong[1]discovered the photosensitive sterile plant Nongken 58S which is sterile in longday conditions and fertile in shortday conditions. Nongken 58S can be used for two purposes， that is， it can be used for propagation under shortday conditions， and for hybrid seed production under longday conditions. This discovery opened the prelude to the breeding of twoline hybrid rice in China. Since then， a number of male sterile lines affected by light and temperature have been found， such as 5460S[2]， Annong S1[3]， and Hengnong S1[4-5]. These new materials， especially Annong S1， have greatly promoted the development of indica twoline hybrid rice in China. Compared with the threeline hybrid rice， the twoline hybrid rice can be freely combined， and has a wide restoring spectrum， and the average yield is increased by 5%-10%. And the sterile line propagation process is greatly simplified， and the promotion area is getting larger and larger. At present， twoline hybrid rice accounts for more than 35% of the total area of hybrid rice in the country， and twoline hybrid rice has an area surpassing that of threeline hybrid rice in Anhui Province and Hunan Province， becoming dominant[6]. Classification of Twoline Male Sterile Lines
According to incomplete statistics， there are more than 40 photo and thermosensitive male sterile rice resources in the world， more than half of which were found in China. These materials can be divided into the following five types according to the main characteristics of their response to light and temperature： photosensitive sterile type （longday sterile， shortday fertile）， such as Nongken 58S， thermosensitive sterile type （hightemperature sterile， lowtemperature fertile）， such as 5460S， Annong S1，Hengnong S， Zhu 1S and TMS10 mutant， antiphotosensitive type （longday fertile， shortday sterile）， such as the CSA mutant and Yi DS， antithermosensitive type （hightemperature fertile， lowtemperature sterile）， such as J207S， G20S， go543S and DiannongS2， and mediumtemperature fertile type （sterile at high temperatures and low temperatures， fertile at medium temperatures）， such as the SokchoMS mutant， which is male sterile at temperatures above 27 ℃ and below 25 ℃ and fertile at temperatures between 25 ℃ and 27 ℃[7]. Of course， the fertility of many materialsis often regulated by the interaction of light and temperature. The division here is based on the most important determinants.
Development of Twoline Male Sterile Lines
Cloned twoline sterile genes
At present， five twoline sterile genes have been cloned， and more than 10 twoline sterile genes have been mapped to different positions on the chromosome[8-10]. The Pms1 and pms3 genes are derived from Nongken 58S， and their products are noncoding RNAs， both of which cause sterility by monobasic mutation. The C→G base mutation of pms3 changes the secondary structure of the noncoding RNA， increases the degree of methylation in the promoter region， reduces the expression level of the noncoding RNA under longday conditions， and initiates the programmed death of anther cells， leading to pollen abortion[11]. Meanwhile， the sterile gene p/tms121 （pms3） of indica Peiai 64S （the offspring of Nongken 58s） was also cloned. The C→G mutation is located at the 11thbase of the 21nt small RNA， which causes the loss of the function of inhibiting the expression of downstream genes， resulting in male sterile[12]. Interestingly， p/tms121 （pms3） is mainly manifested by thermosensitive male sterile in the background of indica rice， but by photosensitive male sterile in japonica rice Nongken 58S. The encoded product of Pms1 is recognized by miR2118 and degraded into 18 pairs of 21nt phasiRNAs. G→A mutation may alter the secondary structure of RNA and further increase the shear efficiency of miR2118， leading to the production of more phasiRNAs under longday conditions. These phasiRNAs may lead to changes in the regulation of downstream gene expression efficiency， thereby participating in photosensitive male sterile regulation[13]. Pms1 and pms3 encode the same product and have similar regulatory mechanisms， but Pms1 is a dominant gene while pms3 is a recessive gene. These studies have broadened peoples understanding on the regulation of plant fertility by noncoding RNAs in response to environmental changes. The CSA gene encodes a R2R3 MYB transcription factor， which regulates the distribution of carbohydrates from vegetative organs to anthers and affects pollen development. Its mutant line （csa， antiphotosensitive） suffers from disturbances in anther nutritional distribution， leading to male sterile， but the fertility can be restored under longlight conditions[14-15]. The tms5 gene is derived from Annong S1 and Zhu 1S and encodes a functionally deficient RNase ZS1 enzyme. The RNase ZS1 of normal rice （TMS5） can degrade the mRNA of UbL40 gene， while the C→A mutation of tms5 that is 71bp from the initiation codon causes early termination of protein synthesis， leading to inactivation of RNase ZS1 enzyme， which allows the mRNA of its target gene UbL40 to accumulate. Under high temperature conditions， UbL40 is expressed at a high level， and the male sterile lines are sterile. Under low temperature conditions， UbL40 is expressed at a low level， and the rice lines are fertile. However， the expression of tms5 itself does not change with temperature[16]. tms5 is currently the most widely used twoline sterile gene， and more than 95% of twoline sterile lines for production contain this gene[17]. The thermosensitive gene TMS10 encodes a leucinerich repeat receptorlike kinases. This gene mutation can cause the lines to be male sterile under high temperature and to be fertile under low temperatures. The TMS10 gene itself is not induced by temperature， but its homologous gene TMS10L is highly expressed at low temperatures， which can compensate for the function of tms10 under low temperature conditions， thereby restoring lowtemperature fertility of plants[18].
The mapping and cloning of sterile genes in these materials has deepened peoples understanding of the molecular mechanism of plant flower organs sensing and responding to light and temperature changes.
Breeding of twoline sterile lines
Moreover， the cloning of these genes also provides gene sources for molecular design breeding of twoline sterile lines. Some scholars have reported new photo/thermosensitive male sterile lines obtained through editing of these fertility genes. In 2016， Zhou et al.[19]used the CRISP/Cas9 system to breed 11 new "transgene clean" thermosensitive genic male sterility lines within a year， accelerating the breeding process of male sterile lines. In 2019， Hirendra Nath Barman et al.[20]used the CRISP/Cas9 system to perform sitedirected mutagenesis on Zhongjiazao17 to obtain twoline sterile line YK17S. Forming combinations with this sterile line， the heterosis performance was excellent. In 2019， Du et al.[21]used the CRISPR/Cas9 technology to edit the TMS5 gene in the background of Wuyunjing 7. The results showed that the mutant individual plants were pollen aborted， but the female gametes were developed normally. These results indicate that thermosensitive male sterile lines can be obtained quickly by editing the thermosensitive male sterile gene TMS5. Based on the position and sequence information of these sterile genes， a batch of efficient molecular markers have been developed. Ding et al.[11]and Zhang et al.[17]， respectively， designed CAPs markers for the pms3 gene based on the mutation site. In 2011， Yang et al.[22]developed an Indel marker SJ001 for tms5 detection， which is located about 5 kb upstream of the functional mutation site of tms5. In 2014， Zhang et al.[23]developed two pairs of dCAPS markers RZ2F1/R and RZ2F2/R directing at the functional mutation site of tms5， by which the male sterile allelotype can be identified by two times of detection. In 2015， Song et al.[24]designed a pair of dCAPS markers， which could identify such three alleles as tms5tms5， TMS5TMS5 and TMS5 tms5 by detection at one time. And they proposed the strategy of selecting tms5 heterozygous （TMS5/tms5） fertile individual plants through molecular markers and selecting tms5 homozygous sterile individual plants after the agronomic traits are stable， so that the selection of new sterile lines is not restricted by light and temperature conditions， thereby shortening the breeding cycle and reducing the workload. Jin et al.[25]developed the functional variation of the tms5 gene into specific molecular markers based on high resolution melting analysis （HRM） （Development and application of functional molecular marker for thermosensitive genic male sterile gene tms5 in rice） in 2018. In 2018， Peng et al.[26]developed a set of SNP molecular markers for rice male sterile gene tms5， which can achieve commercial highthroughput rapid detection. The development of molecular markers for these male sterile genes and the development of new methods for the selection of male sterile lines will accelerate the selection process of the twoline male sterile lines.
Critical sterility inducing temperature and its influencing factors
The critical sterility inducing temperature （or fertility transition temperature） refers to the critical temperature when a sterile line changes from a sterile state to a fertile state （or a fertile line from a fertile state to a sterile state）. The critical sterility inducing temperature is directly related to the security of the twoline seed production. The lower the critical sterility inducing temperature， the longer the security period of the sterile line[27].
In 1989， the unusual low temperature in the Yangtze River Basin caused a large number of twoline sterile lines to selfseed， and people began to realize the important role of temperature in the fertility conversion of twoline sterile lines. Continuous low temperature appeared again in the middle and lower reaches of the Yangtze River in early August in 2001， and Anhui seed quality inspection departments found in 2002 that the unqualified rate of purity in Anhui indica twoline seed production was as high as 40%， and the abnormal plants were mainly sterile lines[29]. Yancheng， Jiangsu was once the largest twoline hybrid rice seed production base in China， accounting for 60% of Chinas twoline hybrid rice seed production area. In 2009-2015， there were 4 years that encountered failure of largearea twoline hybrid rice seed production[30]. The continuous low temperature around 24 ℃ in 2009 led to the failure of nearly 6 700 hm2 of seed production in Jiangsu， Anhui and other places， and a direct economic loss of nearly 100 million yuan， resulting in a reduction in the planting area of twoline hybrid combinations in the next year by more than 1.3 million hm2[31]. The continuous low temperature in the summer of 2014 caused about 90% of twoline male sterile lines in Anhui and Jiangsu to selfseed， resulting in more than 30 million kg of seeds being discarded[32]. The insecurity of seed production has become a major obstacle to the healthy and sustainable development of the twoline rice. One of the main reasons for the insecurity of twoline male sterile lines is the high critical sterility inducing temperature and its easy drift[33]. For the breeding practical twoline sterile lines of rice， low critical sterility inducing temperature is the most important technical indicator[28]. Only when the temperature is lower than or equal to 23.5 ℃ in the Yangtze River basin and lower than or equal to 24 ℃ in the provinces in South China can the effect of low temperature during the summer on seed production be greatly reduced[33-34]. Genetic background affects the critical sterility inducing temperature. The critical sterility inducing temperatures of sterile lines containing the same sterile gene are not necessarily the same. For example， of Annong S1 and Zhu 1S both containing the tms5 sterile gene， Annong S1 has a critical sterility inducing temperature greater than 26 ℃， while that of Zhu 1S is only 22.3 ℃.
At present， most scholars believe that "the critical sterility inducing temperature is a quantitative trait and is controlled by microeffect polygenes"， "the more microeffect genes a material aggregates， the lower critical sterility inducing temperature it has"， and "the impure genetic basis of the sterile lines or genetic heterozygosity is the intrinsic cause for the drift of critical sterility inducing temperature"[35-37]. During the addinggeneration propagation process of sterile lines， individual plants with a higher critical sterility inducing temperature have a higher seed setting rate， so their proportion in the population increases from generation to generation， their critical sterility inducing temperatures will increase from generation to generation， leading to the genetic drift phenomenon， and finally， the sterile line loses its application value[38]. When Peiai 64S passed the examination in 1991， the critical sterility inducing temperature was 23.3 ℃， and in 1993， it had risen to 24.5 ℃. Peiai 64S05， which was introduced by Zeng et al.[39]in 1995， still has a high proportion of lowfertility plants at 25.5 ℃， and exhibits population sterility until 26 ℃， and the critical sterility inducing temperature of some individual plants is up to 28 ℃.
Agricultural Biotechnology 2020
In 1999， He et al.[40]used FEI 64s and 8902s to construct F2 populations to locate QTLs that determine fertility instability. The sterile genes of both FEI 64s and 8902s are derived from Nongken 58s， and the F2 populations showed no fertility segregation when exposed to hightemperature conditions in long sunlight， indicating that Peiai 64s and 8902s have the same sterile gene， so the interference of sterile genes on the mapping results was excluded. However， the fertility stability of the two parents was quite different. Peiai 64s sterility was stable under longday conditions， and it was difficult to propagate it under shortday conditions， while 8902s was prone to selfseeding under longday conditions and could be reproduced easily under shortday conditions. The mapping results showed that such seven QTLs as L2， L3a， L3b， L5， L6， L7 and L10 were detected under longday conditions， and a total of six QTLs， S3a， S3b， S5， S8， and S10 were detected under shortday conditions， in which S3a and S3b overlapped with L3a and L3b in longday conditions. Peiai 64s is more stable in fertility than 8902s， but Peiai 64s contains three loci， L6， S3b， and S8， which can increase fertility instability （increasing the seed setting rate in low temperature environments）， and may be an important reason for the drift of the critical sterility inducing temperature. The results of He et al. showed that the instability of the twoline male sterile lines was determined by multiple loci， and there might be major loci. At present， the molecular mechanism of the critical sterility inducing temperature is still unclear. In the breeding process， the selection of the critical sterility inducing temperature is usually carried out by observing fertility through pollen staining under specific low temperature conditions. This phenotypic selection result is susceptible to factors such as growth period differences. Moreover， the operation processes of staged seeding identification， highaltitude and lowtemperature identification， artificial climate box identification and thermostatic water bath identification are tedious， timeconsuming， laborintensive， and it is difficult to apply them on a large scale. With the deepening of research and the improvement of technical means， the molecular mechanism of the critical sterility inducing temperature will be clarified. At that time， with the help of molecular breeding， the critical sterility inducing temperature could be selected in low generations to eliminate individual plants with high critical sterility inducing temperatures as early as possible， thereby greatly simplifying the breeding process and improving breeding efficiency.
Conclusions
Twoline hybrid rice is an important part of hybrid rice in China， and has made a significant contribution to increasing grain production in China. A number of original achievements have been made in the fields of sterile resource exploration， gene cloning and molecular mechanism revealing， as well as in the fields of breeding and application of twoline rice in China， and worldrenowned achievements have been achieved as well. This paper reviewed the research progress in the discovery， classification， sterile gene cloning， molecular breeding， and critical sterility inducing temperature of photo/thermosensitive male sterile rice， and put forward the prospect of molecular breeding of twoline hybrid rice， especially in the field of critical sterility inducing temperature， in order to provide new ideas for the research and breeding of twoline hybrid rice.
References
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Key words Rice; Male sterile gene; Critical sterility inducing temperature; Molecular breeding
Rice is an important model crop， as well as the staple food grain for nearly half of the worlds population. As the contradiction between increasing population and decreasing cultivated land area becomes increasingly prominent， increasing the yield per unit area of rice is of great significance for ensuring China and even global food security. Since Shi Mingsong discovered Nongken 58S in 1973， twoline hybrid rice has become an important part of hybrid rice in China， and has made a huge contribution to increasing grain production in China. The discovery of a number of major male sterile resources provides a material basis for the invention of hybrid rice. Hybrid rice can be divided into two types， namely， threeline hybrid rice and twoline hybrid rice. Twoline hybrid rice has also become an important part of hybrid rice in China. This paper briefly reviewed the discovery， types and cloning of sterile genes in rice and molecular breeding of twoline rice， and summarized the research progress of critical sterility inducing temperature of twoline rice， aiming to provide new ideas for the research of twoline hybrid rice.
Discovery of Twoline Male Sterile Lines
In 1973， Chinas breeder Shi Mingsong[1]discovered the photosensitive sterile plant Nongken 58S which is sterile in longday conditions and fertile in shortday conditions. Nongken 58S can be used for two purposes， that is， it can be used for propagation under shortday conditions， and for hybrid seed production under longday conditions. This discovery opened the prelude to the breeding of twoline hybrid rice in China. Since then， a number of male sterile lines affected by light and temperature have been found， such as 5460S[2]， Annong S1[3]， and Hengnong S1[4-5]. These new materials， especially Annong S1， have greatly promoted the development of indica twoline hybrid rice in China. Compared with the threeline hybrid rice， the twoline hybrid rice can be freely combined， and has a wide restoring spectrum， and the average yield is increased by 5%-10%. And the sterile line propagation process is greatly simplified， and the promotion area is getting larger and larger. At present， twoline hybrid rice accounts for more than 35% of the total area of hybrid rice in the country， and twoline hybrid rice has an area surpassing that of threeline hybrid rice in Anhui Province and Hunan Province， becoming dominant[6]. Classification of Twoline Male Sterile Lines
According to incomplete statistics， there are more than 40 photo and thermosensitive male sterile rice resources in the world， more than half of which were found in China. These materials can be divided into the following five types according to the main characteristics of their response to light and temperature： photosensitive sterile type （longday sterile， shortday fertile）， such as Nongken 58S， thermosensitive sterile type （hightemperature sterile， lowtemperature fertile）， such as 5460S， Annong S1，Hengnong S， Zhu 1S and TMS10 mutant， antiphotosensitive type （longday fertile， shortday sterile）， such as the CSA mutant and Yi DS， antithermosensitive type （hightemperature fertile， lowtemperature sterile）， such as J207S， G20S， go543S and DiannongS2， and mediumtemperature fertile type （sterile at high temperatures and low temperatures， fertile at medium temperatures）， such as the SokchoMS mutant， which is male sterile at temperatures above 27 ℃ and below 25 ℃ and fertile at temperatures between 25 ℃ and 27 ℃[7]. Of course， the fertility of many materialsis often regulated by the interaction of light and temperature. The division here is based on the most important determinants.
Development of Twoline Male Sterile Lines
Cloned twoline sterile genes
At present， five twoline sterile genes have been cloned， and more than 10 twoline sterile genes have been mapped to different positions on the chromosome[8-10]. The Pms1 and pms3 genes are derived from Nongken 58S， and their products are noncoding RNAs， both of which cause sterility by monobasic mutation. The C→G base mutation of pms3 changes the secondary structure of the noncoding RNA， increases the degree of methylation in the promoter region， reduces the expression level of the noncoding RNA under longday conditions， and initiates the programmed death of anther cells， leading to pollen abortion[11]. Meanwhile， the sterile gene p/tms121 （pms3） of indica Peiai 64S （the offspring of Nongken 58s） was also cloned. The C→G mutation is located at the 11thbase of the 21nt small RNA， which causes the loss of the function of inhibiting the expression of downstream genes， resulting in male sterile[12]. Interestingly， p/tms121 （pms3） is mainly manifested by thermosensitive male sterile in the background of indica rice， but by photosensitive male sterile in japonica rice Nongken 58S. The encoded product of Pms1 is recognized by miR2118 and degraded into 18 pairs of 21nt phasiRNAs. G→A mutation may alter the secondary structure of RNA and further increase the shear efficiency of miR2118， leading to the production of more phasiRNAs under longday conditions. These phasiRNAs may lead to changes in the regulation of downstream gene expression efficiency， thereby participating in photosensitive male sterile regulation[13]. Pms1 and pms3 encode the same product and have similar regulatory mechanisms， but Pms1 is a dominant gene while pms3 is a recessive gene. These studies have broadened peoples understanding on the regulation of plant fertility by noncoding RNAs in response to environmental changes. The CSA gene encodes a R2R3 MYB transcription factor， which regulates the distribution of carbohydrates from vegetative organs to anthers and affects pollen development. Its mutant line （csa， antiphotosensitive） suffers from disturbances in anther nutritional distribution， leading to male sterile， but the fertility can be restored under longlight conditions[14-15]. The tms5 gene is derived from Annong S1 and Zhu 1S and encodes a functionally deficient RNase ZS1 enzyme. The RNase ZS1 of normal rice （TMS5） can degrade the mRNA of UbL40 gene， while the C→A mutation of tms5 that is 71bp from the initiation codon causes early termination of protein synthesis， leading to inactivation of RNase ZS1 enzyme， which allows the mRNA of its target gene UbL40 to accumulate. Under high temperature conditions， UbL40 is expressed at a high level， and the male sterile lines are sterile. Under low temperature conditions， UbL40 is expressed at a low level， and the rice lines are fertile. However， the expression of tms5 itself does not change with temperature[16]. tms5 is currently the most widely used twoline sterile gene， and more than 95% of twoline sterile lines for production contain this gene[17]. The thermosensitive gene TMS10 encodes a leucinerich repeat receptorlike kinases. This gene mutation can cause the lines to be male sterile under high temperature and to be fertile under low temperatures. The TMS10 gene itself is not induced by temperature， but its homologous gene TMS10L is highly expressed at low temperatures， which can compensate for the function of tms10 under low temperature conditions， thereby restoring lowtemperature fertility of plants[18].
The mapping and cloning of sterile genes in these materials has deepened peoples understanding of the molecular mechanism of plant flower organs sensing and responding to light and temperature changes.
Breeding of twoline sterile lines
Moreover， the cloning of these genes also provides gene sources for molecular design breeding of twoline sterile lines. Some scholars have reported new photo/thermosensitive male sterile lines obtained through editing of these fertility genes. In 2016， Zhou et al.[19]used the CRISP/Cas9 system to breed 11 new "transgene clean" thermosensitive genic male sterility lines within a year， accelerating the breeding process of male sterile lines. In 2019， Hirendra Nath Barman et al.[20]used the CRISP/Cas9 system to perform sitedirected mutagenesis on Zhongjiazao17 to obtain twoline sterile line YK17S. Forming combinations with this sterile line， the heterosis performance was excellent. In 2019， Du et al.[21]used the CRISPR/Cas9 technology to edit the TMS5 gene in the background of Wuyunjing 7. The results showed that the mutant individual plants were pollen aborted， but the female gametes were developed normally. These results indicate that thermosensitive male sterile lines can be obtained quickly by editing the thermosensitive male sterile gene TMS5. Based on the position and sequence information of these sterile genes， a batch of efficient molecular markers have been developed. Ding et al.[11]and Zhang et al.[17]， respectively， designed CAPs markers for the pms3 gene based on the mutation site. In 2011， Yang et al.[22]developed an Indel marker SJ001 for tms5 detection， which is located about 5 kb upstream of the functional mutation site of tms5. In 2014， Zhang et al.[23]developed two pairs of dCAPS markers RZ2F1/R and RZ2F2/R directing at the functional mutation site of tms5， by which the male sterile allelotype can be identified by two times of detection. In 2015， Song et al.[24]designed a pair of dCAPS markers， which could identify such three alleles as tms5tms5， TMS5TMS5 and TMS5 tms5 by detection at one time. And they proposed the strategy of selecting tms5 heterozygous （TMS5/tms5） fertile individual plants through molecular markers and selecting tms5 homozygous sterile individual plants after the agronomic traits are stable， so that the selection of new sterile lines is not restricted by light and temperature conditions， thereby shortening the breeding cycle and reducing the workload. Jin et al.[25]developed the functional variation of the tms5 gene into specific molecular markers based on high resolution melting analysis （HRM） （Development and application of functional molecular marker for thermosensitive genic male sterile gene tms5 in rice） in 2018. In 2018， Peng et al.[26]developed a set of SNP molecular markers for rice male sterile gene tms5， which can achieve commercial highthroughput rapid detection. The development of molecular markers for these male sterile genes and the development of new methods for the selection of male sterile lines will accelerate the selection process of the twoline male sterile lines.
Critical sterility inducing temperature and its influencing factors
The critical sterility inducing temperature （or fertility transition temperature） refers to the critical temperature when a sterile line changes from a sterile state to a fertile state （or a fertile line from a fertile state to a sterile state）. The critical sterility inducing temperature is directly related to the security of the twoline seed production. The lower the critical sterility inducing temperature， the longer the security period of the sterile line[27].
In 1989， the unusual low temperature in the Yangtze River Basin caused a large number of twoline sterile lines to selfseed， and people began to realize the important role of temperature in the fertility conversion of twoline sterile lines. Continuous low temperature appeared again in the middle and lower reaches of the Yangtze River in early August in 2001， and Anhui seed quality inspection departments found in 2002 that the unqualified rate of purity in Anhui indica twoline seed production was as high as 40%， and the abnormal plants were mainly sterile lines[29]. Yancheng， Jiangsu was once the largest twoline hybrid rice seed production base in China， accounting for 60% of Chinas twoline hybrid rice seed production area. In 2009-2015， there were 4 years that encountered failure of largearea twoline hybrid rice seed production[30]. The continuous low temperature around 24 ℃ in 2009 led to the failure of nearly 6 700 hm2 of seed production in Jiangsu， Anhui and other places， and a direct economic loss of nearly 100 million yuan， resulting in a reduction in the planting area of twoline hybrid combinations in the next year by more than 1.3 million hm2[31]. The continuous low temperature in the summer of 2014 caused about 90% of twoline male sterile lines in Anhui and Jiangsu to selfseed， resulting in more than 30 million kg of seeds being discarded[32]. The insecurity of seed production has become a major obstacle to the healthy and sustainable development of the twoline rice. One of the main reasons for the insecurity of twoline male sterile lines is the high critical sterility inducing temperature and its easy drift[33]. For the breeding practical twoline sterile lines of rice， low critical sterility inducing temperature is the most important technical indicator[28]. Only when the temperature is lower than or equal to 23.5 ℃ in the Yangtze River basin and lower than or equal to 24 ℃ in the provinces in South China can the effect of low temperature during the summer on seed production be greatly reduced[33-34]. Genetic background affects the critical sterility inducing temperature. The critical sterility inducing temperatures of sterile lines containing the same sterile gene are not necessarily the same. For example， of Annong S1 and Zhu 1S both containing the tms5 sterile gene， Annong S1 has a critical sterility inducing temperature greater than 26 ℃， while that of Zhu 1S is only 22.3 ℃.
At present， most scholars believe that "the critical sterility inducing temperature is a quantitative trait and is controlled by microeffect polygenes"， "the more microeffect genes a material aggregates， the lower critical sterility inducing temperature it has"， and "the impure genetic basis of the sterile lines or genetic heterozygosity is the intrinsic cause for the drift of critical sterility inducing temperature"[35-37]. During the addinggeneration propagation process of sterile lines， individual plants with a higher critical sterility inducing temperature have a higher seed setting rate， so their proportion in the population increases from generation to generation， their critical sterility inducing temperatures will increase from generation to generation， leading to the genetic drift phenomenon， and finally， the sterile line loses its application value[38]. When Peiai 64S passed the examination in 1991， the critical sterility inducing temperature was 23.3 ℃， and in 1993， it had risen to 24.5 ℃. Peiai 64S05， which was introduced by Zeng et al.[39]in 1995， still has a high proportion of lowfertility plants at 25.5 ℃， and exhibits population sterility until 26 ℃， and the critical sterility inducing temperature of some individual plants is up to 28 ℃.
Agricultural Biotechnology 2020
In 1999， He et al.[40]used FEI 64s and 8902s to construct F2 populations to locate QTLs that determine fertility instability. The sterile genes of both FEI 64s and 8902s are derived from Nongken 58s， and the F2 populations showed no fertility segregation when exposed to hightemperature conditions in long sunlight， indicating that Peiai 64s and 8902s have the same sterile gene， so the interference of sterile genes on the mapping results was excluded. However， the fertility stability of the two parents was quite different. Peiai 64s sterility was stable under longday conditions， and it was difficult to propagate it under shortday conditions， while 8902s was prone to selfseeding under longday conditions and could be reproduced easily under shortday conditions. The mapping results showed that such seven QTLs as L2， L3a， L3b， L5， L6， L7 and L10 were detected under longday conditions， and a total of six QTLs， S3a， S3b， S5， S8， and S10 were detected under shortday conditions， in which S3a and S3b overlapped with L3a and L3b in longday conditions. Peiai 64s is more stable in fertility than 8902s， but Peiai 64s contains three loci， L6， S3b， and S8， which can increase fertility instability （increasing the seed setting rate in low temperature environments）， and may be an important reason for the drift of the critical sterility inducing temperature. The results of He et al. showed that the instability of the twoline male sterile lines was determined by multiple loci， and there might be major loci. At present， the molecular mechanism of the critical sterility inducing temperature is still unclear. In the breeding process， the selection of the critical sterility inducing temperature is usually carried out by observing fertility through pollen staining under specific low temperature conditions. This phenotypic selection result is susceptible to factors such as growth period differences. Moreover， the operation processes of staged seeding identification， highaltitude and lowtemperature identification， artificial climate box identification and thermostatic water bath identification are tedious， timeconsuming， laborintensive， and it is difficult to apply them on a large scale. With the deepening of research and the improvement of technical means， the molecular mechanism of the critical sterility inducing temperature will be clarified. At that time， with the help of molecular breeding， the critical sterility inducing temperature could be selected in low generations to eliminate individual plants with high critical sterility inducing temperatures as early as possible， thereby greatly simplifying the breeding process and improving breeding efficiency.
Conclusions
Twoline hybrid rice is an important part of hybrid rice in China， and has made a significant contribution to increasing grain production in China. A number of original achievements have been made in the fields of sterile resource exploration， gene cloning and molecular mechanism revealing， as well as in the fields of breeding and application of twoline rice in China， and worldrenowned achievements have been achieved as well. This paper reviewed the research progress in the discovery， classification， sterile gene cloning， molecular breeding， and critical sterility inducing temperature of photo/thermosensitive male sterile rice， and put forward the prospect of molecular breeding of twoline hybrid rice， especially in the field of critical sterility inducing temperature， in order to provide new ideas for the research and breeding of twoline hybrid rice.
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