Development of cleistogamous rice lines through introduction of the spw1-cls mutation into common chasmogamous cultivars would be unsatisfactory if the cleistogamous lines were phenotypically different from the original cultivars, particularly if agronomic traits were inferior. It is essential that there is little difference between the cleistogamous and the original chasmogamous cultivars, except for the non-flowering character.
Persistence of anthers inside the caryopsis is one of the inevitable features of cleistogamous rice; this characteristic is usually absent in chasmogamous cultivars. Anther persistence might reduce brown rice quality by deforming or staining the endosperm. Anthers remained in the hull throughout the ripening stages of spw1-cls
. However, withering anthers of spw1-cls
do not affect ripening processes (Yoshida et al. 2007
, ). We also demonstrated that brown and polished rice of spw1-cls
are not damaged by persistent anthers. Furthermore, it was possible to remove all remaining anthers by hulling and polishing (). Thus, spw1-cls
maintains brown rice quality and the rice can be handled in the same manner as common cultivars.
Using relatively coarse evaluation procedures, we demonstrated that cleistogamy introduced by the spw1-cls
mutation had little effect on many agronomic traits (Yoshida et al. 2007
). In this study, over a five-year cultivation period, there were some differences in yield-related traits between spw1-cls
and T65; however the estimated yield of spw1-cls
(calculated from yield-related traits) was similar that of T65 and was acceptable (). In this respect, the spw1-cls
mutation has more promise than the d7
mutation, which is accompanied by a dwarfing phenotype and significantly unfavorable agronomic traits, especially low yield (Nagao and Takahashi 1954
, ). In addition, the spw1-cls
lines and individuals are likely to have various second-site mutations that occurred concurrently with the spw1-cls
mutation in the original M1
generation mutant, because they varied in some characteristics (i.e., dwarfism and sterility). It is possible that these mutations lowered the yield of spw1-cls
Because we identified the position of nucleotide change responsible for spw1-cls
, we were able to design DNA markers that detected the spw1-cls
mutation. As a model case for using such DNA markers, we created the cleistogamous backcross lines Yumeaoba-cls using a dCAPS marker (). In Japan, research on GM rice for livestock forage is in progress. Because “Yumeaoba” is one potential cultivar suitable for whole crop silage (Miura et al. 2006
), we selected this variety as the recurrent parent of the cleistogamous backcross line. Yumeaoba-cls had stable cleistogamy in the paddy fields through two years of experiments. Thus, we were able to successfully introduce the spw1-cls
mutation to other cultivars by using a reliable DNA marker. The spw1-cls
mutation was functional in other genetic backgrounds. Although the number of backcrosses was limited, the yield-related traits of Yumeaoba-cls of the BC2
generation in 2009 and the BC2
generation in 2010 were closer to those of the recurrent parent Yumeaoba (), compared with the yield difference between spw1-cls
and T65. The creation of Yumeaoba-cls lines that would be more isogenic with Yumeaoba is likely possible, if we were to increase the number of backcrosses for removing the second-site mutations. On the other hand, the introduced spw1-cls
mutation is not expected to have secondary effects on agronomic traits expressed in the original cultivars.
Our natural crossing tests in the paddy fields clearly showed that spw1-cls
is able to suppress outcrossing (). In all the experiments with T65 as the pollen parent line, we found significant numbers of xenia grains derived from crossings with the pollen parent. The number of xenia grains in the south experimental plot located to the lee of prevailing winds was higher than in the other (i.e., north, east and west) plots. However, we found no xenia grains in experiments with spw1-cls
as the pollen parent line under conditions prevailing when T65 was the pollen parent. Although xenia grains with non-T65 genotype were discovered, we judged that they were derived from crossings other than crossing with spw1-cls
, because the experimental paddy field where the natural crossing tests were conducted was surrounded by other paddy fields where various cultivars and lines other than T65 were cultivated. Crossing rates of rice decrease as distances between plants increase (Sato and Yokoya 2008
, Tanno et al. 2011
). In our experiment, pollen parent lines and seed parent lines were very close to one another. In lines positioned closest together, panicles of individual parents were able to make physical contact. In spite of this close contact, spw1-cls
never crossed with the seed parent lines. Thus, there was almost total suppression of crossing capability in spw1-cls
, at least under the experimental conditions we used. We consider that the difference in whole crossing rate between 2008 and 2010 was related to the length of overlapped heading periods of donor and recipient lines. The temperature in summer 2010 was remarkably high and caused more rapid growth and earlier heading of “Raichou-mochi” than T65 and spw1-cls
. These conditions decreased the overlap period of heading, and thus suppressed the crossing rate in 2010. The number of non-T65 genotype xenia grains employing spw1-cls
as the pollen donor was greater than that employing T65. We hypothesized that this could have been caused by the absence of pollen dispersal from the donor block. Because of the lack of competition in pollination between the pollen donor and other varieties cultivated in neighboring fields, the natural crossing other than with spw1-cls
would have increased.
Although corn, soybean and cotton are the most widely cultivated commercial GM crops worldwide, GM rice cultivars will soon be developed and cultivated widely. GM rice with the spw1-cls
mutation will suppress crossing with non-GM rice cultivars and inhibit gene flow by pollen dispersal. In addition, the mutant also inhibits the non-GM to GM gene flow, which would otherwise eliminate useful characteristics of GM rice. It is important maintain the quality of GM rice, particularly those GM rice traits that are of medical use. The spw1-cls
mutation would also be useful in maintaining “purity of lines” of non-GM rice cultivars. For example, keeping glutinous and non-glutinous lines separate is an issue of concern (Kamagata et al. 1988
). Currently, suppression of such natural crossings is done by separating the heading dates of the cultivars and maintaining adequate distances between cultivated paddy fields of glutinous and non-glutinous cultivars. This entails much effort and is demanding on space. However, the use of glutinous rice cultivars with the spw1-cls
mutation would make it possible to cultivate glutinous and non-glutinous cultivars in adjacent paddy fields without consideration of heading dates.
Although the natural crossing rate of rice is usually affected by the shapes and sizes of stamens and pistils (Kato and Namai 1987a
), cleistogamy in spw1-cls
is unaffected by these factors because the lemma and palea enclose the inner floral organs and do not open. Thus, the spw1-cls
mutation is suitable for cultivars with all shapes and sizes of floral organs.
The molecular lesion of spw1-cls
causes a missense mutation in the SPW1
gene encoding a B-class MADS-box protein. The SPW1 protein forms a heterodimer with MADS2 or MADS4 proteins thus exerting B-class activity to specify lodicule and stamen identities. The spw1-cls
mutation causes reduction in protein-protein interaction between SPW1 and MADS2/4, with consequently reduced B-class activity that results in the transformation of the lodicule to an mrp-like organ (Yoshida et al. 2007
). Yoshida et al.
reported that protein-protein interaction between SPW1cls
and MADS2/4, which causes cleistogamy, is restored by reducing temperature in a yeast two-hybrid system. Therefore, it would be important to determine whether the cleistogamy of spw1-cls
is maintained through the range of climates under which rice is cultivated, particularly where summers are cool. Despite this qualification, we have demonstrated significant advantages and stability of the spw1-cls
mutation and the cleistogamous phenotype. The spw1-cls
mutation is not expected to affect commercial traits, and we were able to reliably introduce the mutation to any cultivar by using DNA markers. We also showed that spw1-cls
cleistogamy effectively inhibits natural crossing in paddy fields. We have thus developed the most practical mechanism for suppressing transgene flow and/or maintaining purity of genetic lines.