| Accurate, rapid and economical method of purity identification hybrids is one of the important measures to protect the safety of hybrid rice production. The period of field identification in Hainan is too long and limit sale. And the results of two identification methods through observing grain shape and phenol staining are not accurate. The protein electrophoresis method also failed to be large-scale used because of poor stability and less polymorphic. Tedious process, expensive equipment, high-quality personnel limit the DNA marker method to be used widely. Another way to identify seed purity is molecular design breeding. Generally the design makes sterile line carry a recessive trait and father carrying dominant trait. This design can effectively distinguish the seeds from sterile line selfing with hybrid Fl seeds. But the design failed to distinguish the seeds coming from mixing and other pollens, and easily lead to high false-positive test results.During we screened heterosis between Indica rice and Japonica rice, "purple complementary" phenomenon was been observed. The genetic mechanisms leading to the phenomenon was analyzed. And applications of the phenomenon in purity identification of hybrid rice were demonstrated by examples. The main conclusions were as follows:(1) Japonica rice GH102 and many Indica rice had non-purple coleoptile, apiculus and stigma. When GH102 crossed with these Indica rice, the F1 had purple lines on coleoptile. F2 seeds showed purple apiculus and stigma. The apiculus and stigma of F1 seeds were not purple because they developped from sterile line and had the same genotype with sterile line. We called the phenomenon as "Purple complementary" that both parents had no purple characters but their progenies had purple characters.(2) Genetic analysis showed that the "purple complementary" traits controlled by the two dominant genes. When the two genes appeared simultaneously, apiculus and stigma showed purple and coleoptile had two purple lines. By map-based cloning method, the two genes were located on Chr.6 and Chr.l. Functional complementation tests identified the two genes were OsCl and OsDFR respectively.(3) Sequence analysis showed for non-purple Indica rice 10bp deletion in third exon of OsCl gene resulted in a frameshift mutation, while OsDFR function properly. GH102 was different with non-purple Indica rice. Its OsCl gene function was normal, while nonsense mutation in the second exon of OsDFR gene caused early termination of protein synthesis. Non-purple Indica rice and GH102 had different mutation genes, so their crossing could produce "complementary purple" phenomenon.(4) Physiological studies indicated that formation of coleoptile purple line needed light. But the type of ligh was not important for formation of purple line. However the blue light could greatly increased the expression of OsC1 and OsDFR genes. Light could induce OsC1 expression, functional OsC1 let OsDFR expression, resulting in anthocyanin synthesis.(5) Stress colud induce the expression of anthocyanin in the leaf sheath through the production of reactive oxygen species (ROS). ROS could improved the expression level of OsCl and OsDFR.(6) "Purple complementary" traits could be used to identify hybid rice purity accurately by two steps. First, by observing apiculus and stigma color of seeds, the seeds with purple apiculus and stigma could be removed. Generally the removed seeds had purple lines on coleoptile plant. Second, to observe coleoptile, the seed with purple lines on coleoptile is true hybrid. The seed with non-purple lines on coleoptile is false hybrid. The purity identification methods with short period, less labor, and low cost can be synchronized with the identification of germination rate.(7) Because OsC1 and OsDFR regulat apperence of purple commonly on apiculus, stigma and coleoptile. For most of rice germplasm, purple on apiculus, stigma and coleoptile either exist, or disappears commonly. But hybrid rice from the crossing between GH102 and non-purple Indica rice is different, F1 apiculus and stigma is colorless, but coleoptile had purple line. So, in the first step of the purity test, by observing apiculus and stigma color, and the seed with purple apiculus and stigma wold be removed which have purple-line coleoptile generally. For the seeds without purple lines on coleoptile, we can separate them in the second step through observing coleoptile.(8) Since apiculus and stigma of parents are colorless, in the process of hybrid seeds production, the mixed plant with purple apiculus and stigma are particularly obvious and removed easily. However mixed plants without purple apiculus and stigma in indica area, have the same genotype with sterile lines, loss function type of OsCl. Even if the mixed plants pollen sterile lines, purity testing by observing coleoptile in the second step can identify false hybrid.(9) We presented a molecular design breeding model that in Indica rice planting area, the genotype of sterile line is Oscl Oscl OsDFR OsDFR and father genotype is OsC1 OsCl Osdfr Osdfr. And theis crossing could product "purple complementary" phenomenon, for accurate, fast, economic identification of hybrids purity. We modified both parents of elit hybrid rice "Hui Liang You 6" by MAS method. The results showed the crossing between two new parents could product "complementary purple" phenomenon. |
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