Coleoptile Purple Line Regulated by A-P Gene System Is a Valuable Marker Trait for Seed Purity Identification in Hybrid Rice

In plants,a large number of anthocyanin biosynthetic genes encoding enzymes and regulatory genes encoding transcription factors are required for anthocyanin synthesis.Coleoptile purple lines are two purple lines on both sides of coleoptiles after seed germination.However,the molecular mechanism of coleoptile purple line is not clear in rice so far.In this study,two major dominant genes,coleoptile purple line 1(OsCPL1,also known as OsC1)and coleoptile purple line 2(OsCPL2),were isolated via map-based cloning,and both of them were required for anthocyanin biosynthesis of coleoptile purple line in rice.The knockout and complementation experiments confirmed that OsC1 was required for purple color in most organs,such as coleoptile line,sheath,auricle,stigma and apiculus,whereas OsCPL2 was just required for coleoptile purple line.OsC1 was predominantly expressed in coleoptiles,flag leaves,and green panicles,and highly expressed in young leaves,whereas OsCPL2 was predominantly expressed in coleoptiles,and extremely lowly expressed in the other tested organs.Loss-of-function of either OsC1 or OsCPL2 resulted in significant reduction of transcript levels of multiple anthocyanin biosynthesis genes in coleoptiles.Coleoptile purple line was further used as a marker trait in hybrid rice.Purity identification in hybrid rice seeds via coleoptile purple line just needed a little water,soil and a small plate and could be completed within 5 d.Molecular marker and field identification analyses indicated that coleoptile purple line was reliable for the hybrid seed purity identification.Our findings disclosed that coleoptile purple line in rice was regulated by two major dominant genes,OsC1 and OsCPL2,and can be used as a simple,rapid,accurate and economic marker trait for seed purity identification in hybrid rice.

Influence of Genetic Drift of Restoring Gene (Rf) on Seed Purity of Yuetai A, a Honglian-Type Cytoplasmic Male Sterile Line in Hybrid Rice

The seed samples of Yuetai A, a Honglian （HL） type cytoplasmic male sterile （CMS） line in hybrid rice were investigated to assess the seed purity and to analyze the cause of off-type plants by imitating the biological contaminant to Yuetai A in Nanjing, Jiangsu Province and Lingshui, Hainan Province during 2004-2006. The seed impurity of Yuetai A mainly resulted from the genetic drift of restoring fertility gene （Rt） after biological contamination but not from its sterility unstability. All of the ten maintainer lines, five restorer lines and three thermo-sensitive genic male sterile lines used in the study could pollinate Yuetai A and Yuetai B to produce F1 plants, directly or indirectly resulting in Rf-gene drifting into Yuetai A and generating ＇iso-cytoplasm restoring-generations＇. Furthermore, high outcrossing rate and similar heading date of Yuetai A with many varieties used in rice production might easily result in the biological contamination. After removing all plants with Rf-gene mixed in Yuetai A and preventing Rf-gene drifting into Yuetai A, the seed purity of Yuetai A and Yuetai B had been raised to 100%.

Analysis on the Different Results of Seed Purity Identification for the Same Hybrid Rice Variety Using Different SSR Primers

Different results of seed purity identification for Gangyou 158, Ⅱ You 808, Wuyou 308 and Tianfengyou 316 were obtained using different SSR primers in our early work. To find out the reasons, the four hybrid combinations were grown in field to identify their purity according to their phenotypic traits. Then, the results of field identification were compared with that of laboratory tests using different SSR primers. The comparison revealed that only sterile lines （female parent） were distin- guished from true hybrids using the primers RM208, RM264, RM242 and RM164 for the purity identification of Gangyou 158, II You 808, Wuyou 308 and Tianfengyou 316, so the results were higher than that of field identification. In contrast, the primers RM341, RM297, RM21 and RM110 were able to distinguish not only the sterile plants but also the cross-pollinated ones from the true hybrids of Gangyou 158,Ⅱ You 808, Wuyou 308 and Tianfengyou 316, and the results of purity identifi- cation using them were close that of field identification, in summary, several pairs of primers should be used for the purity identification of rice hybrids to distinguish all the off-type plants and thus improve the accuracy.