Haplotype of group of genes DTH2 for controlling rice heading period and application of genes DTH2
A technique of haplotype and heading date, applied in the field of genetic engineering
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Embodiment 1D
[0031] Example 1 Construction of DTH2 near-isogenic line
[0032] 1. QTL detection, backcrossing and selection of DTH2
[0033] Using the RILs populations of Asominori and IR24, our previous study detected multiple loci controlling heading date, one of which, DTH2, contained DTH2-a under natural long-day (NLD) conditions. of plants headed earlier than those containing DTH2-i. Using the CSSLs population with Asominori as the background parent and IR24 as the donor parent, we found that CSSL18 was significantly later matured than the background parent Asominori and had stable expression. Compared with the background parent Asominori, CSSL18 only inserted IR24 in some fragments fragment, so the inserted indica fragment IR24 is the root cause of the extremely late ripening of CSSL18. We crossed CSSL18 with Asominori once, backcrossed 4 consecutive times, and then selfed to construct a CSSL18 / Asominori BC containing 374 individuals 4 F 2 population, from which DTH2 was again de...
Embodiment 2
[0039] Example 2 Fine mapping and map-based cloning of DTH2
[0040] 1. Phenotypic identification at heading stage of both parents
[0041] Heading date refers to the date when the first ear of a single plant is drawn; Days of heading date refers to the number of days from sowing to the first ear is drawn from a single plant; Extreme late heading per plant refers to the same or later heading date as the parental NIL (DTH2). Single plant; extremely late heading families refer to families in which the majority of individual headings in a random family are the same or later than the parental NIL (DTH2). Under NLD conditions in Beijing (Dongmen Wang Room, Institute of Crop Science, Chinese Academy of Agricultural Sciences), NIL(DTH2) headed 7.4 days later than Asominori (Table 1, figure 2 C). However, under natural short-day (NSD) conditions in Hainan (Nanfan Base of Nanjing Agricultural University, Lingshui, Hainan), there was no significant difference in heading between the t...
Embodiment 3
[0050] Example 3 Transgenic function complementation experiment of DTH2
[0051] 1. Construction of full-length genome complementary vector
[0052] According to the predicted candidate gene sequence, using the sequence of Nipponbare (English name Nipponbare, a rice variety with complete genome sequencing) as a template, primers were designed to sequence the DTH2 genome derived from Asominori, and it was found that their sequences were consistent in this segment. . Therefore, using the Nipponbare BAC clone OsJNBa33B21 (purchased from the National Genetic Research Center of the Chinese Academy of Sciences) as a template, the whole DTH2 genome was amplified (see Table 4 for primers) to obtain a DNA fragment of 7867 bp. The homologous recombination kit (http: / / bioinfo.clontech.com / infusion / , purchased from Takara, Japan) was used to recombine the fragment into pCAMBIA1305.1 (a publicly reported and used plasmid from Australia, image 3 ) of the SmaI (purchased from Fermentas, C...
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