Physiochemical Analysis Of And Molecular Genetic Studies On The Gelatinization Temperature Character In Rice (Oryza Sativa L.)

Gelatinization temperature (GT) is the temperature at which starch granules irreversibly lose their crystalline order during cooking and is the most important cooking quality character of rice grains. In practical quality evaluation and most research experiments, GT is usually indirectly measured according to the digestibility of milled rice grains in alkali solution and scored as the alkali spreading value (ASV). More sophisticated analysis of the thermal character of rice grains can be done using a differential scanning calorimetry (DSC) instrument, which can determine the temperature at which starch granule starts and ends gelatinization and the heat energy required for gelatinization.In this study, using a low GT mutant (HuangyuB) and other rice varieties of different GT, the relationships of GT and starch physiochemical properties, including amylopectin chains length profile, were analyzed;and the genetic and physiochemical basis of the GT character were investigated using two different genetic populations;based on the findings, SSR markers linked to the SSIIa gene were analyzed in different rice varieties with the aim to develop a strategy for MAS of the GT character;the SSIIa gene of 4 rice varieties of different GT were sequenced and blasted sequences of this gene in Genebank database;one PCR-based SNP marker of the SSIIa gene was developed, and together with one SNP marker of Wx, were used to investigate its relationship with the GT character. Followings are the main results:1) The ASV and DSC parameters of the mutant line HuangyuB, were significantly different from those of II32B. The ASV and the T_o, T_p, T_c of HuangyuB reached to 7.0, and 64.1 ℃, 69.2℃ and 77.8℃, respectively, while those of Ⅱ32 B were 2.0, and 74.0℃, 77.2℃ and 82.5℃, respectively. The ACR (The ratio of amylocpetin chains of DP^12 to those of DP^24) of HuangyuB was 0.235, significantly higher than that of Ⅱ32B (0.157). The ACR was significantly and positively correlated with the ASV, and significantly and negatively with T_o, T_p, T_c in these two lines. X-diffraction result showed that Ⅱ32B had a higher degree ofcrystallinity than HuangyuB and another low GT variety XiushuillO, which implied that the higher GT in II32B might result from the higher degree of crystallinity of starch granules.2) The japonica variety XiushuillO had an ASV similar to those of indica rice varieties Niqingzhan and HuangyuB, but had a significant higher ACR and significant lower Tot Tp, Tc than those of the latter two, which implied that ACR is significantly and negatively correlated with GT in those three varieties and II32B. However, the indica variety, R3027, having a similar ACR to that of Huangyu B, had a GT significantly higher than HuangyuB and Niqingzhan, which indicated that the difference of ACR is not sufficient to explain the GT differences among those varieties.3) Classical genetic studies. HuangyuA and HuangyuB had similar ASV and DSC parameters, which indicated that there was no significant cytoplasmic effect on GT character. The GT of Fi hybrid grains of HuangyuA/II32B was slightly lower than that of II32A/HuangyuB, both are not significantly different from that of II32B, therefore high GT seemed dominant over low GT and was of a triploid endosperm characteristic. The F2 plants, according to the ASV of $2$ grains, segregated in a 1:2:1 ratio in both HuangyuB/II32B and HuangyuB/R3027 population, which indicated that only a single gene each controlled the GT character in these two populations.4) Molecular mapping of GT genes. The loci controlling the GT character, in the F2 population of both HuangyuB/II32B and HuangyuB/R3027, were mapped on the chromosome 6. However, in the former population, the locus was found closely linked with SSR marker RM253, at genetic distances of 2.94 cM, 2.59 cM, 2.22 cM and 1.85 cM, respectively, from RM253, RM276, RM402 RM3077 and RM7088. In the latter population, the locus that controlled the GT variation, alk2(t), was mapped on a locus near the Wx gene at a genetic distance of 3.93 cM.5) The expression of Wx and SSIIa. The amount of GBSSI protein (encoded by the Wx gene) was similar in mature grains of HuangyuB and R3027, but was significantly higher than that of II32B. Little SSIIa protein was detected in rice seeds of HuangyuB and R3027, but considerable amount of SSIIa was detected in II32B.6) Analysis of SSR loci linked with SSIIa. Twenty-six alleles were detected at 8 SSR loci linked with the SSIIa gene (<5 cM) in a total of 104 rice varieties. The RM276 was found to be the most polymorphic locus, having 5 different alleles. Two to three alleles were detected at other SSR loci. In general, those alleles were randomly distributed among rice varieties and showed litlle correlation with the GT character. However, RM402 and RM276 were significantly correlated with the ASV, with coefficients of 0.797 and 0.593, respectively. With those SSR markers, the rice varieties were grouped into indica and japonica types, and all high GT varieties were distinguished from low GT ones, but rice varieties with a medium GT could not be differentiated from either high GT or low GT materials.7). SSIIa sequence. The SSIIa gene of HuangyuB, II32B, R3027 and Pei'ai 64s was sequenced and blasted with other known SSIIa gene sequences obtained from the Genebank database. Consequently, three SNPs in the eighth extron of the SSIIa gene, G/A38io, G/T3915 and GC/TT4341, were identified. These SNPs but G/T3915 resulted in the change of two amino acids, that is, Gly604 and Leu7gi in II32B and Pei'ai 64s and Ser604 and Phe7gi in HuangyuB and R3027.8) Analysis of GC/TT4340 SNP. Two pairs of PCR primer were designed according to the GC/TT4340 SNP and two different SSIIa genotypes, GC and TT, were identified. Co-segregation of the SNP marker with GT was observed in population of F2 Huangyu B/II32B, where plant homozygous for GC had high GT while those with homozygous TT had low GT.9) Relationships between SSIIa and Wx genotype and the GT character in indica rice. In a total of 67 indica rice varieties, all but four high medium and GT (ASV < 5.0) varieties, Iri371, Jiayu948, Ruanzuke and R3027, had the SSIIa SNP genotype of GC, while all 11 low GT (ASV > 5.0) rice materials except Zhou903 had genotype of TT. In combined use of the Wx SNP marker, it was able to further differentiate those five varieties from other counterparts in the same GT group.10) In summary, there were two loci controlling the GT character in rice, one is SSIIa gene, the other is alk2(t), which is closed linked with the Wx gene but not the Wx itself. The GC/TT4340 SNP in the extron of SSIIa might affect the activity of SSIIa.However, different expression level of SSIIa, not the change of SSIIa activity per unit, might be the main reason that caused the GT variations among rice varieties.