Anthranilate synthase allele fragments for increasing rice yield and uses thereof

Abstract

Anthranilate synthase allele fragments for increasing rice yields and uses thereof. A method of differentiating rice materials with a superior allele associated with high yield includes: (1) detecting a genotype of rice to be detected based on a specific gene fragment; where the specific gene fragmentOsASA1 is located in rice genome, and there are typically two allelic forms of OsASA1, OsASA1_a shown as SEQ ID NO. 1 and OsASA1_b shown as SEQ ID NO. 2; and (2) determining and comparing the average yield of a rice population with a genotype of homozygous OsASA1_b and that of a rice population with a genotype of homozygous OsASA1_a under the same growth conditions in different geographical regions. The rice population with the genotype of homozygous OsASA1_b shows a higher average yield than the rice population with the genotype of homozygous OsASA1_a.

Claims

1. A method for breeding rice plants having increased yield, comprising: detecting a genotype of OsASA1 gene in rice plants; selecting a rice plant with a homozygous genotype of OsASA1_b allele; and crossing the selected rice plant with a second rice plant lacking the homozygous genotype of OsASA1_b allele to produce progeny rice plants having increased yield; wherein the nucleotide sequence of the OsASA1_b allele is shown as SEQ ID NO: 2.

2. The method of claim 1, wherein the step of detecting a genotype of OsASA1 gene in rice plants comprises: screening genomes of the rice plants and determining a pair of alleles being OsASA1_a and OsASA1_b; comparing a first yield of the rice plants that carry a homozygous genotype of OsASA1_a allele, with a second yield of the rice plants that carry a homozygous genotype of OsASA1_b allele; based on a compared result that the second yield is higher than the first yield, pre-determining a target homozygous genotype as the OsASA1_b allele; and testing a third yield of rice plants with the OsASA1_b allele under various environments to confirm the rice plants having increased yield; wherein the nucleotide sequence of the OsASA1_a allele is shown as SEQ ID NO: 1.

3. The method of claim 2, wherein the genes of the rice plants are screened by PCR.

4. The method of claim 3, wherein a primer pair for the PCR comprises a first primer shown as SEQ ID NO: 3, and a second primer shown as SEQ ID NO: 4.

5. The method of claim 2, wherein the step of testing a third yield of rice plants with the OsASA1_b allele under various environments to confirm the rice plants having increased yield comprises: cultivating the rice plants with the OsASA1_b allele and rice plants with the OsASA1_a allele simultaneously under various environments; and in response to a determination that the third yield of the rice plants with the OsASA1_b allele is higher than a yield of the rice plants with the OsASA1_a allele, confirming the rice plants having increased yield.

Description

DETAILED DESCRIPTION OF EMBODIMENTS

(1) Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

(2) Unless otherwise specified, materials and reagents used in the following examples are commercially available.

(3) The invention will be further described in detail with reference to the embodiments. These embodiments are merely for illustration, but are not intended to limit the scope of the invention.

Example 1 Design and Synthesis of Primers

(4) Anthranilic acid is an important precursor for the synthesis of tryptophan and guanidine in rice. In the rice genome, at least one gene has been annotated to encode anthranilate synthase (EC 4.1.3.27), called anthranilate synthase alpha 1 (AB022602.1; OsASA1) gene (referred to as OsASA1 gene thereafter). The OsASA1 gene is located on chromosome 3 of rice. Based on a large number of pre-experiments and sequence alignments, it has been found that there were at least two allelic fragments in the OsASA1 gene, OsASA1_a shown as SEQ ID NO. 1 and OsASA1_b shown as SEQ ID NO.2. These allelic fragments were associated with yield per plant of rice.

(5) A specific primer pair consisting of a first primer and a second primer was designed based on the two kinds of allelic fragments.

(6) TABLE-US-00001 Firstprimer(shownasSEQIDNO.3): 5-GTTACTGGAGAGTTGCGTGATG-3. Secondprimer(shownasSEQIDNO.4): 5-CTCATCTACGAATGTAGACTCGGC-3.

Example 2 Establishment of Typing Method Based on the Allelic Fragments in Rice

(7) The method was described as follows.

(8) 1. The specific primer pair consisting of the first primer and the second primer was used to amplify genomic DNA (about 10-100 ng) of rice to be detected by PCR to obtain a PCR amplification product. The PCR amplification was programmed as follows: 35 cycles, for each cycle, 95 C. for 5 minutes, 95 C. for 30 seconds, 61 C. for 1 minute and 72 C. for 1 minute; and then 72 C. for 8 minutes before cooling.

(9) 2. The PCR amplification product was sequenced, and the results were determined as follows. If there was only one type of the PCR amplification product shown as SEQ ID NO. 1, the genotype of the rice was homozygous OsASA1_a; and if there was only one type of the PCR amplification product shown as SEQ ID NO. 2, the genotype of the rice to be detected was homozygous OsASA1_b; and if there were two types of the PCR amplification product respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2, the genotype of the rice was heterozygous OsASA1_a/OsASA1_b.

(10) Due to monoculture of cultivars or landraces and selfing, the proportion of homozygous individuals is typically high.

Example 3 Correlation Analysis Between Genotype of Rice Based on the Allelic Fragment and Yield Per Plant

(11) 1. Statistics on yield per plant of different rice varieties

(12) Numerous rice varieties were planted in Sanya (Hainan) and Beijing in 2014 (see Tables 1-2 for details, where the rice variety names were shown in columns 2; the origins of rice varieties were shown in columns 3; and the 2009 numbers of some rice varieties (according to Biotechnology and Germplasm Resources Institute. Academy of Agricultural Sciences, Yunnan) were shown in column 6. A completely random trial was designed in the field. The mature rice plants were harvested by individual plants, weighed and averaged to obtain the yield per plant (see columns 5 in Tables 1 and 2).

(13) 2. Genotypes of each rice variety were detected according to the typing method in Example 2 (see columns 4 in Tables 1 and 2).

(14) TABLE-US-00002 TABLE 1 Experiments in Sanya Yield per 2009 Number Variety Origin Genotype plant (g) Number 1 Kendao 12 Kiamusze, Heilongjiang OsASA1_a 19.6 2 Kendao 16 Kiamusze, Heilongjiang OsASA1_a 29.2 3 Kendao 25 Kiamusze, Heilongjiang OsASA1_a 37.5 4 Kendao 9 Kiamusze, Heilongjiang OsASA1_a 21.8 5 Kenjiandao 3 Kiamusze, Heilongjiang OsASA1_a 42.4 6 Kenjiandao 6 Kiamusze, Heilongjiang OsASA1_a 23.2 7 Kongyu 131 Kiamusze, Heilongjiang OsASA1_a 41.6 8 Longhua 04426 Kiamusze, Heilongjiang OsASA1_a 31.7 9 Longjiao 04-2411 Kiamusze, Heilongjiang OsASA1_a 41.9 10 Longjing 26 Kiamusze, Heilongjiang OsASA1_a 16.4 11 Longjing 27 Kiamusze, Heilongjiang OsASA1_a 57.2 12 Longjing 29 Kiamusze, Heilongjiang OsASA1_a 32.2 13 Longjingxiang 1 Kiamusze, Heilongjiang OsASA1_a 26.1 14 Longsheng 04042 Kiamusze, Heilongjiang OsASA1_a 22.4 15 Songjing 10 Wuchang, Heilongjiang OsASA1_a 16.3 16 Songjing 11 Wuchang, Heilongjiang OsASA1_a 49.3 17 Songjing 13 Wuchang, Heilongjiang OsASA1_a 48.6 18 Songjing 15 Wuchang, Heilongjiang OsASA1_a 40.6 19 Songjing 16 Wuchang, Heilongjiang OsASA1_a 19.6 20 Songjing 17 Wuchang, Heilongjiang OsASA1_a 32.1 21 Songjing 18 Wuchang, Heilongjiang OsASA1_a 33.3 22 Songzhan 1 Wuchang, Heilongjiang OsASA1_a 38.0 23 Suijing 10 Suihua, Heilongjiang OsASA1_a 22.5 24 Suijing 4 Suihua, Heilongjiang OsASA1_a 28.2 25 Longjin 1 Gongzhuling, Jilin OsASA1_a 12.0 26 Liaoxing 20 Shenyang, Liaoning OsASA1_a 35.6 27 Fengdao 21 Dali, Yunnan OsASA1_a 22.5 28 Xiujing 20 Baoshan, Yunnan OsASA1_a 36.6 29 Yunjing 29 Nanhua Agricultural technical station seed OsASA1_a 48.1 station, Chuxiong, Yunnan 30 Haomuhao Biotechnology and Germplasm Resources OsASA1_a 48.0 1551 Institute, Academy of Agricultural Sciences, Yunnan 31 Jieba Biotechnology and Germplasm Resources OsASA1_a 23.1 1065 Institute, Academy of Agricultural Sciences, Yunnan 32 Mengxinggu Biotechnology and Germplasm Resources OsASA1_a 62.0 1640 Institute, Academy of Agricultural Sciences, Yunnan 33 Songjing 12 Wuchang, Heilongjiang OsASA1_b 25.5 34 Songjing 19 Wuchang, Heilongjiang OsASA1_b 22.2 35 Manglongxichanggu Biotechnology and Germplasm Resources OsASA1_b 75.0 1196 Institute, Academy of Agricultural Sciences, Yunnan 36 Xiaoxigu Biotechnology and Germplasm Resources OsASA1_b 77.2 1651 Institute, Academy of Agricultural Sciences, Yunnan 37 Nuogu Biotechnology and Germplasm Resources OsASA1_b 43.2 1144 Institute, Academy of Agricultural Sciences, Yunnan 38 Changruanmi Biotechnology and Germplasm Resources OsASA1_b 44.5 1140 Institute, Academy of Agricultural Sciences, Yunnan 39 Tianza Biotechnology and Germplasm Resources OsASA1_b 51.9 1282 Institute, Academy of Agricultural Sciences, Yunnan 40 Mengsonggu Biotechnology and Germplasm Resources OsASA1_b 49.4 1012 Institute, Academy of Agricultural Sciences, Yunnan 41 Changbainuo Biotechnology and Germplasm Resources OsASA1_b 39.8 1364 Institute, Academy of Agricultural Sciences, Yunnan 42 Dahonggu Biotechnology and Germplasm Resources OsASA1_b 48.8 1436 Institute, Academy of Agricultural Sciences, Yunnan 43 Menglagu Biotechnology and Germplasm Resources OsASA1_b 40.6 1643 Institute, Academy of Agricultural Sciences, Yunnan 44 Yunhui 290 Biotechnology and Germplasm Resources OsASA1_b 25.3 1638 Institute, Academy of Agricultural Sciences, Yunnan 45 Xiangsigu Biotechnology and Germplasm Resources OsASA1_b 41.2 1017 Institute, Academy of Agricultural Sciences, Yunnan 46 Shuijinghangu Biotechnology and Germplasm Resources OsASA1_b 23.4 1120 Institute, Academy of Agricultural Sciences, Yunnan 47 Xiaohuanggu Biotechnology and Germplasm Resources OsASA1_b 30.9 1655 Institute, Academy of Agricultural Sciences, Yunnan 48 Yidunban Biotechnology and Germplasm Resources OsASA1_b 63.8 1032 Institute, Academy of Agricultural Sciences, Yunnan 49 Mengpenggu Biotechnology and Germplasm Resources OsASA1_b 94.0 1068 Institute, Academy of Agricultural Sciences, Yunnan 50 Ruiligu Biotechnology and Germplasm Resources OsASA1_b 39.4 1192 Institute, Academy of Agricultural Sciences, Yunnan 51 Honggu Biotechnology and Germplasm Resources OsASA1_b 50.4 1381 Institute, Academy of Agricultural Sciences, Yunnan 52 Haonuolong Biotechnology and Germplasm Resources OsASA1_b 34.9 1043 Institute, Academy of Agricultural Sciences, Yunnan 53 Hongxinmo Biotechnology and Germplasm Resources OsASA1_b 25.9 1652 Institute, Academy of Agricultural Sciences, Yunnan

(15) The results in Table 1 indicated that 32 varieties among the 53 random rice varieties had the genotype of homozygous OsASA1_a, and the average yield per plant for these 32 varieties was 33.112.21 g. The rest of 21 varieties among the 53 random rice varieties, with die genotype of homozygous OsASA1_b, exhibited an average yield per plant of 45.114.17 g. The average yield per plant of the varieties with a genotype of homozygous OsASA1_a was significantly different from that of those with a genotype of homozygous OsASA1_b by two-tailed t-test, (P, 0.016).

(16) TABLE-US-00003 TABLE 2 Experiments in Beijing Yield per Number Variety Origin Genotype plant (g) 1 Zhonghua 15 Beijing OsASA1_a 44.54 2 Kendao 12 Kiamusze, Heilongjiang OsASA1_a 14.45 3 Kendao 16 Kiamusze, Heilongjiang OsASA1_a 17.57 4 Kenjiandao 3 Kiamusze, Heilongjiang OsASA1_a 15.41 5 Kenjiandao 6 Kiamusze, Heilongjiang OsASA1_a 29.08 6 Longjiao 04-1963 Kiamusze, Heilongjiang OsASA1_a 13.79 7 Longjing 39 Kiamusze, Heilongjiang OsASA1_a 23.67 8 Longjing 40 Kiamusze, Heilongjiang OsASA1_a 12.10 9 Longjing 41 Kiamusze, Heilongjiang OsASA1_a 17.61 10 Longjingxiang 1 Kiamusze, Heilongjiang OsASA1_a 20.81 11 Longsheng 04042 Kiamusze, Heilongjiang OsASA1_a 13.94 12 Songjing 16 Wuchang, Heilongjiang OsASA1_a 15.92 13 Songjing 18 Wuchang, Heilongjiang OsASA1_a 30.50 14 Songzhan 1 Wuchang, Heilongjiang OsASA1_a 16.49 15 Suijing 10 Suihua, Heilongjiang OsASA1_a 11.63 16 Suijing 4 Suihua, Heilongjiang OsASA1_a 18.48 17 Suijing 8 Suihua, Heilongjiang OsASA1_a 13.34 18 Jijing 810 Jilin OsASA1_a 8.62 19 Jizhan 9 Jilin OsASA1_a 32.36 20 Longjin 1 Gongzhuling, Jilin OsASA1_a 40.71 21 Changbai 22 Changbai Mountain, Jilin OsASA1_a 22.82 22 9600 Jiangsu OsASA1_a 48.44 23 9924 Jiangsu OsASA1_a 22.15 24 Huajing 6 Jiangsu OsASA1_a 13.19 25 Wuyujing 3 Jiangsu OsASA1_a 12.34 26 Zaohua 75 Jiangsu OsASA1_a 28.80 27 Zhen 88 Jiangsu OsASA1_a 30.10 28 Dongshi 15 Liaoning OsASA1_a 61.09 29 Liaojing 287 Liaoning OsASA1_a 25.83 30 Liaoxing 15 Liaoning OsASA1_a 17.74 31 Liaoxing 20 Liaoning OsASA1_a 30.69 32 Liaoxing 9 Liaoning OsASA1_a 41.94 33 Shennong 47 Liaoning OsASA1_a 42.93 34 Shennong 9 Liaoning OsASA1_a 66.49 35 Yanfeng 47-38 Liaoning OsASA1_a 21.98 36 Yanjing 200 Liaoning OsASA1_a 26.25 37 Yanjing 456 Liaoning OsASA1_a 26.28 38 Sheng 102 Shandong OsASA1_a 18.85 39 Aituogu 151 Sichuan OsASA1_a 53.79 40 Xindao 27 Yili, Xinjiang OsASA1_a 39.37 41 Fengdao 21 Dali, Yunnan OsASA1_a 26.66 42 Xiujing 20 Baoshan, Yunnan OsASA1_a 13.76 43 Yunjing 29 Nanhua agricultural technical station-Seed OsASA1_a 19.48 station, Chuxiong, Yunnan 44 Guangzhan 63 Guangdong OsASA1_b 12.30 45 Guiyaug 2 Guangdong OsASA1_b 18.99 46 Wanxian 98 Guangdong OsASA1_b 39.64 47 Zhaoyeqing Guangdong OsASA1_b 71.18 48 Huanglizhanguang 3 Guangdong OsASA1_b 28.30 49 Huanghuazhanguang 4 Guangdong OsASA1_b 57.22 50 Guichao 2 Guangdong OsASA1_b 21.74 51 Huangsiguizhan Guangdong OsASA1_b 38.78 52 Songjing 3 Wuchang, Heilongjiong OsASA1_b 12.31 53 Songjing 13 Wuchang, Heilongjiong OsASA1_b 20.97 54 Xiangaizao 10 Human OsASA1_b 31.34 55 Jinong 316 Jilin OsASA1_b 21.89 56 Changbai 9 Changbaishan, Jilin OsASA1_b 31.85 57 Jijing 105 Jilin OsASA1_b 35.36 58 Jijing 803 Jilin OsASA1_b 45.20 59 Wuyunjing 21 Jiangsu OsASA1_b 14.72 60 9311 Jiangsu OsASA1_b 55.15 61 Zhenxian 96 Jiangsu OsASA1_b 55.90 62 Shuchang 251 Liaoning OsASA1_b 13.58 63 Zhonghua 58 Liaoning OsASA1_b 25.53 64 Luke 3 Sichuan OsASA1_b 28.31 65 Shufeng 101 Sichuan OsASA1_b 51.17 66 Shufeng 101 Sichuan OsASA1_b 51.17

(17) The results in Table 2 showed that 44 varieties among the 66 random rice varieties had the genotype of homozygous OsASA1_a, and the average yield per plant for these 44 varieties was 25.7812.09 g. The rest of 22 varieties among the 66 random rice varieties, with the genotype of homozygous OsASA1_b, exhibited an average yield per plant of 33.25:3.57 g. The average yield per plant of the varieties with a genotype of homozygous OsASA1_a was significantly different from that with a genotype of homozygous OsASA1_b by two-tailed t-test (P, 0.015).

(18) 3. Development of method for screening rice with different yield traits per plant

(19) The method was described as follows.

(20) (1) The genotype of rice to be detected was detected based on a specific gene fragment, where the specific gene fragment, OsASA1, was located in rice genome, and there were at least two allelic forms of OsASA1. OsASA1_a shown as SEQ ID NO. 1 and OsASA1_b shown as SEQ ID NO. 2.

(21) (2) The average yield of a rice population with a genotype of homozygous OsASA1_b and that of a rice population with a genotype of homozygous OsASA1_a were determined and compared under the same growth conditions in different geographical regions. The results indicated that the rice population with the genotype of homozygous OsASA1_b showed a higher average yield per plant than the rice population with the genotype of homozygous OsASA1_a.