时间:2024-08-31
GONG Ke , SUN Lian-ping , ZHANG Ying-xin , CAO Zheng-nan , ZHAO Zhen-dong , ZHOU Zheng , ZHAO Zheng-hong *, ZHAN Xiao-deng *
1. China National Rice Research Institute, National Center for Rice Improvement, Key Laboratory for Zhejiang Super Rice Research, Fuyang 311401, PRC;
2. North Rice Research Center of China Rice Research Institute, Baoqing 155600, PRC;
3. Hunan Rice Research Institute, Changsha 410125, PRC
Abstract In China, the market share of a rice cultivar is mainly determined by its yield and quality. Yuzhenxiang is a high-quality variety with extra-slender grain shape, good quality and unique aroma. In order to investigate its genetic background, 12 pairs of functional markers were used to detect the genotype distribution of eight major grain shape genes including GS3, GW5, GS6, GW6a, GL7/GW7, GLW7, GW8, GS9, one amylase gene Wx and one aroma gene BADH2 in Yuzhenxiang by using Nipponbare, 9311, Huazhan, Zhonghui 8015 and Zhonghui 9308 as controls. The results indicated that the slender grain type of Yuzhenxiang was probably affected by GS3 and GL7/GW7 with the positive co-regulation of multiple grain shape genes, and its outstanding rice quality may be resulted from the cumulative effects of GLW7, GW8 and Wx genotypes, while its unique aroma may be mainly regulated by BADH2-E7 deletion.
Key words Yuzhenxiang; Rice; Functional marker; Grain shape; Rice quality; Aroma
e
.g
. long and extra-slender grains, superior quality, and unique amour) is rarely reported. In order to investigate the genetic background of Yuzhenxiang, the genotype distribution of 8 major grain shape genes (i
.e
.GS3
,GW5
,GS6
,GW6a
,GL7
/GW7
,GLW7
,GW8
, andGS9
), one amylase geneWx
and one aroma geneBADH2
were tested by using Nipponbare, 9311, Huazhan, Zhonghui 8015 and Zhonghui 9308 as controls. The findings can offer theoretical basis for these lection of superior parents and molecular markers in rice genetic breeding.The control materials including Nipponbare, 9311, Huazhan, Zhonghui 8015 and Zhonghui 9308 were provided by the Super Rice Breeding Research Group of China National Rice Research Institute; the original seeds of Yuzhenxiang were provided by Hunan Rice Research Institute.
2.2.1. Grain shape
Collect the grains from the main ears of the testing varieties when they were ripe and golden. Dry the grains in a thermostat (XMTD-8222) at 37℃ for 36 h. Observe the shape of the grains. Select 10 mature and plump-eared grains from each variety; measure the length and width (precision: 0.01 mm) of each grain by Microtek Scan Maker i800 Plus (Type: MRS-9600TFU2L); calculate the length-width ratio. Take 1 000 plump-eared grains of each variety and weigh the monan electronic scales (precision: 0.01g); repeat this step three times; take the mean as the 1 000-grain weight.
2.2.2. DNA extraction and PCR
Fresh and tender leaves of each variety were picked in their seedling stage; the total DNAs were extracted. DNA extraction, PCR amplification, and testing were done according to the method of SUN L Pet al
.. The primer sequences of the functional marker genes in this research were listed in Table 1. PrimerGS6
-InDel,GW6a
-InDel,GL7
-InDel2,GLW7
-InDel,GW5
-InDel andGW8
-InDel were testing markers designed by ZHANG Let al.
according to their research on the alleles of global rice varieties including 257 accessions from the Rice Diversity Panel, and the sequence difference among grain shape genes includingGS6
,GW6a
,GL7
,GLW7
,GW5
andGW8
, by taking Nipponbare as the control. These primers were in accord with the 12 bp insertion at site 714~719 in forwardGS6
, the 6 bp deletion at site 584~589 in forwardGW6a
, the 11 bp deletion atGL7
’s promoter region, the 6 bp deletion atGLW7
’s promoter region, the 1 212 bp deletion at around the 5 kb region ofGW5
, and the 10 bp deletion of GW8’s promoter region. Primer In0919 (GS9
) and Chr701 (GW7
), two gene type markers with good polymorphism, were designed by ZHANG Cet al
.based on a comparison between the 22 bp difference ofGS9
3'UTR region and the 18 bp difference ofGW7
promoter region in 9311 and Nipponbare. Primer SF28 was designed by FAN C Cet al
.according to a specific site related to the early termination ofGS3
translation due to a C–A mutationonGS3’s
2exon. In this research, the grain shape genes of Nipponbare, 9311, Huazhan, Zhonghui 8015 and Zhonghui 9308 were tested using the above functional markers. BLIGH H F Jet al
.found a piece of (CT)n microsatellite sequence inWx
nucleotide sequence and designed a series of functional markers for determining amylase contents; this series was named PrimerWx
-484/485 and could be used to detect the genotype of both high and low amylase-contained rice varieties. WANG Jet al
.createdBADH2
-E2
andBADH2
-E7
based on the 7 bp deletion on the 2exon and the 8 bp deletion on the 7exon ofBADH2
, respectively.BADH2
-E2
can be used to distinguish aromatic and non-aromatic materials by checking if the length of the amplified segment was 100 or 107 bp; the bi-stripe was heterozygous.BADH2
-E7
determined if the gene was aromatic and non-aromatic material by checking if the length of the amplified segment was 196 or 204 bp. If a gene presented deletion for any of these two markers or for both markers, it would be aromatic.Table 1 Marker primers
The grains of Yuzhenxiang were longer and slenderer than the 5 control varieties as shown in Fig. 1; specifically, the grains of Yuzhenxiang were 11.79±0.01 mm in length, 2.11±0.01 mm in width; the length-width ratio was very high, reaching up to 5.50±0.03; the 1 000-grain weight was 26.47±0.09 g (Table 2). Yuzhenxiang was vastly superior to japonica rice Nipponbare in grain shape and 1 000-grain weight; its slender and long grains were also very distinctive among the 4 indica varieties. As shown in Fig. 2, the polished grains of Yuzhenxiang were much better than the controls in terms of appearance and chalkiness.
Table 2 Length, width, and 1 000-grain weight of rice grains
GS3
,GS6
,GLW7
andGL7
/GW7
were the genes regulating the length of rice grains. The testing results of Yuzhenxiang as shown in Fig. 3 indicatedGS6
insertion,GL7
/GW7
insertion,GS3
deletion andGLW7
deletion. Among the 4 grain width genesGW5
,GW8
,GW6a
andGS9,
Yuzhenxiang presentedGW5
insertion,GW8
insertion,GW6a
deletion andGS9
deletion.Fig. 1 Comparison of grain shape[Picture on the left is a comparison of grain width (scale: 0.3 cm); picture on the right is a comparison of grain length (scale: 1 cm).]
Fig. 2 Appearance and quality of polished rice grains
Fig. 3 Testing results of grain shape genes
Fig. 4 Testing results of rice quality gene Wx and aroma gene BADH2
Wx
in Yuzhenxiang was different from those in the other 5 materials and presented the deletion type,i.e
. the low amylase genotype. The results ofBADH2
test indicated that Yuzhenxiang didn’t have the 7 bp deletion in the 2exon ofBADH2
, which was the same as the 5 controls. But Yuzhenxiang presented deletion in markerBADH2
-E7
, proving that the aroma of Yuzhenxiang was mainly caused by the 8 bp deletion in the 7exon.BADH2
, and amylase content geneWx
. All markers were polymorphic inall 6 varieties except markerBADH2
-E2
. MarkerBADH2
-E7
(aroma geneBADH2
) and 485/486 (rice quality geneWx
) presented the same results in all rice varieties except in Yuzhenxiang. It meant the low amylase content and special aroma of Yuzhenxiang could be regulated by these two genes.GS3
,GL7
/GW7
,GLW7
andGS9
were genes related to grain length; their dominant gene types had positive regulation on Yuzhenxiang’s slender grain phenotype. In the test of grain width geneGW5
,GS6
,GW6a
andGW8
, Yuzhenxiang presented the type of insertion, insertion, deletion and insertion, respectively. These gene types had also contributed to the formation of Yuzhenxiang’s slender phenotype.Table 3 Genotype and genetic effects of different rice varieties
i
.e
.GS3
,GW5
,GS6
,GW6a
,GL7
/GW7
,GLW7
,GW8
,GS9
,Wx
, andBADH2
) related to grain shape, rice quality and aroma were tested to investigate the genetic background of Yuzhenxiang by using 12 pairs of functional markers and by taking Nipponbare, 9311, Huazhan, Zhonghui 8015 and Zhonghui 9308 as controls. According to the results, the slender grains of Yuzhenxiang could be caused byGS3
,GL7
andGW7
with the positive coregulation of multiple grain shape genes, among which genotypeGLW7
andGW8
could significantly improve rice quality; the superior rice quality of Yuzhenxiang may be resulted from the combined effects ofGLW7
,GW8
andWx
genotypes, while its unique aroma could be mainly regulated byBADH2
-E7
deletion. The aim of genotype test was to figure out the genetic background of Yuzhenxiang concerning its grain shape, rice quality and unique aroma, thus offering theoretical basis for the pyramiding breeding of multiple proto genes as well as providing some insights on directed breeding of superior rice varieties with molecular markers.The exploration of superior genes is an effective way of improving grain yield and rice quality. The existing research mainly concerns the genetic mechanism of grain shape, rice quality and aroma in mutants. Reports on successful pyramiding breeding of multiple proto genes are extremely rare. Yuzhenxiang, an outstanding rice variety with slender grains, high rice quality, and unique aroma, can be used as donor parent, and the above specific functional markers could be quite useful for screening out the target genes related to grain shape, rice quality, and aroma. Undoubtedly, there might be other proto genes in Yuzhenxiang awaiting further exploration and utilization.
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