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

1. Introduction

In recent years, researchers are focusing on the breeding of good-quality and high-yield aromatic rice cultivar. Yuzhenxiang, a regular medium-maturing late indica rice originally bred by Hunan Rice Research Institute, with long and extra-slender grains, crystal clear appearance, soft and chewy taste, and natural aroma. It is a superior aromatic variety with excellent market potential and breeding value, therefore it is very popular among seed companies and rice corporations. Yuzhenxiang has been widely used by breeding institutes as primal parents to breed high-quality rice cultivar over the past years. Nevertheless, the molecular genetic mechanism of Yuzhenxiang (

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

, and

GS9

), one amylase gene

Wx

and one aroma gene

BADH2

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.

2. Materials and Methods

2.1. Testing materials

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. Testing methods

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 P

et al

.. The primer sequences of the functional marker genes in this research were listed in Table 1. Primer

GS6

-InDel,

GW6a

-InDel,

GL7

-InDel2,

GLW7

-InDel,

GW5

-InDel and

GW8

-InDel were testing markers designed by ZHANG L

et 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 including

GS6

,

GW6a

,

GL7

,

GLW7

,

GW5

and

GW8

, by taking Nipponbare as the control. These primers were in accord with the 12 bp insertion at site 714~719 in forward

GS6

, the 6 bp deletion at site 584~589 in forward

GW6a

, the 11 bp deletion at

GL7

’s promoter region, the 6 bp deletion at

GLW7

’s promoter region, the 1 212 bp deletion at around the 5 kb region of

GW5

, 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 C

et al

.based on a comparison between the 22 bp difference of

GS9

3'UTR region and the 18 bp difference of

GW7

promoter region in 9311 and Nipponbare. Primer SF28 was designed by FAN C C

et al

.according to a specific site related to the early termination of

GS3

translation due to a C–A mutationon

GS3’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 J

et al

.found a piece of (CT)n microsatellite sequence in

Wx

nucleotide sequence and designed a series of functional markers for determining amylase contents; this series was named Primer

Wx

-484/485 and could be used to detect the genotype of both high and low amylase-contained rice varieties. WANG J

et al

.created

BADH2

-

E2

and

BADH2

-

E7

based on the 7 bp deletion on the 2exon and the 8 bp deletion on the 7exon of

BADH2

, 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

3. Results and Analysis

3.1. Grain shape and rice quality

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

3.2. Molecular marker test of grain shape genes

GS3

,

GS6

,

GLW7

and

GL7

/

GW7

were the genes regulating the length of rice grains. The testing results of Yuzhenxiang as shown in Fig. 3 indicated

GS6

insertion,

GL7

/

GW7

insertion,

GS3

deletion and

GLW7

deletion. Among the 4 grain width genes

GW5

,

GW8

,

GW6a

and

GS9,

Yuzhenxiang presented

GW5

insertion,

GW8

insertion,

GW6a

deletion and

GS9

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

3.3. Molecular marker test of rice quality gene Wx and aroma gene BADH2

As shown in Fig 4, the gene type of

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 of

BADH2

test indicated that Yuzhenxiang didn’t have the 7 bp deletion in the 2exon of

BADH2

, which was the same as the 5 controls. But Yuzhenxiang presented deletion in marker

BADH2

-

E7

, proving that the aroma of Yuzhenxiang was mainly caused by the 8 bp deletion in the 7exon.

3.4. Genotyping of Yuzhenxiang

The testing results of 12 functional markers were classified and listed in Table 3 in the order of grain shape genes, aroma gene

BADH2

, and amylase content gene

Wx

. All markers were polymorphic inall 6 varieties except marker

BADH2

-

E2

. Marker

BADH2

-

E7

(aroma gene

BADH2

) and 485/486 (rice quality gene

Wx

) 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

and

GS9

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 gene

GW5

,

GS6

,

GW6a

and

GW8

, 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

4. Conclusion and Discussion

Rice cultivation has a long history in China. The appearance, aroma and taste of rice grains could directly affect consumers’ desire to purchase. Traditional aromatic rice varieties are mainly local cultivars, normally with high plant type, long reproductive cycle, low yield, limited planting region, poor disease resistance and low stress tolerance, which means they are not suitable for massive cultivation and production. In contrast, Yuzhenxiang has competitive advantage in terms of rice quality and grain shape, whereas it is mainly grown as regular rice variety. In this research, the 10 genes (

i

.

e

.

GS3

,

GW5

,

GS6

,

GW6a

,

GL7

/

GW7

,

GLW7

,

GW8

,

GS9

,

Wx

, and

BADH2

) 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 by

GS3

,

GL7

and

GW7

with the positive coregulation of multiple grain shape genes, among which genotype

GLW7

and

GW8

could significantly improve rice quality; the superior rice quality of Yuzhenxiang may be resulted from the combined effects of

GLW7

,

GW8

and

Wx

genotypes, while its unique aroma could be mainly regulated by

BADH2

-

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.