Gu MingHong

Breeding of Selectable Marker-Free Transgenic Rice Lines Containing AP1 Gene with Enhanced Disease Resistance

In order to obtain marker-free transgenic rice with improved disease resistance, the AP1 gene of Capsicum annuum and hygromycin-resistance gene (HPT) were cloned into the two separate T-DNA regions of the binary vector pSB130, respectively, and introduced into the calli derived from the immature seeds of two elite japonica rice varieties, Guangling Xiangjing and Wuxiangjing 9, mediated by Agrobacterium-mediated transformation. Many cotransgenic rice lines containing both the AP1 gene and the marker gene were regenerated and the integration of both transgenes in the transgenic rice plants was confirmed by either PCR or Southern blotting technique. Several selectable marker-free transgenic rice plants were subsequently obtained from the progeny of the cotransformants, and confirmed by both PCR and Southern blotting analysis. These transgenic rice lines were tested in the field and their resistance to disease was carefully investigated, the results showed that after inoculation the resistance to either bacterial blight or sheath blight of the selected transgenic lines was improved when compared with those of wild type.

The Source of Genes Related to Rice Grain Starch Synthesis Among Cultivated Varieties and Its Contribution to Quality

Abstract The property of starch in rice grain endosperm is a very important determinant for rice quality, and it is essential to understand the genetic effect of the genes related to starch synthesis in high-yielding rice varieties for rice quality improvement. The physicochemical properties (e.g., amylose content, gel consistency, and RVA profile) were assessed on 53 rice varieties, including certain typical indica/japonica landraces and certain high-yielding modern varieties. And molecular markers for Sbel, Sbe3 developed on the basis of sequence diversities between the rice subspecies indica and japonica, together with PCR- Acc I marker for Wx gene were used to investigate the genotypes of 53 rice cultivars. The result showed that the developed molecular markers for Wx, Sbel, Sbe3 could distinguish indica or japonica alleles at three loci. Among all the 53 rice cultivars, six genotypes were observed when Sbel, Sbe3 , and Wx loci were considered together, while the genotypes of Wx i Sbel j Sbe3 i and Wx i Sbel j Sbe3 i were absent. In order to explore the genetic effects of the three genes, especially for starch branching enzyme genes, ANOVA and multiple comparison analysis were conducted. The results showed that rice cultivars with different genotypes exhibited different phenotypes, including amylose content, gel consistency and certain RVA characteristics, and the significant differences among the six genotypes were observed. It was concluded that these three genes had randomly recombined during the process of the rice variety development. And the genetic effects of indica and japonica alleles at three gene loci were different, of which, Wx gene plays a major role in determining the starch properties, followed by Sbel and Sbe3 , and the genetic effects of Sbel and Sbe3 in different backgrounds ( Wx i , Wx j ) are different. The results have provided a clue for rice good quality variety development, and the molecular markers will benefit to the improvement in quality of rice.

Variation and Distribution of Vitamin E and Composition in Seeds Among Different Rice Varieties

Vitamin E is an essential nutrient that must be acquired regularly from dietary sources. It is a group of compounds consisting of tocotrienols and tocopherols. The structure of tocotrienols differs from that of tocopherols by the presence of 3 trans-double bonds in the hydrocarbon chain. In this study, a reverse-phase (RP) HPLC method was used to simultaneously measure the contents of either tocopherols or tocotrienols in brown rice, and to compare these parameters among 18 indica and 16 japonica rice (Oryza sativa L.) varieties. The results showed that the proportion of these vitamin E isomers differed substantially between indica and japonica rice varieties. The mean contents of vitamin E and total tocopherol were significantly higher (P < 0.01) in japonica rice than in indica rice, while the total tocotrienol content showed no difference between indica and japonica rice. The principal isomer composition of vitamin E was also different between the 2 subspecies. For example, γ-tocotrienol was the predominant component in indica rice, while the most abundant isomer was α-tocopherol in japonica rice. The ratio of total tocotrienols to tocopherols was also significantly higher (P < 0.01) in indica (1.61) than in japonica rice (0.95). Pearsons correlation analysis demonstrated that the content of α-tocopherol was positively correlated to that of α-tocotrienol in rice. The same relationship was observed for γ-tocopherol and γ-tocotrienol, but there was a negative correlation between α- and γ-isomers. Taken together, the results provide valuable information for studying the vitamin E metabolism or nutritional improvement in rice.

Genetic Effect of PUL Allelic Variation on Rice Cooking and Eating Qualities

Pullulanase (PUL) is a type of starch debranching enzymes involved in starch biosynthesis. To test the effect of PUL allelic variation on cooking and eating qualities in rice (Oryza sativa L.), 2 sets of near-isogenic lines (NILs) for PUL gene were developed by crossing and backcrossing Guichao 2 (indica) and Suyunuo (japonica glutinous) and selected with the help of the gene-specific STS marker on PUL gene. The NILs had no significant differences in amylose content, gel consistency, and starch crystalline structure compared with their corresponding recurrent parents. In contrast, the starch paste viscosity and starch thermal property changed obviously between the NILs and their recurrent parents. It was concluded that PUL gene plays an important role in determining rice grain cooking and eating qualities, and functional differentiation occurred at the PUL locus between indica variety Guichao 2 and japonica variety Suyunuo. The gene-specific molecular marker for PUL gene developed on the basis of genomic sequence diversity could be directly applied in rice good quality breeding program.

Improving the Resistance of Wuyunjing 8 to Rice Stripe Virus via Molecular Marker-assisted Selection: Improving the Resistance of Wuyunjing 8 to Rice Stripe Virus via Molecular Marker-assisted Selection

In this study, a molecular marker-assisted selection (MAS) strategy was used to improve Wuyunjing 8s resistance to rice stripe virus. The Wuyunjing 8 with high yield and erect head selected from 25 late-maturing varieties was used as accepter, and the variety Kuifeng with resistance to rice stripe virus as the donor. Four STS (sequence-tagged site) primers STS11-31, STS11-7, STS11-19, and STS11-43 anchoring the resistance genes were used for assisted selection in the backcross progenies. To 2008, seventy lines in BC3F5 and one hundred and fifteen lines of BC4F4 with stable agronomic traits were obtained with the result of molecular analysis. By appraising agronomic traits, yield, quality and resistance in backcross progenies, teen lines from BC4F5 and two lines from BC3F6, which all carried disease-resistant genes and were similar to Wuyunjing 8 on the integrated traits, were selected. These lines retain the characteristics of high yield and good quality of Wuyunjing 8 and have been prominently improved in resistance to rice stripe virus. Keywords: Japonica; Rice stripe virus; Resistance; Improvement; Molecular marker-assisted selection

Efficiencies of Generating Selectable Marker-Free Transgenic Rice with Different Transformation Methods

To study the efficiency of generating selectable marker-free (SMF) transgenic rice, two transformation methods were employed for four rice varieties (Wuxiangjing 9, Longtefu, Xieqingzao and Zhenshan 97). One method is by using a single twin T-DNA binary vector pYH592 in one Agrobacterium strain, which is composed of two separate T-DNA regions (one carrying an antisense Wx gene and the other carrying a HPT gene). The other one, named as two-strain/two-vector system, is by using two separate binary vectors in two separate Agrobacterium cultures. The results indicated that the average co-transformation frequencies of the antisense Wx gene and the HPT gene were 10.1% and 45.0%, respectively, for the four rice varieties. And the SMF transgenic plants selected from the offsprings of co-transformants were 55.6% and 60.0% in the two-strain/two-vector and twin T-DNA vector binary systems, respectively.

Molecular Cytogenetic Analysis of Spontaneous Interspecific Hybrid Between Oryza sativa and Oryza minuta

Genomic in situ hybridization (GISH) is a powerful tool to characterize parental chromosomes in interspecific hybrids, including the behaviour of autosynapsis and chromosome pairing. It was used to distinguish the chromosomes of Oryza sativa from wild species in a spontaneous interspecific hybrid and to investigate the chromosome pairing at metaphase I in meiosis of the hybrid in this study. The hybrid was a triploid with 36 chromosomes according to the chromosome number investigated in mitosis of root tips. During metaphase I of meiosis in the hybrid, less chromosome pairing was observed and most of the chromosomes existed as univalent. Based on GISH and FISH (Fluorescent in situ hybridization) analyses, the chromosomes of the hybrid were composed of genomes A, B and C. Thus, it was believed that the hybrid was the result of natural hybridization between cultivated rice and wild species O. minuta which was planted in experimental fields.

Genetic Stability of Rice Aneuploid during Its Asexual Propagation

Abstract For undertanding the genetic stability of rice ( Oryza sativa L.) aneuploid during its asexual propagation, a telotetrasome (2 n +·8S+·8S) was selected from the progenies of a rice telotrisome (2 n +·8S), and preserved by asexual reproduction. One of the extra short arms (·8S) was easily lost in the asexual propagation offspring of 2 n +·8S+·8S, resulting in morphological variation. The behaviors of the 2 extra ·8S were observed using fluorescence in situ hybridization (FISH) technique. One of the 2 extra ·8S was found without detectable centromeric satellite repeat (CentO) and centromere-specific retrotransposon (CRR), and failed to be transmitted stably; whereas the other extra ·8S contained CRR but no detectable CentO, which could be transmitted stably during asexual propagation. However, the extra ·8S contained both CentO and CRR sequences in the initial telotrisomic line (2 n +·8S). Therefore, the CentO and CRR sequences of the extra ·8S may be randomly lost during asexual propagation, which results in instable inheritance in aneuploid rice with extra ·8S.

Improvement of Resistance to Rice Stripe Virus in Wuyunjing 8 via Molecular Marker-Assisted Selection

Abstract A molecular marker-assisted selection (MAS) strategy was used to improve resistance to Rice stripe virus (RSV) in rice (Oryza sativa L. subsp. japonica cv. Wuyunjing 8). Rice variety “Aoikaze” with resistance to RSV was used as the donor. Sequence-tagged site (STS) markers STS11-31, STS11-7, STS11-19, and STS11-43 anchoring the resistance loci were used for MAS in the backcross progenies. In 2008, 70 lines of BC3F5 and 115 lines of BC4F4 population were obtained with stable agronomic traits after MAS assay. Further evaluation of agronomic traits, yield, grain quality, and resistance to RSV were carried out in these backcross progenies, and 10 BC4F5 lines and 2 BC3F6 lines showed similar phenotypes to Wuyunjing 8. The 12 lines were finally selected for the improved resistance to RSV and the high-yield and elite-quality of Wuyunjing 8.