Marker-assisted breeding of Indonesia local rice variety Siputeh for semi-dwarf phonetype, good grain quality and disease resistance to bacterial blight

Rice (Oryza sativa L.) is a staple food crop in most of the Asia countries. The introduction of semi-dwarf rice led to record yield increases throughout Asia in the 1960s. The recessive semi-dwarfing gene, sd-1, is one of the most important genes deployed in modern rice breeding. Rice varieties with the sd1 gene have a shortened culm with improved lodging resistance and a greater harvest index, allowing for the increased use of nitrogen fertilizers (Jennings [1964]). The sd1 gene has been cloned from rice and its dominant wild-type allele encodes a gibberellin (GA)-20 oxidase-2 (GA20ox-2) (Ashikari et al. [2002]; Sasaki et al. [2002]; Spielmeyer et al. [2002]). The deletion or substitution mutation of the Os20ox2 gene results in loss of function of the enzyme, which ultimately leads to a deficiency in plant growth hormones gibberellins that control plant height (Ashikari et al. [2002]; Sasaki et al. [2002]; Spielmeyer et al. [2002]). The different alleles of the sd1 gene have remained the predominant semi-dwarf genes in modern rice cultivars (Asano et al. [2007]).

There are two different types of starch, amylose and amylopectin, in rice endosperm and the amylose content affects rice eating and cooking quality (Cruz and Khush [2000]). With content ranging from 5% to 15%, the low-amylose rice is characterized by fluffy texture, glossy appearance of the cooked rice, soft texture of cooled rice, and excellent expansibility for food processing (Zhu et al. [2003]). Genetic studies have shown that the rice Wx gene determines the amylose content in the endosperm. Wx encodes granule-bound starch synthase I (GBSSI), a key enzyme for amylose biosynthesis in the endosperm, and wx mutant endosperm contains almost exclusively amylopectin (Hori et al. [2007]; Sano [1984]; Wang et al. [1995]). In addition to controlling amylose content, Wx also affects gel consistency and gelatinization temperature of rice starch (Su et al. [2011]). Wx^a and Wx^b are two predominantly distributed functional Wx alleles in rice (Sano [1984]; Wang et al. [1995]). Wx^a is widely distributed in indica rice with high amylose content while Wx^b is mainly found in japonica rice with low or intermediate amylose content (Sano [1984]; Wang et al. [1995]). A single nucleotide substitution (G-to-T) at the splice donor site of the first intron in Wx^b potentially alters the splicing site and decrease the splicing efficiency of the first intron of Wx, resulting in the low level of both mature transcript of Wx^b and amylose content (Cai et al. [1998]; Larkin and Park [1999]; Tian et al. [2009]; Wang et al. [1995]). Based on the polymorphism at the splicing site of the first intron, a co-dominant CAPS (cleaved amplified polymorphic sequence) molecular marker PCR-Acc I was developed for selection of Wx alleles in rice breeding (Cai et al. [2002]).

Bacterial blight of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most important bacterial diseases prevalent throughout the world (Gnanamanickam et al. [1999]). Rice yield losses caused by bacterial blight can reduce yield by 20-30% and by up to 50% in some areas of Asia (Mew et al. [1993]). The utilization of host disease resistance (R) genes is the most economic and efficient method for controlling the disease. Xa4 is a dominant R gene that provides durable resistance to bacterial blight. Cultivars with Xa4 conferred resistance to almost all Chinese patho-types of Xoo except for patho-type C5 (Zhang [2009]). Xa21 is another dominant bacterial blight gene, which was originally discovered in wild rice specie Oryza longistaminata (Ikeda et al. [1990]). IRBB21, an Xa21 line in IR24 genetic background, showed resistance to all the known races of Xoo collected from India and the Philippines (Ikeda et al. [1990]). Both Xa4 and Xa21 genes are widely exploited R genes in Asian rice breeding programs for bacterial blight resistance (Datta et al. [2002]; Huang et al. [1997]; Luo et al. [2012]; Luo and Yin [2013]; Singh et al. [2001]; Suh et al. [2013]; Zhang et al. [2006]).

Although modern rice varieties have been planted worldwide, elite local rice varieties are still popular in many rice growing countries due to their good grain quality and adaptation to the local climate and environment. Cultivar Siputeh is a local rice variety still planted in Java and Sumatra islands of Indonesia. Siputeh produces long grain rice with good cooking and eating quality. However, the variety has low yield with tall plant type and long growth duration and is highly susceptible to biotic and abiotic stress (Suhartini [1991]). Here we report the genetic improvement of Siputeh for semi-dwarf phenotype, good grain quality and disease resistance to bacterial blight through marker-assisted breeding.

Marker-assisted selection is a molecular breeding process whereby a molecular marker based on DNA variation is used for indirect selection of an interest agronomic trait. Marker-assisted selection is not affected by environments and can be conducted at any plant growth stage. It is especially useful for selection of recessive genes and biochemical traits in heterozygous plants. In this study, the sd1 gene is a partially recessive gene that controls semi-dwarf phenotype. It is difficult or impractical to select the sd1 gene in the backcrossing progeny according to plant height, especially under greenhouse condition. Similarly, the amylose content in rice grains can only be directly detected after harvesting, however, we need to know the genotype for crossing purpose before flowering. As for the pyramiding of Xa4 and Xa21 for bacterial blight resistance, both R genes have different resistance specificities and their resistance spectrums are over-lapped. It would be impractical to select the two R genes in a single line by conducting disease evaluation and phenotypic selection. To overcome these difficulties, we chose marker-assisted selection approach, which enable us to perform precisely and effectively multiple gene selection and/or pyramiding in limited breeding time.

Compared with Siputeh, the significant change in TS4 is the semi-dwarf plant type, which is mainly resulted from the introduction of the sd1 gene. The semid-warf plant type of rice indiredtly contributes to lodging resistance in the field. It also increases grain-straw ratio, which ultimately results in higher yield. In the present study, TS4 produced higher yields than Siputeh in both field trials conducted in different location (Table 3). The results are consistent with the results of previously studies on the sd1 gene introduced in other varieties (Hedden [2003]; Luo and Yin [2013]; Spielmeyer et al. [2002]). TS4 also showed significant reduction in growth duration than Siputeh. The growth duration of TS4 was about four months when it was grown in Banda Aceh, Indonesia (Table 3), which was similar to most of the typical modern rice varieties grown in tropical region. Short-growth duration makes it easier for farmers to plan growth season, synchronize sowing and harvest time with other varieties and manage diseases and insects in the field.

Like the Xa4 and Xa21 donor line WH421, TS4 provided broad-spectrum resistance to multiple Xoo strains tested in this study. It was noted that WH421 were susceptible or moderate susceptible to 1947, PXO99 and GD1358 while TS4 provided moderate resistance to the three Xoo strains (Table 2). It seems that either Xa4 or Xa21 or both R genes performed better for resistance to Xoo in TS4 than that in WH421. Previously, it was found that the Xa21 gene in IRBB21 conferred complete resistance to PXO99 (Gu et al. [2004]; Ikeda et al. [1990]; Song et al. [1995]), however, in this study, neither WH421 nor TS4 could confer full resistance to the Xoo strain. It has been reported that the R gene-mediated resistance to Xoo in rice may be influenced by rice genetic backgrounds and developmental stages (Cao et al. [2007]). For instance, the Xa21-containing rice line Minghui was still susceptible to PXO99 (Zhang et al. [2006]).

The introduction of the sd1 gene to Siputeh would significantly reduce its plant height and growth duration, which might affect rice grain quality. To overcome this potential shortcoming, we also introduced the Wx^b gene into TS4 through marker-assisted selection. Indeed, the Wx^b gene significantly reduced the amylose content in TS4. Amylose content strongly affects the cooking and eating quality of rice. Rice with amylose content at 25-30% usually tends to be firm and dry after cooking, rice with amylose content at 20-25% tends to be softer and stickier and rice with amylose content at <20% is generally quite soft and sticky. TS4 has amylose content at 15.3-17.8% and shows soft gel consistency. The results indicate that TS4 is generally softer and stickier than Siputeh, which has amylose content at 26.3-28.9% and shows medium to soft gel consistency.

An improved rice line, designed as TS4, has been developed to contain semi-dwarf gene sd1, low amylase content gene Wx^b and bacterial light resistance genes Xa4 and Xa21 through marker-assisted selection. TS4 has semi-dwarf phenotype with reduced growth duration, produces high yield with good grain quality and provides broad-spectrum resistance to Xoo strains. The development of TS4 enriches the diversity of local rice varieties with high yield potential and good grain quality.

YL is the Senior Research Officer of Temasek Life Science Laboratory (TLL) and the Professor of Anhui Rice Research Institute (ARRI), Anhui Academy of Agricultural Sciences (AAAS), with over 25 years of experiences on rice breeding; SZ is the Professor of Agriculture Faculty, Syiah Kuala University (SKU); BB is the Associate Professor of Agriculture Faculty, SKU; TM is the Associate Professor of ARRI, AAAS; ZL is the Director and Professor of ARRI, AAAS; JY is the Professor of ARRI and the President of AAAS; ZY is the Senior Principal Investigator of TLL.