Marker-Assisted Backcross Screening for Vietnamese Aromatic Rice Lines Derived from TLR7/Khaodawkmali105//TLR7
This research focused on genotyping via SSR molecular marker. Backcrossing populations of the combination of TLR7/KhaoDawkMali105//TLR7 was generated to stabilize aromatic genes of donors into rice varieties as acceptors by using an advanced backcrossing method (ABC) with molecular marker assistance. Fifty BC3F2 rice lines were used to evaluate aroma detection through the sensory test, genotypic analysis using microsatellite markers, agronomic characteristics, and other grain quality traits. Sixteen lines having aromatic gene (fgr) locus were found using marker RG28 as homozygous condition in 50 rice lines. The marker RM223 detected lowest number of fgr locus. Two lines (BC3F4-1-8 and BC3F4-1-48) were identified well-defined target for aroma genes having high yield and good quality traits. Thus, these lines were utilized SSR marker for demonstrating marker-assisted selection (MAS) breeding program.
Keywords: Microsatellite Marker; Aroma; Polymorphism; Codominant
The quality of rice is considered based on milling quality, grain size, shape, appearance, aroma and other cooking characteristics [1]. Aromatic rice, also known as fragrant rice, is very popular in Asia. The biochemical basis of aroma was identified as 2-acetyl-1-pyrroline which a fragrant compound being discovered in KhaoDawkmli105, Basmati and Jasmine-type aromatic rice [2,3]. Also, there are pentanol, hexanol, benzaldehyde involving fragrance on rice. Application of results to introgressive a major gene into a high-yielding rice cultivar by continued backcrossing, with assistance of flanking marker. Ahn et al. determined fgr gene positioned on chromosome 8 via RFLP, controlling aroma while Lorieux, et al. observed gene of fgr closely linked with marker RG28 with genetic distance of 4.5cM [3,4]. Another marker RM223 can be possibly used for polymorphism at both of salt gene and aromatic gene.
Recently, molecular markers have been developed for the selection of aromatic plants in rice; they have the advantages of being inexpensive, simple, rapid, requiring small amounts of tissue and highly reliable [5]. Cornell University (USA) researched the original of aromatic rice varieties on genetic base via determined gene of betaine aldehyde dehydrogenase (BADH2) controlled aroma [6]. Yoshihashi, et al. identified a microsatellite marker RM17 that is closely linked to the fgr gene in Khao DawkMali105, a famous aromatic rice cultivar of Thailand [7]. Then a functional BADH2 enzyme inhibits 2AP biosynthesis which is a major component of aroma was reported by Bradbury, et al. [5]. Therefore, in this study, the development of PCR marker for aroma characteristic use in breeding program has made great advance, which made it possible to select rice lines having aroma with good quality and high yield in a backcross population derived from a cross between the aromatic and nonaromatic cultivars.
The cross between high yielding rice TLR7 (a high yield variety) and KhaoDawkMali105 determined aromatic gene as donor were obtained from the High Agricultural Technology Research Institute for Mekong delta, Vietnam (HATRI).
A field experiment was transplanted to an irrigated lowland field in a randomized complete block design in three replications in the field of HATRI. Fifty lines of BC3F2 with their parents were used to evaluate agronomic characteristics and aroma detection through sensory test and genotypic analysis using SSR markers in lab of HATRI. Data on important agronomic traits like plant height, panicle length, filled grains/panicle, unfiled grain/panicle, 1000-grain weight, harvest index and yield were recorded.
Ten randomly selected plants of each genotype were used for agronomic data analysis. Data on plant height (cm), number of effective tillers/plants, panicle length (cm), number of filled grains/panicle, 1000-grain weight (g), days to maturity and grain yield/plant (g) were recorded and subjected to statistical analyses using SAS software. After harvesting, the seeds of each genotype were dehulled for evaluation of the grain quality and aroma. The grains were classified into different types based on their dimension according to Dela Cruz and Khush [8]. Ten seeds of each cultivar removed rice hull and mashed by hand. Take rice powder of each cultivar and put on each experimental tube or petri dish. Add 5ml KOH 1.7% into each petri dish and cover. Store the samples in room temperature for 30 minutes. The samples were scored, corresponding to absence of aroma, slight aroma, moderate aroma and strong aroma, respectively.
DNA isolation was carried out using the mini preparation CTAB method [9]. Two markers RM223 and RG28 (linked to aroma) were used to confirm the presence of fgr gene as described by Garland, et al. and Ahn et al. [10,4]. The details of the primers are given in Table 1. The amplified products were electrophoretic ally resolved on a 3% agarose gel in 0.5xTBE and stained with ethidium bromide 0.5mg/ml and analyzing by Alpha Imager 1220 (Alpha Innotech, CA, USA). The bands representing particular alleles at the microsatellite loci were scored manually on the basis of parental bands like non-aromatic type (TLR7), aromatic type band (KhaoDawMali105), and heterozygotes type band.
SSR marker, which is RG28 and RM223 on chromosome 8 closely associated with aromatic genes, detected these target genes from short-term high-yielding rice varieties and a number of rice varieties carrying aromatic gene.
The agro-morphological data were initially analyzed through examining variance to verify genetic variation in the traits measured. The few traits with insignificant genetic variation, based on the F-test, were not considered for further analyses.
Developing advanced backcrossing (BC3F2) with molecular marker assistance to select individuals carrying aromatic gene in order to cross back with female lines (recurrent lines) and to stabilize aromatic gene in homozygosity was rapid, compared with conventional method. Molecular markers expressed polymorphism clearly, linking to aromatic gene which was recorded on backcrossing populations.
Analysis of BC2F2 showed that primer RG28 confirmed the presence of fragrance gene with size of 200bp to 220bp in 4 lines where the fragrance gene was similar to that of KhaoDawkMali105 (aromatic type) at homozygous and in 16 lines similar to that of TLR7 (non-aromatic type) as homozygous alllele. Rest of the genotypes showed heterozygous condition (Figure 1). In case of RM233, the amplification of DNA was 100% and there were two alleles with size of 200bp to 210bp. Only one line gave aromatic banding pattern similar to that of KhaoDawkMali105. Most of the genotypes showed heterozygous allele.
For primer RG28, the amplification of DNA was 100% and there were two alleles with size from 200bp to 220bp. However, when recorded BC3F2, these lines had gradual decrease of heterozygote by 14%. There were 16 lines that carried aromatic gene: 6, 7, 8, 9, 10, 11, 23, 24, 25, 26, 27, 35, 36, 46, 47, and 48 (Figure 2).
For molecular marker of RM 223, DNA amplification from population BC3F2 accounted for 100% presented bands. This result showed that samples had different bands with two alleles which were commonly clear at position of molecular marker. Difference in quantity and position of bands might show difference in size: 210 bp -220bp. Also, Buu, et al. applied marker RM223 associated with fgr on chromosome 8 with genetic distance of 1.6 cM in breeding high-yielding rice varieties carrying aroma and successfully developed rice varieties of OM4900, OM6161, and OM6162 [11]. The primer RM223 responded best in all the 26 rice genotypes, because RM223 primer could be able to identify aromatic and non-aromatic germplasm effectively which supported the phenotypic results. It can also be said that the RM223 responded best in all the 26 rice lines and it could be readily used in breeding program for releasing aromatic rice variety with considerable yield [12].
Conventional breeding such as genealogy or recurrent selection is to select phenotype between inbred lines or parental lines crossed on basis of test-cross. With such condition, MAS for individuals via using selective index combined molecular evaluation and phenotyping can increase selective effect higher than conventional method. MAS make it possible to enhance selection strength in first generations of genealogy while conventional method is nearly unselective and visible selection to remove the worst lines. Combination between position of molecular marker and QTL will help us to review drought tolerance or pests and diseases at F2 generation. In subsequent generation, selecting can be completed in young plant (seedlings stage) or mature plant which was planted in normal environment reducing the number of trial lines in problemed environment. In this examination, after phenotypic and genotypic observation, it was found that thirteen lines were aromatic gene compared with phenotype (Table 2). In other case, line BC3F2-50-18 had aroma alleles but it still presented non-aromatic in phenotype.
Phenotypic evaluation of aromatic rice lines: The results on agronomic performance in Table 3 showed that the plant height of the aromatic lines ranged from 95 cm (BC3F4-448-96) to 115 cm (BC3F4-1-8). In comparison to P2, there was considerable reduction in plant height in most of the lines. Days to maturity of the aromatic rice lines ranged from 90-105. The effective panicle length varied from 25.6 cm (BC3F4-2-10) to 30.1 (BC3F4-49-47). The number of filled grains/panicle was highest in BC3F4-1-47 (232) and lowest in P2 (165). The 1000-grain weight was highest in BC3F4-49-47 (28.5g) and lowest in BC3F4-2-10 (26.3 g). The HI of the aromatic rice lines ranged from 0.53 to 0.59. Six lines showed higher yield than both the parents (BC3F4-1-6, BC3F4-1-8, BC3F4-1-47, BC3F4-2-10, BC3F4-47-58, and BC3F4-48-96) (Table 3).
Nine advanced lines along with their parents were subjected to quality analysis. Grain quality of rice consists of several components: the milling quality such as head rice and nutritional quality such as protein [13,14]. Cooking and eating qualities are mostly determined by amylose content (AC), gelatinization temperature (GT), and gel consistency (GC) of the grain starch [15]. Appearance quality is mainly specified by grain shape as defined by grain length, grain width, the length–width ratio, and the translucency or chalkiness of the endosperm [16]. As mentioned above, some lines were the first IR64 variety to have both intermediate amylose content and intermediate GT. These traits are considered important for the ideal texture of cooked rice, especially for many rice consumers in South and Southeast Asia [17]. Brown rice percentage varied from 80-84%. The head rice percentage of lines ranged from 41-55%.
Most of the studied lines were found to give non-chalkiness. Four lines were found to give moderate aroma (BC3F4-1-6, BC3F4-1-8, BC3F4-2-10, and BC3F4-48-96), compared to P2. The GL of BC3F4-1-47 recorded highest value of 7.23 mm and BC3F4-47-111 recorded lowest value of 7.05 mm among the parents. Most consumers prefer rice with intermediate amylose content ranged between 20-25%. Amylose content ranged from 16.2% (BC3F4-1-6) to 24% (BC3F4-49-47). Gel consistency (GC) is another major character responsible for the texture of cooked rice. Most of lines had higher GC than the GC of parent’s varieties. The protein content was highest in BC3F4-49-47 (8.7%) while the lowest in BC3F4-1-8 (8.0%) (Table 4).
This presents a real obstacle for DNA extraction and analysis of marker. Using MAS more widely in the first generation will make the procedure less expensive. The use of MAS is ongoing in rice, which is mainly for pyramiding resistant gene and MAB. Increase in the use of the method is expected, especially for MAB [18]. The wider use of MAS is expected with the improvement of methods for analyzing genetic markers and identify candidates for economic traits.
Two molecular markers of RG28 and RM 223 were evaluated with population of BC3 F2 and BC3F4 of TLR7/KhaoDawkMali105//TLR7. Important agronomic characteristics of selected aromatic plants were recorded. Percentage of evaluating trait compared genotype and phenotype showed that percentage of RG28 was higher than RM223. It was found that the aroma trait selected from lines in TLR7/KhaoDawkMali105//TLR7 (lines 3 (BC3F2-1-8) and lines 8 (BC3F2-48-96) had good aroma and superior agronomic. These lines continued to evaluate yield and yield components in the field and also evaluated quality.
This study was supported by Ministry of Science and Technology, Ministry of Agriculture. The authors thank to the staffs of the Division of Genetics and Plant Breeding of High Agricultural Technology Research Institute who facilitated the implementation of the subject.
