1. Introduction
Rice (
Oryza sativa L.) is consumed as a staple food in Asia, especially in the southeast region. In Thailand, the indica rice variety RD6 developed from KDML105 through gamma irradiation is one of the most favorable glutinous rice consumed throughout the northeast of Thailand [
1,
2]. Because of its cooking quality, aroma, and softness, production demand has increased over time. However, its yield of 4.16 ton/ha fails to meet its potential, due to biotic and abiotic stress.
Rice blast disease caused by the fungus
Pyricularia grisea (Cooke) Sacc. leads to crop losses up to 85% of total yield [
3]. Disease symptoms occur in all stages of plant growth, beginning with blast discoloration and wilting of the foliage [
4]. Neck blast can be found at the flowering stage, accelerating plant death [
5]. Severe damage was also observed within areas of intensive planting with high doses of nitrogen application [
6]. Development of new rice varieties resistant to blast fungus is an alternative approach to diminish or control the invasion of this pathogen. The resistance quantitative trait loci (QTL) have been investigated to achieve parental varieties, which are further used for gene pyramiding in breeding programmes. Currently, more than 100 blast-resistant genes have been identified, of which 22 genes structures have been cloned [
7]. In Thailand, few studies of blast resistant genes have been conducted. Noenplab et al. [
8] studied the relationship of leaf blast and neck blast of resistant genes in the Jao Hom Nil (JHN) variety, in which the resistant QTLs were detected on chromosomes 1 and 11. The resistant QTLs conferred resistance to both leaf blast and neck blast. Suwannual et al. [
9] pyramided four blast-resistant QTLs, individually, on chromosomes 2 and 12 within the P0489 variety, and on chromosomes 1 and 11 carried by the JHN variety, resulting in the creation of new RD6 introgression lines. Their results demonstrated that the RD6 introgression lines carrying a high number of QTLs (achieved through pyramiding) reached a broader spectrum of blast resistance to the blast pathogens prevalent in the region.
In addition to rice blast fungus, salt stress is a crucial constraint for RD6 production. Thailand’s northeast region is the country’s largest rice-producing area, and it is comprised of two basins: Sakon Nakhon and Nakhon Ratchasima. In those basins containing an understructure of accumulated salt rock, the salt-affected range covers approximately 1.84 Mha [
10]. Evaporation during the dry season tends to raise salinity from the subsoil to the surface, thereby increasing salinity intensity and increasing salt stress from 2–4 dS/m to 8–16 dS/m [
10,
11,
12]. Rice is a salt-sensitive crop, capable of tolerating salinity at moderate levels of electrical conductivity (4–8 dS/m) [
13]. Therefore, rice produced under rain-fed, lowland conditions is usually exposed to high levels of soil salinity. The RD6 variety, which is well known, was identified as a geographical indication (GI) within the Tung Gula Rong Hai of the Northeast, Thailand. Specifically, RD6 requires optimal soil salinity to enhance rice seed aroma [
14]. However, an abundance of salinity can reduce rice plant growth, tiller number, and seed set-up [
15], and the stress caused by excessive salt can significantly reduce total crop yield and result in plant death [
16].
The Pokkali variety, derived from the International Rice Research Institute (IRRI), has become a well-known source of salinity tolerance worldwide, attributed to the salt-tolerant QTL located on rice chromosome 1 (
Saltol) [
17,
18,
19,
20]. Therefore, several researchers have attempted to develop salt-tolerant rice varieties using the
Saltol QTL [
21,
22,
23,
24,
25,
26].
The marker-assisted backcrossing (MAB) method has been employed to obtain beneficial QTLs from donor parents via introgression between the qualitative and quantitative traits from landraces and wild relatives [
27] due to the precision method with shortened time frame in both foreground and background selection. MAB provides effective gene selection and/ or QTLs for pyramiding multi-genes/QTLs within the rice population. These benefits further support breeding practices for improved resistance and tolerance [
28,
29,
30,
31,
32]. The objective of this study was to determine the blast resistance and salt tolerance levels within the RD6 introgression lines by pyramiding four blast-resistant and one salt-tolerant QTL into the RD6 rice variety in both greenhouse and field conditions.
4. Discussion
Since its release in 1977, the RD6 glutinous rice variety has remained a staple food crop for domestic consumption in Thailand’s north and northeast regions. Comprising 83% of total glutinous rice production in these areas, consumers have developed a preference for its superior characteristics. However, the RD6 variety suffers from several production constraints, including biotic stress responsible for both rice blast [
40] and bacterial blight disease [
41]. Current research has attempted to eliminate sustainable infection-resistant production practices by pyramiding multiple resistant genes [
42]. To date, an RD6 introgression line capable of resisting both biotic and abiotic stress has yet to be developed. Thailand’s salt rock basins of Sakon Nakhon and Nakhon Ratchasima have demonstrated that consistent levels of salinity can enhance the fragrance of the RD6 rice variety [
14] and increase production.
This study proposes the successful introgression of blast-resistant QTLs (qBL 1, 2, 11, and 12) from RGD07005-12-165-1 and the Saltol QTL (Pokkali, chromosome 1) to improve the RD6 rice variety through the MAB method within BC4F4 populations. Trait evaluations were completed for the validation of progenies with desirable traits in each advanced population, based on the introgression of the genetic foregrounds and maintenance of the genetic backgrounds, respectively.
Salt salinity was absent in several areas of northeast Thailand, due to high levels of NaCl [
43], and such factors as precipitation, soil type, and field management. In past research, the evaluation of salt tolerance was typically conducted through salt screening, hydroponic culture, and soil culture, as well as through pot and field methods [
33]. The current study assessed salt tolerance within the breeding populations studied through salt solution, artificial salt culture, and field condition evaluations. Based on the results, salt evaluation under field conditions produced the lowest capability among the tested rice lines (
Table 1,
Table 2 and
Table 3 and 5), due to the inherent difficulties and uncertainties present under field conditions. Kranto et al. [
33] reported that effective alternative screening approaches must be proven to correlate with results produced within the early phases of growth in both greenhouse and field conditions. Within the present study, visual symptom scores of salt stress generated through the salt solution method proved to be the most appropriate method with which to confirm tolerance abilities within a breeding population (
Table 5), suggesting that the salt solution method could, therefore, substitute salt tolerance score analysis in field conditions (
Table 5). However, we acknowledge the necessity to evaluate RD6 plant types, yield performances, and agronomic traits within the field. The introgression lines developed within our study were evaluated for similarity with the original RD6 agronomic traits, namely plant height, panicle length, 4/panicle seed weight, 1000/seed weight, total dry weight, total seed weight, harvest index, seed length, seed width, and seed shape, as well as seed qualities, such as seed morphology (
Table 2 and
Table 4).
The
Saltol QTL on chromosome 1 from the Pokkali rice variety has been commonly used for rice improvement in several studies [
21,
22,
23,
24,
25,
26]. In our results, the
Saltol QTLs from the Pokkali variety produced the greatest salt tolerance within the RD6 introgression lines (
Table 1,
Table 2,
Table 3, and
Table 5). This
saltol QTL also contributed to the maintenance of low Na+, high K+, and low Na+/K+ homeostasis levels in rice stems, further resulting in increased salt tolerance [
24,
44] (
Figure 3). The Pokkali variety was classified to balance the influx of Na+ and K+ for dilution in the mechanism, creating the ability to exclude Na+ from leaf blades and stems [
45,
46]. As the water up-take mechanisms in rice accept both nutrients and salt together, the Pokkali variety thereby demonstrated the highest and most significant differences in leaf, stem, root, and total dry weights when compared with other breeding lines (
Table 5). However, the BC4F4 lines presented the agronomic traits (above) more closely matched to the RD6 than to the Pokkali (
Table 5), due to the advance generation and visual selection of the trait performances (
Table 2 and
Table 4). RD6 performance is very important for farmer acceptance and crop adaptation in our test areas. For example, excessively tall RD6 rice plants present problems in the grain filling stages as a result of heavy wind or rain [
47]. Visual selection may explain the differences in percentages of Na+ of the RD6 introgression lines with those of Pokkali (
Table 5,
Figure 3).
As a photosensitive rice variety, the RD6 grows once a year, during Thailand’s rainy season from late May to November [
48]. These bimodal rain patterns produce favorable conditions for the occurrence of blast disease, causing damage in all stages of growth. Leaf blast generally occurs during the seedling and tilling stages, whereas neck blast usually occurs during the reproductive phase [
4]. In our study, introgression lines were evaluated for blast disease in both the field and upland short-row evaluations.
In this study, the upland short-row method displayed greater incidences of blast disease, due to the favorable microclimate and moisture contents around the experimental plots (
Table 1 and
Table 3) [
49]. The experimental field was influenced by bimodal rain, capable of inducing leaf and neck blast symptoms (
Table 3), further indicating the resistance of the QTLs [
42,
50]. Noenplab et al. [
8] also reported that the blast QTL on chromosome 11 in the JHN variety successfully contributed to leaf and neck blast resistance. Pyramiding of four blast-resistant QTLs through MAS achieved high levels of blast resistance and broad-spectrum resistance to pathogens prevalent in the region [
9]. Moreover, the testing of RD6 introgression lines for durable blast resistance and no-yield penalties were observed [
42]. The results, herein, further demonstrated that neck blast disease caused direct yield loss during the grain filling phase [
51], as well as lower 1000/SW within the original RD6 variety compared with those of the RD6 introgression lines (
Table 4).
The resistance/tolerance abilities of the RD6 introgression lines represent the foreground genetics capable of enhancing plant breeding programs. However, maintaining the background of the original RD6 variety is also desirable; therefore, the quality and performance of the RD6 within the QTL introgression was also a consideration. The BC
4F
4 populations, herein, were achieved through the introgression of blast-resistant QTLs (
qBL 1, 2, 11, and
12) from RGD07005-12-165-1 and
Saltol QTL (Pokkali) and improved the RD6 rice variety through MAB. Consequently, the performance of the RD6 introgression lines was similar to that of the original RD6 variety (
Table 4,
Figure 2). The results indicate that foreground and background selection, together with visual selection, accurately depicts the efficiency of MAB.