Improvement of Morkhor 60-3 Upland Rice Variety for Blast and Bacterial Blight Resistance Using Marker–Assisted Backcross Selection

Abstract

Morkhor 60-3 is an upland rice variety primarily cultivated in northeastern Thailand. This glutinous rice is valued for its adaptability and rich aroma but remains susceptible to significant diseases, particularly blast and bacterial blight. Using resistant varieties represents the most cost-effective approach to address this limitation. This study incorporated the QTLs/genetic markers qBl1, qBl2, and xa5 from Morkhor 60-1 through marker-assisted backcrossing. From the BC₁F₃ population, ten lines were selected based on their parentage and evaluated for blast resistance using a spray inoculation method with 12 isolates of Pyricularia oryzae, and for bacterial blight (BB) resistance using a leaf-clipping method with nine isolates of Xanthomonas oryzae pv. oryzae. Broad-spectrum resistance (BSR) was also assessed in the lines for both diseases. Subsequently, BC₁F₄ lines were evaluated for field performance, including agronomic traits and aroma. Results identified three superior lines, BC₁F₄ 22-7-140-4, BC₁F₄ 22-7-322-5, and BC₁F₄ 22-7-311-9, that demonstrated resistance to both BB and blast pathogens with average BSR values of 0.61 and 1.00, 0.66 and 1.00, and 0.55 and 0.87, respectively. These lines also exhibited enhanced performance in flowering date, plant height, panicle number per plant, grain number per plant, and grain weight. These findings demonstrate the effectiveness of marker-assisted selection (MAS) for gene pyramiding in rice improvement.

1. Introduction

Upland rice is predominantly cultivated in rain-fed areas of Northeast Thailand. Sakon Nakhon is the most widely grown upland rice variety in this region. This variety was developed from a cross between Hom Aom and RD10 varieties. Sakon Nakhon is a glutinous, high-quality, non-photosensitive rice with yields of 2918 kg/ha that thrives in irrigated and rain-fed conditions. However, Sakon Nakhon is susceptible to blast and bacterial blight (BB) diseases and brown plant hopper infestations, which cause significant damage to Thailand’s agricultural sector [1].

Blast is a fungal disease caused by Pyricularia oryzae [2]. Its spores occur naturally and are dispersed by wind. Symptoms can appear on all above-ground plant parts, including leaf blades (leaf blast), leaf sheaths, internodes, nodes, and panicles (neck or panicle blast). Meanwhile, bacterial blight, which is caused by Xanthomonas oryzae pv. oryzae (Xoo), can develop year-round, particularly in high-humidity environments [3]. Both diseases cause severe crop losses throughout the world and Asia [4,5]. While utilizing resistant varieties represents the most effective and economical control strategy, conventional breeding for multiple traits or genes is complex and time-consuming [6]. Therefore, integrating molecular markers with traditional breeding approaches offers an efficient pathway for developing resistant varieties.

DNA markers associated with genes of interest for blast and bacterial blight (BB) resistance that have been well documented have been a significant focus in plant pathology and breeding [7]. To date, researchers have identified more than 122 blast resistance genes, including 66 major genes such as Pi27(t), Pita, Pi54, Pid2, Pi9, Pi2, Pizt, Pi36, Pi37, Pikm, Pi5, Pit, Pid3, pi21, and Pitb [8,9,10]. and 17 minor resistance genes such as Pir2-3, Pirf2-1(t), Pi30(t) Pi31 (t), and Pi32(t) [11,12]. In addition, approximately 39 blast resistance genes have been cloned and characterized, such as Pi54 Pik-p [13,14]. Part of the resistance gene to BB disease has more than 40 resistance genes that have been reported for bacterial blight, including Xa-1, Xa-2, Xa-3, and xa-5 [15,16,17]. These molecular markers linked to resistance genes have been successfully employed in rice breeding programs. For instance, P0489 varieties carrying blast resistance QTLs on chromosomes 1 and 11, flanked by SSR markers RM319/RM212 and RM48/RM207, respectively, have been used to enhance blast resistance. Similarly, SSR markers RM224/RM144 and RM313/RM277, linked to blast-resistant QTLs on chromosomes 2 and 12 in Jao Hom Nil (JHN), have been utilized in blast resistance breeding [18,19]. For BB resistance breeding, the IR62266 variety carrying xa5 on chromosome 5, flanked by markers RM212 and RM159, has proven valuable [20]. These molecular markers have been combined with other resistance genes to achieve broad-spectrum resistance, selecting alleles with significant effects on high-value traits with relatively simple inheritance patterns.

Several advanced techniques are being developed to enhance broad-spectrum disease resistance in plants, especially marker-assisted pyramiding (MAP). MAP effectively combines multiple genes/QTLs for traits of interest (such as disease resistance, insect tolerance, and abiotic stress tolerance). This approach allows breeders to efficiently select for multiple resistance traits simultaneously, thereby improving the durability and overall effectiveness of plant defense mechanisms against a wide range of stresses [21,22]. By pyramiding multiple resistance genes, MAP significantly reduces the risk of resistance breakdown, a common limitation of single-gene resistance strategies [23]. Numerous studies have demonstrated the successful application of this method in integrating multiple resistance genes into elite cultivars. For example, Suwannual et al. [24] combined blast resistance genes in crosses between RD6 × P0489 (qBl2, qBl11) and RD6 × JHN (qBl1, qBl12) through marker-assisted selection (MAS), producing BC₂F₃ 1-13-17-52 and BC₂F₃ 1-28-8-37 lines that exhibited resistance to blast with a broad-spectrum resistance (BSR) of 0.6. Consequently, a near-isogenic line (NIL) of the RD6 variety, BC₂F₃ 6-1/15-1-50, carrying qBl1, qBl2, qBl11, and qBl12, demonstrated strong resistance with high BSR against Thai Xoo isolates [25]. In our previous study, Srichant et al. [26] improved upland rice Sakon Nakhon (SKN) for blast resistance through MAS, resulting in NIL SKN 39-10-19-29-12 BK-6-7, which showed resistance to both blast and neck blast diseases. This improved variety was subsequently certified as “Morkhor 60-3”.

Despite these improvements, Morkhor 60-3 carries only two blast resistance genes, potentially limiting its durability as pathogens adapt. Additionally, it remains susceptible to bacterial blight. Therefore, this study aims to combine genes/QTLs (qBl1, qBl2, and xa5) through marker-assisted backcrossing (MAB) to enhance blast and bacterial blight resistance in Morkhor 60-3.

2. Materials and Methods

2.1. Population Development Using Marker-Assisted Selection

To develop the breeding population, we crossed our line, SKN-39-10-19-29-12-BK-6-7 (Morkhor 60-3) (qBl11 and qBl12), with the donor parent Morkhor 60-1, which possesses additional blast resistance QTLs (qBl1 and qBl2) along with the BB resistance gene xa5 [27]. The resulting F₁ plant was then backcrossed to Morkhor 60-3 to create a BC₁F₁ population. Following this, self-pollination was conducted for one cycle to develop the BC₁F₂ generation. During the development of the F₁, BC₁F₁, and BC₁F₂ populations, marker-assisted selection (MAS) was employed to select for specific alleles. The BC₁F₃ generation was tested for blast and BB resistance under greenhouse conditions. Agronomic traits, yield, and yield components were also evaluated under field conditions. Finally, the BC₁F₄ generation was validated for seed quality and aroma (Figure 1).

MAS was used for selecting resistance alleles. SSR marker franking to four blast resistance QTLs and the BB resistance gene were employed for MAS, including four SSR flanking markers, RM562/RM212 and RM71/RM109, associated with blast resistant QTLs on chromosomes 1 and 2, and two markers, RM164/RM1089, associated with the BB resistant gene on chromosome 5, according to the marker details described by Pinta et al. [27]. BC₁F₁ and BC₁F₂ populations were selected using MAS for blast and BB resistance. Total genomic DNA from young leaves of each rice plant, lines, and their parent were extracted according to the method described by Doyle and Doyle [28] with slight modifications. Polymerase chain reaction (PCR) for SSR markers was carried out at a volume of 15 µL, containing 25 ng of genomic DNA, 1X PCR buffer concentration, 0.2 µM MgCl2, five µM reverse and forward primers, 10 mM dNTP, 7.35 µL dH₂O, and 0.5-unit Taq DNA polymerase. DNA amplification was performed in a DNA thermal cycler for 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 s, primer annealing at 55 °C for 30 s, primer extension at 72 °C for 60 s, and final extension at 72 °C for 7 min. The amplification products were separated on a 4% polyacrylamide gel by electrophoresis. The line selection in the F₁, BC₁F₁, BC₁F₂, and BC₁F₃ generations was performed through MAS for blast and BB resistance. The homozygous resistant genotype of those genes and/or QTLs was identified and allowed to self to deliver BC₁F₃ for greenhouse and field validation.

2.2. Evaluation of Blast and Bacterial Blight Resistance

For blast resistance evaluation, 18 lines/varieties, including a standard (SKN), donor parent (Morkhor 60-1), recurrent parent (Morkhor 60-3), and resistant and susceptible checks (P0489, Jao Hom Nin, IR64, KDML105, and RD6 varieties), were used for blast disease resistance tests at Ubon Ratchathani Rice Research Center, Thailand, in 2023–2024. The experiment was laid out as a factorial arrangement in a completely randomized design (CRD) with three replications. The treatments included genotypes that were analyzed as fixed effects. Soil preparation involved mixing rice husk ash with chemical fertilizer to improve soil structure and nutrient availability. A total of 74 kg N/ha of nitrogen fertilizer was applied in three split doses at 9, 12, and 17 days after planting (DAP).

Then, ten plants were grown with seeds for each replication in plastic boxes (24 × 37 × 12 cm). Blast resistance evaluation was performed at the seedling stage. The twelve P. oryzae isolates from Thailand (STN22063, SRN16371, SRN16381, PSL16358, PRE16415, PSL16362, STN22069, PML16295, UBN21008, STN22068, STN22066, and UBN21028) and (Figure 2) were used for the blast inoculation with the spraying method [29].

To prepare blast inoculum, P. oryzae were grown on rice polish agar (RPA) and incubated for 7–14 days at 25 °C. Sporulation was induced with sterilized rice leaves on a petri dish used to contain mycelium or a spreader and incubated at 25 °C under fluorescent or black light for 3–5 days [30]. Spore suspensions were counted using a hemocytometer (under a compound microscope) after adjusting spore concentrations with water to 5 × 10⁵ spores/mL, and the suspension was mixed with a polyoxymethylene (Tween 20^®) at a ratio of 10 μL/100 mL [31]. The spore suspension was sprayed onto the 24-day-old seedlings using the Badger 150^® sprayer, with a volume of 30 mL. The inoculated seedlings were then incubated in a moist chamber at 25–28 °C and high humidity for 16–18 h. Then, they were relocated to an air-conditioned room for 7 days. The symptoms of blast infection were identified and rated when seedlings began to exhibit signs of infection, following the standard evaluation system for rice (SES) [32]. Blast scores were analyzed for the severity index (SI) using the method described by Sirithanya [33]: highly resistant = 0, resistant (0 < SI ≤ 20), moderate resistant (20 < SI ≤ 40), moderate susceptible (40 < SI ≤ 60), susceptible (60 < SI ≤ 80), and highly susceptible (80 < SI ≤ 100) (Figure 3).

In the case of BB resistance evaluation, ten lines of BC₁F₃ and 7 varieties were included; SKN, Morkhor 60-3 and Morkhor 60-1 (with the standard recurrent and donor parent checks) resistance check varieties; (IR62266 and IRBB5), and (KDML105 and RD6 as susceptible checks) were used for BB resistance evaluation against nine isolates of Xoo distributed across Thailand (SP1-1, NB7-7, NY1-1, CN2-1, CM4-1, UT2-1, NB7-8, MS1-2, and PR5-1) (Figure 2). The experiment was conducted at Khon Kean University, Thailand, in 2022 and laid out by a factorial experiment in a completely randomized design (CRD) with three replications. The inoculum of nine Xoo isolates was prepared with a pure culture of single Xoo isolates that was multiplied on a nutrient agar (NA) medium following the method of a cross-streak plate. The cell suspension of each Xoo isolate was derived by mixing the bacterial inoculum on NA agar with sterile distilled water. The final bacterial colony was suspended in sterilized distilled water and spectrophotometrically adjusted to a concentration of 10⁹ CFU/mL (OD = 0.3 at 600 nm) [34]. The inoculation was conducted at the seedling stage (21 days) using the clipping method [35]. At seven days after inoculation, the plant reaction to BB was measured in terms of lesion length on plants. The plants with BB lesion lengths of 0–5, 5.1–10, 10.1–15, and >15 cm were considered to be resistant (R), moderately resistant (MR), moderately susceptible (MS), and susceptible (S), respectively, in this study [36], as shown in Figure 4.

2.3. Evaluation of Agronomic Traits, Yield Components, and Seed Quality

Thirteen lines and varieties, consisting of 10 near-isogenic lines (NILs), standard variety (SKN), recurrent parent (Morkhor 60-3), and donor parent (Morkhor 60-1), were used for validation. The experiment was laid out in a randomized complete block design (RCBD) with four replications at Khon Kean University’s field crop research station in 2022–2023. Each plot contained two rows, 1.80 m long, spaced 20 cm apart and 20 cm between rows and within rows, respectively. Agronomic traits and yield components, including days to flowering (DF), plant height (PH), panicle length (PL), number of panicles per plant, number of total grains per panicle, 1000-grain weight (GW), harvest index (HI), and yield, were recorded from four plants of each plot.

After harvesting, seeds of each line were assessed for seed quality and aroma. Grain physical quality includes measurements of grain size, taken from the length and breadth of paddy and whole grains. Subsequently, the length/breadth ratio (L/B ratio) is calculated based on the guidelines from IRRI [32]: slender = over 3.0, medium = 2.1 to 3.0, bold = 1.1 to 2.2, and round = less than 1.1 (unit: mm.), and compared with their recurrent parent Mor khor 60-3. In terms of the aromatic test, seed aromatic evaluation was determined through 2-acetyl-1-pyrroline (2AP) content by gas chromatography analysis [37], together with separation mass spectrometer (GC/MS) content following the methods of Sriseadka et al. [38].

2.4. Data Analysis

Disease severity index (SI) for blast was performed according to the method of using the following Formula (1), as described by Sirithanya [33]:

SI = [∑(Ni × Vi)/(V × N)] × 100

(1)

Ni is the number of plants tested in each level, Vi is the disease score at various levels assessed by the plant number, V is the maximum score used in the evaluation, and N is the total number of plants used in the test.

Broad-spectrum resistance (BSR) for blast and BB was calculated using the formula described by Ahn [39].

$$\mathrm{B}\mathrm{S}\mathrm{R}={\displaystyle \frac{\mathrm{S}}{\mathrm{T}}}$$

When S = the number of pathogen isolates to which the rice line shows resistance, T = the total number of pathogen isolates tested. The BSR value ranges from 0 to 1; a BSR of 0 revealed that the rice is susceptible to all isolates. Meanwhile, a BSR of 1 showed that the rice resists all isolates.

Statistical analysis was conducted to evaluate the interaction between isolates of P. oryzae and X. oryzae with different rice strains and was performed according to the method of using the following formula:

Yijk =µ + G_i + l_j + (G l)_ij + ε_ijk

where Yijk is the dependent variable, µ is the overall mean, Gi is the effect of the line (i, treatment ewes and control ewes), lj is the effect of the isolate (j, the day of sponge insertion and removal), and (G × l)ij represents the interaction between line i and isolate j, and eijk is the residual error.

Blast scores, BB disease lesion length, agronomic traits, and seed quality data were analyzed by variance analysis and. According to Gomez and Gomez [40], means comparisons were calculated using the Tukey’s honestly significant difference (HSD) formula:

$$HSD=q\sqrt{{\displaystyle \frac{M{S}_w}{n}}}$$

where $q$ = the standardized range statistic.

$M{S}_w$ = The mean square for within groups from the ANOVA;

$n$ = the number of subjects in each group (all groups must be of equal size).

These were performed by the STATISTIC 10 program (Copyright© 1985–2013, Analytical Software, 2105 Miller Landing Rd., Tallahassee, FL, USA).

3. Results

3.1. Development of Introgression Lines Using Marker-Assisted Selection

This research aimed to establish a population. An F₁ generation was initially created by crossing Morkhor 60-3 and Morkhor 60-1, resulting in 43 plants. From this group, 37 heterozygous individuals were confirmed using MAS (RM319/RM562). Next, 15 of 37 plants used to produce F₁ plants were backcrossed with Mor Khor 60-3, leading to the generation of 108 backcross (BC₁F₁) plants. Eight plants were identified as heterozygous for resistance at two QTLs and one gene. Eight plants were identified, and four were selected to develop the population through self-fertilization. This process generated a total of 1250 plants, designated as BC₁F₂. This group identified ten plants as homozygous for resistance linked to the qBl1, qBl2, and xa5. These ten lines were self-fertilized again, producing the BC₁F₃ and BC₁F₄ generations (Figure 1).

3.2. Validation of Bacterial Blight Resistance

Ten lines of BC₁F₃ of Morkhor 60-3 introgression with a homozygous allele of the BB resistant QTLs and xa5 gene were validated for BB resistance under greenhouse conditions. The data analysis of variance (ANOVA) showed that the genotype (G) and the interaction between genotype and isolate (G × I) were significant (

Table 1. Mean square of selected BC₁F₃ lines and check varieties against twelve isolates of P. oryzae and nine isolates of X. oryzae.

SOV	Blast		BB
	df	MS	df	MS
Isolate (I)	11	2091.8 **	8	45.92 **
Genotype (G)	17	16276.8 **	16	328 **
G × I	187	413.0 **	128	9.36 **
Error	430	260.9	304	4.25
C.V. (%)	92.76		29.68

SOV = source of variation; df = degrees of freedom; MS = mean square; C.V. = coefficient of variation percentage. ** Significant at p ≤ 0.01 by HSD.

) Individuals’ genotypes were directly affected by different BB isolates, demonstrating that the BB resistance is not only dependent on the genetic makeup of the lines but also influenced by the strains of the pathogen.

Evaluating BB resistance in isolates MS1-2 showed lesion lengths of lines BC₁F₃ 22-7-60 and BC₁F₃ 22-11-312 of 9.0 cm and 9.1 cm. In contrast, isolates NB7-8 and MS1-2 exhibited lesion length/reaction on the Morkhor 60-1 variety as 5.5 cm and 6.8 cm (moderate resistance). These results demonstrate that lesion length induced by BB infection is a key indicator of host resistance. Meanwhile, the BC₁F₃ lines carrying the xa5 gene observed varying resistance levels, with BSR values ranging from 0.55 to 0.77. Among these lines, BC₁F₃ 22-11-312, BC₁F₃ 22-11-131, BC₁F₃ 22-15-298, and BC₁F₃ 22-15-311 demonstrated high BSR values of 0.77, 0.72, 0.77, and 0.55, respectively. In comparison, the parental lines, Morkhor 60-1 and Morkhor 60-3, displayed BSR values of 0.88 and 0.38, respectively. Additionally, varieties tested—Sakon Nakhon (standard check), IRBB5 IR62266 (resistant control), and RD6 KDML105 (susceptible control) —showed BSR values of 0.05, 1.00, 0.88, 0.05, and 0.00, respectively. The results indicate that the standard varieties exhibit varying levels of resistance and susceptibility to diseases (

Table 2. Lesion length and reaction to bacterial blight disease of 10 selected BC₁F₃ lines and seven check varieties to nine BB isolates under greenhouse conditions at Khon Kean University research station in 2022.

Lines, Varieties	QTLs Located on Chromosome	Lesion Length (cm) and Reaction to Rice Bacterial Blight Disease									BSR
		SP 1-1	NB 7-7	NY 1-1	CN 2-1	CM 4-1	UT 2-1	NB 7-8	MS 1-2	PR 5-1
BC1F3 22-7-60	qBl1,2,11,12 and xa5	4.2 d (R)	9.0 b (MR)	5.9 b–e (MR)	6.5 de (MR)	5.3 cd (MR)	4.5 de (R)	6.3 c–e (MR)	9.0 bc (MR)	5.3 d–f (MR)	0.61
BC1F3 22-7-128	qBl1,2,11,12 and xa5	4.6 d (R)	5.3 c–f (MR)	7.2 b–d (MR)	7.9 cd (MR)	6.7 b–d (MR)	7.7 a–d (MR)	6.8 c–e (MR)	8.4 c (MR)	3.5 e–f (R)	0.61
BC1F3 22-7-140	qBl1,2,11,12 and xa5	6.0 cd (MR)	7.7 bc (MR)	7.3 b–d (MR)	3.5 e–g (R)	4.5 c–f (R)	6.4 b–d (MR)	8.7 cd (MR)	6.8 cd (MR)	7.3 cd (MR)	0.61
BC1F3 22-7-322	qBl1,2,11,12 and xa5	5.5 cd (MR)	6.7 b–e (MR)	5.1 c–e (MR)	3.7 e–g (R)	6.5 b–d (MR)	5.0 de (R)	4.0 e (R)	8.5 c (MR)	9.5 c (MR)	0.66
BC1F3 22-11-187	qBl1,2,11,12 and xa5	6.0 cd (MR)	8.2 bc (MR)	6.8 b–d (MR)	4.4 e–g (R)	4.7 c–e (R)	4.8 de (R)	6.0 de (MR)	6.1 cd (MR)	7.6 cd (MR)	0.66
BC1F3 22-11-312	qBl1,2,11,12 and xa5	5.8 cd (MR)	4.1 e–g (R)	3.2 e–g (R)	2.8 fg (R)	4.2 d–f (R)	4.7 de (R)	7.4 c–e (MR)	9.1 bc (MR)	9.8 c (MR)	0.77
BC1F3 22-11-131	qBl1,2,11,12 and xa5	5.0 d (R)	8.9 b (MR)	1.7 fg (R)	3.0 fg (R)	4.3 d–f (R)	5.8 d (MR)	6.3 c–e (MR)	6.1 cd (MR)	5.5 d–f (MR)	0.72
BC1F3 22-15-311	qBl1,2,11,12 and xa5	7.8 bc (MR)	4.3 d–g (R)	5.3 b–e (MR)	5.5 d–g (MR)	7.3 bc (MR)	6.1 cd (MR)	7.8 c–e (MR)	6.9 cd (MR)	6.4 c–e (MR)	0.55
BC1F3 22-15-258	qBl1,2,11,12 and xa5	6.7 cd (MR)	7.6 b–d (MR)	7.9 bc (MR)	4.3 e–g (R)	6.3 cd (MR)	7.3 a–d (MR)	6.7 c–e (MR)	8.6 c (MR)	7.2 cd (MR)	0.55
BC1F3 22-15-298	qBl1,2,11,12 and xa5	4.9 d (R)	4.4 d–g (R)	4.4 d–f (R)	6.2 d–f (MR)	6.0 ce (MR)	5.3 d (MR)	5.0 de (R)	8.5 c (MR)	4.6 d–f (R)	0.77
SKN Standard check	-	11.4 a (MS)	8.9 b (MR)	15.1 a (S)	15.4 a (S)	15.8 a (S)	10.2 ab (MS)	15.3 a (S)	12.7 a (MS)	17.7 a (S)	0.05
Morkhor 60-3 recurrent parent	qBl11,12	5.5 cd (MR)	5.0 c–f (R)	8.3 b (MR)	5.1 d–g (MR)	9.4 b (MR)	9.8 a–c (MR)	10.6 bc (MS)	12.3 ab (MS)	14.4 ab (MS)	0.38
Morkhor 60-1 donor parent	qBl1,2,11,12 and xa5	4.8 d (R)	2.8 f–h (R)	3.6 e–f (R)	3.7 f–g (R)	3.2 e–g (R)	1.4 ef (R)	5.5 de (MR)	6.8 cd (MR)	4.9 d–f (R)	0.88
IRBB5 resistance check	xa5	0.1 e (R)	0.7 h (R)	0.9 g (R)	2.5 g (R)	1.6 fg (R)	0.0 f (R)	4.4 de (R)	0.9 e (R)	2.5 f (R)	1.00
IR62266 resistance check	xa5	0.6 e (R)	1.4 gh (R)	0.5 g (R)	5.7 d–g (MR)	1.0 g (R)	0.3 f (R)	5.1 de (MR)	4.7 d (R)	3.0 ef (R)	0.88
KDML105 susceptibility check	-	11.6 a (MS)	15.8 a (S)	14.2 a (MS)	14.3 ad (MS)	14.6 a (MS)	10.1 ab (MS)	14.5 ab (MS)	13.2 a (MS)	13.7 b (MS)	0.00
RD6 susceptibility check	-	9.5 ab (MR)	12.9 a (MS)	16.1 a (S)	10.9 bc (MS)	12.4 a (MS)	10.9 a (MS)	13.5 ab (MS)	13.4 a (MS)	15.3 ab (S)	0.05
Mean		5.87	6.68	6.67	6.19	6.82	5.89	7.86	8.35	8.12
F-test		**	**	**	**	**	**	**	**	**
C.V. %		27.26	29.9	28.43	33.26	25.75	39.28	33.74	25.48	27.25

Note: R = resistant (0–5 cm), MR = moderately resistant (5.1–10 cm), MS = moderately susceptible (10.1–15 cm), S = susceptible (>15 cm), and BSR: 0 = susceptible; 1 = resistance. ** Significant at p ≤ 0.01. The letter after each indicates the significance within each column by HSD. C.V. (%) = coefficient of variation percentage.

).

3.3. Validation of Blast Resistance

The analysis of variance (ANOVA) conducted on the selected BC₁F₃ lines and check varieties revealed significant effects from the isolate (I), genotype (G), and their interaction (G × I). This indicates specific gene-for-gene interactions, resulting in novel phenotypes not observed in the parental lines (Table 1). In BC₁F₃, ten introgression lines demonstrated resistance to blast disease with a BSR of 0.66–1.00 (

Table 3. Severity index (SI), resistance level, and broad-spectrum resistance (BSR) of 10 BC₁F₃ lines and 9 check varieties against 12 blast isolates under greenhouse conditions at Ubon Ratchathani Rice Research Center in 2023.

Lines, Varieties	QTLs Located on Chromosome	SI and Reaction to Rice Blast Disease
		STN 22063	SRN 16371	SRN 16381	PSL 16358	PRE 16415	PSL 16362	STN 22069	PML 16295	UBN 21008	STN 22068	STN 22066	UBN 21028	BSR
BC1F3 22-7-60	qBl1,2,11,12 and xa5	28.13 c–f (MR)	0 d (HR)	0.82 ef (R)	15.67 c–g (R)	9.72 bc (R)	12.98 d–f (R)	0.37 e (R)	0 d (HR)	1.39 c (R)	0 c (HR)	15.56 de (R)	0 c (HR)	0.95
BC1F3 22-7-128	qBl1,2,11,12 and xa5	18.15 d–g (R)	0 d (HR)	0.93 ef (R)	9.88 e–g (R)	4.6 c (R)	18.78 d–f (R)	0 e (HR)	0 d (HR)	0 c (HR)	0 c (HR)	0.74 e (R)	0 c (HR)	1.00
BC1F3 22-7-140	qBl1,2,11,12 and xa5	16.83 d–g (R)	0 d (HR)	0.74 ef (R)	5.88 g (R)	5.71 c (R)	9.84 ef (R)	0 e (HR)	0 d (HR)	0 c (HR)	0 c (HR)	0 e (HR)	0 c (HR)	1.00
BC1F3 22-7-322	qBl1,2,11,12 and xa5	14.07 e–g (R)	0 d (HR)	1.48 ef (R)	11.71 d–g (R)	2.91 c (R)	6.61 f (R)	0 e (HR)	0 d (HR)	1.11 c (R)	0 c (HR)	0 e (HR)	0 c (HR)	1.00
BC1F3 22-11-187	qBl1,2,11,12 and xa5	21.48 d–g (MR)	0 d (HR)	0 f (HR)	20.95 c–f (MR)	8.4 bc (R)	26.77 c–f (MR)	0.62 e (R)	0 d (HR)	2.47 c (R)	0 c (HR)	0 e (HR)	0 c (HR)	0.66
BC1F3 22-11-312	qBl1,2,11,12 and xa5	10.26 fg (R)	0 d (HR)	0 f (HR)	14.83 d–g (R)	3.02 c (R)	25.57 c–f (MR)	0 e (HR)	0 d (HR)	0 c (HR)	0 c (HR)	0 e (HR)	0 c (HR)	0.95
BC1F3 22-11-131	qBl1,2,11,12 and xa5	14.39 e–g (R)	0 d (HR)	0 f (HR)	17.50 c–g (R)	16.4 bc (R)	32.04 b–e (MR)	1.59 de (R)	6.12 cd (R)	0 c (HR)	0 c (HR)	36.51 c–e (MR)	0 c (HR)	0.87
BC1F3 22-15-311	qBl1,2,11,12 and xa5	31.75 b–e (MR)	0 d (HR)	29.63 c–f (MR)	15.82 c–g (R)	11.64 bc (R)	28.57 b–f (MR)	6.42 de (R)	0 d (HR)	1.39 c (R)	7.04 c (R)	3.7 e (R)	3.7 c (R)	0.87
BC1F3 22-15-258	qBl1,2,11,12 and xa5	19.05 d–g (R)	1.11 d (R)	0 f (HR)	7.74 e–g (R)	5.85 bc (R)	18.1 d–f (R)	0.62 e (R)	0 d (HR)	0 c (HR)	0 c (HR)	0 e (HR)	0 c (HR)	1.00
BC1F3 22-15-298	qBl1,2,11,12 and xa5	21.88 d–g (MR)	0 d (HR)	0 f (HR)	6.79 e–g (R)	7.83 bc (R)	18.99 d–f (R)	2.47 de (R)	0 d (HR)	1.23 c (R)	0 c (HR)	0 e (HR)	0 c (HR)	0.95
SKN standard check	-	69.55 a (S)	32.04 bc (MR)	43.70 a–c (MS)	41.9 ab (MS)	73.54 a (S)	69.14 a (S)	74.07 b (S)	64.76 ab (S)	61.11 a (S)	55.46 b (MS)	94.81 ab (HS)	69.63 a (S)	0.04
Morkhor 60-3 recurrent parent	qBl11,12	49.63 ab (MS)	7 cd (R)	33.46 b–f (MR)	29.29 ab (MR)	26.87 b (MR)	44.76 a–c (MS)	8.52 de (R)	18.52 cd (MR)	11.64 c (R)	18.02 c (R)	56.3 b–d (MS)	4.44 c (R)	0.62
Morkhor 60-1 donor parent	qBl1,2,11,12 and xa5	35.56 b–d (MR)	5.49 d (R)	67.20 ab (S)	21.05 c–e (MR)	19.58 bc (R)	25.71 c–f (MR)	3.17 de (R)	3.7 cd (R)	0 c (HR)	6.94 c (R)	59.26 a–c (MS)	3.17 c (R)	0.66
P0489 resistance check	qBl2,12	7.48 g (R)	1.59 d (R)	37.41 b–e (MR)	6.55 fg (R)	6.1 bc (R)	12.98 d–f (R)	5.35 de (R)	0 d (HR)	2.47 c (R)	0 c (HR)	0 e (HR)	1.59 c (R)	0.95
JHN resistance check	qBl1,11	17.69 d–g (R)	9.26 cd (R)	6.17 d–f (R)	7.6 fg (R)	12.43 bc (R)	7.82 ef (R)	15.43 d (R)	7.14 cd (R)	0 c (HR)	0 c (HR)	1.48 e (R)	0 c (HR)	1.00
IR64 resistance check	-	14.74 e–g (R)	0 d (HR)	0 f (HR)	6.39 g (R)	7.41 bc (R)	8.57 ef (R)	3.7 de (R)	2.31 d (R)	7 c (R)	0 c (HR)	2.65 e (R)	10.05 bc (R)	1.00
Azucena resistance check	-	42.06 bc (MR)	48.15 ab (MS)	44.9 a–c (MS)	7.16 e–g (R)	9.79 bc (R)	11.9 d–f (R)	0 e (HR)	38.1 bc (MR)	2.78 c (R)	51.85 b (MS)	68.15 a–c (S)	55.56 a (MS)	0.25
KDML 105 susceptibility check	-	49.63 ab (MS)	66.67 a (S)	77.78 a (S)	48.57 a (MS)	56.61 a (MS)	53.02 ab (MS)	91.11 a (HS)	80.95 a (HS)	62.96 a (S)	100 a (HS)	100 a (HS)	69.73 a (S)	0.00
RD 6 susceptibility check	-	28.26 c–f (R)	40.7 b (MS)	40.41 b–d (MS)	25.71 cd (MR)	9.95 bc (R)	35.24 b–d (MR)	35.19 c (MR)	2.78 cd (R)	38.89 b (MR)	49.21 b (MS)	62.96 a–c (S)	39.81 ab (MR)	0.45
Mean		26.87	11.15	20.24	16.89	15.70	24.60	13.08	11.81	10.23	15.18	26.42	13.55
F-test		**	**	**	**	**	**	**	**	**	**	**	**
C.V. %		45.22	137.34	111.22	51.54	80.84	61.30	67.20	181.13	92.33	107.02	98.60	139.69

Note: HR = highly resistant = 0, resistant (0 < SI ≤ 20), moderately resistant (20 < SI ≤ 40), moderately susceptible (40 < SI ≤ 60), susceptible (60 < SI ≤ 80), and highly susceptible (80 < SI ≤ 100); BSR = broad-spectrum resistance. ** Significant at p ≤ 0.01. The letter after each indicates the significance within each column by HSD. C.V. (%) = coefficient of variation percentage.

). Notably, four of these lines, including BC₁F₃ 22-7-140, BC₁F₃ 22-7-322, and two additional lines (BC₁F₃ 22-15-298 and BC₁F₃ 22-15-311), achieved high BSR values of 1.00, 1.00, 0.95, and 0.87, respectively (Table 3). In comparison, the parental lines, Morkhor 60-1 and Morkhor 60-3, had BSR values of 0.66 and 0.62, respectively. Furthermore, as expected, all resistant check varieties P0489 JHN and IR64 resisted most blast disease isolates with BSR values ranging from 0.95 to 1.00. These high BSR values indicate a high resistance level to the blast disease, making these lines potentially beneficial for further breeding efforts to improve disease resistance in rice. Additionally, RD6 showed resistance to three blast isolates: STN22063, PRE16415, and PML16295 (Table 3). These findings suggest that the introgression of multiple genes and QTLs influences the expression of a gene that plays a significant role in providing resistance to the pathogen.

3.4. Agronomic Traits and Yield Components

Based on the evaluation of agronomic traits and yield components under field conditions, the results revealed significant differences in days to flowering, plant height, panicle length, and 1000-grain weight. However, no significant differences were observed in the number of panicles per plant, total grains per panicle, harvest index, and grain yield (

Table 4. Agronomic traits of 10 BC₁F₄ lines and three check varieties were evaluated under field conditions at Khon Kean University research station in 2022–2023.

Lines, Varieties	QTLs Located on Chromosome	DF (day)	PH (cm)	NPP	PL (cm)	NGP	TGW (g.)	HI	GY (g/plant)
BC1F4 22-7-60-2	qBl1,2,11,12 and xa5	74 g	136 ab	7.25	35.7 a	141	28.96 c–e	0.42	66.88
BC1F4 22-7-128-3	qBl1,2,11,12 and xa5	81 c–e	146 a	6.75	24.5 c	172	28.77 c–e	0.27	60.58
BC1F422-7-140-4	qBl1,2,11,12 and xa5	83 b–d	142 a	7.25	37.5 a	159	28.18 d	0.28	74.49
BC1F4 22-7-322-5	qBl1,2,11,12 and xa5	89 a	146 a	7.75	35.0 ab	160	32.61 ab	0.24	73.83
BC1F4 22-11-187-6	qBl1,2,11,12 and xa5	80 c–f	143 a	6.75	35.2 a	158	30.46 b–d	0.27	56.83
BC1F422-11-312-7	qBl1,2,11,12 and xa5	78 d–g	145 a	7.50	33.7 ab	168	31.21 a–c	0.41	81.57
BC1F4 22-11-131-8	qBl1,2,11,12 and xa5	78 d–g	129 bc	7.25	36.5 a	124	28.07 de	0.36	78.30
BC1F4 22-15-311-9	qBl1,2,11,12 and xa5	88 ab	147 a	6.50	22.7 c	147	28.60 de	0.26	73.12
BC1F4 22-15-258-10	qBl1,2,11,12 and xa5	75 fg	138 ab	8.00	24.7 c	150	30.11 cd	0.30	71.47
BC1F4 22-15-298-11	qBl1,2,11,12 and xa5	77 e–g	138 ab	7.00	25.5 c	153	29.33 c–e	0.30	66.63
SKN standard check	-	87 a–c	146 a	7.75	28.0 bc	129	33.53 a	0.34	99.91
Morkhor 60-3 recurrent parent	qBl11,12	84 b–d	141 ab	6.00	24.2 c	144	27.16 e	0.38	76.90
Morkhor 60-1 donor parent	qBl1,2,11,12 and xa5	73 g	122 b	7.25	21.7 c	136	29.06 c–e	0.40	67.03
Mean		80.82	141.15	7.15	29.59	149.38	0.32	29.69	70.37
F-test		**	**	ns	**	ns	**	ns	ns
C.V. %		4.80	6.00	15.04	17.03	18.71	27.64	5.83	28.32

Note: DF = Days to flowering (day); PH = Plant height (cm); NPP = Number of panicles per plant; PL = Panicle length (cm); NGP = Number of total grains/panicle; HI = Harvest index; TGW = 1000-grain weight (g) GY = Grain yield g/plant. ** Significant at p ≤ 0.01. ns = non-significant difference. The letter after each mean indicates the significance within each column by Tukey’s HSD. C.V. (%) = coefficient of variation percentage.

). These findings suggest that hybrid performance in terms of yield remained relatively stable, despite variability in certain agronomic traits.

Therefore, the selection of significant traits was based on performance not inferior to the check variety, Morkhor 60-3. Several BC₁F₄ lines—including BC₁F₄ 22-7-140-4, BC₁F₄ 22-7-322-5, and BC₁F₄ 22-15-311-9—demonstrated that the improved, early-maturing hybrids achieved statistically higher values in key agronomic traits, indicating superior performance compared to the check variety.

Together, evaluations were focused on the aroma and seed size of paddy and brown grain. All the evaluated traits showed high significance among lines. Additionally, the mean comparison for 2AP content showed that all improved lines were more fragrant than their recurrent parent Morkhor 60-3 (8.42 ppm), as 2AP content of all improved lines ranged from 7.31 to 12.70 ppm (

Table 5. Aroma and seed shape of 10 BC₁F₄ lines and three check varieties.

Lines, Varieties	QTLs Located on Chromosome	2AP (ppm)	Seed Shape
			Paddy Size (mm)			Brown Rice Size (mm)
			Length	Breadth	L/B ratio	Length	Breadth	L/B ratio
BC1F4 22-7-60-2	qBl1,2,11,12 and xa5	8.82	9.72 de	2.58 ab	3.77 d	7.21 b–d	2.14	3.37
BC1F4 22-7-128-3	qBl1,2,11,12 and xa5	10.28	9.79 c–e	2.27 cd	4.31 a–d	6.95 de	2.02	3.44
BC1F4 22-7-140-4	qBl1,2,11,12 and xa5	9.38	9.72 de	2.36 bc	4.12 a–d	7.13 b–e	2.19	3.26
BC1F4 22-7-322-5	qBl1,2,11,12 and xa5	10.47	10.01 b–e	2.60 ab	3.86 de	7.34 a–d	2.26	3.25
BC1F4 22-11-187-6	qBl1,2,11,12 and xa5	10.70	10.13 b–d	2.36 bc	4.29 a–d	7.51 ab	2.07	3.63
BC1F4 22-11-312-7	qBl1,2,11,12 and xa5	10.11	10.72 a	2.29 cd	4.68 a	7.56 ab	1.99	3.80
BC1F4 22-11-131-8	qBl1,2,11,12 and xa5	8.73	9.81 c–e	2.67 a	3.67 e	7.14 b–e	2.11	3.38
BC1F4 22-15-311-9	qBl1,2,11,12 and xa5	10.79	9.76 c–e	2.52 a–c	3.87 de	7.46 ab	2.17	3.44
BC1F4 22-15-258-10	qBl1,2,11,12 and xa5	7.31	10.27 a–c	2.35 bc	4.37 ab	7.44 ab	2.05	3.63
BC1F4 22-15-298-11	qBl1,2,11,12 and xa5	12.70	10.40 ab	2.28 cd	4.56 ab	7.42 ab	2.02	3.67
SKN standard check	-	13.26	10.71 a	2.02 d	4.47 a–d	6.75 e	1.69	4.53
Mor khor 60-3 recurrent parent	qBl1,11	8.42	9.51 e	2.40 bc	3.96 c–e	6.98 c–e	2.13	3.28
Mor khor 60-1 donor parent	qBl1,2,11,12 and xa5	7.33	9.66 de	2.36 bc	4.11 a	6.75 d	2.07	3.26
Mean			10.01	2.39	4.15	7.27	2.10	3.46
F-test			**	**	**	**	ns	ns
C.V. %			3.63	7.39	8.10	4.15	6.39	8.54

Note: 2AP = 2-acetyl-1-pyrroline; L/B ratio: Length/breadth ratio. ** Significant at p ≤ 0.01. ns = non-significant difference. The letter after each indicates the significance within each column by HSD. C.V. (%) = coefficient of variation percentage.

). BC₁F₄ 22-15-311-9 and BC₁F₄ 22-15-298-11 showed the highest 2AP content with 10.79 and 12.70 ppm among improved lines (Table 5). Compared to Morkhor 60-3, the introgressed lines showed higher aromaticity than the donor and recurrent parent, demonstrating that the inheritance of traits results from transgressive segregation.

The selection criteria for seed shape among the 10 BC₁F₄ lines focused on achieving similarity to the recurrent parents. The analysis revealed significant variations in paddy size, particularly concerning the length/breadth (L/B) ratio. Three lines, including BC₁F₄ 22-7-140-4, BC₁F₄ 22-7-322-5, BC₁F₄ 22-15-311-9, and the Morkhor 60-3 variety, exhibited L/B ratios of 4.12, 3.86, 3.87, and 3.96 mm, with brown rice size, grain length, and L/B ratio showing 3.26, 3.25, 3.44, and 3.28 mm, respectively. This indicates that these lines had seed shapes close to the desired phenotype. The favorable L/B ratios suggest potential for improved agronomic performance and alignment with market preferences (Table 5). Moreover, the superior grain quality of the selected lines demonstrated grain characteristics similar to those of the RP Sakon Nakhon (Figure 5).

4. Discussion

Conventional breeding methods, a selective methodology based on superior performance, often rely on weighing, measuring, and visual assessments of phenotypic traits. However, these techniques are not always accurate, as they depend heavily on the breeders’ subjective evaluation and are limited in their ability to assess multiple traits simultaneously [41,42]. In contrast, gene introgression is a vital technique in plant breeding that facilitates the transfer of genetic material between different plant strains. Typically, the process involves multiple rounds of backcrossing, a careful undertaking designed to ensure that the newly developed variety retains the beneficial characteristics of the recurrent parent while incorporating desirable traits from the donor plant [43].

In recent years, advancements in molecular techniques have transformed breeding practices through marker-assisted selection (MAS). MAS is especially effective for complex traits influenced by multiple quantitative trait loci (QTLs), such as disease resistance, drought tolerance, and yield, and significantly speeds up the breeding process [44]. MAS enhances the breeding process by accurately identifying specific gene alleles associated with desirable traits. This allows for a more precise assessment of both the advantages and potential limitations at the genetic level, significantly increasing the likelihood of successfully incorporating beneficial characteristics into new plant varieties [45]. Furthermore, the precision offered by MAS significantly fortifies conventional breeding practices by diminishing reliance on phenotypic screening. This method can be sensitive to variations in environmental conditions and often requires multiple generations to achieve reliable evaluations [46]. This study utilized MAS to achieve genotypic selection for two blast-resistant (QTLs, qBl1 and qBl2) and one bacterial blight (BB)-resistant gene, xa5. This method showed high efficiency in the BC₁F₃ generation, requiring approximately 2.5 to 3 years. Subsequently, disease resistance was evaluated in the selected lines.

The BB disease was artificially inoculated using the clipping method. The rice varieties IRBB5 and IR62266 showed differences in their introgression for BB isolate IR1545, which is well-known as a standard resistant variety in breeding programs [47,48]. Meanwhile, the Morkhor 60-1 variety showed lesion lengths of 5.5 cm and 6.8 cm, demonstrating moderate resistance to the isolates NB7-8 and MS1-2 (Table 2). Several factors contribute to the reduced resistance in plants: (1) Mutations in the xa5 gene affect their effectiveness against certain pathogen strains [49]. Consistent with observations by Ancheta et al. [50], studies have shown that Xoo isolates in central Thailand have successfully adapted to the xa5 resistance gene. (2) The variety Morkhor 60-1 showed a loss of resistance over time, indicating that even well-established resistant varieties may eventually succumb to evolving pathogen populations, increasing disease severity [51]. (3) The isolates used for resistance testing were sourced from various regions (Figure 2). This variability can affect the expression and functionality of resistance genes against diverse pathogen populations [52]. Additionally, the resistance efficacy of rice varieties of the gene-for-gene interaction rule depends on the specific genes present in each rice variety and the type of isolate [53].

Based on the severity index and reaction to rice blast disease results, three resistant check varieties—P0489, Jao Hom Nin, and IR64—showed similar resistance levels to the blast disease. This study focuses specifically on their resistance. The isolates STN22063, PSL16362, and STN22063 demonstrated the susceptibility levels of both the donor and the recurrent parent. In greenhouse conditions, the ten lines exhibited more blast susceptibility rating (BSR) than resistance during the seeding stage, except for BC₁F₃ 22-11-187 and RD6, which showed resistance to most of the blast disease isolates: STN22063, PRE16415, and PML16295 (Table 3), indicating that the introgression of multiple QTLs affects gene expression levels [54]. However, the combined influence of major and minor genes from the donor and recurrent parents may not protect against specific isolates effectively. Moreover, epistatic interactions among these genes can also impact disease resistance [55].

Additionally, the characteristics of seed shape, specifically length and breadth, and aromatic traits in the BC₁F₄ generation were found to exceed those of both the donor and recurrent parents. This enhancement is attributed to transgressive segregation. These observations indicate that backcrossing can effectively introgress and preserve specific traits, leading to significant improvements. Additionally, the selected lines can serve as valuable genetic resources for developing resistance to diseases like blast and BB, making these lines useful for agricultural extension programs.

5. Conclusions

This study has successfully developed an improved Sakon Nakhon variety (Morkhor 60-3) through pyramiding of resistance QTLs (qBl1, qBl2) and gene (xa5) into the BC₁F₃ generation. Selected plants were rigorously evaluated for resistance to blast and bacterial blight diseases under greenhouse conditions. At the same time, the BC₁F₄ generation was assessed for key agricultural quality traits, including seed shape and aroma (2AP content). The results identified three promising lines, BC₁F₄ 22-7-140-4, BC₁F₄ 22-7-322-5, and BC₁F₄ 22-7-311-9, demonstrating effective resistance to blast and bacterial blight diseases. Importantly, these introgression lines maintained agronomic traits, seed shape characteristics, and aroma profiles closely resembling those of Morkhor 60-3 (the Sakon Nakhon improved line carrying blast-resistant QTLs). This successful combination of disease resistance with desirable quality traits represents a significant advancement in developing resilient rice varieties that meet both production challenges and consumer preferences.