1. Introduction
Globally, rice accounts for more than 21% of total food intake; it also provides up to two-thirds of the total calories consumed by more than two billion people across Asia, Africa, and Latin America [
1,
2,
3,
4]. It is estimated that rice production must increase by 0.6–0.9% annually until 2050 in order to meet the demand of an increasing world population [
5]. However, rice yields have plateaued due to biotic and abiotic stresses [
6]. Among these, bacterial blight (BB), which is caused by
Xanthomonas oryzae pv.
Oryzae (
Xoo), is one of the most destructive diseases, limiting rice production around the world. BB was first identified in Japan at the beginning of the twentieth century [
7]. It can cause damage at any stage of the rice-growing cycle. BB causes yield losses generally ranging between 10 and 30%, but which can be as high as 80%, depending on the location, season, weather, crop growth stage, and cultivar [
8,
9,
10,
11]. There are no chemicals or management practices known to reduce the severity of BB [
12]. The development of a BB-resistant rice cultivar through a gene introgression breeding program is critically important [
13,
14,
15].
To date, at least 45 genes across 10 of 12 rice chromosomes have been identified which confer resistance to various strains of
Xoo [
16,
17,
18,
19]. Among these resistance genes, 17 (
xa5, xa8, xa13, xa15, xa19, xa20, xa24, xa25, xa26, xa28, xa31, xa32, xa33, xa34, xa41, xa42 and
xa44) are recessive, while the remaining 28 are dominant [
20,
21,
22]. All the genes are in the series from
Xa1 to
Xa45 [
17]. Out of the 45 resistance genes, nine (
Xa1, Xa3/xa26,
xa5, Xa10, xa13, Xa21, Xa23, xa25 and
Xa27) have been cloned and twelve (
Xa2, Xa4, Xa7, Xa22, Xa30, xa31, xa33,
xa34, Xa38, Xa39,
Xa40 and
Xa42) have been physically mapped [
20,
21,
22,
23,
24,
25,
26,
27,
28,
29]. Some of these resistance genes have been successfully incorporated into rice cultivars that are now extensively cultivated in many rice-growing countries [
13,
15,
30]. Most of the resistance genes follow the classic gene-for-gene concept against the race-specific interaction between rice and
Xoo [
31]. Most of the genes show resistance in all growing stages of rice, while only a few resistance genes, such as
xa13, show resistance to
Xoo only at mature plant stages, and
xa5 and
Xa4 show a broad spectrum of resistance to
Xoo isolates [
32,
33,
34]. The gene
Xa21, which is found in the wild rice species
O. longistaminata, shows resistance against BB at the seedling stage [
35] and has the potential to be highly effective against BB in South and South-East Asia [
32,
33,
34,
36].
Rice lines with multiple BB resistance genes have a wider and more durable level of resistance than those lines that have only a single BB resistance gene [
15,
26,
37,
38,
39]. Large-scale, long-term cultivation of rice cultivars carrying a single BB resistance gene may not be sufficient to beat the BB pathogen and resistance will not persist in a long time. Conventional breeding approaches are not efficient enough for the rapid identification of BB resistance genes [
40,
41,
42].
The use of molecular markers to select a particular trait has numerous advantages over the morphological markers of conventional plant breeding [
14,
39]. We examined the potential for marker-assisted selection to identify rice lines with multiple BB resistance genes (i.e.,
Xa genes). It is possible to develop closely linked molecular markers for each of the resistance genes within the plants, for their easy identification [
15,
30,
43]. Three BB resistance genes (
xa5,
xa13, and
Xa21) were incorporated in the cultivar PR106 through marker-assisted selection (MAS), showing a broad spectrum of resistance against 17
Xoo isolates under field conditions [
15]. This study used phenotypic and molecular markers to identify the BB resistance genes
Xa4,
xa5,
xa13, and
Xa21 in recombinant inbred lines (RILs) derived from a cross between the rice varieties ‘Ciherang’ and ‘IRBB60’.
3. Discussion
More than half of the global population eats rice to meet their daily dietary requirements. The demand for rice production is increasing, and many studies have reported that global rice production needs to double by 2050 to meet this growing demand [
44]. Additionally, there are many biotic and abiotic stresses which affect both the yield quantity and quality of rice crops. To address these constraints and to increase rice production, it is necessary to develop high-yielding cultivars enriched with disease-resistance genes. The development of rice varieties with broad-spectrum resistance against bacterial blight (BB) (caused by the
Xoo bacteria) is hugely challenging due to the presence of several genetically distinct virulent
Xoo strains in different rice growing locations in the world [
45]. There is little published literature on the development of multiple-race-resistant cultivars. To improve the sustainable cultivation of rice we have planned and executed the introgression of four BB (
Xa4,
xa5,
xa13, and
Xa21) resistance genes/QTLs into a Ciherang × IRBB60 cross in order to achieve multiple-BB race resistance in rice. We observed distinct polymorphism with four co-dominant markers linked to the BB resistance genes from the Ciherang (recipient) parent and IRBB60 (donor) parent material (
Figure 4).
The main objective of this study was to identify and successfully demonstrate the function of the four BB resistance genes (
Xa4,
xa5,
xa13, and
Xa21) in the RILs of the F
6 generation. These four target BB resistance genes were identified through bioassays against three BB races (C5, P6, and V) and an effective foreground selection was undertaken using gene-specific markers in 265 RILs in two consecutive generations (F
5 and F
6). These markers were MP, xa5, xa13, and pTA248 for the
Xa4, xa5, xa13, and
Xa21 genes, respectively. PCR products showed approximately 140 bp band for the
Xa4 gene [
46,
47], 198 bp band for the
xa5 gene, 500 bp for the
xa13 gene [
48], and 980 bp for the
Xa21 gene [
46,
49,
50]; these were confirmed by the resistance checks IRBB4, IRBB5, IRBB13 and IRBB21 (
Figure 4).
Of 265 lines, 203 had one or more of the targeted BB resistance genes in different combinations (
Figure 3), and 40, 34 and 11 lines had two, three, or four BB resistance genes. These recombinant lines exhibited a high level of resistance against three virulent BB isolates, which correlates with the results of [
51]. Mundt [
52] also reported that the effectiveness of a combination two or more genes is higher than that of a single gene in defeating simultaneous pathogen mutations for virulence, therefore assembling several resistance genes into a host plant is a viable and practical strategy.
We observed inheritance of some unfavorable characteristics (i.e., biomass growth and grain weight) along with some favorable traits while pyramiding genes/QTLs from the parent IRBB60 variety. A “pull” of undesirable genes from the parent occurred when the
Xa21,
xa13, and
xa5 genes were introgressed from the SS1113 variety [
30]. Ramalingam et al. [
53] assessed the homozygous improved pyramiding lines (BC3F3 generation) that harbored the
xa5 +
xa13 +
Xa21 +
Pi54 +
qSBR7-1 +
qSBR11-1 +
qSBR11-2 genes in terms of physical resistance under greenhouse conditions and suggested that pyramiding three BB resistance genes resulted in higher resistance levels than the lines with only one or two BB resistance genes.
In our bioassay study, 85 pyramided lines which carried at least two BB resistance genes showed a high level of resistance (
Figure 3 and
Figure 4). Eleven pyramided lines (RIL 12, RIL 15, RIL 32, RIL 44, RIL 51, RIL 53, RIL 155, RIL 156, RIL 166, RIL 215 and RIL 232), all of which had
Xa4 + xa5 + xa13 + Xa21 in combination, had higher resistance performance than the RILS with combinations of other genes (
Figure 4) [
51,
54,
55,
56,
57,
58]. These 11 RILs had an average of 1.57 cm of diseased leaf area infected with the three BB races (
Figure 6), which is similar to results from other reports [
41,
47,
51,
57,
59,
60]. The enhanced resistance due to the combination of two or more genes compared to the resistance from a single gene is known as synergistic action, or quantitative complementation [
61]. This result is also further confirmed and explained by our PC analysis.
The pyramided lines were categorized into 11 groups determined by the number and combinations of genes. These pyramided RILs were significantly different in terms of plant height, panicle length, 1000-grain weight, and the spikelet length-to-width ratio, indicating that these pyramided lines have diverse yield potentiality. The field evaluation of improved pyramided lines of the F7 generation demonstrated that selected lines had equivalent yield, agro-morphological, and quality traits and equivalent pyramided genes for BB. This higher level of resistance to BB disease observed in different races without yield penalty is a positive outcome from our approach of integrated genotypic and phenotypic selection methods.
4. Materials and Methods
4.1. Plant Materials
In this study, we used 265 RILs at the F
6 generation of the ‘Ciherang × IRBB60’ cross, which were recombinant of recipient and donor parents, four gene-specific BB resistant cultivars (viz. IRBB4, IRBB5, IRBB13, IRBB21) and one BB susceptible line (IR24) (
Figure 7). Among these genotypes, the recipient parent ‘Ciherang’,
Oryza sativa ssp.
indica, is a variety popular in Indonesia which is susceptible to BB, and the donor parent ‘IRBB60’ was developed by the International Rice Research Institute (IRRI) through the pyramiding of four BB resistance genes (
Xa4,
xa5,
xa13, and
Xa21) into the existing IR24 variety. The plant materials were evaluated up to the F
7 generation for agronomic traits and the segregation of the pyramided BB resistance genes was followed by molecular markers. A rice breeding flowchart is presented in
Figure 8 to illustrate the selection work of each generation of the pyramiding rice lines against three BB races.
4.2. Preparation of Inocula to Infect Rice Plants
Three BB races—C5 (GD1358) and V, which are newly virulent strains in China [
62], and P6 (PX099), which contained TALE PthXo7 [
63] collected from the Philippines—were grown in Wakimoto semi-solid medium (potato 300 g, sucrose 20 g, Na
2HPO
4·2H
2O
2 2 g, Ca(NO
3)
2·4H
2O 0.5 g, agar 25 g,) per liter at 25 °C for 72 h and preserved at 4 °C following the standard methodology [
64,
65,
66]. A single colony was further sub-cultured in Wakimoto liquid medium with agitation at room temperature for 72 h and the cell suspension diluted to 10
8 cells per milliliter of distilled water, which was confirmed by measurement using a spectrophotometer (BOECO MODELS S-200 VIS & S-220 UV/VIS, Hamburg, Germany) with A
600 OD. This sub-culture was used to inoculate the F
7 rice plants grown in the field.
4.3. Inoculation of Rice Plants with BB Races/Isolates
Seeds of the RILs, their parent varieties, and the check varieties were sown in a 50 × 50 cm seedbed. After 22 days, rice seedlings were manually transplanted into experimental plots in Hainan (18.30 N, 109.30 E). Each plot consisted of two rows with 10 plants per row; spacing was 20 cm row-to-row and 17.5 plant-to-plant. The field experiment was conducted using a randomized complete block design with three replicates. The F
5 generation of all varieties and RILs were inoculated with the C5 bacterial race at the reproductive stage (i.e., onset of heading) by clipping 2–3 cm from the tip of the flag leaves and removing all other leaves [
67]. In each plot, four hills per row were randomly selected and inoculated.
Subsequently, the F6 generation of RILs, parents, and check varieties were moved to Beijing (40.20 N, 116.20 E) and inoculated with the V and P6 races. To do this, four hills were randomly selected from the first row of each plot and inoculated with the P6 race; a further four hills were inoculated with the V race. The middle two hills in each row were left without inoculation to avoid contamination of the bacterial races.
4.4. Assessment of Disease Response and Scoring
Disease scoring was conducted two weeks after inoculation, following the protocol recommended by the international rice research institute, IRRI [
68]. The percentage of diseased leaf area (DLA) was calculated as the lesion length (LL) per total leaf length (TLL) × 100. DLA was categorized as resistant (R) if DLA was 5% or less; as moderately resistant (MR) if DLA was between 6and 12%; as moderately susceptible (MS) if DLA was between 13 and 25%; as susceptible (S) if DLA was between 26 and 50%; and as highly susceptible (HS) if DLA was greater than 50% [
68].
4.5. DNA Isolation and PCR Analysis
DNA was extracted from leaf samples of four week old seedlings using the CTAB method [
69]. The gene-specific and gene-linked markers MP1, MP2,
xa5,
xa13 prom, and pTA248, which are linked to genes
Xa4,
xa5,
xa13, and
Xa21, were synthesized by the Open Lab of the Chinese Academy of Agricultural Sciences (
Table 6) and used to confirm the presence of resistance genes [
70]. The polymerase chain reaction (PCR) was performed using 25 µL mixture, which contained 1 µL of 50 ng DNA, 1 µL of 5 µM of each forward and reverse primers, 0.5 µL of 5.0 mM dNTPs, 2.5 µL of 10× PCR buffer (500 mM KCL, 100 mM Tris-HCl pH 8.4, 15 mM MgCl
2, 0.1% gelatin), 1.8 µL of 25 mM MgCl
2, 1.0~0.75 units/µL Taq polymerase, and 16.2 µL sterile distilled H
2O, for the MP1, MP2,
xa13 prom and pTA248 markers.
A reaction mixture of 20 µL, consisting of 5 µL DNA (50 ng/µL), 2 µL of 5 µM of each forward and reverse primers, 0.4 µL of 10 mM dNTPs, 2 µL of 10× PCR buffer, 1.6 µL of 25 mM MgCl2, 0.4 of 5 units/µL Taq polymerase and 6.6 µL sterile distilled H2O, was used for the xa5 marker. For the MP1 and MP2 markers, the PCR profile was followed with the initial denaturation at 94 °C for 4 min followed by 35 cycles of denaturation for 1 min at 94 °C, annealing for 1 min at 56 °C and extension for 2 min at 72 °C, with the final extension for 8 min at 72 °C. The PCR profile was followed with the initial denaturation for 5 min at 94 °C followed by 35 cycles of denaturation for 30 s at 94 °C and annealing for 30 s for the xa13 gene; at 1.4 min for the pTA248 markers targeted to the Xa21 gene at 55 °C with a final extension for 10 min at 72 °C. For the for xa5 gene, the PCR was followed with an initial denaturation for 5 min at 94 °C, followed by 35 cycles of denaturation for 1 min at 94 °C, annealing for 1 min at 68 °C and extension for 1 min at 72 °C, with a final elongation for 4 min at 72 °C.
PCR amplicons were mixed with 5 µL loading dye (loading dye:SYBR = 3:1) and visualized under UV-light after electrophoresis with an 8% polyacrylamide gel for Xa4, with 1.5% agarose gel for the xa13 gene, and a 2% agarose gel for the Xa21 and xa5 genes.
4.6. Data Collection and Analysis
Five plants were sampled from each pyramided line at maturity stage, from which yield and yield-contributing traits were measured. These traits were the number of days until 50% heading was achieved, plant height, the number of effective tillers per hill, panicle length, the number of spikelets per panicle, the percentage of spikelets which were fertile, the spikelet length-to-width ratio, the 1000-grain weight, and the grain yield per plant. Data were analyzed using Microsoft Office Excel 2007 and Statistix 10 software (
http://www.biosci.global/softwar-en/genstat/, accessed on 15 July 2021).