Validation of Functioning
Resistant Genes against Malaysian Biotype of Brown Planthopper in Rice Variety,
Rathu Heenati
Mohamad Bahagia AB Ghaffar1*,
Ruziah Mohd Yusuf2,
Maisarah Mohamad Saad3, Muhammad Fairuz Mohd Yusof4, Shahril
Ab Razak4, Habibuddin Hashim5
and Noor Azmi Shaharuddin2*
1Industrial Crop Research Centre, MARDI Seberang Perai,
13200 Kepala Batas, Pulau
Pinang, Malaysia
2Department of Biochemistry, Faculty
of Biotechnology and Biomolecular Sciences
Universiti
Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
3Paddy and Rice Research Centre, MARDI Seberang Perai,
13200 Kepala Batas, Pulau
Pinang, Malaysia
4Biotechnology &
Nanotechnology Research Centre, MARDI Headquarters, 43400, Serdang, Selangor
5Agric Inov Resources. 08000 Sg Petani, Kedah, Malaysia
*For correspondence: bahagia@mardi.gov.my; noorazmi@upm.edu.my
Contributed equally to this work and are co-first authors
Received 30 June 2020; Accepted 29 November 2021; Published 30 January
2022
Abstract
Rathu Heenati is a donor parent commonly used in the development
of resistant rice varieties against brown planthopper (BPH), Nilaparvata lugens, which
is a devastating insect pest of rice. The variety is reported to have a
broad-spectrum resistance against BPH, through the action of multiple genes and
quantitative trait loci (QTL) including Bph3,
Qbph3, Qbph4 and Qbph10. The
present study was carried out to validate the effectiveness of those genes
against Malaysian BPH population. The study was conducted using an F2 segregating
population which was obtained from a cross between Rathu
Heenati and a Malaysian commercial variety, MR219.
Plant damage score was used to estimate the degree of plant tolerance, while
the amount of honeydew excretion was used to measure the level of antibiosis.
The gene presence in an individual plant was determined based on the
segregation pattern of the flanking microsatellite markers of the respective
reported genes. Results confirmed the role of Bph3 and the three other QTLs
in conferring resistance against the Malaysian biotype of BPH. Marker assisted
breeding can facilitate the monitoring of the introgressed
genes in the plants of a breeding population. © 2022
Friends Science Publishers
Keywords: Oryza sativa L.; Brown plant
hopper (BPH); Resistance gene; Quantitative trait locus; Microsatellite markers
Introduction
Rice (Oryza sativa L.) is one of the most important crops that
feed more than three billion people in the world (Khush
2005; Yudo et al. 2018). It is estimated that
the growing demand of the world population for rice consumption in 2030 would
reach 873 million tons (Purevdorj and Kubo 2000; Yudo et al. 2018). This target however is being
jeopardised by the loss of rice production in the field. Biotic factors contribute to a 52%
reduction of global rice yield (Yarasi et al.
2008) where almost 21% resulted from infestations by various species of insect
pests. One of the major insects that cause a huge problem to rice is Brown
Planthopper (BPH), Nilaparvata lugens (Stεl). Many countries
have reported that BPH greatly damaged the plant and significantly reduced the
rice yield (Huang et al. 1997; Sogawa et
al. 2003; Sun et al. 2005; Shabanimofrad et
al. 2017). In Asia, the annual economic loss caused by BPH is estimated to
be over $300 million (Min et al.
2014; Yuexiong et
al. 2019), with several devastating outbreaks previously reported in China,
Vietnam, Philippines, Indonesia, Thailand, Japan, Korea, India, Bangladesh, and
Malaysia (Heong 2009). BPH has long been identified as
one of the most economically important insect pests of rice in Malaysia.
The utilization of resistant varieties is
regarded as the most effective method in minimizing yield losses. Several BPH
resistant varieties have been bred and released to farmers (Jairin
et al. 2017). Currently, more than 30
BPH resistant genes have been reportedly present in several rice varieties
including in wild type species (Fujita et al. 2013; Sarao
and Bentur 2016). To date, a Sri Lankan rice variety Rathu Heenati has shown high
resistance and is resistant to all four BPH biotypes worldwide (Jairin et al. 2007a; Li et al. 2017), including
the BPH population in Malaysia (Ito et
al. 1994). The resistance of a variety to BPH is measured phenotypically
through the mechanisms of antibiosis, plant tolerance, and antixenosis
(Bhanu et al. 2014; Hu et al. 2016). These mechanisms were used
in the breeding and screening processes for the development of BPH resistant
varieties.
Another breeding approach is currently available
to breeders, i.e. the application of molecular markers in plant breeding,
whereby selection is performed based on the presence or absence of genotypic
markers instead of relying only on the observation of phenotypic expression.
The selection of resistant plants using marker-assisted selection (MAS) is
based on the linkage markers that are located nearby or on the resistant genes themselves.
The introgression of desirable genes from one plant
to another or from the parent plant to their progenies can be monitored through
the presence of those markers (Mekonnen et al. 2017).
The Bph3
gene in Rathu Heenati was
mapped on the short arm of chromosome 6 and is flanked by related markers,
RM589 and RM588 (Jairin et al. 2007a).
Previous studies have also shown that Rathu Heenati's resistance to BPH was also related to the
presence of other genes such as the Bph17 and other minor quantitative
trait loci, QTLs located on various chromosomes (Sun et al. 2005; Jairin et al. 2007a; Kumari et al. 2010; Hu et
al. 2016). A QTL named Qbph3 is located on chromosome 3, positioned
between markers RM313 and RM7. Whereas the second QTL, Qbph4 was found
on the short arm of chromosome 4 between markers RM8213 and RM5953 with a map
distance of 3.6 cM and 3.2 cM.
On the other hand, the Qbph10 was flanked by markers RM484 and RM496 on
chromosome 10 (Sun et al. 2005).
This study aimed to validate the reported gene/QTLs that may have an
association with the resistance of Rathu Heenati to the Malaysian biotype of BPH. While all the four genes/QTLs and their respective markers were known,
the relative resistance expression of each of these resistant factors or
multiple combinations of them is yet to be determined. Previous attempts using Rathu Heenati as donor parent
failed to produce lines with resistance scores comparable to Rathu Heenati. It is then
hypothesized that all the four resistant factors are responsible for the high
resistance of Rathu Heenati.
Assuring the presence of all the four genes in the breeding lines may ensure
the development of highly resistant varieties. Marker-assisted selection could
facilitate the monitoring and introgression of these gene/QTLs in the plants.
Materials and Methods
Plant materials and insect population
A total of 167
F2 progenies were derived from the cross of Rathu
Heenati and MR219. Rathu Heenati (MRGB07637) is a traditional Sri Lankan rice
variety and was used as the BPH resistant donor, whereas MR219 (MRGB11633) is a
commercial high-yielding Malaysian variety that shows susceptibility to the
current field population of BPH. Rice variety Taichung Native One (TN1)
(MRGB01760) was used as the susceptible check variety. The BPH population used
in the study was originally collected from rice fields surrounding the
Malaysian Agricultural Research and Development Institute (MARDI) station in
Seberang Perai, Malaysia. These insects were
subsequently reared and maintained on seedlings of MR219 under greenhouse
conditions. It was previously reported that MR219 harbored the Bph1 gene (Habibuddin
2012) and hence, the insect population used in this study was expected to
represent the Biotype-2 of BPH.
Honeydew test
Honeydew test
is a test for the antibiosis mechanism of resistance. A piece of filter paper
(Whatman No. 1) was dipped into a solution containing 0.02% of bromocresol
green in ethanol (Pathak and Heinrichs
1982; Horgan et al. 2016). The
experiment was conducted in a plant growth room at 22 ± 3ΊC with 60 ± 10% humidity
and artificial photoperiodic lighting of 16h: 8h (light: dark). The dried bromocresol
green-treated filter paper was then placed at the base of individual 40-day-old
plant samples inside a feeding chamber. The filter paper was placed 2 cm above
the soil surface to protect the filter paper from excess humidity of the soil.
Gravid brachypterous BPH females of similar age were starved for 2 h 30 min and
5 brachypterous BPH were subsequently released into the feeding chamber and left to feed on a single tiller for
24 h. Honeydew droplets dropped onto the bromocresol green-treated filter
paper. The area of each blue spot on the bromocresol green-treated filter
papers was measured using a square (mm) grid.
Plant damage score test
Plant damage
score is a measure of plant damage tolerance upon infestation. The 5
brachypterous BPH and their nymphs were left to continue feeding on their
respective test seedlings for 714 days or
until yellowing or death of the MR219 test seedlings were first observed. Each
test plant was scored individually according to the criteria established by the
5th Standard Evaluation System (SES) (IRRI 2013) based on the damage
(scale from 1 to 9) of individual plants as a result of the BPH feeding (Table 1).
Extraction and quantification of DNA samples
Genomic DNA was extracted from
fresh, young leaves of Oryza sativa
using the TacoTM Plant DNA Extraction Kit
(GeneReach Biotechnology Corp, Taiwan) according to
the manufacturers protocol. The purified DNA was stored at -20ΊC. The
concentration and quality of the extracted DNA were determined using NanoQuantTM spectrophotometer (TECAN Infinite
200 PRO, USA).
Evaluation of candidate markers
Sets of
flanking microsatellite markers linked to the respective BPH resistant genes
were evaluated for their polymorphisms on the parental varieties. The selection
of these markers was based on the previous study by Sun et al. (2005) who suggested the involvement of three different QTL
regions in Rathu Heenati,
namely Qbph3, Qbph4/Bph17 and Qbph10
and a major BPH resistant gene (Bph3).
To enhance the density of markers used in this study, 56 microsatellite markers
were further mined from the GRAMENE database (http://www.gramene.org/) based on
their relative position surrounding the above mentioned gene and QTLs (Fig. 1). The best polymorphic, shortest
distance to the respective targeted gene or QTLs were then selected for
subsequent used in the study.
Polymorphism of linked markers on F2 plants
Only 8 polymorphic
markers nearest to the target gene or QTLs were selected based on their
preliminary polymorphism results. The selected polymorphic markers for the
respective gene or QTLs were as follows: a) RM7 and RM1256 for Qbph3, b) RM8213 and RM5473 for Qbph4/Bph17, c) RM8072 and RM588 for Bph3, and d) RM5352 and RM5471 for
Qbph10. Polymorphisms of the respective markers on individual F2
plants were assessed. The results of this test were subsequently merged with
the corresponding data on plant resistance assessments.
The expected presence of resistance loci on F2
plants
The use of two
flanking markers in the MAS breeding program could produce as high as 99
percent selection efficiency as compared to using a single marker (Kelly and Miklas 1998; Collard and MacKill
2008). The expected presence of a resistant gene locus in the individual F2
plants was then based on the segregation pattern of their flanking markers, A
and B, on the left and right-hand sides of the gene, respectively. For example,
AA represented the homozygous genotype of marker A, Aa is heterozygous, and
aa denotes the absence of marker A in a plant, and similarly with the marker
B. Hence, the plants with their representative flanking markers genotypes as
A_B_ are considered to have a gene for resistance at a 99% probability.
Likewise, plants with either combination of A_bb or
aaB_ genotypes may also indicate the presence of
the resistant gene at 95% probability, depending on the proximity of the gene
to the marker alleles A or B of Rathu Heenati. On the other hand, plants with the genotype of aabb of MR219 were classified as individuals without the
gene for resistance.
Statistical analyses
Data analysis
was performed using Microsoft Excel. The Pearsons Chi-square (χ2) test was
performed to evaluate the expected segregation ratio of the F2
population in the Mendelian segregations. The χ2 value was
estimated based on the procedure outlined by Panse
and Sukhatme (2000) as shown below:
χ2=
With the degree
of freedom (df) = k-1, where O = Observed frequency of the class, E = Expected
frequency of the respective class, ∑ = Summation of all classes. The
non-significant χ2 values justified the agreement between the
observed and expected ratio, therefore the null hypothesis is accepted as true.
The presence
of a respective marker A in the individual F2 plants was genotyped
as either AA, Aa or homozygous
aa. The χ2 test was also used to test their expected 1:2:1 segregation
ratio for a dominant factor. A co-segregation ratio of 9:7 was also tested for
the co-segregation of the two-ends flanking markers A and B. The association
between plants having respective resistant genes and their phenotypic
resistance parameters was evaluated using association analysis (SAS version 9.3) where Cramers V coefficient was used
to measure the association strength. The relationship between genes and
susceptible parameter was evaluated using correlation analysis (SAS version
9.1).
Results
Honeydew test and plant damage scores of the parents
Table 1: Modified
damage rating score of the test plants following of a continuous BPH feeding
(IRRI, 1998)
Scale |
Description |
Reaction |
1 |
No damage on
the leaves |
Highly
resistant |
3 |
Very slight
damage on the leaves |
Resistant |
5 |
One to 2
leaves were yellowing |
Moderately
resistant |
7 |
More than
half of the leaves shrank |
Susceptible |
9 |
The plant
died |
Highly
susceptible |
Table 2: Overall mean
scores (± standard errors, SE) for BPH resistance and coefficient of variation
(CV) of parental and control rice varieties based on the honeydew and plant
damage tests
Variety |
N |
Resistance
score (Mean ± SE) |
Reaction |
|||
Honeydew (mm2) |
% CV |
Plant damage
score |
% CV |
|||
Rathu Heenathi MR219 TN1
(Control) |
7 7 7 |
45.71±6.17 185.71±16.74 211.43±22.93 |
40.93 23.85 28.70 |
1.57±0.37 7.86±0.60 8.71± .29 |
62.10 20.03 8.67 |
R S HS |
R, S, and HS denote resistant, susceptible, and highly
susceptible plants, respectively
Fig. 1: Chromosomal
location of BPH resistant genes and QTLs in the F2 population. The
marker names are listed on the right-hand side of the chromosome with the
distance (in cM) displayed on the left-hand side (Mapchart 2.30)
The mean
honeydew excretion (measured as area in mm2) obtained from the release
of 5 gravids BPH on three rice varieties is presented
in Table 2. The honeydew
excretions appeared as a blue-rimmed spot. As expected, the susceptible control
variety (TN1) displayed the highest (211.43 ± 22.93 mm2) mean score
of honeydew spots. Likewise, the mean score of honeydew spots of MR219 was
185.71 ± 16.74 mm2, which was slightly lower than that of TN1. In
contrast, the honeydew droplet area of Rathu Heenati was only 45.71 ± 6.17. Additionally, a low
coefficient of variation (CV) values was observed on TN1 (28.70%) and MR219
(23.85%), while Rathu Heenati
showed a high CV value (40.93%). A similar pattern was also shown for plant
damage measurement. The mean score for susceptible varieties, TN1 and MR219,
was 8.71 ± 0.29 and 7.86 ± 0.60, respectively, while the lowest (1.57 ± 0.37)
score was observed for the resistant donor parent, Rathu
Heenati. The CV value was also recorded higher for Rathu Heenati as compared to
MR219 and TN1. At the end of the study duration, almost all of the MR219 and
TN1 plants were wilted and died due to heavy feedings by the BPH. These results
confirmed the resistance of Rathu Heenati
to BPH which was used as the donor parent and the susceptibility of MR219 as
the recipient parent.
Phenotypic segregation of F2 plants
The frequency
distribution of the 167 F2 plants against the amount of honeydew
excretion displayed a continuous normal distribution, although it was slightly
skewed towards the resistant parent with a mean score of 85.21 mm2 (Fig. 2), which may indicate that this
antibiosis mode of resistance might be controlled by more than one gene. The
majority of the progenies (119 plants) showed score values of ≤100mm2,
while Rathu Heenati and
MR219 had score values of 42.0 mm2 and 181.0 mm2,
respectively. However, the frequency distribution of F2 plants for
plant damage scores is in bimodal distribution (Fig. 3). A total of 36 plants (including Rathu
Heenati) displayed a score of 1. These plants
maintained their green-colored leaves even after 9 days of infestation.
Additionally, 45 F2 plants had a score of 3, and 23 plants were
wilted and die and had a score of 9 (including MR219). Plants with scores of 1,
3, and 5 were pooled and classified as resistant, while plants with the scores
of 7 Table 3: Segregation of the F2
plants of Rathu Heenati x
MR219 for resistance to BPH infestation based on their plant damage scores
Phenotypic
expression of corresponding F2 plants |
Expected F2
genotype |
Observed
number of F2 individuals |
5 ≤
Resistant |
AA, Aa |
130 |
Susceptible
≥ 7 |
aa |
37 |
N=167
Calculated
χ2 value for 3:1 ratio is 0.720 (χ20.05, 1
= 3.841)
The resistant
plants are expected to possess homozygous AA or heterozygous Aa genomes
inherited from Rathu Heenati,
while the susceptible plants are carrying the aa genome of MR219
Table 4: Segregation of marker alleles of eight microsatellite
markers in the F2 progenies derived from the cross of Rathu Heenati and MR219
Markers |
Chr |
Gene/QTL |
Ratio
(1:2:1) |
χ2 |
||
AA |
Aa |
aa |
||||
RM7 |
3 |
Qbph3 |
44 |
69 |
45 |
2.54 |
RM1256 |
40 |
66 |
48 |
3.96 |
||
RM8213 |
4 |
Qbph4 |
47 |
85 |
34 |
2.14 |
RM5473 |
33 |
76 |
49 |
3.44 |
||
RM8072 |
6 |
Bph3 |
98 |
16 |
48 |
135.18 |
RM588 |
26 |
94 |
37 |
7.66 |
||
RM5352 |
10 |
QBph10 |
33 |
59 |
55 |
12.36 |
RM5471 |
33 |
72 |
44 |
1.78 |
AA and aa represent the genotypes of Rathu Heenati and MR219,
respectively. Aa is the heterozygous genotype.
Tabulated
χ20.05, 0.01, 0.001, df2 = 5.991, 9.210
and 13.82, respectively
Fig. 2: Frequency
distribution of the amount of honeydew excreted by BPH feeding on the F2
population of Rathu Heenati/MR219,
N=169
and 9 were
classified as susceptible. In total, 130 resistant plants and 37 susceptible
plants were observed (Table 3).
The χ2 test showed that the phenotypic scoring of plant damage
score was consistent with the expected Mendelian segregation ratio of 3:1, an
indication of the role of a dominant gene effect. A similar result was also
reported by Hu et
al. (2018) who also conducted studies on Rathu
Heenati. The results also showed that BPH faced
difficulties to carry on with durable feeding or ingestion on the majority of
the F2 progenies, suggesting that these plants had been successfully
introgressed with the resistant genes originated from
Rathu Heenati.
Segregation and Co-segregation of the flanking markers
The
segregation of individual markers in the F2 population was analyzed.
Except for marker RM8072, results from the χ2 analysis
show that the segregation of all other markers followed the 1:2:1 segregation
ratio which suggested that each of these
flanking markers was independently inherited as a dominant factor (Table 4). The calculated χ2 value for marker RM8072 was high (135.18), resulting in deviation from
the expected ratio at P<0.001, which could be due to segregation distortion.
This distortion was due to the high
frequency number of AA (98) and the lesser number of the heterozygous Aa (16)
genotypes. The cause of this segregation
distortion could be genetic and environmental factors. This segregation
distortion phenomenon was commonly found during population mapping due to
biological selection or sampling, and this phenomenon usually interferes with
the creation of genetic maps (Xu et al.
1997; Soundararajan et al. 2004).
In this study,
two flanking markers were assumed to be closely linked if they were inherited
in an allelic or co-segregated Table 5: Co-segregation
of the flanking markers of the four putative BPH resistant genes in the F2
progenies of Rathu Heenati
and MR219 cross
Flanking
Markers (Gene) |
RM7/ RM1256 (Qbph3) |
RM8213/ RM5473 (Qbph4) |
RM588/ RM8072 (Bph3) |
RM5471/ RM5352 (Qbph10) |
Segregation
ratio Observed χ2 |
9: 7 101: 53 5.45 |
9: 7 81: 69 0.31 |
9: 7 108: 49 10.03 |
9: 7 64: 71 4.28 |
Tabulated
χ2 0.05, 0.01, 0.001, df1 = 3.841, 6.635
and 10.828, respectively
H0=
The segregation ratio of the flanking markers is in the ratio of 9:7 for the
A_B_: other allele combinations, respectively
Table 6: The association of putative gene or QTLs and the phenotypic expression of
the F2 plants
Gene
combination |
Plant damage
score (Tolerance) |
Honeydew
excretion (Antibiosis) |
||
χ2 |
Cramer's V
coefficient |
χ2 |
Cramer's V
coefficient |
|
Bph3 Qbph3 Qbph4 Qbph10 |
16.1910* 12.260* 13.2328* 12.9218* |
0.4172 0.3442 0.3946 0.3994 |
29.6626* 19.7283* 32.6712* 20.7747* |
0.5588 0.4420 0.5632 0.5064 |
*Significant at P=0.05
Cramers V coefficient: V=0.25-0.75 (moderately strong)
Fig. 3: Frequency distribution
of F2 plants of a cross between Rathu Heenati and MR219 when based on the plant damage scores
following the infestation by BPH
The scores of Rathu Heenati and MR219 as
control varieties were 1 and 9, respectively, N=169
manner, of
which the flanked gene is positioned between them. The plants with genotypes
A_B_ are highly predicted to harbor resistant genes originated from Rathu Henati, while the plants
which co-segregated with the genotype aabb may
indicate the absence of the resistant gene as the alleles were inherited from
MR219. Our results showed that the calculated χ2 value for the
co-segregation of markers RM8213 and RM5473 is 0.31 (Table 5), a value lower
than that of the tabulated value at P>0.05, thus suggesting that the two
flanking markers are closely linked, co-segregated and hence the possibility
for the presence of the gene Qbph4
between them is very high. The calculated χ2 values for the
co-segregation of Qbph3 and Qbph10 markers, respectively were
also low, albeit at a lower confidence level of P>0.01, and may still
support the acceptance of the 9:7 ratio. On the other hand, the χ2
value for the marker set RM588/RM8072 of Bph3 was relatively high
(P>001), due to distortion resulting from a higher number of A_B_ relative
to other genotypes. The co-existing of both the flanking markers in a plant
indicated that they inherited resistant genes from Rathu
Heenati at the particular loci.
Association between the gene presence and phenotypic
expression of the plants
An association
analysis was conducted to estimate the association between the presence of Bph3
gene or the other three respective QTLs in a plant and their phenotypic
expression. All the χ2 values are significant at P<0.05
(Table 6). These results indicated that the resistance expression of a plant is
associated with the respective gene or QTL harbored by them. Plants harboring
any of those resistant genes showed degrees of resistance expressed in the form
of plant damage score and levels of antibiosis, as compared to those plants
without any of the resistant gene or QTL. The strength of association between
genes presence and the expression of the phenotypes is measured by Cramers V
coefficient, which is moderate.
Rathu Heenati as the donor parent
is harboring Bph3 gene and three other QTLs. The variety showed the
lowest plant Table 7: Plant damage score and honeydew excretion on
plants with different number of BPH resistant gene and QTLs
Gene/QTL combination |
No. of gene |
Plant damage score |
Honeydew excretion (mm2) |
||
MR219 (Susceptible parent) No gene (H) Bph3 (A) Qbph3 (B) Qbph4 (C) Qbph10 (D) Bph3/Qbph3 (AB) Bph3/Qbph4 (AC) Bph3/Qbph10 (AD) Qbph3/Qbph4 (BC) Qbph3/Qbph10 (BD) Qbph4/Qbph10 (CD) Bph3/Qbph3/Qbph4 (ABC) Bph3/Qbph3/Qbph10 (ABD) Bph3/Qbph4/Qbph10 (ACD) Qbph3/Qbph4/Qbph10 (BCD) Bph3/Qbph3/Qbph4/Qbph10 (ABCD) Rathu Heenati (Resistant parent) |
0 0 1 1 1 1 2 2 2 2 2 2 3 3 3 3 4 4 |
8.0 7.7 4.4 4.6 1.0 3.7 4.3 7.0 4.3 5.3 1.0 3.8 5.4 5.8 2.8 5.0 2.3 1.8 |
ab a cdef bcde f cdef cdef abc cdef abcde f cdef abcde abcd def abcde def ef |
181.0 143.3 77.9 88.2 30.0 66.7 76.5 110 76.7 113.3 10.0 69.0 94.5 107.5 51.1 107.1 46.4 42.0 |
a ab bcdef bcde ef cdef bcdef bcd bcdef bc f cdef bcde bcd cdef bcd cdef def |
Means within a column with a similar letter are not significantly
different by DMRT
damage score
of 1.80 as compared to the susceptible (without any gene) MR219 with the score
of 8.00, indicating the high level of BPH resistance in Rathu
Heenati (Table 7). A similar pattern of resistance
expression is also demonstrated in the form of honeydew excretion, where the
area of honeydew droplets in Rathu Heenati is 42 mm, significantly lower than that of MR219
(181.0 mm). The F2 plants in groups with
different gene combinations showed variable degrees of resistance, mostly moderate. The F2 plants
in the group ABCD which are having all the four genes showed a low plant damage
score (2.27) and a small honeydew droplet area (46.36mm), which are not
significantly different from that of Rathu Heenati, demonstrated an equivalent level of resistance to
that of Rathu Heenati. A
correlation analysis was also conducted to estimate the relationship between
plant damage score resulted from the BPH feeding and the amount of honeydew
excreted by those feeding BPH. A high correlation coefficient value was
observed between the two parameters (r=0.83009***), which was as expected,
which supported the assumption that excessive feeding, draining of the water, and nutrients from the plants in the
form of honeydew, lead to their wilting and death of the plants.
Discussion
Identification
of suitable resistant candidate genes is one of the most important steps in an
effective resistant breeding program. Therefore, there is a need to validate
all reported genes of interest towards the local BPH population to make sure
their effective function, and that the breeding activity could be carried out
effectively at a reasonable cost. In this study, a Sri Langkan
rice variety, Rathu Heenati,
has been selected to be used as the donor of resistant genes and QTLs for the
development of local rice varieties resistant against BPH, Nilaparvata lugens. The level of BPH resistance in Rathu Heenati was measured and compared to the susceptible check
variety TN1 and the recurrent parent MR219. The measurement was based on the
assessment of the quantity of honeydew excreted area by BPH sucking on the plant (as an antibiosis assessment)
and the plant damage score following BPH infestation (an assessment for plant
tolerance). From the antibiosis perspective, the BPH feeding on Rathu Heenati excreted the lowest
amount of honeydew excretion as compared to those feeding on MR219 or TN1. The
low amount of honeydew excretion on Rathu Heenati could be due to the difficulties in phloem
ingestion by the BPH, thus contributing to the varietys resistance. The higher
coefficient of variation (CV) value recorded in Rathu
Heenati as compared to those on MR219 and TN1 showed
clear evidence of the inconsistency of BPH feeding ability on Rathu Heenati as the proportion
of honeydew droplets of BPH is hugely influenced by its host varieties (Ghaffar
et al. 2011).
Because of the
inability of BPH to consistently feed on the resistant rice plant, the feeding
data as measured by the level of antibiosis has also contributed to plant
tolerance measurement data where Rathu Heenati also showed the lowest plant damage score. This
result was in line with the observation of Akanksha et al. (2019) who showed that the amount of honeydew excretion and
plant damage was highly correlated and significantly affected by host
varieties. It was previously demonstrated that BPH primarily fed on the phloem
of the susceptible varieties, ingesting high-value nutrients such as amino
acids to support its growth and reproduction. During the early phase of stylet
penetration in Rathu Heenati,
undesirable constituent elements in the variety may disturb the BPH and prevent
effective phloem ingestion (Ghaffar et al.
2011). This mechanism thus enables resistant plants to survive BPH attacks. The
inability to consistently feeding on the host plant has caused less injury to
the plant's tissues and hence the potential plant dehydration and loss of
nutrients was prevented. This describes the reason behind the lower plant
damage scores recorded in Rathu Heenati
as compared to the scores on the susceptible control variety TN1 and MR219.
This result confirmed the earlier reports by Habibuddin
(1989) and Jairin et
al. (2007) claiming that Rathu Heenati showed high and broad-spectrum resistance
characteristics to BPH populations in Thailand and Malaysia, respectively. The
variety is thus a very good candidate donor parent for used in the local BPH
resistant breeding program.
However, the
strong resistance of Rathu Heenati
to BPH of variable biotypes, in multiple localities and countries is attributed
to its harboring of at least the Bph3 gene and the three mentioned QTLs, the Qbph3, Qbph4 and Qbph10.
Many attempts in the previous BPH resistance breeding program which utilized Rathu Heenati as the donor parent
produced progenies or new varieties with moderate levels of resistance.
Breeders have difficulty in developing new cultivars having the equivalent
levels of BPH resistance to that of Rathu Heenati. This could be due to the failure to transfer or ensuring the maintenance of all the relevant genes or
QTLs in the newly developed varieties. Our results in Table 7 showed that
different F2 plants may harbor different combinations of BPH
resistant genes or QTLs in the plants, resulting in the variable degree of
resistance to BPH. To ensure the subsequent progenies or generations of the
crosses involving Rathu Heenati
to continuously harbor the desired gene or QTL is a difficult task when
resistance assessment is solely based on phenotypic expression.
Application of
marker assisted selection (MAS) in resistant breeding programs may offer an
opportunity to overcome this limitation. Linked or functional markers could be
used to determine the presence or absence of the genes in the plants. Through
MAS, the introgression of genes would be monitored among breeding lines from
generation to generation until
the new varieties are ready to be selected for released and commercialized.
This study showed that the introgression of the four genes and QTLs could be
ascertained, and newly improved, resistant Malaysian major varieties could be
developed, having a resistance level to BPH which is equivalent to that of Rathu Heenathi.
Conclusion
This study
confirmed that the resistance of Rathu Heenati to BPH is controlled by Bph3 gene and three
other QTLs, namely the Qbph3, Qbph4 and Qbph10. The
introgression of these four resistant factors in the plants and breeding lines
could be monitored through the detection of their flanking microsatellite
markers. Application of marker-assisted selection (MAS) could thus enhance
resistant breeding programs and the development of resistant or improved
popular major cultivars is much easier to achieve.
Acknowledgements
This research
was supported by The Ministry of Energy, Science, Technology, Environment and
Climate Change (MESTECC) through the project grant FLAGSHIP No: TF FP0214B055
(DSTIN) and Malaysia Agriculture Research and Development Institute (MARDI).
Author Contributions
MBAG, RMY and NAS planned the experiments, MBAG, MMS and SAR interpreted
the results, MBAG, HH and RMY made the write up and MF statistically analyzed
the data and made illustrations.
Conflicts of Interest
The
authors declare no conflicts of interest
Data Availability
All data
were presented in this paper and additional information can be obtain from
corresponding author
Ethics Approval
Not
applicable in this paper
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