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Genetic analysis and tagging of gene for brown planthopper resistance in indica rice
M. Mei, C. Zhuang, R. Wan, J. Wu, W. Hu, and G. Kochert
Abstract
Brown planthopper (BPH) is one of the most serious insect pests in south China. About 3,000
cultivars and varieties from this area have been screened for BPH resistance, and more than
100 varieties have been identified to have high or moderate resistance. Genetic analysis of F1
and F2 populations derived from a cross between Sanguizhan, a resistant indica cultivar, and
Taichung Native 1, a highly susceptible cultivar, indicated that resistance was governed by a
single dominant gene. An allelism test revealed that this gene was non-allelic to and
independent of Bph1 and Bph3. For tagging this gene, F3 lines were developed and evaluated
for their resistance to BPH. Restriction fragment length polymorphism (RFLP) markers were
used as probes to survey the bulked DNAs from homozygous resistant lines and homozygous
susceptible lines, and co-segregation between RFLP patterns and resistance in F3 lines was
analyzed. The results obtained so far showed that RZ404, a rice cDNA clone located near the
end of chromosome 9, correlated with resistance. No BPH resistance genes have been
previously reported in this map position.
Introduction
The brown planthopper (BPH) Nilaparvata lugens is one of the most serious pests of rice
throughout Asia. To control this pest through varietal resistance, many investigators have
studied the inheritance of resistance in rice cultivars. At least nine resistant loci have been
reported, which include four dominant genes and five recessive genes (Khush and Brar 1991).
By trisomic analysis, Bph1 and bph2 were mapped to chromosome 4 and Bph3 and bph4 to
chromosome 10.
In south China, the main BPH population is biotype 1. This biotype causes heavy losses in rice
production in this area every year. To evaluate local germplasm for resistance to this insect,
Ceng and Wu (1989) screened about 3,000 accessions from south China based on their
reactions to BPH biotype 1. More than 100 cultivars or varieties were identified to be highly or
moderately resistant.
Studies of the inheritance of resistance indicated that different accessions appeared to have a
single dominant gene, a recessive gene, or two genes (Liu et al 1992). The first molecular map
of rice was published in 1988 (McCouch et al 1988). Recently, two maps with high-density
molecular markers have been constructed simultaneously (Causse et al 1994, Kurata et al
1994). These maps were used to locate a number of important agronomic genes via linkage to
restriction fragment length polymorphism (RFLP) markers.
For BPH resistance, an alien gene introgressed from wild rice Oryza australiensis into cultivated
rice has been mapped to chromosome 12 through molecular tagging (Ishii et al 1994). The
present study was undertaken to detect RFLP markers linked to a BPH resistance gene in
Sanguizhan, a resistant indica cultivar. To verify results from RFLP analysis, allelism tests of the
resistance gene from Sanguizhan with two known resistant loci were also conducted.
Materials and methods
Plant materials and evaluation of BPH resistance Sanguizhan—a domestic cultivar (indica) from
Guangdong Province, China, and identified to have BPH resistance governed by a single
dominant gene (Liu et al 1992) — and Taichung Native 1 (TN1)—a cultivar highly susceptible to
BPH—were used as parents. About 40 F3 lines derived from the F2 individuals were used for the
mapping population. A bulk seeding test was performed to evaluate the population for BPH
resistance (Wu et al 1984). The F3 lines were classified as homozygous resistant, segregating,
or homozygous susceptible.
RFLP analysis
Seedlings from the parents and each F3 line were used for DNA extraction. To speed up
screening, pools of DNA from six homozygous resistant F3 lines (R pool) and six homozygous
susceptible lines (S pool) were made. DNA from parents, pooled samples, and F3 lines were
digested with four restriction enzymes: Eco RI, Eco RV, Hin d III, and Xba l. One hundred and
thirty-seven RFLP markers (rice genomic clones, oat cDNA, and rice cDNA), provided by S.D.
Tanksley of Cornell University, distributed across the 12 chromosomes were screened. After
analysis of preliminary results, more probes were selected from chromosome 9 (including
several clones located on the map constructed by Kurata et al [1994], and rice cDNA clones
from Dr. Uchimiya and mapped by G. Kochert) to survey potentially linked markers.
MAPMAKER Macintosh Version 2.0 (Du Pont Company) was used for segregation data
analysis and genetic distance calculation.
Allele tests
To determine the allelic relationship of resistance gene in Sanguizhan with Bph1 and Bph3,
crosses of Sanguizhan with Mudgo or Rathu Heenati were made. The reaction of F2 progeny
from these two crosses was evaluated by the bulk seedling test. Sanguizhan was the resistant
check and TN1 was the susceptible check.
Results and discussion
Genetic analysis of BPH resistance
Table 1 summarizes the data for resistance of the F1, F2, or F3 progeny to BPH biotype 1 in
three crosses involving Sanguizhan. In the cross with TN1, all the F1 plants were resistant, and
the F2 population segregated in the ratio of 3 resistant: 1 susceptible in the primary study (Liu et
al 1992), indicating that resistance was determined by a single dominant gene. The F3 line from
this cross in the present study segregated in the ratio of 1 resistant: 2 segregating: 1
susceptible, making the same inference, although the value of chi-square was higher than
that from the F2 segregating data. BPH resistance of F2 populations from the cross
Sanguizhan/Mudgo for Bph1 or from Sanguizhan/Rathu Heenati for Bph3 segregated in a ratio
very close to 15 resistant: 1 susceptible, demonstrating that the single dominant gene in
Sanguizhan was nonallelic to and independent of Bph1 and Bph3, the dominant resistance
genes in cultivated rice mapped so far.
The deviations from the 1:2:1 ratio in F3 lines from the cross Sanguizhan/TN1, and from the 15:l
ratio in the F2 populations for the allelic tests may be due to misclassification of a few seedlings
because of unfavorable growing conditions, such as low temperature and low light intensity in
the test room. Under these conditions, some Sanguizhan seedlings also died.
Identification of RFLP markers linked to the resistance gene
Results of screening with 146 RFLP markers indicated that less than 20% of the markers
detected different restriction fragment patterns between Sanguizhan and TN1 (Table 2). Most of
the polymorphisms were detected by only one enzyme with a given probe. However, for a few
clones from chromosomes 4 and 9, two to four enzymes detected RFLP patterns, suggesting
greater variations in the genomic regions between the two parents. When bulked segregant
analysis was performed, polymorphism between the resistant pool (R pool) and the susceptible
pool (S pool) was found with one random amplified polymorphic DNA (RAPD) product which
was then mapped close to one end of chromosome 9 by using a backcross mapping population.
Flanking markers close to the RAPD marker from different maps produced identical restriction
fragment patterns between the parents.
Co-segregation analysis of the BPH resistance phenotype and RFLP patterns of polymorphic
clones RZ404, CD0412, and UCH170 in 40 F3 lines revealed possible linkage between RZ404
and the resistance locus ( = 9.65; P<0.05), but the recombination fraction was high (0.24) (Fig.
1). Markers tightly linked to this resistance locus thus could not be identified. The identification is
difficult because of the small size of the mapping population (some F2 individuals that produced
the F3 lines were infected by diseases) or the misclassification of resistance of some F3 lines
under unfavorable growing condition during the assays. To verify whether the gene controlling
resistance to BPH biotype 1 in Sanguizhan is a new dominant resistance gene and to determine
its location in the genetic map, further co-segregation analysis with a larger mapping population
and allelic tests between this gene and the other reported dominant genes should be
conducted.
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Citation
M. Mei, C. Zhuang, R. Wan, J. Wu, W. Hu, and G. Kochert. 1996. Genetic analysis and tagging
of gene for brown planthopper resistancein indica rice. Pages 590-595. In: GS Khush (ed.) Rice
Genetics III. Proceedings of the Third International Rice Genetics Symposium. Manila
(Philippines): International Rice Research Institute. 1011 p.