Marker assisted pyramiding of Bph6 and Bph9 into elite restorer line 93–11 and development of functional marker for Bph9

Background The brown planthopper (BPH) has become the most destructive and a serious threat to the rice production in Asia. Breeding the resistant varieties with improved host resistance is the most effective and ecosystem-friendly strategy of BPH biological management. As host resistance was always broken down by the presence of the upgrading BPH biotype, the more resistant varieties with novel resistance genes or pyramiding known identified BPH resistance genes would be needed urgently for higher resistant level and more durability of resistance. Results Here, we developed near isogenic lines of Bph9 (NIL-Bph9) by backcrossing elite cultivar 93–11 with Pokkali (harboring Bph9) using marker-assisted selection (MAS). Subsequently, we pyramided Bph6 and Bph9 in 93–11 genetic background through MAS. The resulting Bph6 and Bph9 pyramided line LuoYang69 had stronger antixenotic and antibiosis effects on BPH and exhibited significantly enhanced resistance to BPH than near isogenic lines NIL-Bph6 and NIL-Bph9. LuoYang69 derived hybrids, harboring heterozygous Bph6 and Bph9 genes, also conferred high level of resistance to BPH. Furthermore, LuoYang69 did not affect the elite agronomic traits and rice grain quality of 93–11. The current study also developed functional markers for Bph9. Using functional dominant marker, we screened and evaluated worldwide accessions of rice germplasm. Of the 673 varieties tested, 8 cultivars were identified to harbor functional Bph9 gene. Conclusion The development of Bph6 and Bph9 pyramided line LuoYang69 provides valuable resource to develop hybrid rice with highly and durable BPH resistance. The development of functional markers will promote MAS of Bph9. The identified Bph9 containing cultivars can be used as new sources for BPH resistance breeding programs. Electronic supplementary material The online version of this article (10.1186/s12284-017-0194-x) contains supplementary material, which is available to authorized users.


Background
Rice (Oryza sativa L.), an important cereal crop in Asian-Pacific region, is a stable food resource for more than half the population of the world (Du et al., 2009;Hu et al., 2016). Increasing yield has become the most core goal of rice production. Nevertheless, like the other plants, the growth of rice was disrupted by insect pests attacking, which will lead to significantly yield loss as the major biotic constraint (Myint et al., 2012). Of more than 20 kinds of serious paddy pests known at present, brown planthopper (BPH, Nilaparvata lugens Stål), a migratory and monophagous rice insect, has proved to be the most destructive, especially in the Asia (Sogawa et al., 2003;Brar et al., 2009). BPH not only sucks the phloem sap of rice leaf sheath, but also transmits the viral disease such as rice grassy stunt virus (RGSV), rice ragged stunt virus (RRSV) and rice wilted stunt virus (RWSV) (Heinrichs, 1979;Alam and Cohen, 1998b). Heavy infestation of BPH will result in complete dying of rice, referring the so called "hopperburn" (Watanabe and Kitagawa, 2000).
For preventing the damage caused by BPH, several approaches have been implemented including chemical and biological controlling (Normile, 2008). Recent reports showed the abuse of pesticide or insectcide wrecked the balance of the natural ecosystem by the heavily pollution and would bring about plasticity of BPH to the insecticide (Matteson, 2000;Tanaka et al., 2000;Park et al., 2007;Lakshmi et al., 2010). Comparing with the conventional chemical controlling, developing the host resistance by BPH resistance genes has been regarded as the most economic effective and environmental-friendly solution for controlling it (Matsumura et al., 2009). Seeking BPHresistant germplasm resources from various varieties and utilizing such resistance genes has turned into the most important component of breeding programs (Pathak et al., 1969;Alam and Cohen, 1998a). To date, 31 BPH-resistant genes have been identified in the cultivated rice and wild Oryza species, thirteen of these resistance genes have been cloned by map-based cloning (Ji et al., 2016;Ren et al., 2016;Du et al., 2009;Tamura et al., 2014;Wang et al., 2015;Zhao et al., 2016;Liu et al. 2015;Jing et al., 2017), which provide resistance genes for marker assisted selection (MAS) breeding. However, the varieties bearing single BPH resistance gene were quickly broken down within a few years due to rapid adaptation of BPH or evolution of new biotypes (Jena and Kim, 2010). For instance, the variety of IR26 developed by IRRI with single resistance gene Bph1 showed a moderate resistance to BPH biotype 1 in the early 1970s. However, the resistance was rapidly broken down by BPH biotype 2 after 2-3 years (Khush, 1977). It has been proposed that pyramiding multiple BPH resistance genes is an efficient strategy to develop more durable resistant varieties against BPH. Myint et al., 2012) found that the BPH resistance level of (Bph25 + Bph26)-NILs was significantly higher than either Bph25-NILs or Bph26-NILs. An additive effect occurred after pyramiding three dominant BPHresistance genes (Bph14, Bph15 and Bph18) into the elite indica rice variety 93-11 than the double gene lines and monogenic lines . Bph14, Bph15, Cry1C, and bar were pyramided for an elite restorer lines with processing resistance durability to BPH (Wan et al., 2014). The Bph3 and Bph27(t) single gene introgression japonica lines were inter-crossed following the methods of MAS, and the more durable pyramiding variety with japonica context was developed for crop breeding (Liu et al., 2016). Wang et al., 2016) further improved the BPH-resistance of Huahui938 and its derived hybrids by pyramiding Bph14 and Bph15 using molecular marker-assisted backcrossing (MAB).
The rice variety Swarnalata, carrying BPH resistance gene Bph6 and resisting BPH biotype 4 (Bangladesh BPH population), also exhibited high level of resistance to BPH population in China (biotype 2 being the dominant one). Bph6 in Swarnalata was fine-mapped between the STS markers Y9 and Y19 on the long arm of chromosome 4 (Qiu et al., 2010). Three Sri Lanka varieties, Kaharamana, Balamawee and Pokkali, harboring a dominant BPH resistance gene Bph9, were shown to be resistant to BPH biotypes 1, 2 and 3. Nemoto et al., 1989) firstly mapped Bph9 in Pokkali between RFLP marker OPRO4 and RAPD marker S2545 on chromosome 12. Su et al., 2006) located Bph9 in Kaharamana between SSR markers RM463 and RM5341 on chromosome 12. Recently, Bph9 from Pokkali has been cloned, encoding a rare type of nucleotide-binding and leucine-rich repeat (NLR) gene (Zhao et al., 2016). The cloning of Bph9 revealed that the eight BPH resistance genes clustered on the long arm of chromosome 12 are actually allelic with each other and can be classified into four allelotypes (Zhao et al., 2016). 93-11 is an elite restorer line for two-line and CMS three-line hybrid rice. In present study, we introgressed Bph9 into 93-11 using marker-assisted selection (MAS). We further pyramided Bph6 and Bph9 into 93-11 through MAS. The resulting Bph6 and Bph9 pyramided line LuoYang69 had stronger antixenotic and antibiosis effects on BPH and exhibited significantly enhanced resistance than NIL-Bph6 and NIL-Bph9. Its derived hybrids, harboring heterozygous Bph6 and Bph9 genes, also conferred high level of resistance to BPH. The development of LuoYang69 with Bph6 and Bph9 pyramided provides valuable resource to develop hybrid rice with high BPH resistance and excellent agronomic performance. The current work also developed functional markers for MAS of Bph9 in rice breeding programs. We screened and evaluated worldwide accessions of rice germplasm with functional dominant marker. 8 cultivars of the 673 varieties tested were identified to harbor functional Bph9 gene, providing new sources for BPH resistance breeding programs.

DNA extraction and genotyping
The total genomic DNA was extracted from fresh rice leaves at three-leaf stage using modified CTAB protocol for avoiding the pollution of polysaccharide and polyphenol components (Porebski et al., 1997). The extracted DNA was dissolved in 1 × TE buffer. Targeting sequences were amplified using PCR protocols described by Yang et al., 2002), with minor modifications for different primers. The PCR products of RRGB test were analyzed by 6% denaturing polyacrylamide gel electrophoresis and visualized by silver staining modified by Qiu et al., 2010). The PCR products of MAS were checked by 1.5% agarose gel electrophoresis stained with 2 μL EB, respectively. The wholegenome single nucleotide polymorphsim (SNP) array RICE6K was used to analyze the RRGB of the pyramided line. This SNP array containing 5102 SNP and InDel markers evenly distributed on the 12 chromosomes of rice was developed based on Infinium technology (Yu et al., 2014). The RRGB analysis by RICE6K array was performed at the Life Science and Technology Center, China National Seed Group Co., LTD (Wuhan, China), according to Infinium HD Assay Ultra Protocol (http:// www.illumina.com/).

BPH population and bioassay for evaluation of BPH resistance
The BPH populations used for resistance evaluation were collected from the field in summer at Wuhan University and were fed continuously on Tai-chung Native 1 (TN1, a susceptible indica variety) under greenhouse condition. The Bioassay for BPH resistance evaluation was performed by the a little-modified standard seedling bulk test according to Huang (Huang et al., 2001). The seeds of each rice resistance-improved varieties and susceptible varieties were pre-germinated for the identical developmental stage. Thirty seeds from individual resistance-improved plant were sown in the plastic box (30 length × 18 width × 12 cm height) in rows which were flanked by rows of seeds of the susceptible lines (TN1 or 93-11). The seedlings were thinned to 20 plants evenly distributed per row about 7 days after sowing. At third-leaf stage, the seedlings were infested with 2nd to 3rd instar BPH nymphs at a rate of 20 insects per seedling. After infestation, the boxes were covered by a nylon-gauze finely. When the BPH susceptible varieties or TN1 plants were completely dead, the BPH resistance was evaluated by the six-scale scoring system: 0 = no damage; 1 = very slight damage; 3 = 1st and 2nd leaves of most plants partially yellowing; 5 = pronounced yellowing and stunting or about 10-25% of plants wilting; 7 = more than half of the plants wilting or dead and remaining plants severely stunted or dying; 9 = all plants died. The lower score indicates higher resistance, nevertheless, indicating more susceptible to BPH. The evaluation experiments were repeated three times.

Two-host choice test
A modified protocol was implemented following by Qiu et al., 2010) in host selection behavior experiment. Two 14days-old seedlings (one pyramided plant with one NIL) with same phenotype were sown at opposite ends of roughly perpendicular diagonals in plastic cup (8 cmdiameter, 15 cm-height). Twenty biological replicates were set for each paired group. The soil of such cups was covered by 2 cm-thichness water layer before infesting. At the four-leaf stage, twenty second to third-instar nymphs were released into such cups that completely covered by a nalon-mesh permitting light and air transmitting. The cups were placed in the greenhouse for preventing the artificial disturbance of human-beings. The number of BPH settled on each plant was counted at 3, 6, 18, 24, 48, 72, 96, 120 h, 144 h after releasing for determining the two-host selection number of BPH, respectively.

Survival rate of BPH after feeding
Antibiosis always results in the reduction in insect survival, growth rate or reproduction after ingestion of host tissue (Qiu et al., 2011). To check the insect survival rate after BPH feeding, Seeds of LuoYang69, 2 NILs and 93-11 were grown in the plastic cup under natural condition. At the third-leaf stage, each cup/plant was infested with 10 s to third-instar nymphs and the number of surviving insects was counted from 1 to 9 days after BPH release for 9 days. Sixteen biological replicates were set for the assay. It was noticeable that the position that the cup placed should be same. Then, the number of survived BPH at each check point were divided by 10 total BPH released for the BPH survival rate (%).

Weight gained and honeydew excreted of BPH after feeding
Both the Weight gained of insects (direct) and honeydew excreted by BPH (indirect) after feeding were the important indicators of BPH development. For BPH weight gain and honeydew excretion assay, newly emerged female adults were weighed and enclosed in a pre-weighed Parafilm sachet (each Parafilm sachet contains one female adult) and attached to the leaf sheath of the rice plant. The insects were removed carefully from the sachets after 48 h. Both the insects and each sachet were weighed again. The weight difference of BPH insect was recorded as weight gain of BPH, the weight difference of the Parafilm sachet was recorded as honeydew excretion. These two indicators were measured for 21 biological replicates of the pyramided line, two NILs and the recurrent parent 93-11.
Test of yield-related agronomic traits and rice grain quality Ten agronomic traits: plant height, number of panicles per plant, mean length of panicles, number of grains per panicle, number of filled grains per panicle, number of grains per plant, number of filled grains per plant, number of empty glumes per plant, seed setting rate, and 1000-grain weight related tightly with yield of LuoYang69 and 93-11 were investigated in Hubei Academy of Agricultural Sciences under natural field conditions. LuoYang69 and 93-11 individuals were planted in ten rows with row spacing at 30 cm and plant spacing at 20 cm. Twelve individual plants without BPH damage from middle central rows were selected as biological replicates for measurement.
Mature seeds of LuoYang69 and 9311 were harvested in September and were allowed to dry naturally at room temperature. The following rice grain quality traits were tested by dehulled seeds of LuoYang69 and 93-11: brown rice ratio (BR, grains with the inedible outer hull removed), head rice ratio (HR, unbroken and broken translucent grains with at least 3/4 of a whole grain), chalky rice ratio (CR, grain with an opaque, chalky appearance covering half or more of the body of the grain), chalkiness degree (CD), amylose content (AC), alkali spreading value (ASV), grain length (GL), and ratio of grain length to width (L/W). The assessment criterion of rice grain quality followed the national standard GB/T 17891-1999 and NY/T 593-2013. The test of rice grain quality was performed at Institute of Food Crops, Hubei Academy of Agricultural Sciences (Wuhan, China).

Data analysis
The Chi-square test for goodness-of-fit was done by MS-Excel (Microsoft) and the resistance data and the investigation of agronomic traits were analyzed using statistical one-way ANOVA and comparing the LSD test at a 5% significance level and 1% extremely significance level.

Results
Development of NIL-Bph9 in 93-11 genetic background using MAS 93-11, an elite restorer parent for hybrids, is famous for its good quality, high yield and wide culturing in China. Unfortunately, such elite paddy line was highly susceptible to BPH. To improve BPH resistance of 93-11, 93-11 was used as the recurrent parent to backcross with Pokkali (IRGC 108921, harboring BPH resistance gene Bph9) for seven generations and then self-crossed to produce the population of BC 7 F 2 (Additional file 1: Figure S1) Two flanking markers InD2 and RM28466 tightly linked to Bph9 locus were used to select the positive progenies for continuous backcrossing. Finally, five lines homozygous at Bph9 locus were selected as candidate NILs from BC 7 F 2 populations. A total of 119 polymorphic molecular markers evenly distributed on 12 chromosomes were used to examine the recovery rate of genetic background (RRGB) of candidate NILs. These candidate NILs showed 94.78% to 97.39% genetic identity to 93-11 (Table 1). Then the best individual with the least amount of genetic background noise was selected as NIL-Bph9 and used to produce BC 7 F 2:3 populations. These data indicated that Bph9 has been successfully introgressed into 93-11 through MAS. We further characterized BPH resistance and agronomic traits of NIL-Bph9. The results demonstrated that NIL-Bph9 confers broad-spectrum resistance to BPH without affecting the agronomic performance of the rice plant (Zhao et al., 2016).

Pyramiding of Bph6 and Bph9 in 93-11 genetic background
It has been proposed that some varieties bearing single BPH resistance gene were quickly broken down within a few years due to rapid adaptation of BPH (Khush, 1977). Pyramiding multiple BPH resistance genes is an efficient strategy to develop more durable resistant varieties against BPH. The rice variety Swarnalata contained BPH resistance gene Bph6 and showed a high level of resistance to BPH biotypes 4 and mixed BPH biotypes in China. Previously, we fine-mapped Bph6 and developed NIL-Bph6 in 93-11 genetic background (Qiu et al., 2010). To pyramid Bph6 and Bph9 and develop more durable BPH resistance rice, NIL-Bph6 and NIL-Bph9 were crossed in this study. Two pyramided lines with both homozygous Bph6 and Bph9 were selected by marker H and InD2 from F 2 plants (Additional file 2: Figure S2). The genetic background of these two selected pyramided lineswas assayed using evenly distributed 95 polymorphic markers among the Swarnalata, Pokkali and 93-11 (Table 1). The genetic background of WY17-1-1 was further analyzed by more powerful RICE6K SNP array. The result showed that WY17-1-1 almost had the same genetic background as 93-11, except the locus of Bph6 and Bph9 introgressed from genetic background of Swarnalata and Pokkali (Fig. 1). The introgressed genomic fragments containing Bph6 and Bph9 are about 6.3 Mb (from 20,187,393 to 26,503,057 bp on chromosome 4) and 1.2 Mb (from 22,532,464 to 23,776,103 bp on chromosome 12), respectively (Fig. 1). WY17-1-1 was then designated as LuoYang69 (for pyramiding of Bph6 and Bph9) and subjected to subsequent BPH-resistance evaluation and agronomic traits investigation ( Table 1).

Evaluation of BPH resistance of LuoYang69
To test whether LuoYang69 could improve the BPH resistance than either NIL-Bph6 or NIL-Bph9, firstly we  Fig. 1 Genetic background assay of LuoYang69 using the RICE6K array. The red lines on chromosome 4 and chromosome 12 indicate the SNP loci with homozygous genotypes where genomic fragments of the donor parent Swarnalata (Bph6) and Pokkali (Bph9) were introgressed, respectively evaluated the BPH resistance of these lines at the seedling stage under greenhouse conditions. While the recurrent parent 93-11 and susceptible control TN1 died completely, LuoYang69, along with NIL-Bph6 and NIL-Bph9, showed high levels of resistance to BPH (Fig. 2a). The BPH resistance scores of NIL-Bph6 and NIL-Bph9 were 2.57 ± 0.29 and 2.33 ± 0.24, respectively. Compared with either of NIL-Bph6 and NIL-Bph9, Bph6 and Bph9 pyramided line LuoYang69 conferred significantly enhanced resistance to BPH with resistance score of 1.05 ± 0.12 (P < 0.01), reflecting the additive effect of resistance gene pyramiding (Table 1).
To explore the potential usefulness of LuoYang69 for breeding BPH resistant hybrids, LuoYang69 was crossed with TGMS line Y58S, HL type CMS lines 037A and 203A, and the derived hybrids were evaluated the BPH resistance. The hybrids Y58S/LuoYang69, 203A/LuoYang69 and 037A/LuoYang69 were highly resistant to BPH, with the resistance scores of 1.48, 1.24 and 1.43, respectively (Fig. 2b). More importantly, no significant differences of resistance scores were observed between such hybrids and LuoYang69. Taken together, the results demonstrated that both Bph6 and Bph9 are complete dominance genes and LuoYang69 derived hybrids with heterozygous Bph6 and Bph9 genes could also confer high level of resistance to BPH.
Generally, rice plants have evolved two major resistance strategies against herbivores: antixenosis that affects insect settling, colonization, or oviposition and antibiosis that reduces insect feeding, growth, or survival (Qiu et al., 2011). In two-host choice tests, many more BPH insects preferred to settle on NIL-Bph6 or NIL-Bph9 than on LuoYang69 plants, respectively. Noticeably, the number of settled BPHs on LuoYang69 was constant low (<4 mostly) at all time-points checked. Compared with NIL-Bph6, the numbers of BPH settled on LuoYang69 decreased significantly from the 18 h to 144 h(P < 0.01)after BPH release (Fig. 3a). Compared with NIL-Bph9, the numbers of BPH settled on Luoyang69 plants declined significantly 48 h after BPH release over the study period (P < 0.01) (Fig. 3b). The results above indicated that LuoYang69 has a much stronger antixenotic effect on BPH than either of NIL-Bph6 and NIL-Bph9.
To test whether LuoYang69 improves the antibiosis effect against BPH than NIL-Bph6 or NIL-Bph9, BPH survival rate was investigated on LuoYang69, NIL-Bph6, NIL-Bph9 and 93-11, respectively. As shown in Fig. 3c, no significant differences of BPH survival rate were observed among these plants at the first day of BPH infestation. While the average numbers of surviving BPHs on LuoYang69, NIL-Bph6 and NIL-Bph9 decreased gradually and showed a significant difference in numbers compared with 93-11 from the second day to the last day of BPH infestation. Remarkably, the average numbers of surviving BPHs on LuoYang69 also showed a significant difference compared with either NIL-Bph6 or NIL-Bph9 from the second day of BPH infestation. At the end of investigation, the BPH survival rates of LuoYang69, NIL-Bph6 and NIL-Bph9 dropped to 35.7%, 49.3% and 48.5%, respectively (F = 14.791, P = 0.003 compared by NIL-Bph9 with Luoyang69; F = 11.37, P = 0.004  (Fig. 3c). These findings suggest Bph6 and Bph9 pyramided line LuoYang69 had stronger antibiosis effect on BPH than NIL-Bph6 and NIL-Bph9.
To determine whether LuoYang69 affects BPH growth and development more greatly than NIL-Bph6 or NIL-Bph9, we compared honeydew excretion and weight gain of BPH feeding on these plants. The results indicated that BPH insects fed on LuoYang69 plants showed significantly lower weight gain and honeydew excretion than those on NIL-Bph6 or NIL-Bph9 (Fig. 3d-e). Taken together, these results demonstrate that Bph6 and Bph9 pyramided line LuoYang69 has a much stronger antixenotic and antibiosis effect on BPH than NIL-Bph6 and NIL-Bph9, which contributes to its significantly enhanced resistance against BPH.

The performance of agronomic traits and rice grain quality of LuoYang69
Unexpected linkage drag is a common phenomenon in disease and insect resistance breeding programs, which affects yield and grain quality characteristics of rice cultivars (Yeo and Shon, 2001;Liu et al., 2009). To test whether the agronomic traits and rice grain quality of Bph6 and Bph9 pyramided line LuoYang69 were identical to its recurrent parent 93-11 under normal growth conditions, we evaluated ten agronomic traits of LuoYang69 and 93-11 in Wuhan nearly without BPH damage. Most of agronomic traits such as plant height, panicle number, panicle length, number of filled grains per panicle, total grain number, seed setting rate of LuoYang69 showed no significant difference compared with 93-11. While the 1000-grain weight of LuoYang69 b Settling of BPH insects on LuoYang69 and on NIL-Bph9 in a two-host choice test. c BPH survival rate on LuoYang69, NIL-Bph6, NIL-Bph9 and 93-11 in a non-choice feeding experiment, respectively. d Weight gain of BPH insects after feeding on LuoYang69, NIL-Bph6, NIL-Bph9 and 93-11 for 48 h, respectively. e Measurement of honeydew excreted from BPH insects on LuoYang69, NIL-Bph6, NIL-Bph9 and 93-11, respectively. a and b Bars represent means of 20 replicates. Error bars represent the SD. Means labeled with asterisks are significantly different (P < 0.05). Means labeled with double asterisks are extremely significant different (P < 0.01). c Bars represent means of 16 replicates. Error bars represent the SD. The different letters above the bars are significantly different at P < 0.05 level. d and e Bars represent means of 21 replicates. Error bars represent the SD. The different letters above the bars are significantly different at P < 0.05 level was about 0.54 g heavier than that of 93-11 (P < 0.05) ( Table 2). Moreover, most of the rice grain quality traits (brown rice ratio, head rice ratio, alkali spreading value, amylose content, grain length, grain width, ratio of grain length to width) of LuoYang69 were nearly identical to its recurrent parent 93-11. While Luoyang69 had better rice quality with a lower chalky rice ratio and chalkiness degree compared with 93-11 (Table 3). These results indicated that pyramiding of Bph6 and Bph9 in 93-11 genetic background did not affect the elite agronomic traits and rice grain quality of 93-11. The resulting Luo-Yang69 will be valuable resource to develop hybrid rice with high BPH resistance and excellent agronomic performance.

Development and validation of functional marker for Bph9
In a recent study, we isolated Bph9 and revealed that Bph9 encodes a rare type of nucleotide-binding and leucine-rich repeat (NLR) gene (Zhao et al., 2016). To develop PCR-based functional molecular marker for Bph9, we first compared the genomic sequences of Bph9 alleles in rice varieties Pokalli, 93-11 and Nipponbare. The result showed that a 1.2 kb specific fragment was inserted in intron 1 of Bph9 allele in Pokalli, absent in the corresponding regions of Bph9 alleles in Nipponbare and 93-11. To detect the insertion in intron 1 of Bph9 allele in Pokalli, we designed one forward primer in 3′ sequence of the insertion and one reverse primer spanning the insertion and its flanking sequence. Using this marker, we genotyped the insertion in Pokalli and 20 susceptible cultivars, as well as the original donors of other seven BPH resistance genes (Bph1, bph2, bph7, Bph10, Bph18, Bph21, and Bph26) clustered on the long arm of chromosome 12 and actually allelic with each other (Zhao et al., 2016). As shown in Fig. 4a, a 195-bp fragment of the insertion was amplified by B9D only from Pokalli, while no amplification was obtained from the other cultivars, including those resistant allelotypes of Bph9. B9D might be a dominant Bph9-specific marker which can be used for detecting the presence or absence of Bph9 in rice varieties. As the dominant marker could not differentiate the homozygous and heterozygous status of the alleles, which would limit its application in molecular breeding program. Thus, we developed a codominant InDel marker IR2 according to sequence comparison of Bph9 with its alleles from rice varieties and landraces. Though IR2 itself could not distinguish Bph9 and Bph18 (data not shown), the combination of B9D and IR2 could facilitate MAS of Bph9 in BPH resistance breeding programs.

Discussion
The brown planthopper (BPH) is the most damaging pest of rice (Oryza sativa.L) and a serious threat to rice production in Asia . Host-plant resistance has been proposed as the most economic effective and environmental-friendly approach to reduce BPH damage and increase yield potential of cultivars. The resistance is mainly controlled by major genes and now at least 31 BPH resistance genes have been identified genetically in the cultivated rice germplasm and wild Oryza species sources. Thirteen BPH resistance genes have been cloned via map-based cloning approach (Ji et al., 2016;Ren et al., 2016;Du et al., 2009;Tamura et al., 2014;Wang et al., 2015;Zhao et al., 2016;Liu et al. 2015;Jing et al., 2017). These identified BPH resistance genes provided a solid foundation for marker assisted selection (MAS) in breeding programs. However, the varieties bearing single BPH resistance gene were quickly broken down within a few years due to rapid adaptation of BPH or evolution of new biotypes (Jena and Kim, 2010). Resistant varieties with single resistance gene Bph1 were released in 1973 and saved rice production from massive BPH damage. However, their resistance was broken down in 1976 with the development of a new BPH population (biotype 2). Varieties with bph2 showing effective resistance were then released and widely grown, but again, these varieties were also adapted by another BPH population (biotype 3). Several studies showed some highly and durable resistant varieties carry one or more major genes, along with minor QTLs contributing to BPH resistance. For example, Bph1 carrying rice variety IR64 showed durable and stable resistance after the spread of Bph1-breaking BPH populations (biotype 2), which was conferred by seven minor QTLs detected on chromosomes 1, 2, 3, 4, 6 and 8 (Alam and Cohen, 1998a). Indica cultivar ADR52, highly resistant to BPH, contained two major genes Bph25 and Bph26 along with several associated minor QTLs (Myint et al., 2012, Srinivasan et al., 2015. Progress has been made in pyramiding major BPH resistance genes into elite cultivars. Pyramiding of Bph6 and Bph12 resulted in a higher resistance level than that of near isogenic lines of single gene, showing an additive effect against BPH (Qiu et al., 2012). Bph3 and Bph27(t) pyramided line displayed higher resistance than those lines with single resistance gene (Liu et al., 2016). Bph14 and Bph15 also had a strong dosage effect on the resistance to BPH, are widely used in BPH resistance gene pyramiding breeding practice (Li et al., 2006;Xia et al., 2010;Zhu et al., 2013a;Zhu et al., 2013b;Hu et al., 2012;Hu et al., 2013;Hu et al., 2015;Cai et al., 2015;Wang et al., 2016). These studies demonstrated that pyramiding multiple BPH resistance genes is an efficient strategy to develop higher and more durable resistant varieties against BPH.
Bph6, originally identified in Swarnalata, conferred high level of resistance to BPH biotype 4 (Bangladesh BPH population) and BPH population in China (biotype 2 being the dominant one). Bph6 was fine-mapped on the long arm of chromosome 4 (Qiu et al., 2010). Bph9 in Pokkali was also a high and broad-spectrum BPH-resistance gene, encoding a rare type of nucleotide-binding and leucine-rich repeat (NLR) gene (Zhao et al., 2016). Bph6 and Bph9, which were both explored from the traditional tropical indica cultivars, could be the more adopted genes in rice BPH-resistance pyramiding breeding programs for less linkage drag of unbeneficial traits than those from the wild species. In the present study, we introgressed Bph9 into elite cultivar 93-11 through MAS. Then, Bph6 and Bph9 were pyramided into 93-11 by intercrossing with near isogenic lines NIL-Bph6 and NIL-Bph9. Compared with either of NIL-Bph6 and NIL-Bph9, the resulting Bph6 and Bph9 pyramided line LuoYang69 conferred significantly enhanced resistance to BPH with resistance score of 1.05 ± 0.12 (P < 0.01), reflecting the additive effect of resistance gene pyramiding. The further detailed analyses indicated that Bph6 and Bph9 pyramided line LuoYang69 had a much stronger antixenotic and antibiosis effects on BPH than those near isogeniclines with single resistance gene. The evaluation of agronomic traits and rice grain quality of LuoYang69 showed that most of traits were identical to its recurrent parent 93-11. Noticeably, the 1000-grain weight of LuoYang69 was about 0.54 g heavier than that of 93-11 (P < 0.05). These results demonstrated that Bph6 and Bph9 pyramided line LuoYang69 developed in this study would be valuable resource to develop hybrid rice with high BPH resistance and excellent agronomic performance.
In BPH resistance breeding programs, it is important to make clear whether resistance genes can be used for hybrid improvement and production by checking effects Bph9 are complete dominance genes and combining such both genes would be a practical strategy of hybrid improvement and production for rice breeders. Molecular markers tightly linked to the identified BPH resistance genes are playing the important role in MAS based gene pyramiding breeding programs for the rapid selection of genotypes with targeted gene (Collard et al., 2005;Hayashi et al., 2010). However, the use of such tightly linked markers bears the risk of being lost through genetic recombination between the marker and the target gene, resulting in misdiagnosis of traits of interest. Such problem can be overcome by the application of functional markers (FMs) derived from DNA polymorphisms found in allelic variants of a functional gene. FMs developed from cloned resistance genes are being used in MAS-based breeding (Fjellstrom et al., 2004;Perumalsamy et al., 2010). The combination of markers Bph14P/Bph14N targeting promotor and LRR region of BPH resistance gene Bph14, respectively, were verified in two breeding populations and a Chinese mini core collection of Oryza sativa (Zhou et al., 2013). Our recent work revealed that the eight BPH resistance genes clustered on the long arm of chromosome 12 (Bph1, bph2, bph7, Bph9, Bph10, Bph18, Bph21, Bph26) are actually allelic with each other and can be divided into four allelotypes. We analyzed nucleotide variation of Bph9 alleles from rice varieties and landraces. According to sequence comparison of Bph9 with its alleles, we developed and validated a Bph9-specific functional dominant marker B9D, which can easily distinguish Bph9 with other cultivars, including its resistant allelotypes (Bph1, bph2, bph7, Bph10, Bph18, Bph21, Bph26). Using Bph9-specific functional dominant marker, we screened and evaluated worldwide accessions of rice germplasm. Of the 673 varieties tested, eight were identified to harbor Bph9 and showed resistance to BPH. The sequences of Bph9 alleles in these cultivars were identical to that of Bph9 in Pokkali (IRGC 108921), suggesting that the functional Bph9 locus is derived from a common progenitor. The results also demonstrated that Bph9-specific functional dominant marker B9D is a reliable marker to isolate cultivars harboring the functional Bph9 gene from rice germplasm worldwide. These identified Bph9 containing cultivars can be used as new sources for BPH resistance breeding programs. To differentiate the homozygous and heterozygous status of Bph9, the present work also developed a codominant InDel marker IR2. The combination of B9D and IR2 could either validate the existence of Bph9 or indicate the diploid genotype of paddy lines, which will facilitate MAS of Bph9 in BPH resistance breeding programs.

Conclusion
We pyramided Bph6 and Bph9 into elite restorer line 93-11 through MAS. The resulting Bph6 and Bph9 pyramided line LuoYang69 had stronger antixenotic and antibiosis effects on BPH and exhibited significantly enhanced resistance than near isogenic lines NIL-Bph6 and NIL-Bph9. Its derived hybrids harboring heterozygous Bph6 and Bph9 genes also conferred high level of resistance to BPH. The development of LuoYang69 pyramided with Bph6 and Bph9 provides valuable resource to develop hybrid rice with high BPH resistance and excellent agronomic performance. We also developed functional markers for MAS of Bph9 in rice breeding programs. The 8 identified Bph9 harboring cultivars can be used as new sources BPH resistance breeding programs.