Development of Markers for Identification and Maker-Assisted Breeding of Xa7&nbsp;Gene in Rice (Oryza sativa&nbsp;L.)

doi:10.21203/rs.3.rs-398234/v1

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Development of Markers for Identification and Maker-Assisted Breeding of Xa7 Gene in Rice (Oryza sativa L.)

https://doi.org/10.21203/rs.3.rs-398234/v1

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Utilization of resistance (R) genes to breed resistant cultivars is one of the most effective and economical approach to control rice bacterial blight (BB). Xa7, a dominant, broad-spectrum and durable BB-resistant gene, is an ideal gene resource to improve the resistance of rice varieties to bacterial blight, and this well-known gene with important breeding value has been cloned in our recent study. The isolation of Xa7 will facilitate its application in rice breeding by molecular maker-assisted selection (MAS). In this study, based on the specific sequences in the promoter of Xa7, a functional marker, named as MX7, was developed, which can effectively distinguish the dominant BB-resistant Xa7, the recessive BB-susceptible xa7 and the null allele from different rice varieties. Since MX7 is a dominant marker, it can't tell homozygous from heterozygous, a co-dominant marker closely linked to Xa7, named as M6, was developed simultaneously. After verified by amplification in numerous rice varieties and sequence alignment in RICE 3K database, it is proved that marker M6 is co-segregated with the Xa7 locus. In addition, the effectiveness and accuracy of the two markers were further validated by two F₂ populations. Finally, the designed makers were effectively applied in MAS breeding to improve the BB-resistance of a susceptible variety. This study not only provides reliable functional markers for the identification of Xa7 gene in different rice materials, but also will contribute to the application of Xa7 gene in marker-assisted selection to breed rice varieties with durable disease resistance.

Cellular & Molecular Neuroscience

Rice

Bacterial blight

Disease resistance

Xa7 gene

Functional marker

Marker-assisted selection

Bacterial blight (BB), caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most destructive diseases of rice in the world, and causes serious yield losses (Mew 1987). Since pesticides is expensive and contaminates environment, the most economical and environmental friendly approach to control BB is the utilization of resistance (R) genes to breed resistant varieties (Rao et al. 2002). To date, at least 46 genes conferring host resistance against various Xoo races have been identified (Chen et al. 2020), and sixteen of them have been cloned successfully (Chen et al. 2021). Among those R genes, Xa4 was the first to be widely used in modern rice varieties since the late 1960s, especially with the widespread cultivation of high-yielding rice varieties, such as TN1 and IR8, which caused BB to become a major threat to rice production (Khush 1995). Since then, breeding rice varieties with resistance to BB was extremely valued, and with the identification and cloning of new R genes, as well as the development of available DNA markers for corresponding genes, rice varieties pyramided with different R genes by marker-assisted selection (MAS) have been successfully applied in BB-resistant breeding (Perumalsamy et al. 2010; Bharani et al. 2010; Suh et al. 2013). In fact, with the application of Xa3, Xa4, Xa21 and other resistance genes in rice breeding, bacterial blight had almost disappeared for a short time in China (Zhang 2009). However, large-scale and long-term cultivation of varieties carrying a single R gene resulted in a significant shift in pathogen race frequency, and eventually led to the breakdown of resistance in these cultivars (Rao et al. 2002; Jiang et al. 2020). For example, the most widely deployed BB resistance gene, Xa4, which had been effectively used in rice breeding to control BB for nearly 20 years, but subsequent studies confirmed that its disease resistance has already broke down (Vera-Cruz et al. 2000; Quibod et al. 2019). An effective way to delay the breakdown of BB resistance is to pyramid multiple R genes in a single variety, especially application of R genes with durable disease resistance ability.

Xa7 is a dominant, broad-spectrum and durable BB-resistant gene (Vera-Cruz et al. 2000; White et al. 2009; Zhang et al. 2015). This gene was originally identified from Bangladesh rice variety DV85 (Sidhu et al. 1978), and then by backcrossing with an IRRI rice variety IR24, Xa7 was introduced into IRBB7 (Ogawa et al. 1991). Extraordinarily, according to the results of many years’ field trials, the resistance of Xa7 was proved to be more durable than Xa4 and Xa10 (Vera-Cruz et al. 2000). Up to now, Xa7 is considered to be the most durable resistance gene to bacterial blight. The durable resistance ability of Xa7 is closely related to the specificity of its cognate avirulence genes, both avrXa7 and pthXo3, which are also virulence genes maintaining toxicity of Xoo and are vital for their own survival, so mutations of avrXa7 or pthXo3 risk to be eliminated by natural selection, and this special relationship can help to limit the loss of resistance of Xa7 (Chen et al. 2021; Luo et al. 2021; Vera-Cruz et al. 2000). More interestingly, Xa7-containing rice varieties are more effective at high temperatures, whereas other R genes are less effective (Webb et al. 2010; Cohen et al. 2017; Dossa et al. 2020). In the context of global warming, Xa7 is an ideal R gene for breeding durable disease resistant varieties.

Based on the excellent characteristics of Xa7, mapping based cloning of this gene has been focused for decades by researchers. As early as 1995, Xa7 was initially located at 107.5-cM on the current RGP map (Kaji and Ogawa 1995). Subsequently, Xa7 had been reported to be finely mapped into a 2.7-cM region between marker M1 and M3 on rice chromosome 6, and highly linked with marker M5 speculatively (Porter et al. 2003). And then, Xa7 was further mapped into a 118-kb region flanking with markers GDSSR02 and RM20593 (Chen et al. 2008). After a-decade of hard works, we successfully cloned this gene from a Chinese rice variety Zhen-hui 084 (Chen et al. 2021). Our results showed that Xa7 is a novel executor R gene, it was induced by the corresponding transcription activator-like effectors (TALEs), and the induction was faster and higher under high temperature. In addition, it carries a putative effector binding elements (EBEs) in its promoter (EBE_AvrXa7), which are recognized by its corresponding avirulence genes, and the EBE sequence is essential for its BB resistance in rice (Chen et al. 2021). Based on the cloning and specific sequences analysis of Xa7, here we reported the development and assessment of a functional marker for identification of Xa7 in different rice varieties, and the design and application of a co-dominant marker co-segregated with Xa7 for homozygous analysis of the Xa7 locus. By tested in multiple rice varieties and segregation populations, as well as the collinearity analysis between molecular markers and the Xa7 gene locus by sequence alignment in RICE 3K database, we proved that the developed functional marker can accurately identify whether there is Xa7 gene in different rice varieties, and the combination utilization of the designed functional marker and co-dominant marker is helpful to efficiently application of Xa7 in MAS breeding.

Plant materials

Rice variety Zhen-hui 084 is a Xa7-containing variety, with durable and broad-spectrum resistance to rice bacterial blight disease. Rice variety Chen-hui 448 doesn't contain the Xa7 locus, while rice variety Lao-zao-gu carrying the xa7 allele, both of them is susceptible to Xoo strain PXO86. The seeds of the above rice varieties were sowed in the field. At the booting stage, the stamens of Chen-hui 448 or Lao-zao-gu were emasculated artificially by scissors and crossed with the pollen of Zhen-hui 084, and their F₁ plants were self-crossed to develop the corresponding F₂ populations. All of the other rice varieties used in this study were presented by the Chinese National Rice Research Institute (Hangzhou, China).

Evaluation of bacterial blight resistance

Ten Xoo races were used in this study to evaluate the bacterial blight resistance of rice plants, and all of them were obtained from Chinese Academy of Agricultural Sciences (Beijing, China). The strains were cultured on agar media containing 20 g of sucrose, 5 g of peptone, 0.5 g Ca(NO₃)₂, 0.43 g Na₂HPO₄, and 0.05 g FeSO₄ per liter, and allowed to grow at 28°C for 2 to 3 days. The bacterial colony was suspended in sterile distilled water at an optical density of OD₆₀₀ = 0.5 and immediately used for inoculation. The 60-day-old rice plants were inoculated by using leaf-clipping method (Kauffman et al. 1973). Two weeks later, the lesion length on the cut leaves were measured for evaluation of BB-resistant level (Yin et al. 2000).

DNA extraction and PCR amplification

Total genomic DNA was extracted from rice leaves using Panaud et al. (1996) method. GoTaq® Master Mix (Promega, USA) was used for PCR amplification with 10–20 ng genomic DNA and a suitable concentration of primers according to the manufacturer’s instructions. For marker the functional MX7, three primers were used and equivalently mixed. The forward primer was named as MX7-FP: 5’-AATATATAACCCCCCCCCCCCCAG-3’, the intermediate primer was named as MX7-MP: 5’-ATGGCGGCCGCTGATCATCC-3’, and the reverse primer was named as MX7-RP: 5’-TTAATTGCCACCGATGAGGTAATCC-3’. For the co-dominant marker M6, primers pair are named as M6-FP: 5’-GGGAGAAATTGGCCCCAGTTAGAGAA-3’ and M6-RP: 5’-GCATGTCTGTGTCGATTCGTCCGTACGA-3’. The primers pair of the marker M5 used here was reported by Porter et al. (2003). PCR process was performed as: (1) pre-denatured at 94 ^oC for 5 min; (2) denatured at 94 ^oC for 30 s, annealed at a temperature according to the markers (M6 at 60 ^oC; MX7 at 62 ^oC; M5 at 65 ^oC) for 30 s and extended at 72 ^oC for 60 s, followed by 35 cycles; (3) finally extended at 72 ^oC for more 5 min. The PCR products were examined via 1.5% agarose-gel electrophoresis and stained with ethidium bromide (EB).

Bioinformatic analysis

The alignment of different sequences was carried out by the software named as ClustalX 2.0, and the output of the results was modified by GENEDOC 2.7. The CDS sequences of Xa7 and the amplified sequences of maker M6 were used for sequences similarity analysis. Due to the miss of the Xa7 locus in the reference genomes (both Nipponbare and R498), so sequences alignment were performed with the scaffolds of each cultivar in the RFGB database (http://www.rmbreeding.cn/Blast; Wang et al. 2018). In presenting the sequence alignment results, only the varieties with 100% consistent coding region of Xa7 were listed.

Evaluation of the major agronomic traits

Field trials were tested in the summer season, 30-day-old seedlings of the BC₅F₄ plants derived from the backcross between Zhen-hui 084 and Cheng-hui 448, as well as both the parents were transplanted in the field at 15×25 cm spacing in south China. The experiments were carried out following a randomized complete block design (RCBD) with three replications. The agronomic traits were measured following the standard evaluation system described by Xuan et al. (2019). Ten plants from each entry were recorded as one data replication, including plant height (cm), panicle length (cm), flag-leaf length and width (cm), panicles number per plant, panicle length (cm), grains number per panicle, seed-setting rate (%), grain length and width (mm), and the 1000-grain weight (g). Statistical analysis was performed with independent samples by using t-test for significance between the plants of Zhe-kang 1 and Cheng-hui 448.

Design of molecular markers for identification of Xa7 gene

According to our previous results, the nucleotide sequence of the Xa7 gene is absolutely novel and is not widely existed in rice germplasms (Chen et al. 2021). Therefore, based on the specific coding sequence (CDS) of Xa7, markers directly located in this region could be used for functional identification of this gene in major rice varieties. However, in some susceptible varieties, we also found a completely identical CDS as Xa7, but with variations in its promoter (with an 11-bp fragment insertion and a G to T substitution in the EBE_AvrXa7 region), and in order to distinguish it from the dominant BB-resistant gene Xa7, this type of allele was designated as xa7 (Chen et al. 2021; Fig. 1A). On this basis, a functional PCR marker containing three primers, named as MX7, was designed (Fig. 1A). The forward primer (MX7-FP) was located in the EBE_AvrXa7 region, and the middle primer (MX7-MP) was located in the CDS from the start codon, while the reverse primer was located in the CDS before the stop codon (Fig. 1A). The three primers mentioned above were mixed and used for a single PCR amplification. Theoretically, in rice varieties containing the BB-resistant gene Xa7, a 482-bp fragment (partial promoter + whole CDS) and a 342-bp (only the whole CDS) fragment will be simultaneously amplified, while in rice varieties carrying the xa7 allele, because of the variation in the EBE_AvrXa7 region was unmatched by the forward primer during annealing, only a 342-bp fragment would be amplified. In addition, in varieties absence of the Xa7 locus, no sequence could be amplified at all.

As the functional marker MX7 is more of a dominant marker for most rice varieties, and cannot effectively distinguish the homozygote and heterozygote of Xa7. Therefore, it would be better to develop a co-dominant marker highly linked or co-segregated with Xa7. Previously, according to the reference genome of Nipponbare, we finally mapped Xa7 into a 51-kb region flanking with markers M5 and M10 (Chen et al. 2021), which are more closely linked to Xa7 than the reported markers used in the fine mapping of Xa7 (Porter et al. 2003; Chen et al. 2008; Fig. 1B). Actually, several co-dominant molecular markers of Xa7 have been developed and applied in rice breeding, among them, M5 is more closely linked to Xa7 (Porter et al. 2003; Chen et al. 2008; Perez et al. 2008; Zhang et al. 2010; Huang et al. 2012; Yap et al. 2016; Mi et al. 2018). However, the distance between M5 and Xa7 is still 92.3-kb on its true physical map (Fig. 2B), so that it may not be able to co-segregated with Xa7. By sequence alignment, fortunately, a 227-bp fragment insertions and deletions (indels) was found between IRBB7 and IR24 in the region only 16.6-kb away from Xa7 (Fig. 1C, D). Based on this indels, a co-dominant molecular marker, named as M6, was designed, the predictive fragments amplified from IRBB7 and IR24 are 306-bp and 531-bp, respectively (Fig. 1D).

Verification of the designed markers in different types of rice varieties

Eighteen rice varieties were used for verification of the designed markers, including six varieties containing the dominant resistant gene Xa7, seven varieties carrying the recessive susceptible gene xa7, and the other five varieties without this locus at all (Chen et al. 2021; Fig. 2). The amplification results of MX7 were consistent with the expected results, the 342-bp and 482-bp fragments (partial promoter + whole CDS) were simultaneously amplified from all of the Xa7 containing varieties, but only the 342-bp fragment (whole CDS) was amplified from all of the xa7 carrying varieties, and no fragment was amplified from all of the null-allele varieties (Fig. 2A). These results indicated that MX7 is a dominant marker, and could be used to distinguish Xa7 from xa7 in rice cultivars. For marker M6, the 306-bp fragment was amplified from the varieties containing Xa7 or xa7, while the 531-bp fragment was amplified from the null-allele varieties (Fig. 2B). As a comparison, marker M5 was also analyzed, the amplification result of marker M5 was similar to that of marker M6, except for the polymorphisms of M5 were less conserved in the Xa7 null-allele varieties (Fig. 2C). These results suggested that both M5 and M6 have co-dominant polymorphisms between Xa7 (or xa7) and the null allele, but cannot distinguish Xa7 from xa7.

To verify whether marker MX7, M6 and M5 could be widely used for identification of Xa7 in rice varieties, a total of 167 Chinese rice varieties were assessed by the three markers and inoculated with PXO86, a Xoo strain containing the corresponding avirulence gene avrXa7 and can be used in the identification of Xa7 (Table S1). The identification results of MX7 showed that no varieties were detected containing Xa7, and twenty-one varieties were detected carrying the xa7 allele, while the other 146 varieties were detected as the null-allele of the Xa7 locus. The bacterial blight inoculation results were consistent with MX7, none of the 167 varieties were highly resistant to PXO86. For maker M6, an about 306-bp fragment were amplified from all of the xa7 carrying varieties, while an 531-bp fragment were amplified from all of the null-allele varieties (Table S1). However, compared to M6, there is no such good consistent polymorphism of marker M5 in these varieties (Table S1). These results were further confirmed by varieties from other countries, as shown in Table 1, maker MX7 can accurately identify the Xa7 gene in varieties from different countries, and in terms of the linkage with the Xa7 locus, maker M6 is more precise than M5.

Table 1

Information of the varieties with *Xa7*, *xa7* or null allele.
No.	Variety name	Origin	^a M5	^b M6	^c MX7	^d PXO86	^eLesion length (cm)
1	IRBB7	Philippines	+	+	Xa7	HR	0.6 ± 0.29
2	DV85	Bangladesh	+	+	Xa7	HR	0.4 ± 0.15
3	DV86	Bangladesh	+	+	Xa7	HR	0.5 ± 0.23
4	AUS 242	Bangladesh	+	+	Xa7	HR	0.3 ± 0.06
5	AUS 299	Bangladesh	+	+	Xa7	HR	0.4 ± 0.08
6	AUS 308	Bangladesh	+	+	Xa7	HR	0.4 ± 0.07
7	Zhen-hui 084	China	+	+	Xa7	HR	0.5 ± 0.12
8	Lao-zao-gu	China	+	+	xa7	S	29.2 ± 2.50
9	ARC 10100	India	+	+	xa7	S	37.1 ± 2.37
10	DANGAR	India	+	+	xa7	S	26.6 ± 1.99
11	BARI SUTAR	India	+	+	xa7	S	33.2 ± 5.78
12	MHARAKA	Kenya	+	+	xa7	S	34.4 ± 2.22
13	MANSARA DHAN	Nepal	+	+	xa7	S	31.3 ± 0.92
14	MOTIA	Pakistan	+	+	xa7	S	28.0 ± 3.67
15	CHAO HAI	Laos	-	+	xa7	S	30.4 ± 2.47
16	KHAO SIM	Thailand	-	+	xa7	S	28.0 ± 1.54
17	Lu-cai-hao	China	-	+	xa7	S	24.7 ± 1.68
18	Jin-bao-yin	China	-	+	xa7	S	24.1 ± 1.82
19	Xiang-wan-xian 3	China	+	-	Null	S	27.7 ± 1.06
20	Zhen-xian 232	China	+	-	Null	S	27.0 ± 1.39
21	Hong-ai-nuo	China	+	-	Null	S	24.9 ± 2.09
22	Shan-jiu-gu	China	+	-	Null	S	22.2 ± 3.56
23	76 − 1	China	-	-	Null	MR	5.2 ± 1.17
24	IR24	Philippines	-	-	Null	S	27.2 ± 1.73
25	Zhong-hua 11	China	-	-	Null	S	25.9 ± 0.52
26	Nipponbare	Japan	-	-	Null	MS	7.8 ± 0.48
27	9311	China	-	-	Null	S	22.3 ± 0.64
28	Cheng-hui 448	China	-	-	Null	S	26.9 ± 0.45
^a Genotypes of the marker M5 in the varieties. ‘+’ refers to a IRBB7-like fragment and ‘-’ refers to other types fragments. ^b Genotypes of the marker M6 in the varieties. ‘+’ refers to the 306-bp fragment and ‘-’ refers to the 531-bp fragment. ^c Genotypes of the alleles identified by the functional marker MX7. Xa7 refers to the BB-resistant dominant gene, xa7 refers to the recessive susceptible allele with an identical CDS of Xa7 but variations in the promoter, and null refers to an allele without the Xa7 CDS and its promoter. ^d High resistant (HR), medium resistant (MR), medium susceptible (MS) or susceptible (S) phenotype of the varieties inoculated by the Xoo strain PXO86; ^e the lesion length of infected leaves 2 weeks after inoculated with POX86. The value of traits is measured as mean ± S

In order to further analyze whether marker M6 is co-segregated with Xa7/xa7 allele, the sequences of Xa7/xa7 and marker M6 were used for the collinear analysis in the database of 3010 cultivated rice varieties. As listed in Table S2, total 410 rice varieties were found to contain alleles with a 100% identity of the Xa7 CDS, but were not sure to be Xa7 or xa7 due to the poor quality of sequencing in the EBE region (poly C or G). Meanwhile, there are 401 varieties containing an identical sequence with the 306-bp fragment amplified from IRBB7, and the sequence similarity of the other 9 varieties are also more than or equal to 95% (Table S2). In addition, no Xa7 or xa7 allele was found in more than 2500 rice varieties, and most of them containing an identical sequence with the 531-bp fragment (data not shown). The above results proved that maker M6 is co-segregated with the Xa7 locus.

The information of some representative varieties contained the Xa7, xa7 or null alleles were listed in Table 1, including genotypes of the markers and phenotypes of the BB resistance to PXO86. Our results indicated that the marker MX7 could be used for functional identification of Xa7 in rice varieties, and the marker M6 is co-segregated with the Xa7 locus better than marker M5. Thus, combination of the functional marker MX7 and the co-dominant marker M6 together can be effectively used for the identification and marker-assisted breeding of Xa7.

Verification of the designed markers in segregated populations

In addition, in order to evaluate the accuracy of the designed primers in segregated populations, the Xa7-containing variety Zhen-hui 084 was crossed with the null-allele variety Chen-hui 448, and the F₁ progenies were self-crossed to build the F₂ segregated population. The F₂ plants were individually amplified with marker MX7, and inoculated with PXO86. The 342-bp and 482-bp bands of MX7 were both amplified in the resistant plants, but were not amplified in the susceptible plants (Fig. 3A). Although marker MX7 could functionally identify Xa7 from the resistant plants, obviously, it could not distinguish homozygote and heterozygote of the Xa7 locus (Fig. 3A). Therefore, marker M6 was used for further analysis of these plants, and it clearly displayed the difference among dominant homozygote, heterozygote and recessive homozygote of the Xa7 locus (Fig. 3A). An F₂ segregated population derived from the cross between the Xa7-containing variety Zhen-hui 084 and the xa7-carrying variety Lao-zao-gu was also built, subsequently analyzed by both the designed markers and POX86. In this situation, M6 was useless due to no polymorphism between the two parents, but MX7 could still identify the resistant plants by the specific 482-bp band in spite of the 342-bp bands existed in all of the F₂ plants (Fig. 3B). The above results indicate that for the identification of Xa7 in rice varieties, only the functional maker is credible, and there are a large number of false positives when utilization of molecular markers closely linked to or co-segregated with the Xa7 locus. Unfortunately, a large number of previous studies have used these unreliable closely linked molecular markers to identify whether there is Xa7 gene in rice varieties (Deo et al. 2013; Majumder et al. 2020; Prasannakumar et al. 2021; Ullah et al. 2012).

Application of the designed markers in MAS breeding

The rice variety Cheng-hui 448, a Chinese indica restorer line with wide compatibility and good qualities, was bred from a cross between American rice Lemont and Chinese restorer Line 871028 (Ren et al. 1999). This variety is highly resistant to Magnaporthe grisea, but is generally susceptible to Xoo (Fig. 5). To improve its BB resistance, Cheng-hui 448 was selected as a recurrent parent and backcrossed with the Xa7-containing variety Zhen-hui 084 (Fig. 4). At that time, as the Xa7 gene has not been isolated, all the plants of BC₁F₁, BC₂F₁, BC₃F₁, BC₄F₁ and BC₅F₁ were amplified by marker M6, and the PXO86-resistant plants with the 306-bp fragment of M6 were further backcrossed with Cheng-hui 448 (Fig. 4). And then, the homozygous BC₅F₁ plants selected by M6 were further self-crossed for three generations to get the BC₅F₄ plants, which were finally identified by the functional marker MX7 after we cloned Xa7. Finally, through three times of background selection, the BC₅F₄ plants was regarded as the near isogenic lines containing Xa7 gene, we named it as Zhe-kang 1. All of the Zhe-kang 1 plants could amplified the 342-bp and 482-bp bands of MX7 (data not shown) and displayed a broad-spectrum of BB resistance (Fig. 5). Moreover, there was no significant difference of the mainly agronomic traits between Cheng-hui 448 and the improved near isogenic line Zhe-kang 1 (Table 2).

Table 2

The major agronomic performance of the improved line Zhe-kang 1 and the recurrent parent Cheng-hui 448.
Variety	Plant height (cm)	Flag-leaf length (cm)	Flag-leaf width (cm)	No. of tillers per plant	Panicle length (cm)	No. of grains per panicle	Spikelet fertility (%)	Grain length (mm)	Grain width (mm)	1000-grain weight (g)
Cheng-hui 448	105.72 ± 0.97	29.92 ± 0.19	1.58 ± 0.12	10.17 ± 0.75	28.18 ± 0.17	144.67 ± 8.07	77.74 ± 1.75	10.39 ± 0.38	2.76 ± 0.07	25.53 ± 0.25
Zhe-kang 1	106.54 ± 0.42	30.28 ± 0.47	1.68 ± 0.08	10.10 ± 0.63	28.10 ± 0.14	147.40 ± 8.44	77.32 ± 1.51	10.47 ± 0.46	2.80 ± 0.12	25.68 ± 0.18
Data shown as Mean ± SD. Statistical analysis was performed by a two-tailed t-test for paired samples, and there were no significant difference in all of the tested traits.

Host-plant resistance is a cost-effective and environmentally safe approach to reduce yield loss caused by rice bacterial blight disease. However, the acquisition of host-plant resistance depends on the identification and application of resistance genes. In fact, among the 46 identified BB-resistance genes, only few of them have been widely used in rice breeding, such as Xa3、Xa4、Xa7、Xa21 and Xa23 (Zhang et al. 2009; Mi et al. 2018). The main reason is that some genes have poor resistance, or narrow-spectrum, and part of them are recessive genes, which limited them to be widely used in hybridization breeding. At the same time, whether the resistance of the R genes can be sustained also needs to be considered. Many varieties with Xa4 have been cultivated widely in Asia to control BB disease; the large-scale and long-term cultivation of those varieties carrying Xa4 resulted in significant shifts in the race frequency of Xoo, and eventually led to the decline of resistance to bacterial blight. In this study, according to the resistance characteristics of Xa7, such as broad-spectrum (Ogawa et al. 1991), long-lasting (Vera-Cruz et al. 2000) and heat-tolerant (Webb et al. 2010), we selected the newly cloned Xa7 as the gene resource to develop broad-spectrum and durable-resistance rice variety. However, although the resistance of Xa7 has been proved to be more durable, whether it can escape the migration of pathogenic population needs to be further studied.

Transgenic technology and molecular maker-assisted selection are the main ways to utilize disease resistance genes in breeding. Due to the evaluation and supervision of transgenic crops are very strict, MAS is still the main way for resistance improvement in rice (Wong et al. 2016; Jin et al. 2019). Therefore, it is of great significance to development molecular markers that can be used in MAS breeding. In the present study, based on the specific sequence of the Xa7 gene, a functional marker of Xa7 and a co-dominant marker that co-segregated with Xa7 were developed. The main reason of designing two molecular markers for breeding application of Xa7 is the particularity of its own alleles in different rice cultivars. Unlike most of the other R genes, there are three types of the Xa7 locus in rice varieties, which are Xa7-containing-resistant type, xa7-containing susceptible type, and the null-allele susceptible type. Sequence difference between Xa7-containing-resistant type and xa7-containing susceptible type only existed in the EBE region of the Xa7 promoter, and whether this difference is the same in different rice varieties is still difficult to identify (Chen et al. 2021), therefore, it is not available to design a co-dominant marker at this site. Moreover, as shown in Table S2, only a small proportion of rice materials containing the Xa7 or xa7 alleles, while more than 2500 rice varieties belong to the null-allele susceptible type. As homozygote and heterozygote in MAS breeding cannot be distinguished only by the functional marker MX7, hence, combination of the functional marker MX7 and the co-dominant marker M6 together will be more effectively for the identification and marker-assisted breeding of Xa7. For identification of Xa7, it is worth noting that utilization of molecular markers closely linked to or co-segregated with the Xa7 locus is not reliable.

The application of molecular markers makes it more convenient to transfer resistance genes into a variety. However, the accuracy and validity of the designed molecular markers should be evaluated before be used in breeding. In this study, in order to estimate the efficiency of the designed molecular markers, firstly we analyzed the physical distance between different markers and the target gene Xa7 (Fig. 1B and Fig. 1C). Subsequently, utilization of different rice cultivars from both China (Table S1) and all over the world (Table 1), the polymorphisms of the designed markers in those varieties were identified. As exhibited in Fig. 2 and Table 1, consistent with the result of Xoo inoculation, the functional marker MX7 can specifically identify Xa7 gene from the Xa7-containing rice varieties, while the co-dominant marker M6 showed a better linkage with the Xa7 locus when compared to previously used marker M5. And then, the above results were further confirmed by a more large population based on the high throughput data information of the 3010 rice accessions (Table S2). In addition, the designed makers were verified in practical breeding to improve the bacterial blight resistance of a BB-susceptible variety Cheng-hui 448. The analysis of major agronomic traits revealed that the introduction of Xa7 gene enhanced the BB-resistance of Cheng-hui 448, but did not impact the yield and other main agronomic traits (Fig. 5). Thus, the makers developed in this study can not only be used to identify Xa7 gene from different germplasm, but also guide the application of Xa7 in molecular marker-assisted breeding.

In this study, the functional marker MX7 for identification of Xa7 and the co-dominant marker M6 that co-segregated with the Xa7 locus were developed. And combination of the functional marker MX7 and the co-dominant marker M6 together can be effectively used for the identification and marker-assisted breeding of Xa7.

AcknowledgmentsWe sincerely thank Prof. Zhang Hongsheng (Nanjing Agricultural University) and Gao Zhengyu (China National Rice Research Institute, Chinese Academy of Agricultural Sciences) for kindly providing rice materials.

Authors' contributionsJBM and XFC designed and supervised the experiments. PCL, LM and LMH performed the experiments. YLX and YTZ contributed to MAS breeding of Zhe-kang 1. QQ, XMZ and DLZ provided rice varieties and revised the manuscript. CPL, LM and XFC wrote the manuscript with input from all other co-authors. All the authors have read and approved the final manuscript.

Funding This work was supported by the Ministry of Agriculture of China (2016ZX08009003-001), the National Natural Science Foundation of China (32071987, 31871605), and the Natural Science Foundation of Zhejiang Province (LD19C130001).

Compliance with ethical standards

Conflict of interest All the authors declare that they have no competing interests.

Ethical approval We declare that the present experiments comply with the ethical standards of

the country in which they were performed.

Data Availability All data related to this study have been submitted.

Animal Research (Ethics) Not applicable in this study.

Consent to Participate (Ethics) Not applicable in this study.

Consent to Publish (Ethics) Not applicable in this study.

Plant Reproducibility The plants used in this study can be reproduced.

Clinical Trials Registration Not applicable in this study.

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Development of Markers for Identification and Maker-Assisted Breeding of Xa7 Gene in Rice (Oryza sativa L.)

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Abstract

Figures

Introduction

Material And Methods

Plant materials

Evaluation of bacterial blight resistance

DNA extraction and PCR amplification

Bioinformatic analysis

Evaluation of the major agronomic traits

Results

Verification of the designed markers in different types of rice varieties

Verification of the designed markers in segregated populations

Application of the designed markers in MAS breeding

Discussion

Conclusions

Declarations

References

Supplementary Files

Status:

Version 1