Backcrossing with Marker Assistance to Introduce Broad-Spectrum Bacterial Leaf Blight Resistance in the Malaysian Elite Variety MR297

Round 1

Reviewer 1 Report

Marker assisted backcross breeding (MABB) is a reliably and effectively method in germplasm innovation. Here, the authors used MABB method to introduce bacterial leaf blight resistant genes Xa21, xa13, xa5, and Xa4 from the IRBB60 to the high-yielding Malaysian MR297. Three obtained germplasm showed both high levels of resistance and high yielding. I have some concerns about this work and this manuscript:

Major concerns:

Q1: In the Introduction section, please add some introductions about of the disease resistance and susceptibility genes to bacterial blight of rice. It’s very important here, because you try to develop bacterial leaf blight resistant rice germplasm.

Q2: Please give some information about the variety MABB and IRBB60, such as their alleles of bacterial leaf blight resistant genes.

Q3: Please move all the figures and tables from the Materials and Methods section into the Results section. Table 1 and Figure 1 and 2 are important results, not methods.

Q4: It’s better to give the distribution of all selected markers along whole genomes (not in a table) as well as the position of targeted genes Xa21, xa13, xa5, and Xa4. Thus it would be clear to the readers.

Q5: Would you please add pictures of the obtained germplasm with high levels of resistance and high yielding? 

Minor concerns:

Q1: There are many clerical errors throughout the manuscript. Please check them carefully and revise them.

Q2: This is not correct in abstract. Xoo resistance genes from MR297 or IRBB60? Introgression of four Xoo resistance genes from the high-yielding Malaysian MR297 lineage Xa21, xa13, xa5, and Xa4 from the IRBB60 lineage.

Author Response

Comments and Suggestions for Authors

Marker assisted backcross breeding (MABB) is a reliably and effectively method in germplasm innovation. Here, the authors used MABB method to introduce bacterial leaf blight resistant genes Xa21, xa13, xa5, and Xa4 from the IRBB60 to the high-yielding Malaysian MR297. Three obtained germplasm showed both high levels of resistance and high yielding. I have some concerns about this work and this manuscript:

 

Reviewer-1# Comments and Suggestions for Authors.

 

Major concerns：

 

Concern # 1:

In the Introduction section, please add some introductions about of the disease resistance and susceptibility genes to bacterial blight of rice. It is very important here, because you try to develop bacterial leaf blight resistant rice germplasm.

 

Authors’ response:

 

• We would like to thank the reviewer for this comment. We agree with the Reviewer that it is a very important aspect. Indeed, after a careful examination there are currently 42 R genes known to protect rice from Xoo, and this number is rising. These R genes are selective in their effectiveness against particular Xoo strains or races, which are collections of strains that share incompatibility with designated sets of R genes because of co-evolution and selection pressure between Xoo and rice.

 

Authors’ action:

• The revised text was started with line No. 50 page 2 till line No. 53 page 2. In the following, we had copied the text inserted into the paper.

 

Concern # 2:

Please give some information about the variety MABB and IRBB60, such as their alleles of bacterial leaf blight resistant genes.

 

Authors’ response:

• We would like to thank the reviewer for this comment. We agree with the Reviewer that it is a very important aspect. Indeed, after a careful examination, the parents in this research was MR297 developed at the Malaysian Agricultural Research and Development Institute (MARDI) On March 13, 2016, MR297 is Padi MARDI (MRQ76 P446)/P446 (Razak et al., 2019). Variety MR297 has a high yield. In addition, IRBB60 was gathered from the Philippines-based International Rice Research Institute (IRRI). The rice leaf blight caused by bacteria is not harmful to the IRBB60 cultivar. It is made up of the BLB R-genes Xa4, xa5, xa13, and Xa21.The dominant genes are Xa4 and Xa21, while the recessive genes are xa5 and xa13. During hybridization and backcrossing, MR297 acted as the female parent (receiver) and later as the recurrent parent, while IRBB60 was only used as the male parent (donor) during hybridization, which produced F1 plants. For planting spacing, weed control, fertilizer use, insect and disease control, and other management techniques, the Malaysian Agricultural Research and Development Institute (MARDI) handbook was used as a guide.

 

 

 

Authors’ action:

• The revised text was started with line No. 102 page 3 till line No. 107 page 3. In the following, we had copied the text inserted into the paper.

 

Concern # 3:

Please move all the figures and tables from the Materials and Methods section into the Results section. Table 1 and Figure 1 and 2 are important results, not methods.

 

Authors’ response:

• We appreciate the reviewer's attention. We agree with the reviewer that it is a very significant feature. Indeed, after a careful examination, for Table 1 and figure 1 but for Figure 2, it is the scheme for experiment stile in materials and method.

 

Authors’ action:

• The revised text was started with line No. 194 page 5 till line No. 199 page 6. In the following, we had copied the text inserted into the paper.

 

Concern # 4:

It is better to give the distribution of all selected markers along whole genomes (not in a table) as well as the position of targeted genes Xa21, xa13, xa5, and Xa4. Thus, it would be clear to the readers.

 

Authors’ response:

• We agree with the reviewer that it is an important factor. In fact, a detailed study revealed that, the selection of suitable polymorphic markers is important in a MABB for maximum recovery of the recurrent parent genome and reduction of the donor parent genome. It also makes the recovery of the recurrent parent genome faster. 475 rice SSR markers were screened for polymorphism between MR297 and IRBB60 parental varieties, and polymorphic markers were selected while considering the number of repeat motifs of markers and their locations across the 12 chromosomes of rice. Following effective PCR amplification and the cycling conditions used in this investigation, 83 polymorphic markers between the two parents were found. The 475 markers examined revealed a total amount of polymorphism spread across the 12 chromosomes of 17.47%. The outcome revealed that there were between four (on chromosomes 1 and 10) and ten verified polymorphic markers (on chromosomes 2 and 8). Chromosomes 5 and 12 had nine polymorphic markers, while chromosome 4 had seven polymorphic markers, and chromosomes 3, 6, 7, 9, and 11 had six polymorphic markers each (Table 1). Through the MABB program, the 83 polymorphic background markers were used for background selection to find out what percentage of the recurrent parent genome was found in the BC₁–BC₂ offspring that had multiple BLB resistance genes added.

 

Authors’ action:

• The revised text was started with line No. 176 page 5 till line No. 195 page 5. In the following, we had copied the text inserted into the paper.

 

 

 

Concern # 5:

Would you please add pictures of the obtained germplasm with high levels of resistance and high yielding? 

Authors’ response:

• We agree with the reviewer that it is an important factor the we added pictures of the obtained germplasm with high levels of resistance and high yielding.

Figure 8. MR297, IRBB60, and the best BC₂F₂.

 

Figure 9. The best BC₂F₂ lines during vegetative phase.

 

Figure 10. The best BC₂F₂ lines during ripening phase.

 

Authors’ action:

• The pictures added as Figures 8, 9, and 10 with line No. 359 page 12 till line No. 369 page 13. In the following, we had inserted into the paper.

 

Reviewer-1# Comments and Suggestions for Authors.

Minor Concern

Concern # 1:

There are many clerical errors throughout the manuscript. Please check them carefully and revise them.

 

Authors’ response:

• We would like to thank the reviewer for this comment; indeed, the comments were considered and revised.

 

Authors’ action:

• The revised text was started with all manuscript.

 

Concern # 2:

This is not correct in abstract. Xoo resistance genes from MR297 or IRBB60? Introgression of four Xoo resistance genes from the high-yielding Malaysian MR297 lineage Xa21, xa13, xa5, and Xa4 from the IRBB60 lineage.

 

Authors’ response:

• We would like to thank the reviewer for this comment; indeed, the comments were considered and revised.

Authors’ action:

• The revised text was started with line No. 20 page 1.

 

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Minor concerns：

1. All gene names (Xa21, xa13, xa5, Xa4) and Latin abbreviations (XOO) should be italic.

2. All numbers in BC₁F₁, BC₂F₁ and BC₂F₂ should be converted into subscript.

3. Convert the number "2" in "cm²" in line 283 and table 5 should be converted to superscript.

4. The abbreviation "FLOWER" in Table 5 is inconsistent with the description in the note.

5. Remove "s" from "BLBs" in line 78. The connection symbol "-" could be removed from "Ac-coding", "11-mm" and "2.78-mm" in line 388 and line 293 respectively.

 

Major concerns：

6. The main purpose of this study is the introduction of disease resistance genes, but there are no phenotype pictures of improvement lines in the text. I suggest the author provide some necessary pictures.

7. The transcriptional activator like effectors (TALEs) of bacterial blight directly interact with Xa5 to activate rice susceptibility genes, which is conducive to the growth and reproduction of pathogenic bacteria in rice. When Xa5 is mutated into xa5, TALEs and xa5 have no interaction and cannot activate the expression of susceptible genes, thus inhibiting the transfer of pathogenic bacteria. Xa13 is one of the important susceptible genes activated by Xa5 through the invasion of bacterial blight pathogen, and is also a key regulator response to rice pollen development and grain filling. Therefore, is it necessary to pyramid xa5 and xa13? Does xa13 affect yield traits? Xa5 is required to participate in the transcription of Xa23, Xa27 and Xa7 in rice. Loss of function of Xa5 will inhibit the role of Xa23, Xa27 and Xa7. Does xa5 affect the resistance of several other disease resistance genes in this study? Therefore, my suggestion is to analyze the resistance and agronomic traits of different combinations of disease resistance genes.

8. This study does not consist any research of rice blast resistance, hence the conclusions in lines 388 to 390 are inaccurate.

Author Response

Reviewer-2# Comments and Suggestions for Authors.

 Minor Concern

Concern # 1:

All gene names (Xa21, xa13, xa5, Xa4) and Latin abbreviations (XOO) should be italic.

 

Authors’ response:

• We would like to thank the reviewer for this comment; indeed, the comments were considered and revised.

Authors’ action:

• The revised text was started for All gene names (Xa21, xa13, xa5, Xa4) and Latin abbreviations (Xoo) for all manuscript.

 

Concern # 2:

All numbers in BC₁F₁, BC₂F₁ and BC₂F₂ should be converted into subscript.

 

Authors’ response:

• We would like to thank the reviewer for this comment; indeed, the comments were considered and revised.

Authors’ action:

• The revised text was started all numbers in “BC₁F₁, BC₂F₁and BC₂F₂” were converted into subscript with all manuscript text.

Concern # 3:

Convert the number "2" in "cm²" in line 283 and table 5 should be converted to superscript.

 

Authors’ response:

• We would like to thank the reviewer for this comment; indeed, the comments were considered and revised.

Authors’ action:

• The revised text was started with line table 5 page 12.

 Concern # 4:

The abbreviation "FLOWER" in Table 5 is inconsistent with the description in the note.

 

Authors’ response:

• We would like to thank the reviewer for this comment; indeed, the comments were considered and revised.

Authors’ action:

• The revised text was started with line table 5 page 12.

 Concern # 5:

Remove "s" from "BLBs" in line 78. The connection symbol "-" could be removed from "Ac-coding", "11-mm" and "2.78-mm" in line 388 and line 293 respectively.

 

Authors’ response:

• We would like to thank the reviewer for this comment; indeed, the comments were considered and revised.

Authors’ action:

• The revised text was started with line No. 78 page 2 and line No. 323 page 12. In the following, we had copied the text inserted into the paper.

Reviewer-2# Comments and Suggestions for Authors.

Major Concern

 Concern # 6:

The main purpose of this study is the introduction of disease resistance genes, but there are no phenotype pictures of improvement lines in the text. I suggest the author provide some necessary pictures.

 

Authors’ response:

• We would like to thank the reviewer for this comment; indeed, the comments were considered and revised.

Authors’ action:

• The pictures added as Figures 8, 9, and 10 with line No. 359 page 12 till line No. 368 page 13. In the following, we had inserted into the paper.

 

 

 

Figure 8. MR297, IRBB60, and the best BC₂F₂.

Figure 9. The best BC₂F₂ lines during vegetative phase.

Figure 10. The best BC₂F₂ lines during ripening phase.

 

Concern # 7:

The transcriptional activator like effectors (TALEs) of bacterial blight directly interact with Xa5 to activate rice susceptibility genes, which is conducive to the growth and reproduction of pathogenic bacteria in rice. When Xa5 is mutated into xa5, TALEs and xa5 have no interaction and cannot activate the expression of susceptible genes, thus inhibiting the transfer of pathogenic bacteria. Xa13 is one of the important susceptible genes activated by Xa5 through the invasion of bacterial blight pathogen, and is also a key regulator response to rice pollen development and grain filling. Therefore, is it necessary to pyramid xa5 and xa13? Does xa13 affect yield traits? Xa5 is required to participate in the transcription of Xa23, Xa27 and Xa7 in rice. Loss of function of Xa5 will inhibit the role of Xa23, Xa27 and Xa7. Does xa5 affect the resistance of several other disease resistance genes in this study? Therefore, my suggestion is to analyze the resistance and agronomic traits of different combinations of disease resistance genes.

 

Authors’ response:

• We would like to thank the reviewer for this important point. I have examined my new varieties selected BC₂F₂ plants by test them for BLB resistance using phenotypic data (Isolation and multiplication of Xoo).Procedure for challenging of rice germplasm for  Eight separate sites were used to gather the sick rice samples. A single cell was extracted from the slimy, yellowish bacterial colony that had grown around the infected samples using a sterilized inoculating wire loop, and it was then zigzagged streaked onto a plate of nutritional agar. Plates were streaked, and then incubated for two days at 25 to 26°C. Each culture plate was filled with 5 ml of distilled water, the bacterial colonies were suspended, and the MacFarlane standard was used to adjust the inoculum concentration to 108 cfu/ml.

Using Scoring for length of lesion (LL)

“No.”	“Lesion length (cm)”	“Description”
1	“< 5.0”	“R = resistant”
2	>5.0	“S = susceptible”

 

 

 

Also using Scoring system for percentage disease leaf area (%DLA)

“Disease score”	“Lesion area (%)”	“Disease reaction”
1	“1-5”	“R”
3	“6 - 12”	“MR”
5	“13 – 25”	“MS”
7	“26 – 50”	“S”
9	“51 – 100”	“HS”

 

 

Authors’ action:

 

• We are now evaluating and analysing the data from this experiment in preparation for publication.

 

 

Concern # 8:

This study does not consist any research of rice blast resistance; hence, the conclusions in lines 388 to 390 are inaccurate.

 

Authors’ response:

• We would like to thank the reviewer for this comment; indeed, the comments were considered and revised.

 

Authors’ action:

• The revised text was started with line No. 423 page 16.

 

Author Response File: Author Response.pdf