Genotyping the High Protein Content Gene NAM-B1 in Wheat (Triticum aestivum L.) and the Development of a KASP Marker to Identify a Functional Haplotype
Round 1
Reviewer 1 Report
Increase of protein content is one of the major breeding objects. The previous studies reported that NO APICAL MERISTEM-B1 (NAM-B1) gene can regulate wheat grain protein content, and three haplotypes were found in this gene. In this study, authors aimed to identify new resources with high protein content and developed new molecular maker for MAS breeding. Among 165 genetic associations, three of them carried the wild type NAM-B1 allele. This study provided the information on selection of valuable genetic resources with high protein content and reliable KASP molecular markers, but some format or typo mistakes need to be resolved in the references. In addition, authors should also try to develop new STS or KASP makers to detect the presence of NAM-B1 instead of SSR markers.
Minor editing of English language was required.
Author Response
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Reviewer 2 Report
Dear Editor,
I have reviewed the manuscript titled “Identifying a Functional Haplotype of the High Protein Content Gene NAM-B1 in Wheat (Triticum aestivum L.) using a Novel KASP Marker” by Cha et al.
Wheat is one of the major staples for humans and it has been cultivated in many parts of the world. Wheat grain quality is the major breeding focus after yield, in which, protein content is the key trait. The protein content of the harvested wheat grains plays an important role in determining the market class and price in many countries. Thus, often, the farmers would choose a variety with excellent protein content and quality over slight yield compromise in some parts of the wheat growing regions. Identification and map-based cloning of the “High Grain Protein Content-1” (GPC-B1) locus in wheat motivated wheat breeders throughout the world to introgress this gene to increase protein content in their germplasm. Normally, improved wheat varieties contain grain protein between 8-12%, but lines introgressed with GPC-B1 allele often produce up to 18%. However, many later reports showed that lines with introgressed GPC-B1 locus negatively affected test weight and significantly reduced grain yield. Breeders still use the GPC-B1 in combination with other quality and/or yield related alleles in their program to improve overall grain quality without compromising the yield.
The GPC-B1 locus was cloned in 2006 and found three major alleles viz. a wild type-functional, a +1bp insertion-non-functional, and a deletion (failure of marker amplification-to be more precise) assumed to be non-functional. Since then number of studies have used the GPC-1 markers for allelic diversity analysis and marker assisted selection. So far, no reliable marker has been developed for the detection of the deletion allele.
In this manuscript, the authors used SSR markers to screen 165 wheat cultivars and identified 41 varieties that showed GPC-B1. They used Sanger sequencing methods to identify the casual ‘NAM-B1’ allele in the 41 wheat cultivars. Additionally, they developed and validated a KASP marker for identifying the NAM-B1 wildtype and +1 bp insertion allele to use in high throughput genotyping. The manuscript was written clearly and coherently. The introduction and method were detailed enough to understand, and the results were well written. The presence of GPC-B1 in < 25% of the varieties is not surprising and the identification of one variety with wild type allele in this study is the new finding.
Although the idea of developing a high throughput marker for this locus is needed and will be highly useful to the community, the way the experiments were conducted need a major revision. The authors did not include No Template Control (NTC) in their experiment, which is necessary for the KASP assay analysis. The fluorescent values for the varieties with GPC-B1 deletions should fall close to NTC and not with functional wild type alleles. I would suggest the authors repeat the reaction with appropriate controls.
Minor comments or questions:
1. The authors used two SSR markers (Xucw108 and Xucw109) that are downstream of the causal NAM-B1 gene for the PCR analysis from the Uauy et al. (2006). Why did the authors not use markers upstream of the gene?
2. Why didn’t the authors use water controls for contamination or marker control for detecting DNA degradation in their PCR screening?
3. The bibliography needs rearrangement as some of the citations do not match with the references.
Author Response
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Reviewer 3 Report
The paper screened the haplotype of the NAM-B1 gene in 165 wheat cultivars. Among them, 41 carried the GPC-B1 locus. However, only 3 out of 41 contain the wild-type NAM-B1 while the remaining showed a 1-bp insertion in the gene. The authors finally proved that utilizing the KASP assay which uses allele-specific primers could discriminate different haplotypes of NAM-B1. The experiments were properly designed, and the results supported their idea. I don’t have major concerns and only have some minor questions/suggestions.
In line 48, is that normal for wheat to have such a big range (2.1-72 g/kg) of GPC? In the same line, compared to “what value of GPC” in the recurrent parents?
Author Response
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Reviewer 4 Report
The manuscript entitled "Identifying a Functional Haplotype of the High Protein Content Gene NAM-B1 in Wheat (Triticum aestivum L.) using a Novel KASP Marker” described the work of screening 41 out of 165 wheat cultivars carrying GPC-B1 locus, and then through sequencing gene region identified three cultivars carried wild type NAM-B1 gene and 38 carried the variant that has a 1-bp insertion in the NAM-B1 gene. A KASP marker for detecting the variation was then developed. Even though this is for a brief report, a major revision is required:
1) The title of the manuscript is misleading:
a) The wild type or mutant of NAM-B1 or even the GPC-B1 locus were not identified using the KASP marker, but by the sequencing method.
b) I never saw someone says a novel marker since any marker, if developed, are novel, and that’s why we develop markers for traits, and no one will put effort on developing a marker if the marker is already existed.
c) What does “Functional Haplotype” mean? I didn’t see any work in this research was done related to gene function; how can you say a Functional Haplotype?
2) More detailed background information of NAM-B1 need to be added in the introduction.
3) This research only did gene sequencing but have no information about protein content of all tested cultivars. It will be easy to test protein concentration of cultivars that have no GPC-B1, have GPC-B1 but a mutant NAM-B1, or have GPC-B1 with a wild-type NAM-B1. Does the cultivar with a wild-type NAM-B1 gene certainly have higher protein concentration than the others without? How can you claim the higher protein concentration is totally due to a wild-type NAM-B1? What’s the performance of cultivar ‘Benhur’ for protein content? You claim it is a novel cultivar (see line 151), does that mean it has unique background from the other cultivars?
4) According to cloning of NAM-B1, markers Xucw108 and Xucw109 both are on the right side of gene NAM-B1. Is it possible the recombination could occur between NAM-B1 and Xucw108? If this is true, markers Xucw108 and Xucw109 won’t be able to detect gene NAM-B1.
5) Full length of NAM-B1 is 1542 bp, but sequencing area in this research is 555 bp (Table 1), why not to compare the whole sequence but only partially?
6) The KASP marker seems classified nonfunctional (blue in Fig. 3) and wild-type/gene deletion (red in the same figure), so how can this marker for differentiating wild-type and gene deletion?
English is fine for this manuscript.
Author Response
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Author Response File: Author Response.pdf
Round 2
Reviewer 4 Report
I recommend to publishing this manuscript.
