Mapping QTLs underpin nutrition components in aromatic rice germplasm

As rice is an important staple food globally, research for development and enhancement of its nutritional value it is an imperative task. Identification of nutrient enriched rice germplasm and exploiting them for breeding programme is the easiest way to develop better quality rice. In this study, we analyzed 113 aromatic rice germplasm in order to identify quantitative trait loci (QTL) underpinning nutrition components and determined by measuring the normal frequency distribution for Fe, Zn, amylose, and protein content in those rice germplasm. Comparatively, the germplasm Radhuni pagal, Kalobakri, Thakurbhog (26.6 ppm) and Hatisail exhibited the highest mean values for Fe (16.9 ppm), Zn (34.1 ppm), amylose (26.6 ppm) and protein content (11.0 ppm), respectively. Moreover, a significant linear relationship (R2 = 0.693) was observed between Fe and Zn contents. Cluster analysis based on Mahalanobis D2 distances revealed four major clusters of 113 rice germplasm, with cluster III containing a maximum 37 germplasm and a maximum inter-cluster distance between clusters III and IV. The 45 polymorphic SSRs and four trait associations exhibited eight significant quantitative trait loci (QTL) located on eight different chromosomes using composite interval mapping (CIM). The highly significant QTL (variance 7.89%, LOD 2.02) for protein content (QTL.pro.1) was observed on chromosome 1 at 94.9cM position. Also, four QTLs for amylose content were observed with the highly significant QTL.amy.8 located on chromosome 8 exhibiting 7.2% variance with LOD 1.83. Only one QTL (QTL.Fe.9) for Fe content was located on chromosome 9 (LOD 1.24), and two (QTL.Zn.4 and QTL.Zn.5) for Zn on chromosome 4 (LOD 1.71) and 5 (LOD 1.18), respectively. Overall, germplasm from clusters III and IV might offer higher heterotic response with the identified QTLs playing a significant role in any rice biofortification breeding program and released with development of new varieties.

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Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation characteristics of epigenetic variation at different levels in both varieties and mutant 23 groups were examined. The results showed that: (1) the global genomic DNA 24 methylation level of the 91 bud mutants of 'Fuji' ranged from 29.120%-45.084%, with 25 an average of 35.910%. Internal cytosine methylation was the main DNA methylation 26 pattern. Regarding the variation of methylation patterns of 'Fuji' mutants, the vast 27 majority of loci maintained the original methylation pattern existed in 'Fuji'. CHG 28 methylation variation was the main type of variation. (2) The variation in methylation 29 patterns between the mutant groups was greater than that of methylation levels. Among 30 these patterns, the variation in CHG methylation patterns (including CHG 31 hypermethylation and CHG demethylation) was expected to be dominant. The observed 32 variation in methylation levels was more important in the Color mutant group; however, 33 the variation in methylation patterns was more obvious in both the early maturation and 34 Spur mutant groups. Moreover, the range of variation in the Early-maturation group 35 was much wider than that in the Spur mutant group. Malus domestica Borkh. cv. 'Fuji' is one of the most economically important apple 44 varieties in China and the world. 'Fuji' apple is prone to mutation, through which 45 abundant bud mutants have been derived, the majority of which have been adapted as 46 superior varieties in production [1]. Studies have shown that epigenetics is an important 47 cause of bud mutations in fruit crops, but it has not yet to be systematically studied in 48 a large sample size. The study of the characteristics of epigenetic effects in bud mutants 49 is the premise for further studying the epigenetic mechanism of mutations and carrying 50 out epigenetic breeding. 51 Epigenetics studies the heritable changes in gene function that eventually lead to 52 phenotypic variation with no changes in the underlying DNA sequence [2]. Epigenetics 53 is involved in the regulation of gene expression, and changes are dynamic with respect 54 to the endogenous and/or external environmental stimuli, thus affecting the phenotypic 55 plasticity and environmental adaptability of organisms [3]. Epigenetic variation 56 enhances biodiversity and complexity, especially in asexual organisms without gene 57 recombination, which is helpful to the promotion of functional phenotypic diversity and 58 has a greater impact on variation and evolution and more survival significance [4]. 59 DNA methylation, representing the most important form of epigenetic modification, 60 is ubiquitous in higher plants [5]. predominately in many plant species [11]. 70 The current research on DNA methylation is mainly focused on local and global 71 aspects. The former mainly targets specific traits, starting with the changes directly 72 related gene methylation in promoter or gene core sequence, and has more advantages 73 in revealing the mechanism of occurrence of specific phenotypic variation; The latter 74 studies the changes in the overall methylation of the genome, which is more conducive 75 to the comprehensive analysis of the role of epigenetics in the variation and diversity. 76 Many techniques have been developed to analyze global DNA methylation and its 77 alterations [12]. The method of methylation-sensitive amplified polymorphism (MSAP) 78 is one of the most efficient, economical, and widely one used for the detection of DNA 79 methylation events [13][14]. This approach is a modified version of the amplified 80 fragment length polymorphism (AFLP) method based on the differential sensitivity of 81 isoschizomeric restriction endonucleases to site-specific cytosine methylation [15]. 82 MSAP analysis employs two types of cleaved enzymes EcoRI (rare cutter) and 83 HpaII/MspI (frequent cutters) which recognize similar tetranucleotide 5'-CCGC 84 representing a different sensitivity to methylation at the inner or outer cytosine. If one 85 or both cytosines are methylated at both DNA strands, HpaII is inactive. If one or both 86 cytosines are methylated in only one strand they are cleaved by HpaII. In contrast, MspI 87 reacts when only the internal cytosine is hemi-or fully-(double strand) methylated [16]. 88 So, on the basis of variant band patterns resulting from differential digestion of the 89 genome by HpaII/MspI isozymes, variation in the DNA methylation level and pattern 90 in the whole genome is detected [15,17]. MSAP has been successfully applied for the 91 analysis of the variation in methylation levels and patterns in a variety of plant species 92 [13]. 93 In general, bud mutations are an important source of new varieties of fruit crops. 94 Characteristics such as a perennial nature, long juvenile phase, heterozygosity, and 95 sexual incompatibilities in fruit crops hamper their improvement through conventional 96 breeding [18]. Compared with conventional hybridization, the selection of bud mutants 97 gives the advantages of shortening the breeding cycle and reducing the workload and 98 costs. Such an approach can be used to obtain excellent varieties by modifying 99 individual traits without changing the desirable qualities of the parent plant [19]. A 100 variety of perennial fruit trees of economic importance originated from bud changes 101 [20][21]. However, Spontaneous bud mutation occurs at a very low frequency, moreover, 102 many of these mutations may be deleterious, making the organism less adapted to its 103 environment, and in some cases may even be lethal [18]. Therefore, mutants that 104 survive in adverse environments and even present excellent phenotypes are considered 105 to have good characteristics for adaption. Epigenetic regulation mediated by DNA 106 methylation is considered as one of the important mechanisms in plant adaptive 107 procedure [14,22]. Studying the molecular mechanism of bud mutation at the DNA 108 level is thus of great significance. 109 Fuji is the most representative apple cultivar in which plenty of new bud mutations 110 arise [23]. Multiple bud mutants generated from the standard cultivar Fuji. Those 111 represent highly similar genetic backgrounds and have abundant types of variation. 112 Additionally, Fuji bud mutants were frequently reported to be available in orchards at 113 unusual locations, such as under dense high-voltage lines, at high altitudes, subjected 114 to an abnormal climate, severe drought, waterlogging, frost, or sudden diseases or insect 115 pest infestations [24]. This means that the conditions for induction of bud mutation are 116 consistent with the factors inducing epigenetic effects. Therefore, in a summary of the 117 above mentioned, Fuji mutation line could be regarded as ideal sets for research on 118 epigenetic regulation. However, to our knowledge, global DNA methylation in this line 119 has not been reported yet. 120 A general understanding of the mechanisms of genome-wide DNA methylation in 121 'Fuji' mutants is a prerequisite for their utilization in epigenetic mechanism studies or 122 breeding. Therefore, in the present study, nearly one hundred 'Fuji' bud mutants were 123 used as study materials for the first time. Then, through genetic variation as control, we 124 focused on the analysis of variations in DNA methylation levels and patterns within 125 groups and between groups as well as epigenetic diversity. 126 We were interested in the following questions: (1)  The primer sequences of MSAP and AFLP were listed in the S1 Table. Based on   155 previous pilot tests, we selected 23 optimal primer combinations for MSAP analysis 156 (S2 Table) and 19 optimal primer combinations for the AFLP analysis (S3 Table). HM-, 157 E-, and M-corresponded to the sequence of H/M00, E00, and M00, respectively (S1 158   Table). All types of methylation 285 variation patterns (15 subclasses) were detected in the test varieties.    Table, CHG-hypo, CHG-hyper, CG-hyper, and CG-hypo displayed differences 300 among different tested varieties, but the general trend basically showed the following 301 correlations: CHG-hypo>CHG-hyper>CG-hyper>CG-hypo (S2 Fig.). (CHG-302 hypo+CHG-hyper) was significantly higher than (CG-hypo+CG-hyper) (P<0.01). The 303 relative trend between the total demethylation frequency and the hypermethylation 304 frequency in different varieties also exhibited diversity, including the following 305 findings: (1) the demethylation frequency was higher than the frequency of methylation, 306 (2) the hypermethylation frequency was higher than that of demethylation, and (  Color mutant groups was very significant (Fig 2). Taken together, the above results 375 showed that among the three 'Fuji' mutant groups, the Spur and Early-maturation 376 groups showed similar epigenetic patterns; however, between the Color groups and 377 either the Spur group or the Early-maturation group, epigenetic differences were 378 apparent.  Table) showed that the epigenetic similarity coefficient between varieties ranged from 388 0.515 to 0.807; the lowest similarity was found between 'Yishuizhongqiu' and 'Wengao  Table). The mean similarity coefficient 438 between the standard 'Fuji' variety and its mutants was 0.80, ranging from 0.684 439 (between 'Fuji' and 'Su Fuji') to 0.877 (between 'Fuji' and 'Yanfu No.7'), indicating 440 that the genetic variation of the mutants differed from that of 'Fuji''. 441 The UPGMA results showed (Fig 3(B showed that most of the genetic variation existed within the mutant groups (95%); a 480 very small portion (5%) existed between the populations (P< 0.01).  reports present results show that the proportion of the former is greater than of the latter 613 [11]. In this study, the levels of CG and CHG methylation were found to be 22.311% 614 and 13.599%, respectively. The level of the former was significantly higher than that In conclusion, the present study uncovered abundant changes in methylation levels 663 and patterns between not only bud mutants and their mother 'Fuji' but also bud mutants,                  Table). HM-, E-, and M-corresponded to the sequence of H/M00, 310 E00, and M00, respectively (S1 Table).    (Table   418 1). The number of amplified methylated loci ranged from 1793 to 2136, and that of     Table).   Table. 439  Table). All types of methylation variation patterns (15 subclasses) were detected in 459 the test genotypesvarieties.

Analysis of DNA methylation levels and variation patterns at
460 461   The CVs of each parameter were compared between the three mutant groups and 537 analyzed (Table 3). It was shown that the emphases of epigenetic variation were  Table) showed that the epigenetic The UPGMA results showed (Fig 3(A)) that nearly 84% (43/51) of the 603 genotypesvarieties varieties in the color Color mutant group clustered closely together 604 to form an independent cluster (Cluster 1). The-e Early-maturation mutant group, the The UPGMA results showed (Fig 3(B) The analysis of molecular variance (Table 5) showed that most of the variation occurred within 691 the mutant groups (88%), and only a very small portion (12%) occurred among the mutant groups. 692 The input binary distance matrix was used to calculate the value of PhiPT, and locus-by-locus 693 AMOVA showed ( than that in the other two intervals, but these loci were highly differentiated, accounting for 10.53% 700 and 8.92% of the total number of loci, respectively. 701 702  accounting for 0.92915% of the total DNA methylation-sensitive loci (Fig 6(A)). The differentiation between the three 'Fuji' mutant groups (Fig 6(B)). By comparing the results of AMOVA with the AFLP and MSAP results (Table 5), it 846 could be seen that the mutant groups showed a certain degree of differentiation 847 according to both markers, and the epigenetic differentiation ratios within and between 848 the groups were greater than the genetic differentiation ratios. The group mean PhiPT 849 results (genetic: 0.05; epigenetic: 0.12) showed that the degree of epigenetic 850 differentiation was also higher than that of genetic differentiation. 851 The results of pairwise PhiPT (Table 5) between groups showed that the degree of 852 differentiation between the three mutant groups was similar, but the epigenetic variation 853 was significant. The epigenetic differentiation value (0.03) between the spur and-early-

Dear editor:
Thank you for the thoughtful and thorough review. We are very grateful for your and reviewer's nice comments on our manuscript. According to the advices, we amended the relevant parts in manuscript (No. PONE-D-20-01600). The editorial decision on our previous manuscript was major revision. Moreover, some problems in language were existed. So, in general, the present manuscript has been greatly revised in contents as well as language. Some problems in grammar raised by reviewers were corrected and rephrased; some were removed due to the adjustment in contents. Please see the new revision for details. The followings are mainly answers to the questions raised by reviewers. Thank you very much!

To #1 reviewer： Comment
Response ■ -Text is wordy and language not well structured. English should be improved throughout.
The section of the Introduction and Discussion have been rewritten. Also, we asked AJE: English Editing & Author Services for Research Publication as well as our friends oversea to help do correction in the revised manuscript. The certificate from AJE company will be attached in this manuscript. We are really sorry for the troubles caused to you because of our poor English.
■ line 91-94. Not clear. Score '1' we also get by digestion by either HpaII, MspI enzymes due to no methylation (band present in both). This is done in order to distinguish between MSLs (methylation-susceptible) and NMLs (nonmethylated) data in the MSAP amplification results since only the MSP data are meaningful. We use the R package msap for relevant statistics, not manual score. Therefore, in the revised manuscript, avoiding wordy, the explanation of this part was removed in the introduction section. Actually, the '1' here refers to two situations that a certain sample is cut by HpaII and MspI. For situation of H0M1 or H0M1, when the band is scored to make MSLs matrix, both will be recorded '1'. ■Is not clear if Fuji mutants are considered varieties or genotypes. The repeatability of banding patterns assessed by conducting two sets of independent MSAP and AFLP analyses and only the consistent bands were included.

Response to Reviewers
(see line 159-160 in the new version manuscript) ■line 157. How were leaves stored?
Young leaves immediately frozen in liquid nitrogen and then stored at -80℃ prior to DNA isolation. (see line 142 in the new version manuscript) ■lines 157-158. Which were these developmental and phenological stages? Perhaps authors could be more specific.
We sampled plant materials with the same age on the same dates (12 June 2016) when they were bearing fully expanded leaves. (see line 138 in the new version manuscript)

Content
Comment Response Introduction ■it seems that more than one author wrote the introduction. English must be improved in several parts (especially the first half). In the new revised version, we have tried our best doing complementary in statistically significant analysis for items that could be conducted for significant analysis. But we are really sorry.
Because we used one mixed sample per variety in our study, for some items, like CVs between different mutant groups, it was impossible to perform significance analysis. ■line278. what statistical analysis have you conducted to say that??
In the new revised draft, we performed a statistical analysis of the significant difference between CG and CHG methylation levels. ■line291. In how many cases do they occur these changes in the patterns of cytosine methylation?
We have tabulated the statistical results in the supplementary materials section. (See new added S5 Table) ■line303. 91 varieties???
Yes. It should be 91 varieties. ■line305. this paragraph better fits to the discussion section. line313. also this paragraph better fits to the discussion section.
The relevant content in these two paragraphs has been simplified and doing statement in the discussion section.
Line447. not a clear seperation between the different groups Yes. Because this is the clustering result based on AFLP data. The results showed that, unlike the epigenetic results, there was not a clear separation between the different groups genetically. They just presented local clustering and mixed phenomena. ■Line452. tested you have to be consistent using either genotypes, samples or varieties Thank for your advice very much. We have changed all of them to variety as a unified statement.
Discussion The discussion should be more focused on results. It's not clear If the epigenetic differences are significantly different between the three groups.
 We adjusted the content in the discussion section to make our discussion more focus on results. Moreover, we did difference significance analysis of epigenetic differences between the three groups. The result was significantly different between the three groups.


The discussion is very big and some points are very assumptive. it should be shortened and provide a clear conclusion which correlates directly with the results.
According to your advice, discussion was shorted. Additionally, we also added conclusions in the discussion section.
■line667. Can you provide any literature? Done. Please see literature 39-56. ■line716. is it statistically higher? please verify Yes. We performed a statistical analysis of the significant difference between CG and CHG methylation levels. The result exhibited significant difference (P<0.01). ■line720-729. Is there any literature supporting these results too?