Identification of Maize Rf4-Restorer Lines and Development of a CAPS Marker for Rf4
by
,
Yongming Liu
1,2,3,†
,
Ling Zhang
1,3,†,
Xiaowei Liu
1,†,
Peng Zhang
1,
Zhuofan Zhao
1,
Hongyang Yi
1 and
Moju Cao
1,* 1
Key Laboratory of Biology and Genetic Improvement of Maize in Southwest Region of Ministry of Agriculture, Maize Research Institute, Sichuan Agricultural University, Chengdu 611130, China
2
Institute of Urban Agriculture, Chinese Academy of Agricultural Sciences, Chengdu National Agricultural Science and Technology Center, Chengdu 610213, China
3
Hainan Yazhou Bay Seed Laboratory, Sanya 572025, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2022, 12(7), 1506; https://doi.org/10.3390/agronomy12071506
Submission received: 5 May 2022
/
Revised: 13 June 2022
/
Accepted: 18 June 2022
/
Published: 23 June 2022
Abstract
:Rf4 is one of the dominant restorer genes for maize C-type cytoplasmic male sterility (CMS-C), which has significant value in hybrid maize seed production. However, the highly complex fertility restoration mechanism of CMS-C makes it difficult to screen Rf4-restorer lines, and insufficient Rf4-restorer lines limit its use in current agricultural production. To search for Rf4-restorer lines, in this study, the genotypes of eighteen inbred maize lines at the Rf4 locus were analyzed based on the male fertility investigation of hybrid F1, the genetic analysis of F2 populations, molecular marker mapping, allelic tests, and Rf4 genomic sequence analysis. Our results indicated that of the eighteen maize inbred lines, ten were able to completely rescue CMS-C line CHuangzaosi (CHZS) male sterility. A genetic analysis showed that DAN598, PHT77, 78551S, and LH212Ht only contained one dominant restorer gene each, and the molecular-marker mapping indicated that their restorer genes were located at the short arm of chromosome 8. The allelic testing of the fertility of the restorer (Rf) demonstrated that the restorer gene of twelve inbred lines, including DAN598, PHT77, 78551S, and LH212Ht, was allelic to one restorer gene of A619. Furthermore, the genomic sequence alignment of Rf4 revealed that there were two different amino acids in the coding sequence between the A619 (Rf4Rf4) restorer lines and four CMS-C lines (rf4rf4). For the crucial S1596 site variation (TTT/TAC), DAN598, PHT77, 78551S, and LH212Ht shared the same bases (TTT) with A619 and encoded phenylalanine, while the four CMS-C sterile lines had the TAC and encoded tyrosine. Our results revealed that these tester lines, DAN598, PHT77, 78551S, and LH212Ht, were the Rf4-restorer lines. Additionally, derived from the sequence variants of Rf4, 39 possible Rf4-restorer lines from 129 inbred maize lines were detected. Furthermore, we developed a Cleaved Amplified Polymorphism Sequences (CAPS) marker based on the S1596 variations. The PCR amplification product of S1596 (TAC) was digested by the TatI endonuclease into two bands with sizes of ~260 bp and ~100 bp. In comparison, when S1596 was TTT, the PCR product could not be digested. In conclusion, in this study, we identified various Rf4-restorer lines for maize CMS-C and developed a molecular marker for Rf4. The reported results will contribute to the popularization and application of Rf4 in hybrid maize-seed production.
1. Introduction
Plant cytoplasmic male sterility (CMS) is a natural, widespread phenomenon in higher plants that is characterized by maternal inheritance and the inability to generate pollen grains while still producing normal female gametes. CMS is an imperative driver of heterosis utilization in plants, as it enhances the purity of hybrid seeds by eliminating the need for artificial emasculation [1,2]. Certain nuclear genes, termed restorers-of-fertility (Rf), can rescue CMS male fertility. CMS/Rf systems have been successfully used for high-efficiency hybrid seed production in various crops [3].
Maize is an important food and cash crop, and its demand is increasing around the world. The three-line system uses cytoplasmic male sterility lines, maintainer lines, and restorer lines, and it has significant application potential in commercial hybrid maize seed production [4]. According to the characteristics of the mitochondrial genome and the pattern of fertility restoration, sterile maize cytoplasms can be divided into three major types: T (Texas), S(USDA), and C (Charrua) [5,6]. CMS-T sterile lines are no longer widely used in commercial hybrid seed production because of their susceptibility to Bipolaris (Helminthosporium) maydis race T, which causes southern corn leaf blight [7,8]. S-type cytoplasmic sterility (CMS-S) is a form of gametophytic sterility, and its fertility can be easily affected by climatic factors [9]. In comparison, C-type cytoplasmic sterility (CMS-C) is becoming one of the most attractive tools for hybrid maize-seed production due to its resistance to southern corn leaf blight and its stable sterility [9]. Furthermore, CMS-C has a positive effect on grain yield [10,11].
The male sterility of CMS-C is caused by the chimeric mitochondrial gene atp6c [12], and its fertility restoration is controlled by multiple loci, including the major restorer-of-fertility (Rf) and the quantitative trait locus (QTL). Currently, the major reported restorer genes for CMS-C include Rf4 and Rf5 [13,14]. Rf4 is located at the end of the short arm of chromosome 8, and Rf5 is located at the long arm of chromosome 5 [14,15,16]. Furthermore, an inhibitor gene, named Rf-I, which can constrain the function of Rf5 but not Rf4, was mapped to chromosome 7 [17]. Rf5 has not been cloned yet, while Rf4 has been proven to encode a bHLH transcription factor (GRMZM2G021276) [18]. The allele of the Rf4 restorer gene is also annotated as Male Sterile23, an essential gene for tapetal development [19]. Through the single-cell RNA sequencing of meiocytes and microspores, it was found that Rf4 plays an important role in the maintenance of redox homeostasis in pollen formation [20]. Comparative transcriptomic and proteomic analyses revealed that Rf4 might boost energy generation to restore pollen fertility [21,22].
In addition to those major restorer genes, the male fertility recovery of maize CMS-C can be affected by several QTLs. Based on the study of weak restorer lines and translocation lines, some QTLs were found on the long arm of chromosome 3, the short arm of chromosome 4, the long arm of chromosome 5, and chromosome 9 [23,24]. Kohls et al. mapped three QTL loci with a high contribution rate that were distributed in bin 2.09, bin 3.06, and bin 7.03 [25]. Recently, we detected a major locus, qRf8-1, at the long arm of chromosome 8 in the inbred line A619 by QTL-seq [26]. Additionally, we found that there may be minor restoration sites in the nuclear genome of male sterile lines. Although these loci cannot restore CMS-C when they exist alone, they can participate in fertility restoration through polymerization in the hybrid offspring of male sterile lines and tester lines [27].
As a major restorer gene for CMS-C, Rf4 has a powerful restoring ability and can restore almost all CMS-C lines. However, very few Rf4-restorer lines have been identified so far, even though they have significant application potential in the utilization of CMS-C. Jaqueth et al. [18] reported that the mutation of Y187F enables Rf4 to restore the male fertility of CMS-C. Unexpectedly, we found that a few maize inbreds, such as 48-2 and Zi330, encoding phenylalanine at the functional site could not restore CMS-C lines [27]. Therefore, the genotyping of the Rf4 locus by analyzing its sequence features alone is not enough to determine the resilience of a maize line to CMS-C. In this study, to accurately identify strong Rf4-restorer lines, various investigative methods, including sterility restoring–maintaining relationship determination, genetic analysis, molecular-marker mapping, and allelic tests were performed. Moreover, a Cleaved Amplified Polymorphism Sequences (CAPS) marker based on Rf4 variations was developed for molecular-marker-assisted breeding.
2. Materials and Methods
2.1. Plant Materials
The plant materials used in this experiment comprised the maize CMS-C line C-Huangzaosi (CHZS, rf4rf4), inbred lines A619 (Rf4Rf4rf5rf5) and Guang10-2 (Rf4Rf4rf5rf5), 18 tester lines (Table 1), and 129 maize varieties (Table S1). Among these plant materials, A619 was provided by Prof. Jihua Tang of Henan Agricultural University, and the rest were provided by the Chinese Maize Industry Technology System or Maize Research Institute of Sichuan Agricultural University. In this research, all plant materials were numbered and routinely managed in the field.
2.2. Phenotyping Male Fertility
In this experiment, the male fertility of maize anther was investigated in the field with the methods reported by Kohls [25] and Feng [28]. Anther fertility was investigated every other day, from when tiller tassels began to branch until the end of flowering. Based on the degree of anther exertion and the amount of scattering pollens, plant male fertility was graded on a scale from I to V: (I) 0–5% of anthers exerted without any pollen, (II) 5–25% of anthers exerted with less pollen, (III) 26–50% of anthers exerted with normal scattering pollens, (IV) 51–75% of anthers exerted with normal scattering pollens, and (V) over 75% of anthers exerted with normal scattering pollens. Plants with scores of I or II were viewed as sterile, while scores of III, IV, and V were recorded as fertile plants [29].
2.3. Investigation of F1 Male Fertility
In the spring of 2015, 18 tester lines were pollinated to the CMS line CHZS in Wenjiang, Sichuan province to obtain hybrid F1 seeds. In the spring of 2016, about 28 plants were planted for each F1 cross in Wenjiang, Sichuan province, and a male fertility investigation was conducted in the field.
2.4. Genetic Analysis and Molecular Marker Mapping
In the summer of 2016, the F1 with completely restored male fertility were self-pollinated to harvest F2 seeds. In the spring of 2017, the male fertility of several F2 populations was investigated in Wenjiang, Sichuan province. In addition, the fertility recovery of A619 to CHZS was closely analyzed in this experiment. In the spring of 2017, the (CHZS × A619)F2 and CHZS × (CHZS × A619) populations were planted in Wenjiang, Sichuan province. In the summer of the same year, the (CHZS × A619)F2 population was also planted in Xinxiang, Henan province, and the male fertility investigation was carried out in the field.
The male fertility patterns of some F2 populations are not a typical normal distribution but a single or multi-peak distribution, indicating the existence of major genes. Accordingly, the maximum likelihood method was used to estimate the number (K) of major genes: K = (logN-logn)/0.6021, where N and n are the number of total plants and sterile plants (grade I and II) in the F2 population, respectively [30]. The chi-squared test was conducted after statistical analysis. Subsequently, molecular-marker analysis was conducted on the F2 populations with a segregation ratio of male fertile plants to male sterile plants of 3:1. Firstly, total genome DNA was extracted from individual plants with extreme phenotypes (grade I and grade V) in the F2 populations by the CTAB method [31] for subsequent analysis. Secondly, based on the reports of Kolhs [32] and Shao [33], 40 pairs of DNA markers at the top of the short arm of chromosome 8 were screened for polymorphic primers (see Table S2 for details). Finally, these polymorphic primers were selected for the genotyping of F2 individuals with extreme phenotypes. PCR amplification was performed using Master Mix and the following reaction conditions: initial denaturation at 94 °C for 6 min; 35 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 30 s; and a final extension at 72 °C for 2 min.
2.5. Allelic Tests
The population construction processes for Rf4 allelic testing are shown in Figure 1. If the genotype of a tester line was Rf4Rf4, the allele test population was entirely made up of fertile plants without fertility segregation. If the genotype of a tester line was rf4rf4, both fertile and sterile plants appeared in the allelic test population. Specifically, in the spring of 2015, the F1 generation was obtained by crossing the inbred line A619 or Guang10-2 with several tester lines in Wenjiang, Sichuan province. In the winter of 2015, the harvested F1 seeds were sown in Xishuangbanna, Yunnan province, and the pollen of F1 materials was collected to pollinate the CMS-C line CHZS for hybridization. In the spring of 2016, harvested allelic test population seeds were sown in Wenjiang for investigation of male fertility in the field.
2.6. Correlation Analysis between Rf4 Variants and Fertility Restoration Ability
The genomic sequence of Rf4, including its 5′ and 3′ untranslated regions (UTRs), was amplified by high-fidelity KOD FX DNA polymerase (Toyobo, Japan) with the primers (5′-GGAAGGAGGAAACCAAGTCG-3′, 5′-TGTAACGAGCAAGCGGATTTA-3′) [34], and PCR was performed as follows: 94 °C for 3 min; 40 cycles of 98 °C for 10 s, 58 °C for 30 s, and 68 °C for 3 min; and a final extension at 68 °C for 10 min. The amplified fragment was purified and then sequenced with an ABI 3730 xl DNA analyzer. After ambiguous sequences were manually deleted, the sequences of Rf4 among several inbred maize lines were analyzed using CodonCode Aligner 5.1 software (CodonCode Corporation, Dedham, MA, USA). In addition, in order to analyze the sequence information of 129 inbred maize lines at the key mutation sites of the Rf4 gene, a 785 bp fragment of Rf4 was amplified with the following primers: 5′-TGTTCGTGTGTTTCAGTTCAGC-3′ and 5′-AGATCACCGTCGCCCTATCA-3′. PCR amplification was performed using Master Mix and the following reaction conditions: initial denaturation at 95 °C for 5 min; 35 cycles of denaturation at 95 °C for 30 s, annealing at 52 °C for 30 s, and extension at 72 °C for 30 s; and a final extension at 72 °C for 8 min.
2.7. Molecular Marker Development for Rf4
A CAPS molecular marker was developed based on the sequence polymorphisms of Rf4 at S1596 (TTT/TAC). Firstly, the online software NEB cutter (http://nc2.neb.com/NEBcutter2/, (accessed on 25 May 2017)) was used to compare the digestion sites between TTT and TAC. Subsequently, a pair of PCR primers (5′-TACTGCGACGGCCACTACCC-3′, 5′-TGAAACGATGGACGAACATAAGAG-3′) was designed, and the amplified fragment contained the TTT/TAC variation with a size of ~360 bp. PCR amplification was performed using Vazyme Taq Mix and the following reaction conditions: initial denaturation at 94 °C for 5 min; 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s; and a final extension at 72 °C for 8 min. After the PCR products were confirmed by sequencing, they were digested by the TatI restriction enzyme. The enzyme digestion system comprised 30 μL: 17 μL of ddH2O, 2 μL of 10 × FastDigest Green Buffer, 10 μL of PCR amplification products (~200 ng DNA), and 1.0 μL of TatI FastDigest enzyme (Thermo Scientific™, Waltham, MA, USA). The enzyme digestion reaction was performed at 65 ℃ for 90 min, and a loading buffer was subsequently added to terminate the reaction. The digested products were detected with 3% agarose gel electrophoresis.
3. Results
3.1. Male Fertility Preformation of F1
The male fertility investigation results of the hybrid F1 showed that A619 (Rf4Rf4rf5rf5) and Guang10-2 (Rf4Rf4rf5rf5) completely rescued the male fertility of the CMS-C line, CHZS; among the other 18 tester lines, 10, 2, and 6 lines fully restored, partially restored, and did not restore the male fertility of the CHZS, respectively (Figure 2 and Table 2).
3.2. Genetic Analysis and Restorer Gene Mapping
The hybrid F1 crosses showing completely restored male fertility were then self-pollinated to obtain the F2 populations, and the male fertility of each F2 population was assessed (Table 3). By analyzing the male fertility grade distribution of each F2 population, we found that the male fertility grades in each F2 population were continuously distributed. Except for the (CHZS × PHW79)F2 and (CHZS × PHR55)F2 populations, most of the individuals were completely fertile in the other nine F2 populations. Hence, the number of restorer genes of these nine F2 populations was estimated using the maximum likelihood method. The chi-squared test showed that DAN598, PHT77, 78551S, and LH212Ht contained one dominant restorer gene and PHN82 contained two pairs of major restorer genes. The number of main restorer genes could not be estimated for other materials (Table 4). It is worth noting that A619 showed two pairs of restorer genes for CHZS when the (CHZS × A619)F2 were planted in Xinxiang city, Henan province (35°37′ N latitude, 113°90′ E longitude), while the restorer genes in A619 did not conform to the above-mentioned results when the (CHZS × A619)F2 and CHZS × (CHZS × A619) populations were planted in Wenjiang, Sichuan province (30°68′ N latitude, 103°85′ E longitude), which indicates that the external environment has an impact on the fertility-restoring function of A619.
To identify whether the dominant restorer genes in four inbred lines—DAN598, PHT77, 78551S, and LH212Ht—were allelic to Rf4, a linkage analysis was carried out on the corresponding F2 population with molecular markers at the top of the short arm of chromosome 8. Among the 40 pairs of molecular markers (see Table S2 for details), two SSR markers, b0329 (5′-CTCCGAACCTGATCCGAGTA-3′, 5′-AGGGGAGAGGTCCCAGAATA-3′) and S-4 (5′-ATGGCAGCAGGGCTTCAG-3′, 5′-CTGGGACCACCTGTTCACTG-3′), showed polymorphisms between CHZS and the four tester lines. Next, the genotyping analysis of extreme phenotypic individuals in four F2 populations was carried out using the b0329 and S-4 markers. It was found that there was obvious segregation distortion between the amplified product bands and the plant fertility (Figure 3 and Table 5). At the same time, for the four F2 populations, the number of recombinant plants detected by the b0329 (chr8: 245652) marker was lower than that detected by the S-4 (chr8: 1109020) marker. These results showed that the dominant restorer genes of DAN598, PHT77, 78551s, and LH212Ht were located at the top of the short arm of chromosome 8, which was consistent with the physical position of Rf4, indicating that these restorer genes might be allelic to Rf4.
3.3. Allelic Analysis
In order to explore the genotypes of each tester line at the Rf4 locus, 18 tester lines and A619 (Rf4Rf4rf5rf5) and Guang10-2 (Rf4Rf4rf5rf5) were used to construct allelic analysis populations (Table 6 and Table 7). In the allelic analysis, 19 populations were fertile plants, indicating that the corresponding tester lines contained the same dominant restorer gene as A619. Conversely, fertility segregation occurred in the other seven populations tested for allelism, with completely sterile, partially fertile, and completely fertile plants, indicating that they did not contain a dominant restorer gene or that their restorer genes were not allelic to those in the A619.
3.4. Correlation Analysis between Rf4 Sequence Variations and Fertility Restoration Ability
The Rf4 genomic sequences of five restorer lines (A619, DAN598, PHT77, 78551S, and LH212Ht) and four CMS-C lines (CHZS, C478, C698-3, and CRP128) were compared and analyzed. As shown in Table S3 and Figure S1, 34 nucleotide variations were detected in the Rf4 genomic sequences between the five restorer lines and four sterile lines, twenty-two of which were in non-coding regions and twelve of which were in exonic regions. Of the twelve sequence variations located in the exonic regions, four caused amino acid changes: Asn/HisS1343, Ala insertionS1433, Phe/TyrS1596, and Leu/ProS1957. Interestingly, five restorer lines displayed TTT at the S1596 locus, whereas four male sterile lines showed TAC at this locus. These results showed that the TTT/TAC variation at the S1596 locus was closely related to the fertility recovery function of Rf4. Furthermore, we analyzed the variation in the S1596 locus in 129 inbred maize lines and found that 39 varieties were TTT at this locus (Table S1), indicating that they might be strong restorers of CMS-C. However, this finding requires further verification.
3.5. Development of a CAPS Marker Based on the Rf4 S1596 Variations
Based on the restriction enzyme cleavage site analysis, it was found that there is a TatI enzyme cleavage site when the S1596 site is TAC. The TatI enzyme can specifically recognize the W^GTACW sequence (W is A or T), but the S1596 site is TTT and cannot be recognized by this enzyme (Figure 4). Therefore, TatI was selected as an endonuclease for the CAPS marker development. Furthermore, a ~360 bp fragment containing the S1596 locus was amplified and then digested by the TatI endonuclease. According to the results of the gel electrophoresis analysis (Figure 5), only when the S1596 site was TAC could the PCR product be digested into two fragments by TatI endonuclease, of which one was ~260 bp and the other was ~100 bp. These results showed that a CAPS marker based on the S1596 variant site was successfully developed for maize Rf4, which will facilitate the cultivation of Rf4-restorer lines using molecular-marker-assisted technologies.
4. Discussion
The male fertility restoration mechanism of CMS-C in maize is very complex. In addition to the role of restorer genes, the nuclear–cytoplasmic interaction and nuclear–nuclear interaction between the sterile and restorer lines may affect the fertility restoration of maize CMS-C [27]. The complexity of the fertility restoration of maize CMS-C makes it difficult to identify strong restorer lines. Currently, restorer lines are generally obtained via the continuous backcrossing of restorer genes. In order to enrich the germplasm resources of the CMS-C restorer lines, the authors of this study identified four Rf4-restorer lines with different pedigrees, DAN598, PHT77, 78551S, and LH212Ht, through a series of genetic analyses combined with molecular markers. The identification of these restorer lines should significantly facilitate maize-breeding programs utilizing the C-type cytoplasm.
The nuclear genome of male sterile lines may have an influence on the fertility restoration function of restorer lines [6]. For example, the inbred line 18Bai can restore the male fertility of the CMS-C line C48-2, but not CHZS; by contrast, the inbred line Zi330 can restore CHZS, but not C48-2 [27]. At the same time, according to the results of previous studies [13,16,24,35,36], Fengke1 shows one restorer gene for the CMS-C lines, CHZS and CENES (C-Ernanersi), and two pairs of restorer genes for CMo17 and C237. Similarly, Guang10-2 exhibits one restorer gene for CHZS and CENES and two Rf genes for CMo17. In addition, the major fertility-restoring gene Rf5 can only restore CMS-C lines lacking Rf5-I [17]. These results show that the restoring ability of restorer lines may be affected by the nuclear background of the sterile line. Hence, in the process of breeding or using restorer lines, the influence of the nuclear background of male sterile lines should be considered. In this experiment, DAN598, PHT77, 78551S, and LH212Ht were shown to completely restore the pollen fertility of CHZS, but their restoration function for other CMS-C lines may change. In future research, the recovery ability of these restorer lines needs to be further investigated.
In this study, A619 (Rf4Rf4) was used to determine the genotype alleles at the Rf4 locus. The results of the work of Chen et al. [13], Tang et al. [14], and Sisco [37] all showed that Rf4 is the only restorer gene in A619. However, in our previous study, we found that A619 had two pairs of restorer genes in the fertility restoration of CMS-C C48-2 [38]. According to Huang et al. [36], the dominant restorer gene in A619 may be located on chromosome 7, and the inbred line A619 may possess another dominant restorer gene in addition to Rf4. Furthermore, Sisco [37] not only mapped the restorer gene Rf4 to chromosome 8 but also inferred that Rf4 had a duplicate on chromosome 3 in A619. Because A619 may contain an extra dominant restorer gene, its allelic test alone cannot directly identify Rf4-containing restorer lines. Therefore, in this research, several F2 populations were further used to estimate the number of restorer genes in DAN598, PHT77, 78551S, and LH212Ht to CHZS, and molecular markers were used to map them. This experimental design ensured that the fertility restoration of CHZS by the four materials was only controlled by Rf4. The allelic test populations of the seven inbred lines (K10, PHW79, L127, L139, PHJ75, PHN82, and PHR55) showed full male fertility. This indicates that they contain dominant restorer genes; however, the restorer gene allele in A619 must be determined.
Similar to the (C48-2 × A619)F2 population we observed [27], the (CHZS × A619)F2 in this study showed different male-fertility grade distribution patterns at different sites. Tracy et al. [39] found that the proportion of fertile plants in the same cross population was significantly different in different years. Liu et al. [40] pointed out that cool and high-humidity external conditions are more conducive to the fertility restoration of CMS-C stamens. Chen et al. [41] argued that weak QTLs are more vulnerable to external conditions than major restorer genes. combined with our results, these studies indicate that the fertility recovery of CMS-C can be affected by environmental factors.
In this study, by comparing the Rf4 genomic sequence between several male sterile lines (rf4rf4) and restorer lines (Rf4Rf4), we found that there are four amino acid variations in the coding region of Rf4, of which the variation of the S1596 locus (TTT/TAC) tends to be more critical. The S1596 site encodes phenylalanine (TTT) in five restorer lines, and it encodes tyrosine (TAC) in four CMS-C lines. These results are consistent with previous reports and suggest that this amino acid residue encoded by S1596 is located within the core of the four-helix bundle, a region that is critical for stabilizing dimer conformation and influencing interaction partner selection [18]. Functional CAPS markers are especially helpful for marker-assisted selection, and they are widely used in the breeding of various crops and the rapid selection of novel restorer lines [42,43]. With the S1596 variations, we successfully developed a CAPS molecular marker within Rf4. Compared to the TaqMan marker developed by Jaqueth et al. [18], the CAPS marker tends to be more rapid, economical and reliable in practical application. Based on our results, these molecular markers can be used for the preliminary screening of Rf4-restorer lines; cross tests would then be required to further confirm the male-fertility-restoring ability of tester lines.
5. Conclusions
Based on the results of the male-fertility investigation, genetic analysis, molecular marker application, and allelic tests, this study proved that four maize inbreds, DAN598, PHT77, 78551S, and LH212Ht, are restorer lines containing Rf4. At the same time, based on the variations in the Rf4 genomic sequence, 39 possible strong restorer lines for CMS-C from 129 inbred maize lines were found. In addition, this study successfully developed a CAPS marker based on the variation of Rf4 at S1596. These results could promote the application of CMS-C in maize-breeding programs and provide the basis for further research to increase the understanding of the complexity of CMS-C fertility restoration.
Supplementary Materials
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy12071506/s1. Table S1. Genotype analysis of Rf4 among 129 maize inbreds. S1343, S1433, and S1596 are polymorphic sites that can cause amino acid changes between restorer lines and CMS-C lines. Table S2. DNA markers on the short arm of chromosome 8 used in this study. The underline indicates the markers reported by Shao Keke (2011) or Kohls (2010), and the physical location of each marker is based on the first base-pair location on the reference genome of maize B73 v4. Table S3. Genomic sequence alignment of Rf4 among several maize inbreds. The sites mean the base-pair locations in the Rf4 genomic sequence. Figure S1. Comparison of Rf4 genomic sequences between restorer lines and CMS-C lines. The four sites, S1343, S1433, S1596, and S1957, that can cause amino acid changes are marked.
Author Contributions
Y.L.: investigation, original draft, and funding acquisition; L.Z.: investigation and writing—review and editing; X.L.: investigation and formal analysis; P.Z.: methodology and visualization; Z.Z.: investigation; H.Y.: investigation; M.C.: conceptualization, project administration, and funding acquisition. All authors have read and agreed to the published version of the manuscript.
Funding
This research was funded by Sichuan Science and Technology Program: 2021YFYZ0017, 2020JDRC0044; Hainan Yazhou Bay Seed Laboratory: B21Y10210; Central Public-Interest Scientific Institution Basal Research Fund: S2022003.
Institutional Review Board Statement
Not applicable.
Informed Consent Statement
Not applicable.
Data Availability Statement
The data supporting the results of this study are included in the manuscript and its additional files.
Acknowledgments
We thank the Chinese Maize Industry Technology System for providing inbred maize lines for the experiments, with the aid of Guangtang Pan and Lujiang Li. We also thank Jihua Tang (Henan Agricultural University) and Shibin Gao (Sichuan Agricultural University) for providing several inbred maize lines.
Conflicts of Interest
The authors declare no conflict of interest.
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Figure 1.
Flowchart of allelic tests of the CMS-C restorer gene Rf4.
Figure 2.
Fertility performance of F1 crosses of CHZS and tester lines.
Figure 3.
Electrophoresis analysis of b0329 marker in some F2 individuals, derived from the cross between CHZS and the tester lines. F, fertile individuals; S, sterile individuals; P1, fertile parents; P2, the CMS-C line, CHZS.
Figure 3.
Electrophoresis analysis of b0329 marker in some F2 individuals, derived from the cross between CHZS and the tester lines. F, fertile individuals; S, sterile individuals; P1, fertile parents; P2, the CMS-C line, CHZS.
Figure 4.
Analysis of restriction enzyme digesting sites of S1596.
Figure 5.
Gel electrophoresis of the CAPS marker variants of the Rf4 gene in maize lines.
Table 1.
Names and pedigree information of 18 maize tester lines used in this experiment.
| No. | Inbreds | Pedigree | No. | Inbreds | Pedigree |
|---|---|---|---|---|---|
| 1 | DAN598 | (Dan340/Danhuang11) × (Danhuang02/Dan599) | 10 | L127 | P3901 × W117 |
| 2 | K10 | (Chang3/5003)/Chang3 | 11 | L139 | P3901 × P3780 |
| 3 | ZH03 | Unknown | 12 | E8501 | 387 × FRMo17 |
| 4 | ZJ102 | Unknown | 13 | PHJ75 | (207/G96) × 12241 |
| 5 | ZJ302 | Unknown | 14 | PHN82 | (PHG29/HD38) × 5333X |
| 6 | ZJ402 | Unknown | 15 | LH212Ht | LH123Ht × (LH123Ht × LH24) |
| 7 | PHT77 | (814/995) × 8111XX | 16 | PHR55 | PH005 × PHG84 |
| 8 | 78551S | 78060A × LH38 | 17 | PHR58 | PH383 × PHG16 |
| 9 | PHW79 | (PHT90/PH595) × 211XX | 18 | LH215 | R177 × Mo17C2 |
Table 2.
Fertility ratings of F1 progenies derived from the cross between CHZS and the tester lines.
Table 2.
Fertility ratings of F1 progenies derived from the cross between CHZS and the tester lines.
| Male Fertility of F1 | Corresponding Inbred Lines |
|---|---|
| Grade V (Completely fertile male) | A619, Guang10-2, DAN598, PHT77, 78551S, PHW79, L127, L139, PHJ75, PHN82, LH212Ht, PHR55 |
| Grade II–IV (Partially fertile male) | K10, E8501 |
| Grade I (Completely sterile male) | ZH03, ZJ102, ZJ302, ZJ402, PHR58, LH215 |
Table 3.
Male fertility grade distribution of related F2 and BC1 populations.
| Year and Place | Populations | Male Fertility Grades | Total Plants | ||||
|---|---|---|---|---|---|---|---|
| I | II | III | IV | V | |||
| 2017, Spring, Wenjiang | (CHZS × DAN598)F2 | 19 | 6 | 3 | 3 | 103 | 134 |
| 2017, Spring, Wenjiang | (CHZS × PHT77)F2 | 26 | 10 | 4 | 2 | 104 | 146 |
| 2017, Spring, Wenjiang | (CHZS × 78551S)F2 | 22 | 11 | 2 | 6 | 92 | 133 |
| 2017, Spring, Wenjiang | (CHZS × PHW79)F2 | 34 | 29 | 11 | 8 | 54 | 136 |
| 2017, Spring, Wenjiang | (CHZS × L127)F2 | 14 | 5 | 2 | 1 | 135 | 157 |
| 2017, Spring, Wenjiang | (CHZS × L139)F2 | 13 | 6 | 6 | 4 | 103 | 132 |
| 2017, Spring, Wenjiang | (CHZS × PHJ75)F2 | 13 | 7 | 4 | 13 | 98 | 135 |
| 2017, Spring, Wenjiang | (CHZS × PHN82)F2 | 7 | 4 | 2 | 2 | 145 | 160 |
| 2017, Spring, Wenjiang | (CHZS × LH212Ht)F2 | 20 | 9 | 10 | 9 | 82 | 130 |
| 2017, Spring, Wenjiang | (CHZS × PHR55)F2 | 69 | 6 | 16 | 9 | 57 | 157 |
| 2017, Spring, Wenjiang | (CHZS × A619)F2 | 3 | 4 | 15 | 6 | 223 | 251 |
| 2017, Summer, Xinxiang | (CHZS × A619)F2 | 9 | 11 | 11 | 2 | 369 | 402 |
| 2017, Spring, Wenjiang | CHZS × (CHZS × A619) | 42 | 22 | 13 | 11 | 84 | 172 |
Table 4.
Evaluation of the number of major restorer gene by the chi-squared test.
| Populations | Total Plants | No. of Sterile Plants | No. of Rf | χ2 | ρ |
|---|---|---|---|---|---|
| (CHZS × DAN598)F2 | 134 | 25 | 1 | 2.547 | 0.110 |
| (CHZS × PHT77)F2 | 146 | 36 | 1 | 0.000 | 1.000 |
| (CHZS × 78551S)F2 | 133 | 33 | 1 | 0.003 | 0.960 |
| (CHZS × PHN82)F2 | 160 | 11 | 2 | 0.027 | 0.870 |
| (CHZS × LH212Ht)F2 | 130 | 29 | 1 | 0.369 | 0.543 |
| (CHZS × A619)F2 | 402 | 20 | 2 | 0.908 | 0.341 |
Table 5.
Percentage of recombinants (%) in the F2 populations, assessed by b0329 and S-4 markers.
| Populations | No. of Fertile Plants | No. of Sterile Plants | No. of Recombinants | Percentage of Recombinants (%) | ||
|---|---|---|---|---|---|---|
| b0329 | S-4 | b0329 | S-4 | |||
| (CHZS × DAN598)F2 | 89 | 19 | 11 | 11 | 10.2 | 10.2 |
| (CHZS × PHT77)F2 | 99 | 28 | 0 | 3 | 0 | 2.4 |
| (CHZS × 78551S)F2 | 90 | 22 | 7 | 11 | 6.3 | 9.8 |
| (CHZS × LH212Ht)F2 | 75 | 17 | 9 | 13 | 9.8 | 14.1 |
Table 6.
The segregation of fertility in BC1 populations derived from the cross between the CHZS, A619, and tester lines.
Table 6.
The segregation of fertility in BC1 populations derived from the cross between the CHZS, A619, and tester lines.
| Tester Line | Combination | Fertility Grade of Tassel | Total Plants | ||||
|---|---|---|---|---|---|---|---|
| I | II | III | IV | V | |||
| Guang10-2 | CHZS × (Guang10-2 × A619) | 0 | 0 | 0 | 0 | 177 | 177 |
| DAN598 | CHZS × (A619 × DAN598) | 0 | 0 | 0 | 0 | 107 | 107 |
| K10 | CHZS × (A619 × K10) | 0 | 0 | 0 | 0 | 69 | 69 |
| ZH03 | CHZS × (ZH03 × A619) | 7 | 7 | 3 | 4 | 46 | 67 |
| ZJ102 | CHZS × (ZJ102 × A619) | 20 | 14 | 8 | 0 | 140 | 182 |
| ZJ302 | CHZS × (ZJ302 × A619) | 15 | 4 | 6 | 2 | 79 | 106 |
| ZJ402 | CHZS × (ZJ402 × A619) | 9 | 3 | 8 | 5 | 121 | 146 |
| PHT77 | CHZS × (A619 × PHT77) | 0 | 0 | 0 | 0 | 145 | 145 |
| 78551S | CHZS × (78551S × A619) | 0 | 0 | 0 | 0 | 165 | 165 |
| CHZS × (Guang10-2 × 78551S) | 0 | 0 | 0 | 0 | 190 | 190 | |
| CHZS × (78551S × Guang10-2) | 0 | 0 | 0 | 0 | 188 | 188 | |
| PHW79 | CHZS × (A619 × PHW79) | 0 | 0 | 0 | 0 | 177 | 177 |
| L 127 | CHZS × (L127 × A619) | 0 | 0 | 0 | 0 | 29 | 29 |
| CHZS × (A619 × L127) | 0 | 0 | 0 | 0 | 152 | 152 | |
| L 139 | CHZS × (A619 × L139) | 0 | 0 | 0 | 0 | 174 | 174 |
| CHZS × (L139 × A619) | 0 | 0 | 0 | 0 | 238 | 238 | |
| E8501 | CHZS × (A619 × E8501) | 3 | 10 | 11 | 4 | 104 | 132 |
| PHJ75 | CHZS × (A619 × PHJ75) | 0 | 0 | 0 | 0 | 185 | 185 |
| CHZS × (Guang10-2 × PHJ75) | 0 | 0 | 0 | 0 | 185 | 185 | |
| CHZS × (PHJ75 × Guang10-2) | 0 | 0 | 0 | 0 | 175 | 175 | |
| PHN82 | CHZS × (A619 × PHN82) | 0 | 0 | 0 | 0 | 130 | 130 |
| LH212Ht | CHZS × (A619 × LH212Ht) | 0 | 0 | 0 | 0 | 202 | 202 |
| PHR55 | CHZS × (A619 × PHR55) | 0 | 0 | 0 | 0 | 201 | 201 |
| CHZS × (PHR55 × A619) | 0 | 0 | 0 | 0 | 143 | 143 | |
| PHR58 | CHZS × (A619 × PHR58) | 7 | 14 | 25 | 13 | 136 | 195 |
| LH215 | CHZS × (A619 × LH215) | 8 | 6 | 17 | 8 | 158 | 197 |
Table 7.
Summary of the allelic tests in the populations derived from the cross between the CHZS, A619, and tester lines.
Table 7.
Summary of the allelic tests in the populations derived from the cross between the CHZS, A619, and tester lines.
| Male Population Fertility of Allelic Testing Crossing with A619 | Inbreds |
|---|---|
| All fertile plants | Guang10-2, DAN598, K10, PHT77, 78551S, PHW79, L127, L139, PHJ75, PHN82, LH212Ht, PHR55 |
| Coexistence of fertile and sterile plants | ZH03, ZJ102, ZJ302, ZJ402, E8501, PHR58, LH215 |
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Liu, Y.; Zhang, L.; Liu, X.; Zhang, P.; Zhao, Z.; Yi, H.; Cao, M. Identification of Maize Rf4-Restorer Lines and Development of a CAPS Marker for Rf4. Agronomy 2022, 12, 1506. https://doi.org/10.3390/agronomy12071506
AMA Style
Liu Y, Zhang L, Liu X, Zhang P, Zhao Z, Yi H, Cao M. Identification of Maize Rf4-Restorer Lines and Development of a CAPS Marker for Rf4. Agronomy. 2022; 12(7):1506. https://doi.org/10.3390/agronomy12071506
Chicago/Turabian StyleLiu, Yongming, Ling Zhang, Xiaowei Liu, Peng Zhang, Zhuofan Zhao, Hongyang Yi, and Moju Cao. 2022. "Identification of Maize Rf4-Restorer Lines and Development of a CAPS Marker for Rf4" Agronomy 12, no. 7: 1506. https://doi.org/10.3390/agronomy12071506
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