Identification and Characterization of LaSCL6 Alleles in Larix kaempferi (Lamb.) Carr. Based on Analysis of Simple Sequence Repeats and Allelic Expression
State Key Laboratory of Tree Genetics and Breeding, Key Laboratory of Tree Breeding and Cultivation, National Forestry and Grassland Administration, Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, China
*
Author to whom correspondence should be addressed.
Forests 2020, 11(12), 1296; https://doi.org/10.3390/f11121296
Submission received: 12 November 2020
/
Revised: 28 November 2020
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Accepted: 1 December 2020
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Published: 1 December 2020
(This article belongs to the Section Forest Ecophysiology and Biology)
Abstract
:Simple sequence repeats (SSRs) are widely used as markers for the assessment of genetic diversity and marker-assisted breeding. In a previous study, two SSRs (GCA and CCA), were found in the genomic sequence of Larix (La) SCL6, which plays important roles in the growth and development of Larix kaempferi (Lamb.) Carr. In this study, we analyzed the polymorphisms of these two SSRs in the L. kaempferi population. We found that each SSR had five different polymorphisms, among which (GCA)7 and (CCA)7 were predominant. In addition, 12 haplotypes were detected, with (GCA)7(CCA)7 having the highest frequency. Furthermore, we detected the haplotypes of LaSCL6 in mature trees and their seeds and analyzed the relationships between parents and offspring. The expression patterns of five LaSCL6 alleles were analyzed and they showed balanced expression during vegetative development. Taken together, these findings not only provide more genetic information on LaSCL6, but also provide a candidate marker for genetic studies and breeding.
1. Introduction
Simple sequence repeats (SSRs) are a class of DNA sequences consisting of short, tandem-repeated motifs (1–6 bp in length) [1]. SSR sequences are highly polymorphic, and their presence results in allelic variants with different frequencies at the population level [2,3,4]. In addition, some allelic variants show differential expression during plant growth and development [5,6,7]. Due to their advantages of being co-dominant, multi-allelic, reliable, PCR-based, and abundant in plant genomes [8], SSRs have been used as markers for the assessment of genetic diversity [9,10], genetic structure [11], parentage analyses [12], pedigree and mating system analyses [13], and marker-assisted breeding [14].
Larix kaempferi (Lamb.) Carr, a forest tree of important ecological and economic value and widely-grown in the northern hemisphere, is monoecious and mainly wind-pollinated [15]. In recent years, SSRs have been used to investigate the pollen contamination rate and paternal contributions of L. kaempferi, which affects the seed productivity and severely limits the improvement of seed orchard yield and the construction of clonal seed orchards [16,17].
SCARECROW-LIKE6 (SCL6), a member of the GRAS (GAI-RGA-SCR) family that is regulated by microRNA171 at the post-transcriptional level, is involved in many aspects of growth and development, such as shoot branching [18], meristem maintenance [19], and somatic embryogenesis [20,21]. In a previous study, we identified the homologue of SCL6 in L. kaempferi, and we found two SSRs (GCA and CCA) in the genomic sequence of LaSCL6 [22]. Understanding the genetic variation of a specific gene in a population is important for the study of its functions and for future breeding improvements.
In this study, we analyzed the polymorphism of SSR sequences of LaSCL6 in the L. kaempferi population, and we studied the specific expression of five alleles. Our results not only provide more genetic information on LaSCL6, but also provide a candidate marker for genetic studies and breeding.
2. Materials and Methods
2.1. Plant Materials
All the materials were collected from a Dagujia seed orchard (42°22′ N, 124°51′ E), Liaoning Province, in Northeast China. Endosperms were used to assess the frequency of SSRs and they were separated from 200 mature seeds; after immersion in water for one day, the seed coats were removed, and then the endosperms were isolated for DNA extraction. The needles and seeds of three mature trees were used to study the genetic information delivery from parents to offspring, and endosperms and embryos were sampled separately; the needles, endosperms, and embryos were used for DNA extraction. Six-month-old seedlings were used to study the expression of alleles. After analyzing the alleles of 40 seedlings, the needles, stem, and the root from a single seedling were sampled for RNA extraction. All the samples used for DNA and RNA extraction were frozen in liquid nitrogen and stored at −80 °C.
2.2. DNA Extraction and Polymerase Chain Reaction (PCR) Amplification
The genomic DNA of L. kaempferi was isolated with the CTAB plant genome DNA rapid extraction kit (Aidlab Biotech, Beijing, China) according to the manufacturer’s protocols. The primers 5′-AGCGAGGTCAAGAAAGAAGAGC-3′ and 5′-TTGGGAACGAATGGCGTAGGG-3′ were used to amplify the sequence fragment containing two SSR loci from the DNA template with Platinum® Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA). The PCR products were purified with a gel extraction kit (Tiangen, Beijing, China), ligated into the pEASY®-T1 simple cloning vector (TransGen Biotech, Beijing, China), and sequenced. Multiple sequence alignments were performed with ClustalX [23]. The tertiary structures were predicted by SWISS-MODEL (https://swissmodel.expasy.org/).
2.3. RNA Extraction and Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR)
Total RNAs extracted from the needle, stem, and root were isolated with the EasyPure RNA kit (TransGen Biotech, Beijing, China) according to the improved manufacturer’s protocol and then reverse-transcribed into cDNA with the TransScript® Ⅱ one-step gDNA removal and cDNA Synthesis SuperMix kit (TransGen Biotech, Beijing, China). TB Green® Premix Ex Taq™ (Tli RNase H Plus) (Takara, Shiga, Japan) was used to assess the expression levels of alleles with the allele-specific primers. LaEF1A1 was used as the internal control [21]. The qRT-PCR was performed with three biological replicates and the data are shown as mean ± SD. Statistical analysis was performed with SPSS19.0 using analysis of variance.
3. Results and Discussion
3.1. SSRs in LaSCL6 Show High Levels of Polymorphism
In 200 endosperms, four types of GCA repeats and three types of CCA repeats were detected, and the frequencies of (GCA)7 (161/200, 80.5%) and (CCA)7 (197/200, 98.5%) were the highest (Table 1). In the 40 seedlings, five types of GCA repeats and five types of CCA repeats were detected, and the repeats (GCA)7 (51/80, 63.8%) and (CCA)7 (62/80, 77.5%) were also most frequent (Table 1).
Furthermore, the haplotypes of two SSR loci of LaSCL6 were analyzed, and 12 haplotypes occurred, among which (GCA)7(CCA)7 was predominant (206/280, 73.6%) (Table 2, Figure 1). The occurrence of polymorphism of LaSCL6 helps to study the diversity of the L. kaempferi population.
Notably, in the repeat region of GCA, there were two single-nucleotide polymorphisms (SNPs) (G-A and C-T) (Figure 1, Table 1 and Table 2). When G is changed to A, the codon CAG changes to CAA, but this is a synonymous SNP without changing the amino-acid. When C is changed to T, the codon CAG changes to the termination codon TAG, resulting in the termination of LaSCL6 translation and a change in the protein length, and this reveals the regulation of LaSCL6 expression at the genomic level [22].
3.2. SSRs Affect LaSCL6 Structure and Function
Protein has a defined three-dimensional structure after the folding of the primary structure. The three-dimensional structures of 12 haplotypes of LaSCL6 were predicted and the results showed that they differed in at least at five places (Figure 2). The GCA and CCA repeat regions in LaSCL6 result in repeats of the CAG and CCA codons, which code for polyglutamine and proline, respectively, and this might affect the structure and function of LaSCL6.
SSRs in the exon affect protein structure and thus lead to a change of protein function [24]. In human genes, tandem repeats of polyglutamine cause incurable neurodegenerative diseases [25,26]. In Arabidopsis thaliana, polyglutamine length in EARLY FLOWERING 3 has dramatic effects on flowering time and circadian clock-related phenotypes [24]. Poly proline affects the binding of profilin and may have consequences for the regulation of actin cytoskeletal dynamics in plant cells [27]. The SSRs in the exon of LaSCL6 change the primary structure of its protein, and this affects its three-dimensional structure, which might result in the functional diversification of LaSCL6; further work is needed to test this.
3.3. Parent-Offspring Relationships Can Be Analyzed by SSRs in LaSCL6
L. kaempferi is monoecious and mainly wind-pollinated [15]. Accurate information about parent-offspring relationships is important for Larix breeding programs [10,16]. Three mature trees were used to study the genetic information delivery of LaSCL6 from parents to offspring based on the haplotypes of the two SSR loci in LaSCL6.
Tree 1 was heterozygous and had two haplotypes: (GCA)7(CCA)7 and (GCA)4(CCA)7 (Table 3). The haplotypes of endosperms and embryos from 13 seeds were determined. All the haplotypes of 13 endosperms and 92.3% (24/26) of the haplotypes of 13 embryos were found in tree 1, while 7.7% (2/26) were not found in tree 1 (Table 3).
Tree 2 was heterozygous and had two haplotypes: (GCA)7(CCA)7 and (GCA)6(CCA)7 (Table 3). The haplotypes of endosperms and embryos from 7 seeds were determined. All the haplotypes of 7 endosperms and 78.6% (11/14) of the haplotypes of 7 embryos were found in tree 2, while 21.4% (3/14) were not found in tree 2 (Table 3).
Tree 3 was homozygous and had one haplotype (GCA)7(CCA)7 (Table 3). The haplotypes of endosperms and embryos from 9 seeds were determined. All the haplotypes of 9 endosperms and 66.7% (12/18) of the haplotypes of 9 embryos were found in tree 3, while 33.3% (6/18) were not found in tree 3 (Table 3).
In the seeds of L. kaempferi, the embryo (2n) is diploid and its two haplotypes come from the female and male parents; the endosperm (n) is haploid and its haplotype is from the mother tree and the same as one haplotype of the embryo. Here, we took LaSCL6 as a case study and found that the haplotype of an endosperm was the same as one haplotype of the mother tree and the embryo. For most seeds, the two haplotypes of an embryo were the same as those of the female parent (Table 3), indicating that the female and male parents had the same haplotype. For some seeds, one haplotype of an embryo differed from those of the female parent (Table 3), indicating that it was from the male parent that had a different genetic component from the female parent. Notably, the same haplotype occurred in female and male parents, suggesting that these two SSR loci in LaSCL6 make no contribution to self-incompatibility.
3.4. The Expressions of LaSCL6 Alleles Have the Same Patterns
Allelic expression imbalance has been studied in several plant species, and sometimes it can result in a phenotypic change [5,6,7,28]; to determine whether it occurs in LaSCL6, the expression patterns of five LaSCL6 alleles in three heterozygous L. kaempferi seedlings were analyzed by qRT-PCR assay with allele-specific primers (Table 4).
The haplotypes of seedling 1 were (GCA)7(CCA)7 and (GCA)6(CCA)8 (Figure 3a). The expression patterns of LaSCL6 with (GCA)7(CCA)7 were detected with primer sequences containing the (GCA)7 or (CCA)7 repeat motif, and those of LaSCL6 with (GCA)6(CCA)8 were detected with primer sequences containing the (GCA)6 or (CCA)8 repeat motif (Figure 3a, Table 4). For LaSCL6 with (GCA)7(CCA)7, the same expression patterns were detected by the (GCA)7 and (CCA)7 primers (Figure 3a, Table 4). For LaSCL6 with (GCA)6(CCA)8, the same expression patterns were detected by the (GCA)6 and (CCA)8 primers (Figure 3a, Table 4). For two LaSCL6 alleles, the same expression patterns were detected by the primers (GCA)7 and (GCA)6, or (CCA)7 and (CCA)8 (Figure 3a, Table 4).
The haplotypes of seedling 2 were (GCA)4(CCA)8 and (GCA)6(CCA)8. Almost the same expression patterns for two LaSCL6 alleles were detected by the primers (GCA)4 and (GCA)6 (Figure 3b, Table 4). The haplotypes of seedling 3 were (GCA)4(CCA)7 and (GCA)7(CCA)7, and almost the same expression patterns for two LaSCL6 alleles were detected by the primers (GCA)4 and (GCA)7 (Figure 3c, Table 4).
Taken together, all five alleles were strongly expressed in stems, weakly in roots, and showed almost the same expression patterns and balanced expression during the process of vegetative development.
4. Conclusions
In summary, the SSRs in LaSCL6 show high levels of polymorphism and might affect the protein structure and function. Based on an analysis of the haplotypes of LaSCL6 in mother trees and their offspring, the parent-offspring relationships were determined. These results provide a candidate marker for the L. kaempferi genetic studies and breeding.
Altogether, we have obtained new information on LaSCL6, especially its regulation by microRNA171 and its genomic structure. For future studies, constructing the LaSCL6 network by mining its interacting proteins and target genes will help to reveal the molecular mechanisms underlying the growth and development of L. kaempferi.
Author Contributions
Q.-L.Z. carried out the study, analyzed the data, and wrote the manuscript. X.-Y.L. performed the isolation of endosperm and DNA extraction. W.-F.L. designed the study, analyzed the data, and revised the manuscript. L.-W.Q. provided suggestions on the experimental design and analyses. All authors have read and agreed to the published version of the manuscript.
Funding
This work was supported by the National Natural Science Foundation of China (31770714), the Basic Research Fund of Research Institute of Forestry (RIF2014-07), and the National Transgenic Major Program (2018ZX08020-003).
Acknowledgments
The authors thank IC Bruce (Peking University) for critical reading of the manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
Data Archiving Statement
The full-length cDNA sequences of LaSCL6 and LaSCL6-variant1 have been deposited in GenBank with the accession number JX280920 and MK501379, respectively.
References
- Sharma, P.C.; Grover, A.; Kahl, G. Mining microsatellites in eukaryotic genomes. Trends Biotechnol. 2007, 25, 490–498. [Google Scholar] [CrossRef] [PubMed]
- Ahmed, H.G.M.D.; Khan, A.S.; Khan, S.H.; Kashif, M. Genome wide allelic pattern and genetic diversity of spring wheat genotypes through SSR markers. Int. J. Agric. Biol. 2017, 19, 1559–1565. [Google Scholar] [CrossRef]
- Sajjad, M.; Khan, S.H.; Shahzad, M. Patterns of allelic diversity in spring wheat populations by SSR-Markers. Cytol. Genet. 2018, 52, 155–160. [Google Scholar] [CrossRef]
- Sawamura, Y.; Takada, N.; Yamamoto, T.; Saito, T.; Kimura, T.; Kotobuki, K. Identification of parent-offspring relationships in 55 japanese pear cultivars using S-RNase allele and SSR markers. J. Jpn. Soc. Hortic. Sci. 2008, 77, 364–373. [Google Scholar] [CrossRef] [Green Version]
- Qiu, D.; Bai, S.; Ma, J.; Zhang, L.; Shao, F.; Zhang, K.; Yang, Y.; Sun, T.; Huang, J.; Zhou, Y.; et al. The genome of Populus alba x Populus tremula var. glandulosa clone 84K. DNA Res. 2019, 26, 423–431. [Google Scholar] [CrossRef]
- Shao, L.; Xing, F.; Xu, C.; Xu, C.; Zhang, Q.; Che, J.; Wang, X.; Song, J.; Li, X.; Xiao, J.; et al. Patterns of genome-wide allele-specific expression in hybrid rice and the implications on the genetic basis of heterosis. Proc. Natl. Acad. Sci. USA 2019, 116, 5653–5658. [Google Scholar] [CrossRef] [Green Version]
- Zhang, X.; Borevitz, J.O. Global analysis of allele-specific expression in Arabidopsis thaliana. Genetics 2009, 182, 943–954. [Google Scholar] [CrossRef] [Green Version]
- Powell, W.; Morgante, M.; Andre, C.; Hanafey, M.; Vogel, J.; Tingley, S.; Rafalski, A. The comparison of RFLP, RAPD, AFLP and SSR (microsatellite) markers for germplasm analysis. Mol. Breed. 1996, 2, 225–238. [Google Scholar] [CrossRef]
- Oreshkova, N.V.; Belokon, M.M.; Jamiyansuren, S. Genetic diversity, population structure, and differentiation of Siberian larch, Gmelin larch and Cajander larch on SSR-markers data. Russ. J. Genet. 2013, 49, 178–186. [Google Scholar] [CrossRef]
- Yu, X.M.; Zhou, Q.; Qian, Z.Q.; Li, S.; Zhao, G.F. Analysis of genetic diversity and population differentiation of Larix potaninii var. chinensis using microsatellite DNA. Biochem. Genet. 2006, 44, 483–493. [Google Scholar] [CrossRef]
- Lind, J.F.; Gailing, O. Genetic structure of Quercus rubra L. and Quercus ellipsoidalis E. J. Hill populations at gene-based EST-SSR and nuclear SSR markers. Tree Genet. Genomes 2013, 9, 707–722. [Google Scholar] [CrossRef]
- Grattapaglia., D.; Diener, P.S.D.A.; Santos, G.A.D. 12. Grattapaglia. D.; Diener, P.S.D.A.; Santos, G.A.D. Performance of microsatellites for parentage assignment following mass controlled pollination in a clonal seed orchard of loblolly pine (Pinus taeda L.). Tree Genet. Genomes 2014, 10, 1631–1643. [Google Scholar] [CrossRef]
- Funda, T.; Chen, C.; Liewlaksaneeyanawin, C.; Kenawy, A.M.A.; El-Kassaby, Y.A. Pedigree and mating system analyses in a western larch (Larix occidentalis Nutt.) experimental population. Ann. For. Sci. 2008, 65, 705. [Google Scholar] [CrossRef] [Green Version]
- Gupta, P.K.; Varshney, R.K. The development and use of microsatellite markers for genetic analysis and plant breeding with emphasis on bread wheat. Euphytica 2000, 113, 163–185. [Google Scholar] [CrossRef]
- Nishimura, M.; Setoguchi, H. Homogeneous genetic structure and variation in tree architecture of Larix kaempferi along altitudinal gradients on Mt. Fuji. J. Plant Res. 2011, 124, 253–263. [Google Scholar] [CrossRef]
- Chen, X.; Sun, X.; Dong, L.; Zhang, S. Mating patterns and pollen dispersal in a Japanese larch (Larix kaempferi) clonal seed orchard: A case study. Sci. China Life Sci. 2018, 61, 1011–1023. [Google Scholar] [CrossRef] [PubMed]
- Hansen, O.K. Mating patterns, genetic composition and diversity levels in two seed orchards with few clones—Impact on planting crop. For. Ecol. Manag. 2008, 256, 1167–1177. [Google Scholar] [CrossRef]
- Wang, L.; Mai, Y.X.; Zhang, Y.C.; Luo, Q.; Yang, H.Q. MicroRNA171c targeted SCL6-II, SCL6-III, and SCL6-IV genes regulate shoot branching in Arabidopsis. Mol. Plant 2010, 3, 794–806. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Fan, T.; Li, X.; Yang, W.; Xia, K.; Ouyang, J.; Zhang, M. Rice osamiR171c mediates phase change from vegetative to reproductive development and shoot apical meristem maintenance by repressing four OsHAM transcription factors. PLoS ONE 2015, 10, e0125833. [Google Scholar] [CrossRef] [Green Version]
- Li, H.; Zhang, J.; Yang, Y.; Jia, N.; Wang, C.; Sun, H. miR171 and its target gene SCL6 contribute to embryogenic callus induction and torpedo-shaped embryo formation during somatic embryogenesis in two lily species. Plant Cell Tissue Organ Cult. 2017, 130, 591–600. [Google Scholar] [CrossRef]
- Li, W.F.; Zhang, S.G.; Han, S.Y.; Wu, T.; Zhang, J.H.; Qi, L.W. The post transcriptional regulation of LaSCL6 by miR171 during maintenance of embryogenic potential in Larix kaempferi (Lamb.) Carr. Tree Genet. Genomes 2014, 10, 223–229. [Google Scholar] [CrossRef]
- Zang, Q.L.; Li, W.F.; Qi, L.W. Regulation of LaSCL6 expression by genomic structure, alternative splicing, and microRNA in Larix kaempferi. Tree Genet. Genomes 2019, 15, 57. [Google Scholar] [CrossRef] [Green Version]
- Thompson, J.D.; Gibson, T.J.; Plewniak, F.; Jeanmougin, F.; Higgins, D.G. The CLUSTAL_X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 1997, 25, 4876–4882. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Undurraga, S.F.; Press, M.O.; Legendre, M.; Bujdoso, N.; Bale, J.; Wang, H.; Davis, S.J.; Verstrepen, K.J.; Queitsch, C. Background-dependent effects of polyglutamine variation in the Arabidopsis thaliana gene ELF3. Proc. Natl. Acad. Sci. USA 2012, 109, 19363–19367. [Google Scholar] [CrossRef] [Green Version]
- Gatchel, J.R.; Zoghbi, H.Y. Diseases of unstable repeat expansion: Mechanisms and common principles. Nat. Rev. Genet. 2005, 6, 743–755. [Google Scholar] [CrossRef]
- Orr, H.T. Polyglutamine neurodegeneration: Expanded glutamines enhance native functions. Curr. Opin. Genet. Dev. 2012, 22, 251–255. [Google Scholar] [CrossRef] [Green Version]
- Gibbon, B.C.; Zonia, L.E.; Kovar, D.R.; Hussey, P.J.; Staiger, C.J. Pollen profilin function depends on interaction with proline-rich motifs. Plant Cell 1998, 10, 981–993. [Google Scholar] [CrossRef] [Green Version]
- Guo, M.; Rupe, M.A.; Zinselmeier, C.; Habben, J.; Bowen, B.A.; Smith, O.S. Allelic variation of gene expression in maize hybrids. Plant Cell 2004, 16, 1707–1716. [Google Scholar] [CrossRef] [Green Version]
Figure 1.
Information on simple sequence repeats in the genomic DNA sequence of LaSCL6. The GCA and CCA repeat regions are underlined. The arrows indicate the loci of single nucleotide polymorphisms (SNPs) (# haplotypes with SNPs; * identical residues).
Figure 1.
Information on simple sequence repeats in the genomic DNA sequence of LaSCL6. The GCA and CCA repeat regions are underlined. The arrows indicate the loci of single nucleotide polymorphisms (SNPs) (# haplotypes with SNPs; * identical residues).
Figure 2.
Prediction of protein tertiary structures of different haplotypes of LaSCL6. Numbers indicate the potential differences among the structures.
Figure 2.
Prediction of protein tertiary structures of different haplotypes of LaSCL6. Numbers indicate the potential differences among the structures.
Figure 3.
The expression patterns of LaSCL6 alleles. Expression patterns of LaSCL6 alleles in needle, stem, and root of Larix kaempferi (Lamb.) Carr seedling 1 (a), seedling 2 (b), and seedling 3 (c) assayed by qRT-PCR with LaEF1A1 as the internal control. The qRT-PCR was performed with three biological replicates, and the data are shown as the mean ±SD.
Figure 3.
The expression patterns of LaSCL6 alleles. Expression patterns of LaSCL6 alleles in needle, stem, and root of Larix kaempferi (Lamb.) Carr seedling 1 (a), seedling 2 (b), and seedling 3 (c) assayed by qRT-PCR with LaEF1A1 as the internal control. The qRT-PCR was performed with three biological replicates, and the data are shown as the mean ±SD.
Table 1.
Polymorphism of simple sequence repeats in LaSCL6 in the population of Larix kaempferi (Lamb.) Carr.
Table 1.
Polymorphism of simple sequence repeats in LaSCL6 in the population of Larix kaempferi (Lamb.) Carr.
| Repeat Motif | Number of Repeats | Frequency in Endosperms | Frequency in Seedlings |
|---|---|---|---|
| GCA | 4 | 5/200 (2.5%) | 6/80 (7.5%) |
| 6 | 2/200 (1.0%) + 15/200 (7.5%) a | 13/80 (16.3%) + 4/80 (5.0%) a | |
| 7 | 161/200 (80.5%) | 48/80 (60.0%) + 3/80 (3.8%) b | |
| 8 | – | 1/80 (1.3%) | |
| 9 | 17/200 (8.5%) | 5/80 (6.3%) | |
| CCA | 6 | – | 1/80 (1.3%) |
| 7 | 197/200 (98.5%) | 62/80 (77.5%) | |
| 8 | 2/200 (1.0%) | 14/80 (17.5%) | |
| 9 | 1/200 (0.5%) | 1/80 (1.3%) | |
| 12 | – | 2/80 (2.5%) |
a (GCA)6#; b (GCA)7#; – not detected.
Table 2.
Haplotypes of two simple sequence repeats in LaSCL6 in the population of Larix kaempferi (Lamb.) Carr.
Table 2.
Haplotypes of two simple sequence repeats in LaSCL6 in the population of Larix kaempferi (Lamb.) Carr.
| Haplotype | Frequency in Endosperms | Frequency in Seedlings |
|---|---|---|
| (GCA)4 (CCA)7 | 3/200 (1.5%) | 2/80 (2.5%) |
| (GCA)4 (CCA)8 | 1/200 (0.5%) | 2/80 (2.5%) |
| (GCA)4 (CCA)9 | 1/200 (0.5%) | 1/80 (1.3%) |
| (GCA)4 (CCA)12 | – | 1/80 (1.3%) |
| (GCA)6 (CCA)7 | 2/200 (1.0%) + 15/200 (7.5%)a | 4/80 (5.0%) + 4/80 (5.0%) a |
| (GCA)6 (CCA)8 | – | 9/80 (11.3%) |
| (GCA)7 (CCA)6 | – | 1/80 (1.3%) |
| (GCA)7 (CCA)7 | 160/200 (80.0%) | 43/80 (53.8%) + 3/80 (3.8%) b |
| (GCA)7 (CCA)8 | 1/200 (0.5%) | 3/80 (3.8%) |
| (GCA)7 (CCA)12 | – | 1/80 (1.3%) |
| (GCA)8 (CCA)7 | – | 1/80 (1.3%) |
| (GCA)9 (CCA)7 | 17/200 (8.5%) | 5/80 (6.3%) |
a (GCA)6# (CCA)7; b (GCA)7# (CCA)7; – not detected.
Table 3.
Haplotypes of two simple sequence repeats in LaSCL6 of mother trees and their seeds.
| Mother Tree | Seed | ||
|---|---|---|---|
| Number | Endosperm | Embryo | |
| Tree 1 | 1 | (GCA)7 (CCA)7 | (GCA)7 (CCA)7 (10/10) (100.0%) |
| (GCA)7 (CCA)7 (23/54, 42.6%) | 2 | (GCA)4 (CCA)7 | (GCA)7 (CCA)7 (5/10) (50.0%) |
| (GCA)4 (CCA)7 (31/54, 57.4%) | (GCA)4 (CCA)7 (5/10) (50.0%) | ||
| 3 | (GCA)7 (CCA)7 | (GCA)7 (CCA)7 (5/10) (50.0%) | |
| (GCA)4 (CCA)7 (5/10) (50.0%) | |||
| 4 | (GCA)4 (CCA)7 | (GCA)7 (CCA)7 (7/10) (70.0%) | |
| (GCA)4 (CCA)7 (3/10) (30.0%) | |||
| 5 | (GCA)7 (CCA)7 | (GCA)7 (CCA)7 (10/10) (100.0%) | |
| 6 | (GCA)4 (CCA)7 | (GCA)7 (CCA)7 (4/10) (40.0%) | |
| (GCA)4 (CCA)7 (6/10) (60.0%) | |||
| 7 | (GCA)4 (CCA)7 | (GCA)7 (CCA)7 (4/10) (40.0%) | |
| (GCA)4 (CCA)7 (6/10) (60.0%) | |||
| 8 | (GCA)4 (CCA)7 | (GCA)7 (CCA)7 (6/10) (60.0%) | |
| (GCA)4 (CCA)7 (4/10) (40.0%) | |||
| 9 | (GCA)7 (CCA)7 | (GCA)9 (CCA)7 (4/10)(40.0%) | |
| (GCA)7 (CCA)7 (6/10) (60.0%) | |||
| 10 | (GCA)7 (CCA)7 | (GCA)6 (CCA)8 (4/10)(40.0%) | |
| (GCA)7 (CCA)7 (6/10) (60.0%) | |||
| 11 | (GCA)7 (CCA)7 | (GCA)7 (CCA)7 (10/10) (100.0%) | |
| 12 | (GCA)4 (CCA)7 | (GCA)7 (CCA)7 (5/10) (50.0%) | |
| (GCA)4 (CCA)7 (5/10) (50.0%) | |||
| 13 | (GCA)4 (CCA)7 | (GCA)7 (CCA)7 (10/10) (100.0%) | |
| Tree 2 | 1 | (GCA)7 (CCA)7 | (GCA)7 (CCA)7 (8/10) (80.0%) |
| (GCA)7 (CCA)7 (58/60, 96.7%) | (GCA)6 (CCA)7 (2/10) (20.0%) | ||
| (GCA)6 (CCA)7 (2/60, 3.3%) | 2 | (GCA)7 (CCA)7 | (GCA)7 (CCA)7 (5/10) (50.0%) |
| (GCA)6 (CCA)8 (5/10)(50.0%) | |||
| 3 | (GCA)7 (CCA)7 | (GCA)7 (CCA)7 (10/10) (100.0%) | |
| 4 | (GCA)7 (CCA)7 | (GCA)7 (CCA)7 (10/10) (100.0%) | |
| 5 | (GCA)7 (CCA)7 | (GCA)7 (CCA)7 (9/10) (90.0%) | |
| (GCA)8 (CCA)7 (1/10)(10.0%) | |||
| 6 | (GCA)7 (CCA)7 | (GCA)7 (CCA)7 (8/10) (80.0%) | |
| (GCA)7 (CCA)6 (2/10)(20.0%) | |||
| 7 | (GCA)7 (CCA)7 | (GCA)7 (CCA)7 (9/10) (90.0%) | |
| (GCA)6 (CCA)7 (1/10) (10.0%) | |||
| Tree 3 | 1 | (GCA)7 (CCA)7 | (GCA)7 (CCA)7 (9/10) (90.0%) |
| (GCA)7 (CCA)7 (60/60, 100.0%) | (GCA)7 (CCA)6 (1/10)(10.0%) | ||
| 2 | (GCA)7 (CCA)7 | (GCA)7 (CCA)7 (9/10) (90.0%) | |
| (GCA)7 (CCA)6 (1/10)(10.0%) | |||
| 3 | (GCA)7 (CCA)7 | (GCA)7 (CCA)7 (8/10) (80.0%) | |
| (GCA)4 (CCA)8 (2/10)(20.0%) | |||
| 4 | (GCA)7 (CCA)7 | (GCA)7 (CCA)7 (9/10) (90.0%) | |
| (GCA)6 (CCA)7 (1/10)(10.0%) | |||
| 5 | (GCA)7 (CCA)7 | (GCA)7 (CCA)7 (5/10) (50.0%) | |
| (GCA)6 (CCA)7 (5/10)(50.0%) | |||
| 6 | (GCA)7 (CCA)7 | (GCA)7 (CCA)7 (10/10) (100.0%) | |
| 7 | (GCA)7 (CCA)7 | (GCA)7 (CCA)7 (10/10) (100.0%) | |
| 8 | (GCA)7 (CCA)7 | (GCA)7 (CCA)7 (10/10) (100.0%) | |
| 9 | (GCA)7 (CCA)7 | (GCA)7 (CCA)7 (9/10) (90.0%) | |
| (GCA)6 (CCA)8 (1/10)(10.0%) | |||
The haplotypes of embryos different from that of their mother trees are shown in bold. The numbers in parentheses refer to the occurrence frequencies of the haplotypes in the sequenced clones.
Table 4.
Primers for allele-specific quantitative reverse transcription-polymerase chain reaction.
| Primer Name | Primer Sequence (5′-3′) |
|---|---|
| (GCA)4 | F-AGAAGAGCCGCAGCAGCAGCAGAGCCGAGC |
| R-AGGCGGAGGGAGGTCTTT | |
| (GCA)6 | F-CCCTGCACGTTGGTATCCT |
| R-GCTCGGCTCTGCTGCTGCTGCTGCTGCGGCTCTTCT | |
| (GCA)7 | F-CCCTGCACGTTGGTATCCT |
| R-CGGCTCTGCTGCTGCTGCTGCTGCTGCGGCTCT | |
| (CCA)7 | F-CCTCCGCCACCACCACCACCACCACCATTTGCC |
| R-CCGAGGCCATGTTAAAGC | |
| (CCA)8 | F-CCTCCGCCACCACCACCACCACCACCACCATTTGCC |
| R-CCGAGGCCATGTTAAAGC |
F, forward primer; R, reverse primer; the repeat regions in primers are shown in bold.
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Zang, Q.-L.; Li, X.-Y.; Qi, L.-W.; Li, W.-F. Identification and Characterization of LaSCL6 Alleles in Larix kaempferi (Lamb.) Carr. Based on Analysis of Simple Sequence Repeats and Allelic Expression. Forests 2020, 11, 1296. https://doi.org/10.3390/f11121296
AMA Style
Zang Q-L, Li X-Y, Qi L-W, Li W-F. Identification and Characterization of LaSCL6 Alleles in Larix kaempferi (Lamb.) Carr. Based on Analysis of Simple Sequence Repeats and Allelic Expression. Forests. 2020; 11(12):1296. https://doi.org/10.3390/f11121296
Chicago/Turabian StyleZang, Qiao-Lu, Xiang-Yi Li, Li-Wang Qi, and Wan-Feng Li. 2020. "Identification and Characterization of LaSCL6 Alleles in Larix kaempferi (Lamb.) Carr. Based on Analysis of Simple Sequence Repeats and Allelic Expression" Forests 11, no. 12: 1296. https://doi.org/10.3390/f11121296
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