1. Introduction
Triticale (x
Triticosecale Wittmack) is a plant genus comprised of synthetic amphidiploid crops combining the genomes of wheat (
Triticum) and rye (
Secale). Triticale was created in order to obtain a crop with the productivity of wheat and the durability of rye. The chromosome set of triticale consists of wheat (AABB or AABBDD) and rye (RR) chromosomes and its ploidy varies from tetraploid (2n = 2x = 14, AARR) to octaploid (2n = 8x = 56, AABBDDRR). The hexaploid level (2n = 6x = 42, AABBRR) is the most common in commercial cultivars due to its excellent combination of traits and genomic stability [
1]. Currently, triticale is grown in more than 40 countries around the world because it produces high and stable yields in a wide range of soil and climatic conditions [
2,
3]. As with other cereals, reduced plant height improves lodging resistance and it is a desirable trait in breeding programs. A number of height-reducing genes are currently known in wheat and rye genomes, and the most effective genes are exploited in the breeding of semi-dwarf cultivars of these cereals and triticale. Primarily, these are the dwarfing alleles
Rht-B1b and
Rht-D1b of the wheat gene
Rht1 (from the B and D genomes, respectively), which encode DELLA protein participating in gibberellin signaling [
4,
5]. The dominant dwarfing allele of rye
Ddw1 (formerly known as
Hl) was discovered in the rye genome in a study of natural rye dwarf mutant EM-1 [
6]. Recent data suggest that
Ddw1 encodes gibberellin 2-oxidase that destroys gibberellin [
7].
Ddw1 has attracted most attention in rye and triticale breeding programs because of its strong effect on plant height. However, beside reducing plant height, the dwarfing alleles
Ddw1 may also reduce the grain weight per spike and the weight of 1000 grains. This negative effect of
Ddw1 can be compensated for by the presence of
Rht-B1b [
8,
9,
10].
The dominant gain-of-function effect of
Ddw1 complicates the reliable differentiation of homozygous pure lines from heterozygotes based on plant phenotype. Marker-assisted selection (MAS) is a powerful approach to cereal breeding, however there is currently no reliable marker for dominant
Ddw1 homozygous plants. Currently, markers targeting microsatellite locus
REMS1218 are used for
Ddw1 genotyping.
REMS1218 (GenBank accession BE587316) contains (AG)
8 repeats and was originally discovered by the analysis of cDNA sequences in GenBank. It was mapped at the end of a long arm of chromosome 5R in rye population obtained by crossing cv. Steel and cv. Monstrous. Initially, several
REMS1218 were revealed [
11]. The position of
REMS1218 is close to the restriction fragment length polymorphism (RFLP) marker
Xwg199, which co-segregates with
Ddw1. The distance between
Xwg199 and
Ddw1 has been estimated as 5.6 cM, and as 5.3 cM between
Xwg199 and
REMS1218 in the same direction [
11,
12]. In a study by Tenhola-Roininen and Tanhuanpa, a combination of SSR and SNP markers for
REMS1218 was suggested to distinguish dominant dwarfing
Ddw1 from recessive neutral
ddw1 alleles, however, this was not able to distinguish
Ddw1 homozygous from
Ddw1 ddw1 heterozygotes is some cases. In addition, this approach involves analytical discrimination of PCR products with a difference of only 4 bp (317 and 321 bp), which requires either an expensive capillary electrophoresis system or labor-consuming manual polyacrylamide gel electrophoresis procedures [
13].
Originally, PCR amplification of REMS1218 was found to produce two product (amplicons) patterns based on their size: a combination of 317 bp and 321 bp products associated with the dwarfing
Ddw1 allele, and a 317 bp product associated with the neutral
ddw1 allele [
13]. Amplicon 317 bp in
ddw1 ddw1 plants is shown as a single peak when analyzed using capillary electrophoresis. In fact, 317 bp amplicon consists of two products that are equal in size but polymorphic in nucleotide sequence. In our previous study using a collection of 86 triticale accessions in plants homozygous for neutral
ddw1 alleles, we observed several REMS1218 PCR products of various lengths, but in all cases, they were produced together with 317 bp product [
5]. Thus, all alleles of the
Ddw1 gene found to date produce the conservative 317 bp product, which makes it impossible to distinguish heterozygous and dominant
Ddw1 homozygous plants using capillary electrophoresis only. The purpose of this study was to (1) simplify the protocol for identification of
Ddw1 alleles and make it useful for screening in conventional breeding laboratories, and (2) to improve the method so that it detects 100% of the dominant allele of the
Ddw1 gene.
For this work, we used winter triticale Khongor and Avangard as cultivars homozygous for the dominant allele (
Ddw1 Ddw1). The dwarfing
Ddw1 allele in this cultivar was transferred from triticale cultivar AD-Zelenyi, the latter inherited
Ddw1 from rye EM-1. Two spring triticale cultivars Solovei-Kharkovskii and Dublet were used as carriers of the recessive (
ddw1 ddw1) genotype. Previously, in a study of 86 triticale cultivars and breeding lines we revealed that plant height of semi-dwarf cultivars of spring triticale is reduced due to only one gene, wheat-derived
Rht-B1b, and they do not carry
Ddw1 [
5].
In this study, we applied a cleaved amplified polymorphic sequence (CAPS) approach and developed a marker that identifies Ddw1 dwarfing and ddw1 neutral alleles, and distinguishes reduced height heterozygous Ddw1 ddw1 plants from homozygotes Ddw1 plants.
2. Materials and Methods
2.1. Plant Material
Plants (names of cultivars are according to
http://wheatpedigree.net) with known dominant dwarfing (
Ddw1) or recessive neutral (
ddw1) alleles of
Ddw1 gene were used for
Ddw1 CAPS marker development: (1) winter triticale Khongor (
Ddw1 Ddw1 Rht-B1b Rht-B1b); (2) winter triticale Avangard (
Ddw1 Ddw1 Rht-B1a Rht-B1a); (3) winter triticale Valentin 90 (
Ddw1 Ddw1); (4) winter triticale AD Zelenyi (
Ddw1 Ddw1); (5) spring triticale Solovei Kharkovskii (
ddw1 ddw1 Rht-B1b Rht-B1b); (6) spring triticale Dublet (
ddw1 ddw1 Rht-B1b Rht-B1b); (7) winter triticale Dozor (
ddw1 ddw1).
Plant of two F2 populations were partially used for testing Ddw1 CAPS marker and for assessing the association of Ddw1 allele with the plant height. One population designated Kh/D segregating in ddw1/Ddw1 alleles was obtained by crossing Khongor (Ddw1 Ddw1 Rht-B1b Rht-B1b) and Dublet (ddw1 ddw1 Rht-B1b Rht-B1b). Another population designated A/SKh segregating in ddw1/Ddw1 and Rht-B1a/Rht-B1b was obtained by crossing Avangard (Ddw1 Ddw1 Rht-B1a Rht-B1a) and Solovei Kharkovskii (ddw1 ddw1 Rht-B1b Rht-B1b). F1 uniform heterozygotes and their F2 segregating progenies were grown in a greenhouse at 22–26 °C, with 5 plants per vegetative pot. Ninety-one F2 plants of Kh/D population and seventy-seven F2 plants of A/SKh population were genotyped and used to assess the association between theDdw1 allele and plant height.
2.2. DNA Isolation and PCR Amplification of REMS1218 Microsatellite Fragment
Genomic DNA was isolated from individual plant leaves using the cetyltrimethylammonium bromide (CTAB) method [
14] with some modifications and adaptations for 96-well deep plates. Briefly, 15–50 mg of dried grinded leaves was extracted by 0.5 mL buffer containing 0.7 M NaCl, 1% CTAB, 25 mM Tris-HCl pH 8.0 and 10 mM EDTA. The suspension was incubated for 1 h at 65 °C with shaking, extracted at room temperature with 2/3 volume of chloroform/isoamyl alcohol (24:1), and centrifuged 30 min at 500×
g using a swing-out rotor for deep-well plates. Aqueous phase (200 μL) was collected and DNA was precipitated with 360 µL isopropyl alcohol at −20 °C for 30 min followed by 1 h centrifuge at 500×
g. The DNA pellet was rinsed with 300 μL 70% ethanol, dried and dissolved in 150 µL H
2O.
The allelic variant of
Ddw1 was determined by PCR using primers for microsatellite marker
REMS1218 (forward: 5′-CGC ACA AAC AAA AAC ACG AC-3′, reverse: 5′-CAA ACA AAC CCA TTG ACA CG-3′) [
13]. PCR reactions were performed using a DNA Engine Tetrad 2 (Bio-Rad, USA) thermocycler with the following conditions: initial denaturation at 94 °C for 5 min; 35 cycles of 94 °C for 30 s, 60 °C for 30 s and 72 °C for 1 min, and a final extension step of 5 min at 72 °C. PCR products were analyzed by capillary electrophoresis using ABI 3130xl DNA analyzer (Applied Biosystems, Life Technologies, CA, USA).
2.3. Cloning and Sequencing REMS1218 PCR Products
REMS1218 PCR products were cloned into pGEM-T Easy vector (Invitrogen, USA) according to the manufacturer’s instructions. The resulting plasmids were transformed into
E. coli strain DH10B, bacterial colonies of interest were selected based on blue-white screening and based on the appropriate size of PCR product after amplification using M13 primer. DNA sequencing was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit (Nimagen, Netherlands) and 3130xl Genetic Analyzer (Applied Biosystems, Life Technologies, CA, USA) according to the manufacturer’s instructions. Sequences were analyzed using GeneDoc software [
15].
2.4. CAPS Analysis of REMS1218
REMS1218 PCR amplification products were cleaved by restriction endonucleases MnlI (New England Biolabs, MA, USA), Bso31I, RsaI (SibEnzyme Ltd., Moscow, Russian Federation) according to the instructions of the manufacturer of the enzymes. Restriction fragments were separated using 2% agarose gel in Tris-borate buffer with EDTA (TBE) and ethidium bromide at 6 V/cm and visualized in UV-transilluminator Gel Doc XR+ (Bio-Rad Laboratories, Inc., CA, USA). To evaluate our Ddw1 CAPS marker, we (1) compared the results of Ddw1 genotyping by capillary electrophoresis and by CAPS marker on the triticale cultivars (Khongor, Avangard, Valentin 90, AD Zelenyi, Solovei Kharkovskii, Dublet, and Dozor), the mix of DNA from two plants homozygous for alternative Ddw1 alleles, and F2 crosses of Khongor (Ddw1 Ddw1) and Dublet (ddw1 ddw1); and (2) performed two model MAS experiments on breeding two different triticale cultivars homozygous for Ddw1 with two different triticale cultivars homozygous for ddw1 with the analysis of the association of plant height with Ddw1 genotype using a 77-plant sample from A/SKh and a 91-plant sample from the Kh/D F2 population.
2.5. Rht-B1b Genotyping
Rht1 genotyping was carried out by PCR amplification using primer pairs BF, MR1 and BF, WR1 (Syntol Ltd., Russian Federation) and conditions previously reported in [
16]. PCR products were run on 2% agarose gel. The presence of dwarfing
Rht-B1b allele in plant genome was assigned if primers BF and MR1 yielded PCR product of 237 bp. The absence of
Rht-B1b allele (the presence of
Rht-B1a allele) was assigned if similar product was amplified using BF and WR1 primers.
2.6. Statistical Analysis
The differences in plant height between homozygous Ddw1, ddw1 and heterozygous Ddw1 ddw1 plants for the Kh/D F2 population, which is homogeneously homozygous in the Rht1 gene (Rht-B1b), were accessed using one-way ANOVA. Two-way ANOVA was used for the analysis of the plant height of the A/SKh F2 population, which segregated into two semi-dwarfing alleles, Ddw1 and Rht-B1b. The genotype for each of the genes (Ddw1 and Rht-B1b) was considered as a factor with 3 levels, depending on the allelic state of the gene (dominant homozygous, heterozygous, and recessive homozygous). No interactions between the factors were considered. Bonferroni post-hoc test was used for the comparisons between groups with different allelic states. Statistically significant difference was assumed for p < 0.05. The calculations were done using Statistica 10 software (Statsoft Inc., Tulsa, OK, USA).
4. Discussion
The introduction of the dwarfing
Ddw1 allele increases lodging resistance and productivity of triticale cultivars. MAS significantly facilitates the breeding process, however, there is currently no cheap and reliable marker for the allelic state of the
Ddw1 gene.
REMS1218 marker, which is tightly linked to
Ddw1 was improved by its combination with
REMS1218 SNP marker by Tenhola-Roininen and Tanhuanpa, but was still unable to differentiate
Ddw1 homozygotes from
Ddw1 ddw1 heterozygotes in some cases [
13]. Recently, fine mapping of
Ddw1 in rye has been reported [
7], however, no practical method for
Ddw1 genotyping was provided. In our study we used more types, varieties and a higher number of plants compared to Tenhola-Roininen and Tanhuanpa [
13], and by using capillary electrophoresis we observed one more pattern of
REMS1218 PCR product (Type 3 in the current paper). Types 1 and 2 in our amplifications were consistent with the PCR products observed by Tenhola-Roininen and Tanhuanpa. Despite the differences in PCR product profiles on capillary electrophoresis between the dwarfing
Ddw1 allele and neutral
ddw1 alleles, the
REMS1218 PCR marker cannot distinguish
Ddw1 ddw1 heterozygote from
Ddw1 Ddw1 homozygotes. Indeed, the peak 317 bp is present in all tested genotypes, thus it cannot differentiate dominant
Ddw1 from recessive
ddw1.
Ddw1-specific peak 321 bp appears in both homo- and heterozygous plants.
In our study, we cloned and sequenced
REMS1218 PCR products, and revealed three restriction endonucleases,
Mnl1,
RsaI and
Bso31I, that in combination are able to distinguish polymorphisms of
REMS1218. All variants of restriction fragments of
REMS1218 PCR products that are well-detected in 2% agarose gel are summarized in
Table 1. The
REMS1218 CAPS marker, which is based on
Mnl1,
RsaI and
Bso31I restrictases, allows us to unequivocally identify the
Ddw1 genotype. An example of the universal algorithm for
Ddw1 genotyping of plants with unknown parental lines is shown in
Figure 4. In the case of MAS for
Ddw1, the type of
ddw1 allele (Type 1 or Type 3 of
REMS1218 PCR products) for some cultivars is known. In the current study, Type 1 allele was observed in spring triticale (cv. Dublet and cv. Solovei Kharkovskii), and Type 3 allele was observed in winter triticale (cv. Dozor). For cultivars with an unknown type of
ddw1 allele, this can be quickly determined by capillary electrophoresis since it requires examination of just a few plants. Then, depending on the
ddw1 allele in the parent line, just
Mnl1 (in case of breeding with Type 1) or two restrictases,
RsaI and
Bso31I (in case of breeding with Type 3) is enough for CAPS genotyping. The one-step protocol for
Ddw1 genotyping for MAS is shown in
Table 3. It is worth noting, that
ddw1 ddw1 plants are easily phenotypically determined by height. Thus, in MAS of Type 3
ddw1 lines, the identification of
Ddw1 homozygotes and
Ddw1 ddw1 heterozygotes is essentially enough for further breeding. In this case, the restrictase,
RsaI, is sufficient for MAS of Type 3 plants.
Genotyping by the Ddw1 CAPS marker was identical with genotyping by capillary electrophoresis, and low plant height was statistically significantly associated with homozygous dwarfing Ddw1 genotype in both model experiments, and with heterozygous Ddw1 ddw1 genotype in one of the experiments, perhaps because of the higher number of plants.