Development of genome-specific primers for homoeologous genes in allopolyploid species: the waxy and starch synthase II genes in allohexaploid wheat (Triticum aestivum L.) as examples

Abstract

Background: In allopolypoid crops, homoeologous genes in different genomes exhibit a very high sequence similarity, especially in the coding regions of genes. This makes it difficult to design genome-specific primers to amplify individual genes from different genomes. Development of genome-specific primers for agronomically important genes in allopolypoid crops is very important and useful not only for the study of sequence diversity and association mapping of genes in natural populations, but also for the development of gene-based functional markers for marker-assisted breeding. Here we report on a useful approach for the development of genome-specific primers in allohexaploid wheat.

Findings: In the present study, three genome-specific primer sets for the waxy (Wx) genes and four genome-specific primer sets for the starch synthase II (SSII) genes were developed mainly from single nucleotide polymorphisms (SNPs) and/or insertions or deletions (Indels) in introns and intron-exon junctions. The size of a single PCR product ranged from 750 bp to 1657 bp. The total length of amplified PCR products by these genome-specific primer sets accounted for 72.6%-87.0% of the Wx genes and 59.5%-61.6% of the SSII genes. Five genome-specific primer sets for the Wx genes (one for Wx-7A, three for Wx-4A and one for Wx-7D) could distinguish the wild type wheat and partial waxy wheat lines. These genome-specific primer sets for the Wx and SSII genes produced amplifications in hexaploid wheat, cultivated durum wheat, and Aegilops tauschii accessions, but failed to generate amplification in the majority of wild diploid and tetraploid accessions.

Conclusions: For the first time, we report on the development of genome-specific primers from three homoeologous Wx and SSII genes covering the majority of the genes in allohexaploid wheat. These genome-specific primers are being used for the study of sequence diversity and association mapping of the three homoeologous Wx and SSII genes in natural populations of both hexaploid wheat and cultivated tetraploid wheat. The strategies used in this paper can be used to develop genome-specific primers for homoeologous genes in any allopolypoid species. They may be also suitable for (i) the development of gene-specific primers for duplicated paralogous genes in any diploid species, and (ii) the development of allele-specific primers at the same gene locus.

Figure 1

Alignment of partial genomic DNA sequences of three homoeologous SSII genes from hexaploid wheat indicating the position of genome-specific primers. The exon and intron regions are shown with gray and white color, respectively. Deletions are indicated by dashes. The sequence of the forward primer and complementary sequences of the reverse primers are boxed.

Figure 2

A comparison between unspecific (SSII-7A-F3/SSII-7A-R3) and specific (SSII-7A-F3/SSII-7A-R3a) primer sets. Each primer set was used to amplify Chinese Spring (CS), N7AT7D, N7BT7D and N7DT7B. M = 1 kb plus DNA ladder.

Figure 3

Genome-specific PCR amplification for the three homoeologous Wx loci. Each primer set was used to amplify Chinese Spring (CS, lane 1), N7AT7D (lane 2) and N4AT4D (lane 3) and N7DT7B (lane 4). M = 1 kb plus DNA ladder. PCR amplification with (A) 7A-specific primer sets, (B) 4A (B genome)-specific primer sets, and (C) 7D-specific primer sets.

Figure 4

Genome-specific PCR amplification for the three homoeologous SSII loci. Each primer set was used to amplify Chinese Spring (CS, lane 1), N7AT7D (lane 2) and N7BT7D (lane 3) and N7DT7B (lane 4). M = 1 kb plus DNA ladder. PCR amplification with (A) 7A-specific primer sets, (B) 7B-specific primer sets, and (C) 7D-specific primer sets.

Figure 5

PCR amplification for distinguishing null alleles of the waxy genes and wild type in wheat. (A) PCR results with primer set Wx-7A-F1 and Wx-7A-R1a to distinguish the wild type (Wx-A1a) and null allele (Wx-A1b) of the Wx-A1 gene. (B) PCR results with primer set Wx-7B-F2 and Wx-7B-R2 to distinguish the wild type (Wx-B1a) and null allele (Wx-B1b) of the Wx-B1 gene. (C) PCR results with primer set Wx-7D-F3 and Wx-7D-R3a to distinguish the wild type (Wx-D1a) and null allele (Wx-D1b) of the Wx-D1 gene. M = 1 kb plus DNA ladder. Lanes: 1, CS; 2, Sturdy; 3, Fujimi Komugi; 4, Gabo; 5, Gamenya; 6, Santanta; 7, Bai Huo; 8, Kanto 107; 9, NSGC 8645; 10, NSGC 8646.

Figure 6

PCR amplification of 18 diploid (Triticum and Aegilops species) and 13 tetraploid (wild and domesticated forms) progenitors of hexaploid wheat using the primer set SSII-7B-F2/SSII-7B-R2. M = 1 kb plus DNA ladder. Lanes 1-2, T. monococcum accessions; lanes 3-5, T. boeoticum accessions; lanes 6-8, T. urartu accessions; lanes 9-13, T. turgidum ssp. durum accessions; lanes 14-18, T. turgidum ssp. dicoccon accessions; lanes 19-21, T. turgidum ssp. dicoccoides accessions; lanes 22-25, Ae. speltoides accessions; lanes 26-31, Ae. tauschii accessions.