Expression divergence of TaMBD2 homoeologous genes encoding methyl CpG-binding domain proteins in wheat (Triticum aestivum L.)

Most hexaploid wheat genes are present as triplicate homoeologs derived from the ancestral species. Previously, we isolated six wheat cDNAs with open reading frame, encoding methyl CpG-binding domain proteins (MBDs). In this study, the genomic and cDNA sequences of three TaMBD2 homoeologous genes were obtained and mapped on chromosomes 5A, 5B and 5D, respectively. These sequences showed a very high conservation in the coding region and the exon/intron structure, but the cDNA sequences are distinguishable by a 9-bp insertion in coding region and a size polymorphism in the 3'-untranslated region (UTR). The expression patterns of each homeologous gene in different tissues of various developmental stages and in response to abiotic stress were analyzed by using real-time PCR. Relative mRNA abundance of the three homoeologs varied considerably in different developmental stages from seedling to developing seeds. Most notably, TaMBD2-5B and TaMBD2-5D were highly responsive to salt stress and TaMBD2-5B was specifically upregulated by low temperature in the seedling leaves. These results provide further evidence for the expression variation of genes duplicated in allopolyploids. Moreover, the variation of TaMBD2 homoeologous gene expression in response to environmental stress may enable plants to better cope with stresses in their natural environments.     

小麦tae-MIR156前体基因的克隆及其靶基因TaSPL17多态性分析

Squamosa-promoter binding protein (SBP)-box基因是植物特有的一类转录因子, 广泛参与植物生长发育, 其部分成员受miR156调控。文章克隆了小麦(Triticum aestivum) tae-MIR156前体基因, 转录后能够形成茎环结构。小麦10个SBP-box基因中, 仅TaSPL3和TaSPL17在编码区存在tae-miR156识别位点。SPL17在普通小麦的A基因组供体种乌拉尔图小麦(Triticum urartu, AA) UR209和B基因组供体种拟斯卑尔脱山羊草(Aegilops speltoides, BB) Y2001中均为多拷贝(SPL17-A1、SPL17-A2和SPL17-A3; SPL17-B1、SPL17-B2和SPL17-B3), 在D基因组供体种粗山羊草(Aegilops tauschii, DD) Ae38中仅检测到一种序列(SPL17-D); SPL17-A2与SPL17-B2, SPL17-A3与SPL17-B3、SPL17-D两两之间序列的一致性程度均大于99%, 且与普通小麦(中国春、衡观35和双丰收)的TaSPL17序列具有较高的一致性, 提示它们可能来源于共同的祖先基因, 并且在进化过程中高度保守。靶基因TaSPL17中的tae-miR156识别位点非常保守, 在根据单株穗数和基因型多样性挑选的SubP1和SubP2群体中均未检测到tae-miR156识别位点存在变异碱基。     

Proteome analysis of diploid,tetraploid and hexaploid wheat: towards understanding genome interaction in protein expression

Hexaploid wheat (Triticum aestivum L.) is derived from a complex hybridization procedure involving three diploid species carrying the A, B and D genomes. The proteome patterns of diploid, tetraploid and hexaploid wheat were analyzed to explore the genome interaction in protein expression. At least two species from each of the diploid and tetraploid were used to compare their proteome maps with a hexaploid wheat cv. Chinese Spring. The ancestral cultivars were selected based on their history of closeness with the cultivated wheat. Proteins were extracted from seed flour and separated by two-dimensional electrophoresis (2-DE) with isoelectric focusing of pH range from 4-10. 2-DE maps of cultivated and ancestral species were analyzed by computer assisted image analyzer. The region of high molecular weight glutenin subunits of hexaploid wheat showed similarity with those of the diploid donors, BB and DD genomes. The omega gliadin, which is controlled by B genome in common wheat, was assumed to have evolved as a result of interaction between AA and BB genomes. The low molecular weight glutenins and alpha and beta gliadin regions were contributed by the three genomes. This result suggests that the function of donor genomes particularly in the expression of proteins in hexaploid wheat is not totally independent; rather it is the product of interactions among the diploid genomes in the hexaploid nuclear constitutions. The expression of nonstorage proteins was affected substantially due to the removal of the D genome from hexaploid constitution. Location of the structural gene controlling one of the alpha amylase inhibitor proteins in the nonstorage protein region was identified in the short arm of chromosome 3D.     

从CIMMYT引进的人工合成六倍体小麦D染色体组微卫星分子标记的遗传差异

采用微卫星（SSR）分子标记技术,选用23个D染色体组特异性引的对来自CIMMYT的26份人工合成六倍体小麦D染色体组的遗传多样性进行了分析。研究发现,26份材料在D染色体组上存在丰富的等位基因变异（92个）,平均每个基因座为4个。遗传距离计算结果也显示,26份材料D染色体组之间具有较大的遗传差异,平均遗传距离高达0．4955。因此,人工合成六倍体小麦D染色体组中存在丰富的遗传多样性,可以作为拓宽普通小麦遗传基础的新的遗传变异来源。研究还发现,由同一个粗山羊草基因型与不同硬粒小麦杂交合成的人工合成六倍体小麦（如合成种17和18）在所用检测的23个基因座中有3个存在差异,说明小麦在多倍化后,供体基因组在重复序列区域会发生遗传分化。     

Lr34 multi-pathogen resistance ABC transporter: molecular analysis of homoeologous and orthologous genes in hexaploid wheat and other grass species

The Triticum aestivum (bread wheat) disease resistance gene Lr34 confers durable, race non-specific protection against three fungal pathogens, and has been a highly relevant gene for wheat breeding since the green revolution. Lr34, located on chromosome 7D, encodes an ATP-binding cassette (ABC) transporter. Both wheat cultivars with and without Lr34-based resistance encode a putatively functional protein that differ by only two amino acid polymorphisms. In this study, we focused on the identification and characterization of homoeologous and orthologous Lr34 genes in hexaploid wheat and other grasses. In hexaploid wheat we found an expressed and putatively functional Lr34 homoeolog located on chromosome 4A, designated Lr34-B. Another homoeologous Lr34 copy, located on chromosome 7A, was disrupted by the insertion of repetitive elements. Protein sequences of LR34-B and LR34 were 97% identical. Orthologous Lr34 genes were detected in the genomes of Oryza sativa (rice) and Sorghum bicolor (sorghum). Zea mays (maize), Brachypodium distachyon and Hordeum vulgare (barley) lacked Lr34 orthologs, indicating independent deletion of this particular ABC transporter. Lr34 was part of a gene-rich island on the wheat D genome. We found gene colinearity on the homoeologous A and B genomes of hexaploid wheat, but little microcolinearity in other grasses. The homoeologous LR34-B protein and the orthologs from rice and sorghum have the susceptible haplotype for the two critical polymorphisms distinguishing the LR34 proteins from susceptible and resistant wheat cultivars. We conclude that the particular Lr34-haplotype found in resistant wheat cultivars is unique. It probably resulted from functional gene diversification that occurred after the polyploidization event that was at the origin of cultivated bread wheat.     

The Ha locus of wheat: identification of a polymorphic region for tracing grain hardness in crosses.

The grain softness proteins or friabilins are known to be composed of three main components: puroindoline a, puroindoline b, and GSP-1. cDNAs for GSP-1 have previously been mapped to group-5 chromosomes and their location on chromosome 5D is closely linked to the grain hardness (Ha) locus of hexaploid wheat. A genomic DNA clone containing the GSP-1 gene (wGSP1-A1) from hexaploid wheat has been identified by fluorescent in situ hybridization as having originated from the distal end of the short arm of chromosome 5A. A genomic clone containing the gene (wGSP1-D1) was also isolated from Aegilops tauschii, the donor of the D genome to bread wheat. There are no introns in the GSP-1 genes, and there is high sequence identity between wGSP1-A1 and wGSP1-D1 up to 1 kb 5' and 300 bp 3' to wGSP1-D1. However, regions further upstream and downstream of wGSP1-D1 share no significant sequence identity to corresponding sequences in wGSP1-A1. These regions therefore identified potentially valuable sequences for tracing the Ha locus through assaying polymorphic DNA sequences. The sequence from 300 to 500 bp 3' to wGSP1-D1 (wGSP1-D13) was mapped to the Ha locus in a mapping population. wGSP1-D13 was also tightly linked to genes for puroindoline a and puroindoline b which have been previously mapped to be at the Ha locus. In addition wGSP1-D13 was used to detect RFLPs between near isogenic soft and hard Falcon lines and in a random selection of soft and hard wheats.     

Nucleotide diversity and molecular evolution of the WAG-2 gene in common wheat (Triticum aestivum L) and its relatives

In this work, we examined the genetic diversity and evolution of the WAG-2 gene based on new WAG-2 alleles isolated from wheat and its relatives. Only single nucleotide polymorphisms (SNP) and no insertions and deletions (indels) were found in exon sequences of WAG-2 from different species. More SNPs and indels occurred in introns than in exons. For exons, exons+introns and introns, the nucleotide polymorphism π decreased from diploid and tetraploid genotypes to hexaploid genotypes. This finding indicated that the diversity of WAG-2 in diploids was greater than in hexaploids because of the strong selection pressure on the latter. All dn/ds ratios were < 1.0, indicating that WAG-2 belongs to a conserved gene affected by negative selection. Thirty-nine of the 57 particular SNPs and eight of the 10 indels were detected in diploid species. The degree of divergence in intron length among WAG-2 clones and phylogenetic tree topology suggested the existence of three homoeologs in the A, B or D genome of common wheat. Wheat AG-like genes were divided into WAG-1 and WAG-2 clades. The latter clade contained WAG-2, OsMADS3 and ZMM2 genes, indicating functional homoeology among them.     

Molecular mapping of wheat. Homoeologous group 3.

A prerequisite for molecular level genetic studies and breeding in wheat is a molecular marker map detailing its similarities with those of other grass species in the Gramineae family. We have constructed restriction fragment length polymorphism maps of the A-, B-, and D-genome chromosomes of homoeologous group 3 of hexaploid wheat (Triticum aestivum L. em. Thell) using 114 F7-8 lines from a synthetic x bread wheat cross. The map consists of 58 markers spanning 230 cM on chromosome 3A, 62 markers spanning 260 cM on 3B, and 40 markers spanning 171 cM on 3D. Thirteen libraries of genomic or cDNA clones from wheat, barley, and T. tauschii, the wheat D genome donor, are represented, facilitating the alignment and comparison of these maps with maps of other grass species. Twenty-four clones reveal homoeoloci on two of the three genomes and the associated linkages are largely comparable across genomes. A consensus sequence of orthologous loci in grass species genomes is assembled from this map and from existing maps of the chromosome-3 homoeologs in barley (Hordeum spp.), T. tauschii, and rice (Oryza spp.). It illustrates the close homoeology among the four species and the partial homoeology of wheat chromosome 3 with oat (Avena spp.) chromosome C. Two orthologous red grain color genes, R3 and R1, are mapped on chromosome arms 3BL and 3DL.     

Analysis of the functions of TaGW2 homoeologs in wheat grain weight and protein content traits

GW2 is emerging as a key genetic determinant of grain weight in cereal crops; it has three homoeologs (TaGW2‐A1, ‐B1 and ‐D1) in hexaploid common wheat (Triticum aestivum L.). Here, by analyzing the gene editing mutants that lack one (B1 or D1), two (B1 and D1) or all three (A1, B1 and D1) homoeologs of TaGW2, several insights are gained into the functions of TaGW2‐B1 and ‐D1 in common wheat grain traits. First, both TaGW2‐B1 and ‐D1 affect thousand‐grain weight (TGW) by influencing grain width and length, but the effect conferred by TaGW2‐B1 is stronger than that of TaGW2‐D1. Second, there exists functional interaction between TaGW2 homoeologs because the TGW increase shown by a double mutant (lacking B1 and D1) was substantially larger than that of their single mutants. Third, both TaGW2‐B1 and ‐D1 modulate cell number and length in the outer pericarp of developing grains, with TaGW2‐B1 being more potent. Finally, TaGW2 homoeologs also affect grain protein content as this parameter was generally increased in the mutants, especially in the lines lacking two or three homoeologs. Consistent with this finding, two wheat end‐use quality‐related parameters, flour protein content and gluten strength, were considerably elevated in the mutants. Collectively, our data shed light on functional difference between and additive interaction of TaGW2 homoeologs in the genetic control of grain weight and protein content traits in common wheat, which may accelerate further research on this important gene and its application in wheat improvement.     

黄淮麦区地方小麦品种籽粒颜色相关基因Tamyb10-1等位变异检测?

Tamyb10-1基因属于MYB家族的一种转录因子,决定着小麦种皮的颜色,同时对穗发芽抗性也具有一定影响。本研究以来自我国黄淮麦区的地方小麦品种为材料,利用功能标记对参试小麦品种Tamyb10-1基因位点在3A、3B和3D染色体上的等位变异类型进行了检测。结果表明,参试材料中上述每一位点均有2种等位变异类型,由此形成了7种基因型组合,分别为Tamyb10-A1a/Tamyb10-B1a/Tamyb10-D1a、Tamyb10-A1a/Tamyb10-B1a/Tamyb10-D1b、Tamyb10-A1a/Tamyb10-B1b/Tamyb10-D1a、Tamyb10-A1b/Tamyb10-B1a/Tamyb10-D1a、Tamyb10-A1b/Tamyb10-B1b/Tamyb10-D1a、Tamyb10-A1b/Tamyb10-B1a/Tamyb10-D1b和Tamyb10-A1b/Tamyb10-B1b/Tamyb10-D1b,其分布频率分别为38.0%、15.0%、1.0%、8.0%、1.0%、33.0%和4.0%。进一步研究结果表明,种皮颜色为白色时,Tamyb10-1基因在3个位点均为野生型,而当任何一个位点发生突变时均表现为红色。由于该基因也影响穗发芽的抗性,且子粒颜色与其抗氧化能力密切相关,因此本研究对以子粒颜色性状为育种目标的优异种质资源筛选具有一定参考价值。     

Genetic characterization and mapping of the Rht-1 homoeologs and flanking sequences in wheat

The introgression of Reduced height (Rht)-B1b and Rht-D1b into bread wheat (Triticum aestivum) varieties beginning in the 1960s led to improved lodging resistance and yield, providing a major contribution to the ‘green revolution’. Although wheat Rht-1 and surrounding sequence is available, the genetic composition of this region has not been examined in a homoeologous series. To determine this, three Rht-1-containing bacterial artificial chromosome (BAC) sequences derived from the A, B, and D genomes of the bread wheat variety Chinese Spring (CS) were fully assembled and analyzed. This revealed that Rht-1 and two upstream genes were highly conserved among the homoeologs. In contrast, transposable elements (TEs) were not conserved among homoeologs with the exception of intronic miniature inverted-repeat TEs (MITEs). In relation to the Triticum urartu ancestral line, CS-A genic sequences were highly conserved and several colinear TEs were present. Comparative analysis of the CS wheat BAC sequences with assembled Poaceae genomes showed gene synteny and amino acid sequences were well preserved. Further 5′ and 3′ of the wheat BAC sequences, a high degree of gene colinearity is present among the assembled Poaceae genomes. In the 20 kb of sequence flanking Rht-1, five conserved non-coding sequences (CNSs) were present among the CS wheat homoeologs and among all the Poaceae members examined. Rht-A1 was mapped to the long arm of chromosome 4 and three closely flanking genetic markers were identified. The tools developed herein will enable detailed studies of Rht-1 and linked genes that affect abiotic and biotic stress response in wheat.     

Origin, dispersal and genomic structure of a low-copy-number hypervariable RFLP clone in Triticum and Aegilops species

The genome of common wheat has evolved through allopolyploidization of three ancestral diploid genomes. A previously identified restriction fragment length polymorphism (RFLP) marker, pTag546, has the unique feature of showing hypervariability among closely related common wheat cultivars. To understand the origin and the mode of dispersal of this hypervariable sequence in the wheat genome, the distribution and structure of the homologous sequences were studied using ancestral diploid species, tetraploid disomic substitution lines and synthetic hexaploid lines. Comparative Southern blot and PCR analyses suggested that pTag546 homologs in the tetraploid and hexaploid wheat were derived from the S genome of Aegilops speltoides. Some pTag546 homologs were found to have transposed to A and D genomes in polyploid wheat. Evidence of transposition and elimination in some synthetic hexaploid lines was also obtained by comparing their copy numbers with those in the parental lines. Southern blot analysis of a genomic clone using a contiguous subset of sequences as probes revealed a core region of hypervariability that coincided with the region containing pTag546. No obvious structural characteristics that could explain the hypervariability, however, were found around the pTag546 sequence, except for accumulation of small repetitive sequences at one border. It was concluded that pTag546 increased its copy number through yet unknown mechanism(s) of transposition to various chromosomal locations over the period of allopolyploid evolution and during the artificial genome manipulation in wheat.     

Retention of D genome chromosomes in pentaploid wheat crosses

The transfer of genes between Triticum aestivum (hexaploid bread wheat) and T. turgidum (tetraploid durum wheat) holds considerable potential for genetic improvement of both these closely related species. Five different T. aestivum/T. turgidum ssp. durum crosses were investigated using Diversity Arrays Technology (DArT) markers to determine the inheritance of parental A, B and D genome material in subsequent generations derived from these crosses. The proportions of A, B and D chromosomal segments inherited from the hexaploid parent were found to vary significantly among individual crosses. F(2) populations retained widely varying quantities of D genome material, ranging from 99% to none. The relative inheritance of bread wheat and durum alleles in the A and B genomes of derived lines also varied among the crosses. Within any one cross, progeny without D chromosomes in general had significantly more A and B genome durum alleles than lines retaining D chromosomes. The ability to select for and manipulate this non-random segregation in bread wheat/durum crosses will assist in efficient backcrossing of selected characters into the recurrent durum or hexaploid genotype of choice. This study illustrates the utility of DArT markers in the study of inter-specific crosses to commercial crop species.     

Recurrent deletions of puroindoline genes at the grain hardness locus in four independent lineages of polyploid wheat

Polyploidy is known to induce numerous genetic and epigenetic changes but little is known about their physiological bases. In wheat, grain texture is mainly determined by the Hardness (Ha) locus consisting of genes Puroindoline a (Pina) and b (Pinb). These genes are conserved in diploid progenitors but were deleted from the A and B genomes of tetraploid Triticum turgidum (AB). We now report the recurrent deletions of Pina-Pinb in other lineages of polyploid wheat. We analyzed the Ha haplotype structure in 90 diploid and 300 polyploid accessions of Triticum and Aegilops spp. Pin genes were conserved in all diploid species and deletion haplotypes were detected in all polyploid Triticum and most of the polyploid Aegilops spp. Two Pina-Pinb deletion haplotypes were found in hexaploid wheat (Triticum aestivum; ABD). Pina and Pinb were eliminated from the G genome, but maintained in the A genome of tetraploid Triticum timopheevii (AG). Subsequently, Pina and Pinb were deleted from the A genome but retained in the A(m) genome of hexaploid Triticum zhukovskyi (A(m)AG). Comparison of deletion breakpoints demonstrated that the Pina-Pinb deletion occurred independently and recurrently in the four polyploid wheat species. The implications of Pina-Pinb deletions for polyploid-driven evolution of gene and genome and its possible physiological significance are discussed.