长穗偃麦草分子育种基础研究进展

长穗偃麦草(Elytrigiaelongata)为禾本科小麦族偃麦草属植物,是一种丛生的多年生冷季型牧草。长穗偃麦草原产于欧洲南部、小亚细亚和俄罗斯南部,在美国、加拿大和澳大利亚等国大面积种植。其引入我国后,1956年开始用作小麦(Triticumaestivum)远缘杂交的野生亲本,鲜有作为牧草大面积种植的报道。长穗偃麦草具有耐盐碱、耐涝和抗病等特点,可作为耐盐碱牧草用于建设“滨海草带”,利于避免草粮争地/争水,实现碳中和,保障我国粮食安全。全世界已育成推广了10余个长穗偃麦草品种,但我国尚无引种或自主选育品种,不利于“滨海草带”的建立。长穗偃麦草遗传背景复杂,基础研究薄弱,育种技术远远落后于稻麦等粮食作物。该文对长穗偃麦草分子育种基础,如育种历程、加速育种、资源创新、组织培养、基因组序列及分子标记进行综述,以期为长穗偃麦草品种选育和“滨海草带”建设提供参考。     

长穗偃麦草Actin基因片段克隆及表达模式分析

根据已报道的其他植物肌动蛋白(Actin,ACT)基因的保守序列设计一对简并性引物,以长穗偃麦草根中总RNA为模板,采用RT-PCR方法克隆出ACT基因,命名为EeACT。结果表明,该基因片段长度598 bp,编码198个氨基酸,与其他植物ACT氨基酸序列同源性较高,达90%以上。低温、盐及干旱处理12 h,EeACT在其地上部与根中的表达水平没有显著差异,说明EeACT表达稳定。     

小麦×长穗偃麦草的受精和早期胚胎发育

用石蜡切片法,对小麦（Ｔｒｉｔｉｃｕｍａｅｓｔｉｖｕｍ）和长穗偃麦草（Ｅｌｙｔｒｉｇｉａｅｌｏｎｇａｔａ）杂交的受精和早期胚胎发育进行了观察。结果表明,长穗偃麦草花粉在小麦柱头上萌发良好,花粉管可顺利长入花柱和胚囊。观察的１７０个小麦子房中,１７６５％发生了双受精,产生了胚和胚乳;９４１％发生了单卵受精,只产生胚而无胚乳;４７１％发生了单极核受精,只产生胚乳而无胚;总受精率为３１７７％;成胚率为２７０６％。由于胚乳的缺乏或发育异常及败育,最终难以获得有生活力的种子。为小麦与长穗偃麦草远缘杂交提供了细胞胚胎学证据。     

小麦x长穗偃麦草的受精和早期胚胎发育

用石蜡切片法,对小麦(Triticum aestivum)和长穗偃麦草(Elytrigia elongata)杂交的受精和早期胚胎发育进行了观察。结果表明,长穗偃麦草花粉在小麦柱头上萌发良好,花粉管可顺利长人花柱和胚囊。 观察的170个小麦子房中,17.65％发生了双受精,产生了胚和胚乳;9.41％发生了单卵受精,只产生胚而无胚乳;4.71％。发生了单极核受精,只产生胚乳而无胚;总受精率为31.77％;成胚率为27.06％。由于胚乳的缺乏或发育异常及败育,最终难以获得有生活力的种子。为小麦与长穗偃麦草远缘杂交提供了细胞胚胎学证据。     

长穗偃麦草优异基因的染色体定位及应用

长穗偃麦草比较公认的有2个种,即二倍体长穗偃麦草(Thinopyrum elongatum,2n=2X)和十倍体长穗偃麦草(Thinopyrum ponticum,2n=10X),是重要的小麦近缘种,具有抗病、抗寒、抗旱、耐盐碱等优良性状。因其基因组中蕴含许多对小麦品种改良极为有用的基因,且易与小麦杂交等优势,多年来长穗偃麦草一直作为小麦遗传改良的优良种质资源而备受关注。本文对长穗偃麦草的基因组研究及其在小麦的抗逆、抗病和提高光合能力、产量及高分子量谷蛋白(HMW-GS)含量等方面的应用做了综述,为其基因组中优异基因的进一步开发和利用提供了理论依据。     

利用生化及分子标记确定长穗偃麦草（Elytrigia elongatum,EE.2n=14） …

利用限制性片段长度多态性（ＲＦＬＰ）及等电聚焦（ＩＥＦ）技术确定普通小麦中国春－二倍体长穗偃麦草７个异附加系所附加的外源染以体与小麦染色体的部分同源性,共有８个生化标记,１３个ＲＦＬＰ标记在亲本间揭示了多态性,结果表明：长穗偃麦草的１Ｅ,２Ｅ,３Ｅ,４Ｅ,５Ｅ,６Ｅ,７Ｅ７条染色体分别与小麦染色体的１、２、３、４、５、６、７７个部分同源群具有部分同源关系,偃麦草的１Ｅ与７Ｅ、５Ｅ与７Ｅ染色体间可能     

长穗偃麦草中E和E1基因组高分子量麦谷蛋白基因启动子部分序列的进化分析

通过PCR克隆的方法,获得了分别来自二倍体长穗偃麦草的E基因组和四倍体长穗偃麦草的E_1基因组的4个高分子量麦谷蛋白亚基(HMW-GS)基因启动子的部分序列。序列分析表明,它们之间的同源性较高,两个x型亚基启动子序列之间只有1个碱基的差异,而两个y型亚基启动子序列完全相同,x和y型亚基启动子序列之间的长度和部分碱基位点都有差异。推测四倍体长穗偃麦草中的E_1基因组可能起源于二倍体的E基因组。与来自小麦族的A、B、D和G基因组部分亚基基因的启动子序列比较表明,小麦族的这一区域在进化上是相当保守的,不同基因组来源的序列同源性都在90％以上。经过对这些序列的聚类分析,表明长穗偃麦草的y型HMW-GS基因与其他亚基基因的进化关系较远,而x型亚基基因与一个来自小麦1B染色体的亚基基因关系最近。     

长穗偃麦草基因组中与耐低磷营养胁迫有关的基因的染色体定位

以中国春－长穗偃麦草二体异附加系和二体异代换系为材料，对其耐低磷营养胁迫特性进行鉴定和遗传分析，结果表明（１）长穗偃麦草的４Ｅ一＾ 色体携有耐低营养胁迫的基因，且其效应远远超过背景亲本中国春。     

长穗偃麦草DREB类基因EeAP2.2 的克隆与序列分析

由于一些基因的特殊碱基序列限制,使得应用一种技术获得基因的全长cDNA序列比较困难。本研究结合RACE和Genome Walking技术从十倍体长穗偃麦草（Elytrigia elongata,2n=70）中克隆了AP2家族的一个全长cDNA序列,命名为EeAP2.2。序列分析表明,该基因具有一个837bp的开放阅读框,编码279个氨基酸残基,含有一个保守的AP2结构域,是AP2大家族的一个新成员。该基因编码的氨基酸序列与GenBank已有的普通小麦AP2家族两个同源基因编码蛋白TaDREB1和TaDREBW50（登录号分别为：AAL01124.1和AAY44605.1）具有98%的氨基酸序列一致性, 与大麦AP2蛋白HvDREB1-a（登录号AAY25517.1）,高羊茅AP2蛋白FaDREB2A （登录号CAG30547.1） 及水稻OsDREB2.2（登录号AY064403）的氨基酸序列一致性分别为 93%、86%、69%。说明该基因与小麦AP2家族基因的同源性最高。本研究除获得了长穗偃麦草一个重要抗逆转录因子基因EeAP2.2的全长cDNA序列外,也提供了一种快速、有效克隆功能基因的方法。     

将Thinopyrum bessarabicum和Thinopyrum elongatum的种质导？…

２个双倍体即Ｃ．Ｓ－Ｔｈｉｎｏｐｙｒｕｍ ｂｅｓｓａｒａｂｉｃｕｍ（ＡＡＢＢＤＤＪＪ ２ｎ＝８ｘ＝５６）和ＧＨＫ－Ｔｈｉｎｏｐｙｒｕｍ ｅｌｏｎｇａｔｕｍ（ＡＡＢＢＤＤＥＥ ２ｎ＝８ｘ＝５６）与普通小麦“中国春”杂交,获得了２个七倍体杂种。对２３个双倍体、中国春、杂种Ｆ１和部分Ｆ２进行麦谷蛋白ＳＤＳ－Ｐａｇｅ电泳,及结合染色体分带技术和田间赤霉病抗性鉴定筛选出由Ｔｈ．ｂｅｓｓａｒａｂｉｃｕｍ和Ｔｈ     

Genome-wide identification and characterization of replication origins by deep sequencing

Background

DNA replication initiates at distinct origins in eukaryotic genomes, but the genomic features that define these sites are not well understood.

Results

We have taken a combined experimental and bioinformatic approach to identify and characterize origins of replication in three distantly related fission yeasts: Schizosaccharomyces pombe, Schizosaccharomyces octosporus and Schizosaccharomyces japonicus. Using single-molecule deep sequencing to construct amplification-free high-resolution replication profiles, we located origins and identified sequence motifs that predict origin function. We then mapped nucleosome occupancy by deep sequencing of mononucleosomal DNA from the corresponding species, finding that origins tend to occupy nucleosome-depleted regions.

Conclusions

The sequences that specify origins are evolutionarily plastic, with low complexity nucleosome-excluding sequences functioning in S. pombe and S. octosporus, and binding sites for trans-acting nucleosome-excluding proteins functioning in S. japonicus. Furthermore, chromosome-scale variation in replication timing is conserved independently of origin location and via a mechanism distinct from known heterochromatic effects on origin function. These results are consistent with a model in which origins are simply the nucleosome-depleted regions of the genome with the highest affinity for the origin recognition complex. This approach provides a general strategy for understanding the mechanisms that define DNA replication origins in eukaryotes.     

Identification and characterization of trait-specific SNPs using ddRAD sequencing in water buffalo

Single Nucleotide Polymorphism (SNP) is one of the important molecular markers widely used in animal breeding program for improvement of any desirable genetic traits. Considering this, the present study was carried out to identify, annotate and analyze the SNPs related to four important traits of buffalo viz. milk volume, age at first calving, post-partum cyclicity and feed conversion efficiency. We identified 246,495, 168,202, 74,136 and 194,747 genome-wide SNPs related to mentioned traits, respectively using ddRAD sequencing technique based on 85 samples of Murrah Buffaloes. Distribution of these SNPs were highest (61.69%) and lowest (1.78%) in intron and exon regions, respectively. Under coding regions, the SNPs for the four traits were further classified as synonymous (4697) and non-synonymous (3827). Moreover, Gene Ontology (GO) terms of identified genes assigned to various traits. These characterized SNPs will enhance the knowledge of cellular mechanism for enhancing productivity of water buffalo through molecular breeding.     

Genome-wide identification of the Phaseolus vulgaris sRNAome using small RNA and degradome sequencing

Background

MiRNAs and phasiRNAs are negative regulators of gene expression. These small RNAs have been extensively studied in plant model species but only 10 mature microRNAs are present in miRBase version 21, the most used miRNA database, and no phasiRNAs have been identified for the model legume Phaseolus vulgaris. Thanks to the recent availability of the first version of the common bean genome, degradome data and small RNA libraries, we are able to present here a catalog of the microRNAs and phasiRNAs for this organism and, particularly, we suggest new protagonists in the symbiotic nodulation events.

Results

We identified a set of 185 mature miRNAs, including 121 previously unpublished sequences, encoded by 307 precursors and distributed in 98 families. Degradome data allowed us to identify a total of 181 targets for these miRNAs. We reveal two regulatory networks involving conserved miRNAs: those known to play crucial roles in the establishment of nodules, and novel miRNAs present only in common bean, suggesting a specific role for these sequences. In addition, we identified 125 loci that potentially produce phased small RNAs, with 47 of them having all the characteristics of being triggered by a total of 31 miRNAs, including 14 new miRNAs identified in this study.

Conclusions

We provide here a set of new small RNAs that contribute to the broader knowledge of the sRNAome of Phaseolus vulgaris. Thanks to the identification of the miRNA targets from degradome analysis and the construction of regulatory networks between the mature microRNAs, we present here the probable functional regulation associated with the sRNAome and, particularly, in N₂-fixing symbiotic nodules.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1639-5) contains supplementary material, which is available to authorized users.     

Improved Heritability Estimation from Genome-wide SNPs

Estimation of narrow-sense heritability, h², from genome-wide SNPs genotyped in unrelated individuals has recently attracted interest and offers several advantages over traditional pedigree-based methods. With the use of this approach, it has been estimated that over half the heritability of human height can be attributed to the ∼300,000 SNPs on a genome-wide genotyping array. In comparison, only 5%–10% can be explained by SNPs reaching genome-wide significance. We investigated via simulation the validity of several key assumptions underpinning the mixed-model analysis used in SNP-based h² estimation. Although we found that the method is reasonably robust to violations of four key assumptions, it can be highly sensitive to uneven linkage disequilibrium (LD) between SNPs: contributions to h² are overestimated from causal variants in regions of high LD and are underestimated in regions of low LD. The overall direction of the bias can be up or down depending on the genetic architecture of the trait, but it can be substantial in realistic scenarios. We propose a modified kinship matrix in which SNPs are weighted according to local LD. We show that this correction greatly reduces the bias and increases the precision of h² estimates. We demonstrate the impact of our method on the first seven diseases studied by the Wellcome Trust Case Control Consortium. Our LD adjustment revises downward the h² estimate for immune-related diseases, as expected because of high LD in the major-histocompatibility region, but increases it for some nonimmune diseases. To calculate our revised kinship matrix, we developed LDAK, software for computing LD-adjusted kinships.     

Synthesis and cytological characterization of trigeneric hybrids involving durum wheat,Thinopyrum bessarabicum,and Lophopyrum elongatum

Summary In an attempt to transfer genes for salt tolerance and other desirable traits from the diploid wheatgrasses, Thinopyrum bessarabicum (2n=2x=14; JJ genome) and Lophopyrum elongatum (2n=2x=14; EE genome), into durum wheat cv Langdon (2n=4x=28; AABB genomes), trigeneric hybrids with the genomic constitution ABJE were synthesized and cytologically characterized. C-banding analysis of somatic chromosomes of the A, B, J, and E genomes in the same cellular environment revealed distinct banding patterns; each of the 28 chromosomes could be identified. They differed in the total amount of constitutive heterochromatin. Total surface area and C-banded area of each chromosome were calculated. The B genome was the largest in size, followed by the J, A, and E genomes, and its chromosomes were also the most heavily banded. Only 25.8% of the total chromosome complement in 10 ABJE hybrids showed association, with mean arm-pairing frequency (c) values from 0.123 to 0.180 and chiasma frequencies from 3.36 to 5.02 per cell. The overall mean pairing was 0.004 ring IV + 0.046 chain IV + 0.236 III + 0.21 ring II + 2.95 rod II + 20.771. This is total pairing between chromosomes of different genomes, possibly between A and B, A and J, A and E, B and J, B and E, and J and E, in the presence of apparently functional pairing regulator Ph1. Because chromosome pairing in the presence of Ph1 seldom occurs between A and B, or between J and E, it was inferred that pairing between the wheat chromosomes and alien chromosomes occurred. The trigeneric hybrids with two genomes of wheat and one each of Thinopyrum and Lophopyrum should be useful in the production of cytogenetic stocks to facilitate the transfer of alien genes into wheat.     

Genome-wide selection of tag SNPs using multiple-marker correlation

MOTIVATIONS: The tag SNP approach is a valuable tool in whole genome association studies, and a variety of algorithms have been proposed to identify the optimal tag SNP set. Currently, most tag SNP selection is based on two-marker (pairwise) linkage disequilibrium (LD). Recent literature has shown that multiple-marker LD also contains useful information that can further increase the genetic coverage of the tag SNP set. Thus, tag SNP selection methods that incorporate multiple-marker LD are expected to have advantages in terms of genetic coverage and statistical power. RESULTS: We propose a novel algorithm to select tag SNPs in an iterative procedure. In each iteration loop, the SNP that captures the most neighboring SNPs (through pair-wise and multiple-marker LD) is selected as a tag SNP. We optimize the algorithm and computer program to make our approach feasible on today's typical workstations. Benchmarked using HapMap release 21, our algorithm outperforms standard pair-wise LD approach in several aspects. (i) It improves genetic coverage (e.g. by 7.2% for 200 K tag SNPs in HapMap CEU) compared to its conventional pair-wise counterpart, when conditioning on a fixed tag SNP number. (ii) It saves genotyping costs substantially when conditioning on fixed genetic coverage (e.g. 34.1% saving in HapMap CEU at 90% coverage). (iii) Tag SNPs identified using multiple-marker LD have good portability across closely related ethnic groups and (iv) show higher statistical power in association tests than those selected using conventional methods. AVAILABILITY: A computer software suite, multiTag, has been developed based on this novel algorithm. The program is freely available by written request to the author at ke_hao@merck.com     

Genome-wide SNPs reveal the fine-scale population structure of Laodelphax striatellus in China using double-digest restriction site-associated DNA sequencing

The small brown planthopper (SBPH), Laodelphax striatellus (Fallén) is one of the most destructive rice pests and has caused serious economic losses in China. To clarify the genetic differentiation and population genetic structure of this insect pest, we investigated the genomic polymorphisms, genetic differentiation, and phylogeography of 31 SBPH populations from 28 sampling sites from three climatic zones of China using double-digest restriction site-associated DNA sequencing (ddRADseq). In total, 2,813,221,369 high-quality paired-end reads from 306 individuals and 1925 single nucleotide polymorphisms (SNPs) were obtained. Low levels of genetic diversity and significant genetic differentiation were observed among the SBPH populations, and three genetic clusters were detected in China. Neutrality tests and bottleneck analysis provided strong evidence for recent rapid expansion with a severe bottleneck in most populations. Our work provides new insights into the genetics of the SBPH and will contribute to the development of effective management strategies for this pest.