A post-labeling method for multiplexed and multicolored genotyping analysis of SSR, indel and SNP markers in single tube with bar-coded split tag (BStag)

<p>Abstract</p> <p>Background</p> <p>Genotyping analysis using capillary DNA sequencing with fluorescently labeled primer pairs obtained by polymerase chain reaction (PCR) is widely used, but is expensive. The post-PCR labeling method using fluorescently labeled short oligonucleotides and nested PCR of the amplified product obtained from unlabeled primer pairs is a simple and inexpensive alternative. However, previously reported protocols often produced spurious peaks or inconsistent amplification under multiplexed analysis as a result of simultaneous progress of both the amplification and labeling reactions and local homology of the attached tag sequence.</p> <p>Results</p> <p>A set of 16 bp-long oligonucleotide sequences termed bar-coded split tag (BStag), comprising a common basal region, a three-nucleotide 'bar-code' sequence, and a mismatched nucleotide at the middle position were designed for selective post-PCR labeling. The BStag was attached at the 5' end of the forward primer of interest. The melting temperature of the BStag was low enough to separate the labeling reaction from initial PCR amplification, and each sequence was minimally divergent but maintained maximum selectivity. Post-PCR labeling of the amplified product was achieved by extending for three cycles at a lower annealing temperature after the conventional amplification program with the appropriate fluorescently labeled BStag primer. No amplification was confirmed with BStag primers for 12 plant species. The electropherogram of the labeled product obtained using this method was consistent with that of prelabeled primer, except for their apparent size.</p> <p>Conclusions</p> <p>BStag enabled multiplexed post-PCR labeling of simple sequence repeat or insertion/deletion markers with different dyes in a single tube. BStag in conjunction with locus specific oligo and allele specific oligo was also useful for single nucleotide polymorphism analysis. The labeling protocol was simple and no additional operation was required. Single-tube multiplexed post-PCR labeling is useful for a wide variety of genotyping studies with maximal flexibility and minimal costs.</p

Breeding New Premium Quality Cultivars by Citrus Breeding 2.0 in Japan: An Integrative Approach Suggested by Genealogy

Developing varieties with diverse features that satisfy varied commercial needs, improving overall fruit quality, and quickly releasing them, are prerequisites in citrus breeding. However, these three goals require trade-offs in conventional breeding, even with the application of the marker-assisted selection technique. Conventional breeding cannot achieve these three goals simultaneously and it has been regarded as a breeding trilemma. Integrating a genomics-assisted breeding (GAB) approach that relies on quantitative trait locus detection by genome-wide association study and genome-wide prediction of a trait by genomic selection using enriched marker genotypes enhances breeding efficiency and contributes to eliminating the trilemma. Besides these efforts, the analysis of the genealogy of indigenous citrus varieties revealed that many high-quality indigenous varieties were selected within a few generations. It suggested that selecting a new premium quality hybrid is possible by integrating it with the GAB technique and helps avoid the trilemma. This review describes how a new approach, “Citrus Breeding 2.0” works for rapidly developing new, premium quality hybrids and introduces three applications of this technique, specifically, rebreeding, complementary breeding, and mimic breeding based on the ongoing citrus breeding program in NARO, Japan

マイクロアレイを用いた長期低温遭遇時のウメ休眠芽のトランスクリプトーム解析

Bud dormancy is a critical developmental process for perennial plant survival, and also an important physiological phase that affects the next season’s growth of temperate fruit trees. Bud dormancy is regulated by multiple genetic factors, and affected by various environmental factors, tree age and vigor. To understand the molecular mechanism of bud dormancy in Japanese apricot (Prunus mume Sieb. et Zucc.), we constructed a custom oligo DNA microarray covering the Japanese apricot dormant bud ESTs referring to the peach (P. persica) genome sequence. Because endodormancy release is a chilling temperature-dependent physiological event, genes showing chilling-mediated differential expression patterns are candidates to control endodormancy release. Using the microarray constructed in this study, we monitored gene expression changes of dormant vegetative buds of Japanese apricot during prolonged artificial chilling exposure. In addition, we analyzed seasonal gene expression changes. Among the 58539 different unigene probes, 2345 and 1059 genes were identified as being more than twofold up-regulated and down-regulated, respectively, following chilling exposure for 60 days (P < 0.05). Cluster analysis suggested that the expression of the genes showing expression changes by artificial chilling exposure were coordinately regulated by seasonal changes. The down-regulated genes included P. mume DORMANCY-ASSOCIATED MADS-box genes, which supported previous quantitative RT-PCR and EST analyses showing that these genes are repressed by prolonged chilling exposure. The genes encoding lipoxygenase were markedly up-regulated by prolonged chilling. Our parametric analysis of gene-set enrichment suggested that genes related to jasmonic acid (JA) and oxylipin biosynthesis and metabolic processes were significantly up-regulated by prolonged chilling, whereas genes related to circadian rhythm were significantly down-regulated. The results obtained from microarray analyses were verified by quantitative RT-PCR analysis of selected genes. Taken together, we have concluded that the microarray platform constructed in this study is applicable for deeper understanding of the molecular network related to agronomically important bud physiology, including dormancy release.永年性作物において越冬芽の休眠は, 環境に適応するために進化した成長制御機構のひとつであり, かつ翌年の成長を左右する重要な農業形質のひとつである. 芽の休眠には多くの遺伝的要因が関わるばかりでなく, 多岐にわたる環境要因や樹勢, 樹齢の影響もうけており, 多くの遺伝子の発現制御が休眠に関与していると考えられる. そこで本研究では, 休眠関与候補遺伝子の単離を目的に, カスタムマイクロアレイを用いたウメ休眠芽のトランスクリプトーム解析をおこなった. まず, 我々が先行研究で獲得したウメ芽 EST 配列情報をもとに, ゲノム全体をできるだけ偏りなく網羅するように選びだした 58539 ユニジーンに相当するプローブを搭載したマイクロアレイを構築した. 次いで, 休眠覚醒を誘導する長期の低温処理を施したウメ休眠芽の遺伝子発現変動を調査した. その結果, 有意に 2 倍以上発現が上方制御あるいは下方制御されるプローブが 2345 個あるいは 1059 個同定された. これらの変動遺伝子のなかには季節的な発現同調性がみられるものがあった. 下方制御される遺伝子のなかには, 先行研究で休眠への関与が示唆されている DORMANCY ASSOCIATED MADS-box が含まれており, 上方制御される遺伝子にはリポキシゲナーゼが含まれていた. Parametricanalysis of gene set enrichment 解析の結果, 長期の低温遭遇によってジャスモン酸やオキシリピン生合成・代謝の GO タームが有意に上昇し, 概日リズムの GO タームが有意に減少した. これらの GO タームに含まれる遺伝子について定量 RT-PCR を行った結果, マイクロアレイで検出された発現変動パターンとほぼ同様であった. 以上の結果より, 本研究で構築したマイクロアレイはウメ休眠芽の網羅的な遺伝子発現解析に有効であることが示された. 本実験では, ウメの休眠覚醒に関与する遺伝子ネットワークあるいは新規候補遺伝子の探索に活用した

Draft Sequencing of the Heterozygous Diploid Genome of Satsuma (Citrus unshiu Marc.) Using a Hybrid Assembly Approach

Satsuma (Citrus unshiu Marc.) is one of the most abundantly produced mandarin varieties of citrus, known for its seedless fruit production and as a breeding parent of citrus. De novo assembly of the heterozygous diploid genome of Satsuma (“Miyagawa Wase”) was conducted by a hybrid assembly approach using short-read sequences, three mate-pair libraries, and a long-read sequence of PacBio by the PLATANUS assembler. The assembled sequence, with a total size of 359.7 Mb at the N50 length of 386,404 bp, consisted of 20,876 scaffolds. Pseudomolecules of Satsuma constructed by aligning the scaffolds to three genetic maps showed genome-wide synteny to the genomes of Clementine, pummelo, and sweet orange. Gene prediction by modeling with MAKER-P proposed 29,024 genes and 37,970 mRNA; additionally, gene prediction analysis found candidates for novel genes in several biosynthesis pathways for gibberellin and violaxanthin catabolism. BUSCO scores for the assembled scaffold and predicted transcripts, and another analysis by BAC end sequence mapping indicated the assembled genome consistency was close to those of the haploid Clementine, pummel, and sweet orange genomes. The number of repeat elements and long terminal repeat retrotransposon were comparable to those of the seven citrus genomes; this suggested no significant failure in the assembly at the repeat region. A resequencing application using the assembled sequence confirmed that both kunenbo-A and Satsuma are offsprings of Kishu, and Satsuma is a back-crossed offspring of Kishu. These results illustrated the performance of the hybrid assembly approach and its ability to construct an accurate heterozygous diploid genome

Segregation and Heritability of Male Sterility in Populations Derived from Progeny of Satsuma Mandarin

<div><p>Male sterility derived from Satsuma mandarin (<i>Citrus unshiu</i>) has been used in Japanese citrus breeding programs to obtain seedless cultivars, which is a desirable trait for consumers. Male sterility has often been evaluated by anther development or pollen fertility; however, the inheritance and heritability of male sterility derived from Satsuma is poorly understood. In this study, we investigated the mode of inheritance and broad-sense heritability of male sterility derived from Satsuma. Initially, we evaluated the total number of pollen grains per anther and apparent pollen fertility, as indicated by lactophenol blue staining, in 15 citrus cultivars and selections to understand the male sterility of Satsuma. The results indicated that male sterility was primarily caused by decreased number of pollen grains per anther in progeny of Satsuma. We also evaluated these traits in three F₁ populations (hyuganatsu × ‘Okitsu No. 56’, ‘Okitsu No. 46’ × ‘Okitsu No. 56’ and ‘Okitsu No. 46’ × ‘Kara’), of which the parents are derived from Satsuma. Individuals in these populations showed strong segregation for number of pollen grains per anther. The apparent fertility of pollen also showed segregation but was almost constant at 70%–90%. The estimated broad-sense heritability for the number of pollen grains per anther was as high as 0.898 in the ‘Okitsu No. 46’ × ‘Okitsu No. 56’ and ‘Okitsu No. 46’ × ‘Kara’ populations. These results indicated that the number of pollen grains per anther primarily determined male sterility among progeny of Satsuma, and this trait was inherited by the progeny. Development of DNA markers closely linked to male sterility using the F₁ populations of ‘Okitsu No. 46’ × ‘Okitsu No. 56’ and ‘Okitsu No. 46’ × ‘Kara’ is expected to contribute to the breeding of novel seedless citrus cultivars.</p></div

Distribution of pollen grains per anther and apparent pollen fertility of three F₁ populations.

<p>(A) Distribution for number of pollen grains per anther and (B) distribution for apparent pollen fertility. Black arrows indicate the number of pollen grains per anther for each parents in each F₁ population. a: ‘Okitsu No. 56’ in 2014, b: ‘Okitsu No. 56’ in 2015, c: hyuganatsu in 2014, d: hyuganatsu in 2015, e: ‘Okitsu No. 46’ in 2014, f: ‘Okitsu No. 46’ in 2015, g: ‘Kara’ in 2014, h: ‘Kara’ in 2015. Gray bars represent evaluations in 2014, and white bars represent data obtained in 2015.</p

Broad-sense heritability of number of pollen grains per anther and apparent pollen fertility of three F₁ populations.

<p>Broad-sense heritability of number of pollen grains per anther and apparent pollen fertility of three F₁ populations.</p

Association between apparent pollen fertility and <i>MS-F1</i>.

<p>The apparent pollen fertility is examined in ‘Okitsu No. 46’ × ‘Okitsu No. 56’ (O46-O56) population and it is based on the average of three years. (A) Association between apparent pollen fertility and alleles of NSX156, TSRA107, and SSR08B32. Average of apparent pollen fertility in 8–17 seedlings is shown. (B) Enlarged genetic linkage maps for O46-O56 near <i>MS-F1</i>. The loci with the highest logarithm of the odds score in the range of <i>MS-F1</i> and the flanking markers are shown. The alleles linked with <i>MS-F1</i> and <i>ms-f1</i> are shown on the right side and left side of LG6a, respectively. (C) Selection efficacy of the <i>MS-F1</i> haplotype block in the O46-O56 population. <i>MS-F1</i>/<i>ms-f1</i> shows individuals with both “207” allele at NSX 156 and “102” allele at SSR08B32 (<i>n</i> = 8). <i>ms-f1</i>/<i>ms-f1</i> indicates individuals without both the “207” and the “102” allele (<i>n</i> = 17). Underlines indicate the alleles linked with <i>MS-F1</i> and derived from ‘Okitsu No. 46’. Asterisk (****) shows significance level at <i>p</i> < 0.0001. Bars indicate standard deviation.</p

Quantitative trait loci (QTLs) for the number of pollen grains per anther (NPG) and apparent pollen fertility (APF) in ‘Okitsu No. 46’ × ‘Okitsu No. 56’ population obtained by multiple QTL mapping (MQM) for three consecutive years.

<p>Quantitative trait loci (QTLs) for the number of pollen grains per anther (NPG) and apparent pollen fertility (APF) in ‘Okitsu No. 46’ × ‘Okitsu No. 56’ population obtained by multiple QTL mapping (MQM) for three consecutive years.</p