甜玉米是控制玉米淀粉合成途径中的一个或几个基因发生突变造成淀粉合成受阻、糖类物质积累而成。糖分主要以还原糖、蔗糖和水溶性多糖的形式存在。育种过程中发现并被利用的甜玉米突变基因有su1、sh2、bt1、bt2、du1和se1等，这些基因的隐性纯合突变体在玉米籽粒的表型特征上呈现出明显的差异。鲜食玉米育种实践中，不同甜质基因以及它们与waxy基因的组合创新利用一直在不断尝试[7-
爽甜糯（XT）玉米是海南绿川种苗有限公司在鲜食玉米品种选育过程中发现的一种基因突变体，经过6年的深入研究，被认定为一种具有应用潜力的玉米新种质，还创制出一批新自交系，并育成鲜食玉米新品种美玉爽甜糯501。该品种在国家鲜食玉米品种区域试验中表现出色，赢得广泛好评。XT玉米在乳熟期的含糖量达到9%~10%，与普甜玉米相当。进入乳熟后期，其籽粒转为糯性。这种玉米以支链淀粉为主，具有高含糖量，口感独特，嫩食偏甜，稍老则兼具甜糯，故被称为爽甜糯。遗传分析表明，爽甜糯籽粒的胚乳特性是由甜质基因和糯质基因共同控制[13]。尽管如此，控制爽甜糯玉米甜质特性的目标基因尚未被定位和克隆。本研究克隆XT玉米中甜质调控关键基因，并开发目标基因的功能标记，加快分子标记辅助育种技术在XT玉米种质创新和品种选育中的应用，提高育种效率和精准度。
1 材料与方法
1.1 试验材料
XT玉米品种由海南绿川种苗有限公司培育而成，其外观特征明显区别于传统的普通玉米、普甜玉米（su1su1）以及超甜玉米（sh2sh2）。目前，XT玉米以及本研究中所使用的其他玉米自交系均已收录于北京市农林科学院玉米研究所种质资源库中，可供研究和育种工作使用。
1.2 试验方法
1.2.1 玉米籽粒中可溶性糖和淀粉含量测定
将籽粒自然晾干后，采用可溶性糖测定试剂盒（北京索莱宝科技有限公司）测定可溶性糖含量。利用直链/支链/总淀粉含量（酶法）试剂盒（苏州格锐思生物科技有限公司）测定总淀粉及直链淀粉含量，每个样品3次生物学重复。
1.2.2 爽甜糯玉米XT甜质调控基因的BSR-seq定位
在2021年12月，于北京市农林科学院玉米研究所海南南滨农场的育种基地（18°23′21.51″ N，109°11′09.13″ E）进行XT玉米与普通玉米自交系京2416和京724的正反交试验，随后进行自交，构建XT×京2416、XT×京724、京2416×XT和京724×XT F2代分离群体。针对每个分离群体，挑选饱满和皱缩的籽粒进行催芽，并分别采集30个单株叶片后等量混合，使用天根生化科技（北京）有限公司总RNA提取试剂盒提取总RNA，从而制备饱满粒和皱缩粒的2个RNA混池。这些RNA混合池随后被用于BSR-seq分析，该分析依照Data2Bio公司的标准操作流程进行[14-15]。BSR-seq的原始数据已被提交至NCBI的SRA（Sequence Read Archive）数据库中，项目登录号为PRJNA770099。
1.2.3 候选基因DNA和cDNA序列分析
表1 du1基因DNA扩增引物
Table 1
| 引物名称Primer name | 5′→3′ |
|---|---|
| DU3F | CGTTTGCTGGGAAGTGGTTC |
| DU3R | AGTCTTGGCGCATAGTTGCT |
| DU4F | GCTCCAAGAAGAGCGAACAC |
| DU4R | ATAGCCAGCAACGGATTGGATT |
| DU5F | TCCAATCCGTTGCTGGCTAT |
| DU5R | GCCACAGCTGATAGATCACGA |
| DU6F | CGTGATCTATCAGCTGTGGC |
| DU6R | AGCAGGGCAAACGGTATTCT |
| DU7F | GACCATTTGGGCCATTCATCAC |
| DU7R | AGCACGTGAAAGACTAGTGAC |
| DU8F | GGCACCAATCGCAAAGGTTA |
| DU8R | GAGGGCTTCCCTTGCTGTATTT |
| DU9F | GCCTTTCCTATTTGGCAGCAC |
| DU9R | TTTTCCTTGTGTAGCCAGGCA |
| DU10F | GCGAAGTTGCACTGTTAGCTT |
| DU10R | TGTCAGGCGAGTGACGATTC |
| DU11F | GATGTCCCCGTCGTAGGAAT |
| DU11R | TACATTGGCCGTTGGTTGGAT |
| DU12F | CAACCAACGGCCAATGTAGT |
| DU12R | GGGTGAGAAGACAAAGATACCCT |
表2 du1基因cDNA扩增及KASP分子标记的引物
Table 2
| 引物名称Primer name | 5′→3′ |
|---|---|
| cDNA扩增cDNA amplification | |
| DU3F | CGTTTGCTGGGAAGTGGTTC |
| DU4R | ATAGCCAGCAACGGATTGGATT |
| DX18F | ATAGTTCACTTCCCTGAGCC |
| DX6R | TCAACGCTGCCATCATAGGT |
| DX16F | CAAGTCCCGTCATATGTGCT |
| DX4R | AGATGAATGCGGTGGTCGTAG |
| DX10F | CTACGACCACCGCATTCATCT |
| DX2R | TCAAGGTACCATCCTGGAGCT |
| DX6F | TTGAGGTTGAGTATCGTCCG |
| DX1R | GTAGTAGTCTGCCATTCTGGAC |
| DUX5F | CTGCACGACGTATTTCATGTGG |
| DUX5R | CCATTCTGTATGCCTGCTTAGG |
| DU6F | CGTGATCTATCAGCTGTGGC |
| DU9R | TTTTCCTTGTGTAGCCAGGCA |
| DX17F | GTCACTAGTCTTTCACGTGCTG |
| DU11R | TACATTGGCCGTTGGTTGGAT |
| KASP分子标记KASP molecular marker | |
| AlleleX | AAGAGAACCAACAATGGACTGGTG |
| AlleleY | AAGAGAACCAACAATGGACTGGTC |
| Common1 | TCCAATCCATCACCAGTTACGATAAACTA |
| Common2 | TCCGTGGTAGGGATCGTATTCCTAT |
1.2.4 目的基因KASP分子标记开发及基因分型检测
1.3 数据处理
利用GraphPad Prism 5（
2 结果与分析
2.1 XT玉米的籽粒形态及可溶性糖含量分析
图1
图1
XT玉米籽粒表型特征和可溶性糖含量
(a) 自然晾干籽粒形态。(b) 籽粒透射光观察图。(c) 籽粒横剖面图。(d) 籽粒纵剖面图。标尺=1 cm。“*”代表P < 0.05，“****”代表P < 0.0001。
Fig.1
Phenotypic appearance and soluble sugar contents of XT grains
(a) Naturally dried grain morphology. (b) Observation diagram of grain transmission light. (c) Grain cross-section diagram. (d) Grain longitudinal section diagram. Scale bar=1 cm.“*”represents P < 0.05,“****”represents P < 0.0001.
2.2 XT玉米籽粒淀粉含量分析
表3 自然晾干玉米籽粒的可溶性糖、总淀粉及直链淀粉含量
Table 3
| 编号 Number | 样品名称（玉米类型） Sample name (maize type) | 可溶性糖含量 Soluble sugar content (mg/g) | 总淀粉含量 Total starch content (mg/g) | 直链淀粉含量 Amylose content (mg/g) | 直链淀粉比例 Amylose ratio (%) |
|---|---|---|---|---|---|
| 1 | 京724（普通玉米） | 19.32±0.11d | 537.20±3.00b | 153.20±1.11b | 29.50±0.18a |
| 2 | 京2416（普通玉米） | － | 676.00±4.18a | 172.80±1.33a | 26.44±0.15b |
| 3 | SH251（超甜玉米） | 192.80±2.59a[14] | 229.60±3.38h | 50.21±1.47c | 22.62±0.47c |
| 4 | T9（普甜玉米） | 154.30±2.63b[14] | 524.40±2.96c | 45.53±1.07d | 8.98±0.19d |
| 5 | 219M（糯玉米） | － | 502.20±3.79d | 8.51±1.11f | 1.75±0.22f |
| 6 | XT（爽甜糯玉米） | 62.82±0.11c | 460.60±1.24e | 21.25±0.41e | 4.78±0.08e |
| 7 | XT选系1 | － | 330.50±2.27g | 7.66±0.72f | 2.39±0.21f |
| 8 | XT选系2 | － | 387.00±1.07f | 6.38±0.72f | 1.70±0.19f |
| 9 | XT选系3 | － | 457.10±2.50e | 6.80±0.84f | 1.54±0.18f |
不同小写字母表示差异显著（P < 0.05）。
Different lowercase letters indicate significant differences (P < 0.05).
分析直链淀粉比例发现，超甜玉米SH251的直链淀粉比例（22.62%）与普通玉米（京724、京2416）接近；普甜玉米T9的直链淀粉比例偏低（8.98%），为偏糯质型；XT玉米及其选系的支链淀粉比例均高于95%，为糯质型。籽粒含糖量和淀粉含量测定结果揭示了XT玉米兼具甜味和糯性的内在原因。
2.3 XT玉米甜质调控基因的BSR-seq定位
皱缩表型是甜质基因表达的关键特征，因此被用作对XT玉米中甜质基因遗传定位的依据。基于皱缩表型，我们对4个F2代分离群体进行了BSR- seq分析，结果（图2）显示，在连锁概率高于0.05的SNP中，90%以上聚集在第10号染色体。我们通过滑动窗口法进一步优化定位区间（每个窗口包含20个SNP，窗口移动步长为5个SNP），并综合4个分离群体的定位数据，将候选基因区域缩小至10号染色体的24~68 Mb区间（参考B73 RefGen_v3基因组版本）。根据B73基因组序列的基因功能注释，我们推测GRMZM2G141399（du1）可能是控制该性状的候选基因。GRMZM2G141399编码的淀粉合成酶SSII发生突变，将会导致植物体内糖类物质的积累[18-
图2
图2
XT玉米甜质调控基因BSR-seq定位结果
Fig.2
BSR-seq mapping results for the sweetness regulating gene in XT
2.4 候选基因的DNA和cDNA序列分析
为了确认候选基因du1是否为控制XT玉米甜质特性的目标基因，我们对XT玉米与普通玉米B73中的Du1基因全长DNA序列进行了比较分析。分段扩增结果（图3a）揭示，使用Du5F/Du5R引物会在B73中产生1105 bp的扩增片段，而在XT玉米中，相应的扩增片段长度显著扩展至约8 kb，推测XT玉米du1基因的第三外显子区域发生了大片段插入突变。PCR产物测序分析进一步（图3b）显示，在XT玉米du1基因第三外显子的1455 bp处之后，存在5839 bp的大片段插入。经CENSOR（
图3
图3
XT玉米与普通玉米B73中Du1基因DNA序列比较
(a) Du5F/Du5R引物对在XT玉米及其3个选系（XT1、XT2、XT3）与普通玉米B73间的扩增结果对比。
Fig.3
Comparison analysis of Du1 gene DNA sequences between XT and B73
(a) Discrepancy in the PCR amplification products between XT maize and its three inbred lines (XT1, XT2, XT3) compared to ordinary maize B73 utilizing the Du5F/Du5R primer pair.
图4
图4
XT玉米与普通玉米B73中Du1基因cDNA扩增产物比较
(a) cDNA扩增引物在du1基因上的位置。(b) cDNA分段扩增产物的差异比较。
Fig.4
Comparison analysis of Du1 gene cDNA amplification products between XT and B73
(a) The location of cDNA amplification primers on the du1 gene. (b) Comparison analysis of the differences in cDNA segment amplification products.
2.5 du1自然突变体的KASP分子标记
通过与先前利用Mu转座子插入技术创制的玉米du1突变体[22]进行序列比较，以及在NCBI数据库进行BLAST搜索，发现我们克隆的XT玉米中du1突变基因是一个新的等位变异。针对du1突变体第3外显子上的5839 bp大片段插入，开发了一个位点特异性的KASP分子标记，命名为du1- KASP。
利用此分子标记对XT玉米及其3个选系（XT1、XT2和XT3）、4份糯玉米自交系（京科糯219F、京科糯219M、京6和BN2）、3种普通玉米自交系（京724、京2416和B73）、20份携带su1su1sh2sh2双突变基因的微胚乳玉米自交系（Huada122、Huada164、Huada165、Huada180、Huada227、Huada254、Huada415、Huada491、Huada512、Huada515、Huada517、Huada534、Huada536、Huada548、Huada595、Huada599、Huada600、Huada618、HuajianF和HuajianM）以及杂交种“XT×京6”和“XT×BN2”进行基因分型检测，结果显示，在XT玉米及其3个选系中成功检测到TGTTATNACCAGT/TGTTATNACCAGT插入序列，而其他玉米自交系均显示为-/-，即无插入突变。在杂交种“XT×京6”和“XT×BN2”中，检测到了TGTTATNACCAGT/-的杂合型（图5）。检测结果与各材料的遗传背景一致，说明du1- KASP分子标记能够精准高效地筛选出携带du1突变的玉米单株。
图5
图5
du1-KASP分子标记的基因分型检测结果
黑点为空白对照，红点为无插入突变的纯合基因型，蓝点为插入突变的纯合基因型，绿点为杂合基因型。
Fig.5
Illustration of genotyping assay utilizing the du1-KASP molecular marker
Black dots represent the blank control, red dots represent the homozygous genotype without insertion mutation, blue dots represent the homozygous genotype with insertion mutation, and green dots represent heterozygous genotypes.
3 讨论
3.1 Du1基因在玉米品质改良中的核心作用
Du1基因的鉴定和功能解析对于作物育种具有极其重要的意义。水稻中，Du1基因编码的新型Prp1蛋白主要在穗部表达，通过影响Wx(b)前体mRNA的剪接效率来调节wx基因的表达，从而调控淀粉的生物合成[25-26]。这一发现为水稻品质的遗传改良提供了全新的策略。在水稻育种实践中，du1突变体所展现的典型形态和农艺性状已被有效地利用[27]。这些突变体不仅提升了水稻的食用品质，而且为深入研究淀粉合成的分子机制提供了宝贵的遗传资源。利用Mu转座子插入技术，研究人员[20,22]成功在玉米中创制了du1突变体，基于基因表达产物的大小和模式，推测玉米中的Du1基因负责编码淀粉合成酶SSII，并在du1突变中观察到淀粉合酶SSII和淀粉分支酶SBEIIa的活性显著降低，这一变化对玉米淀粉的合成和结构产生了重要影响，为玉米品质的遗传改良开辟了新的途径。此外，du1与胚乳隐性基因waxy存在显著的相互作用。du1waxy双基因突变体展现出独特的淀粉组成，不仅包含高分子量的支链淀粉，还包含40%的中间成分[28]，这种特殊的淀粉组成为提升玉米的食用品质提供了新的遗传学策略和改良途径。本研究中的XT玉米具有独特的风味，其嫩食偏甜，稍老食则甜糯一体[13]。本研究可溶性糖含量测定显示，XT玉米籽粒的可溶性糖含量显著高于普通玉米，但显著低于普甜（su1）玉米，这一特性与其独特的遗传背景紧密相关。基因定位和分子克隆结果表明，XT玉米du1基因发生了自然突变，该突变在第3外显子的1455 bp后存在5839 bp的Gypsy类LTR反转座子插入，导致了氨基酸编码的异常和功能缺失，为玉米品质的遗传改良提供了新的基因资源。
3.2 du1-KASP分子标记将成为爽甜糯玉米遗传育种的新工具
KASP分子标记是一种结合了PCR扩增和荧光检测的方法，具有操作灵活、高准确性、高通量和自动化等特点而备受青睐[29]。这种技术在农作物的分子标记辅助育种中已经得到了广泛地应用，极大地提高了育种工作的效率和精确度。在水稻育种中，基于籽粒镉积累调控基因的功能性位点，开发的KASP分子标记LCd-38能够高效区分不同水稻品种的籽粒镉积累水平[30]，这对筛选低镉含量的水稻品种、保障粮食安全具有重要意义。在小麦育种领域，江苏省农业科学院的小麦遗传育种团队开发的双重KASP分子标记能够在单一反应中同时鉴定2个基因，显著提升了小麦分子标记辅助育种的效率[31]。玉米籽粒的表型特征受到环境和遗传因素的共同影响，这使得仅通过籽粒表型来识别与淀粉合成相关的突变基因变得复杂。针对du1基因的功能性突变位点，本研究开发了位点特异性KASP分子标记，能够有效区分du1du1、du1Du1和Du1Du1等不同基因型的玉米，为爽甜糯玉米的分子标记辅助育种提供了强有力的工具。开发这种分子标记，不仅会加速爽甜糯玉米的育种过程，同时也为玉米品质性状的遗传改良开辟了新的途径。
4 结论
XT玉米的甜质特性源于du1基因的自然突变，该du1突变体在其第3外显子的1455 bp后发生了5839 bp的Gypsy类LTR反转座子插入，这一插入导致了du1基因的转录异常和功能丧失。该基因自然突变体为du1基因的一个新等位变异。针对du1突变位点开发的KASP分子标记能够高效精准地检测玉米基因组中的du1纯合、杂合突变，对爽甜糯玉米的分子标记辅助育种具有重要的应用价值。
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随着基因测序技术的发展,植物基因组数据越来越丰富,其中的单核苷酸多态性（single nucleotide polymorphism,SNP）数据由于具有高密度、高通量和易于自动化分析等特点而被广泛用于分子标记的开发和应用。竞争性等位基因特异性PCR（kompetitive allele-specific PCR,KASP）技术是近些年来发展起来的一种主要基于SNP的高通量基因分型技术。该技术由于其高通量、低成本和可操作性强等优点而在农作物性状改良等领域具有很大的应用潜力。本文介绍了KASP技术的发展、原理和方法步骤,综述了该技术在主要农作物的种质资源鉴定、分子标记辅助育种、基因定位和种子纯度鉴定等遗传育种中的应用,并讨论了该技术的优缺点,以期为今后农作物育种研究提供参考依据。
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[本文引用: 2]
Cadmium (Cd) accumulation in maize grains is detrimental to human health. Developing maize varieties with low-Cd contents is important for maize grains safe consumption. However, the key genes controlling maize grain Cd accumulation have not been cloned. Here, we identified one major locus for maize grain Cd accumulation (qCd1) using a genome-wide association study (GWAS) and bulked segregant RNA-seq analysis with a biparental segregating population of Jing724 (low-Cd line) and Mo17 (high-Cd line). The candidate gene ZmHMA3 was identified by fine mapping, which encodes a tonoplast-localized heavy metal P-type ATPase transporter. An EMS mutant analysis and an allelism test confirmed that ZmHMA3 influences maize grain Cd accumulation. A transposon in intron 1 of ZmHMA3 is responsible for the abnormal amino acid sequence in Mo17. Based on the natural sequence variations in ZmHMA3 gene of diverse maize lines, four PCR-based molecular markers were developed, which were successfully used to distinguish five haplotypes with different grain Cd contents in the GWAS panel and to predict grain Cd content levels of widely used maize inbred lines and hybrids. These molecular markers can be used to breed elite maize varieties with low grain Cd contents.© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.
Sequence polymorphism of the waxy gene in waxy maize accessions and characterization of a new waxy allele
玉米淀粉品质遗传改良研究的进展
Evidence for independent genetic control of the multiple forms of maize endosperm branching enzymes and starch synthases
DOI:10.1104/pp.67.6.1141
PMID:16661824
[本文引用: 2]
Soluble starch synthase and starch-branching enzymes in extracts from kernels of four maize genotypes were compared. Extracts from normal (nonmutant) maize were found to contain two starch synthases and three branching enzyme fractions. The different fractions could be distinguished by chromatographic properties and kinetic properties under various assay conditions. Kernels homozygous for the recessive amylose-extender (ae) allele were missing branching enzyme IIb. In addition, the citrate-stimulated activity of starch synthase I was reduced. This activity could be regenerated by the addition of branching enzyme to this fraction. No other starch synthase fractions were different from normal enzymes. Extracts from kernels homozygous for the recessive dull (du) allele were found to contain lower branching enzyme IIa and starch synthase II activities. Other fractions were not different from the normal enzymes. Analysis of extracts from kernels of the double mutant ae du indicated that the two mutants act independently. Branching enzyme IIb was absent and the citrate-stimulated reaction of starch synthase I was reduced but could be regenerated by the addition of branching enzyme (ae properties) and both branching enzyme IIa and starch synthase II were greatly reduced (du properties). Starch from ae and du endosperms contains higher amylose (66 and 42%, respectively) than normal endosperm (26%). In addition, the amylopectin fraction of ae starch is less highly branched than amylopectin from normal or du starch. The above observations suggest that the alterations of the starch may be accounted for by changes in the soluble synthase and branching enzyme fractions.
Integration of omic networks in a developmental atlas of maize
DOI:10.1126/science.aag1125
PMID:27540173
[本文引用: 1]
Coexpression networks and gene regulatory networks (GRNs) are emerging as important tools for predicting functional roles of individual genes at a system-wide scale. To enable network reconstructions, we built a large-scale gene expression atlas composed of 62,547 messenger RNAs (mRNAs), 17,862 nonmodified proteins, and 6227 phosphoproteins harboring 31,595 phosphorylation sites quantified across maize development. Networks in which nodes are genes connected on the basis of highly correlated expression patterns of mRNAs were very different from networks that were based on coexpression of proteins. Roughly 85% of highly interconnected hubs were not conserved in expression between RNA and protein networks. However, networks from either data type were enriched in similar ontological categories and were effective in predicting known regulatory relationships. Integration of mRNA, protein, and phosphoprotein data sets greatly improved the predictive power of GRNs. Copyright © 2016, American Association for the Advancement of Science.
Characterization of dull1, a maize gene coding for a novel starch synthase
Du1,encoding a novel Prp1 protein, regulates starch biosynthesis through affecting the splicing of Wxb pre-mRNAs in rice (Oryza sativa L.)
dull:rice mutants with tissue-specific effects on the splicing of the waxy pre-mRNA
水稻品质的遗传与育种改良研究进展
DOI:10.13560/j.cnki.biotech.bull.1985.2021-1598
[本文引用: 1]
随着人民生活水平的提高,在追求水稻高产的同时,优质已成为育种家的育种目标和消费者的关注重点。水稻品质包含碾磨品质、外观品质、蒸煮食味品质和营养品质四类指标,本文从水稻品质的遗传与改良两方面展开论述,回顾了水稻品质的遗传与育种改良研究进展,介绍了已克隆的稻米品质相关基因或QTL的功能及其在稻米品质改良中的应用。最后,在此基础上分析了现阶段水稻品质的遗传与改良存在的问题并对未来研究方向作了展望。
Observations on the α‐amylolysis pattern of some waxy maize starches from inbred line Ia453
基于高通量测序开发玉米高效KASP分子标记
DOI:10.3724/SP.J.1006.2019.83067
[本文引用: 1]
SNP (Single Nucleotide Polymorphism)在基因组中数量多、分布广, 适用于大规模、自动化基因型检测。本研究利用205份不同来源的玉米自交系全基因组重测序数据鉴定出一系列多态性高的二态性SNP位点并开发出700个KASP分子标记。其中, 202个在46个玉米代表系中得到验证的KASP标记进一步用于系统进化树构建及群体结构分析。结果显示, 开发成功的KASP标记在染色体上分布均匀, 平均PIC为0.463, 平均MAF为0.451。基于KASP标记位点和总SNP位点的聚类分析结果高度吻合。KASP标记位点与总SNP位点的遗传距离相似性系数高达89.5%, 能成功区分玉米的杂种优势群。该KASP标记可在玉米种质资源分析、连锁群构建以及杂种优势群划分等方面发挥重要作用。
水稻籽粒镉积累KASP分子标记LCd-38的开发与利用
DOI:10.13304/j.nykjdb.2021.1037
[本文引用: 1]
水稻（Oryza sativa L.）是我国主粮作物之一，稻米镉污染对我国粮食安全造成一定威胁。镉低积累水稻品种的选育可有效降低水稻籽粒镉污染风险，高效准确的镉积累相关分子标记在镉低积累水稻品种选育中具有至关重要的作用。以水稻微核心种质为材料，基于籽粒镉积累关联基因的功能性SNP位点开发了KASP分子标记LCd-38。选取安全利用类镉污染土壤为试验地，利用该标记对当地主栽水稻品种进行基因分型和镉低积累水稻品种筛选。开发的分子标记LCd-38能够有效地将不同水稻品种分为籽粒高镉积累基因型（CC）和低镉积累基因型（TT）。以水稻苗期叶片为材料，快速鉴定到试验区5个低镉积累水稻品种和5个高镉积累水稻品种，与成熟期实测籽粒镉含量结果一致。综上所述，分子标记LCd-38可高效准确地预测不同水稻品种的籽粒镉积累特性，可应用于镉低积累水稻品种的早期筛选和分子标记辅助选择育种。
Cost-effective duplex Kompetitive Allele Specific PCR markers for homologous genes facilitating wheat breeding
DOI:10.1186/s12870-023-04116-y
PMID:36855097
[本文引用: 1]
Owing to successful cloning of wheat functional genes in recent years, more traits can be selected by diagnostic markers, and consequently, effective molecular markers will be powerful tools in wheat breeding programs.The present study proposed a cost-effective duplex Kompetitive Allele Specific PCR (dKASP) marker system that combined multiplex PCR and KASP™ technology to yield twice the efficiency at half the cost compared with the common KASP™ markers and provide great assistance in breeding selection. Three dKASP markers for the major genes controlling plant height (Rht-B1/Rht-D1), grain hardness (Pina-D1/Pinb-D1), and high-molecular-weight glutenin subunits (Glu-A1/Glu-D1) were successfully developed and applied in approved wheat varieties growing in the middle and lower reaches of the Yangtze River and advanced lines from our breeding program. Three markers were used to test six loci with high efficiency. In the approved wheat varieties, Rht-B1b was the most important dwarfing allele, and the number of accessions carrying Pinb-D1b was much greater than that of the accessions carrying Pina-D1b. Moreover, the number of accessions carrying favorable alleles for weak-gluten wheat (Null/Dx2) was much greater than that of the accessions carrying favorable alleles for strong-gluten wheat (Ax1 or Ax2/Dx5). In the advanced lines, Rht-B1b and Pinb-D1b showed a significant increase compared with the approved varieties, and the strong-gluten (Ax1 or Ax2/Dx5) and weak-gluten (Null/Dx2) types also increased.A cost-effective dKASP marker system that combined multiplex PCR and KASP™ technology was proposed to achieve double the efficiency at half the cost compared with the common KASP™ markers. Three dKASP markers for the major genes controlling PH (Rht-B1/Rht-D1), GH (Pina-D1/Pinb-D1), and HMW-GS (Glu-A1/Glu-D1) were successfully developed, which would greatly improve the efficiency of marker-assisted selection of wheat.© 2023. The Author(s).
