Porotnikov I V, Mitrofanova O P, Antonova O Yu
Federal Research Center the N.I. Vavilov All-Russian Institute of Plant Genetic Resources (VIR), St. Petersburg, Russia.
Vavilovskii Zhurnal Genet Selektsii. 2022 Mar;26(2):128-138. doi: 10.18699/VJGB-22-16.
Mutant alleles of the Rht-B1 and Rht-D1 (Reduced height) genes are widely used in bread wheat breeding for the development of intensive-type cultivars. These genes and their f lanking regions have been sequenced and the point mutations leading to the nonsense codons (Rht-B1b, Rht-B1e, Rht-B1p and Rht-D1b alleles) and various insertions (Rht-B1c, Rht-B1h and Rht-B1i-1) associated with a change in plant height have been described. DNA-markers based on the allele-specif ic PCR have been developed to identify single-nucleotide changes. However, the use of such technique imposes stringent PCR conditions, and the resulting data are not always unambiguous. An alternative can be found in the CAPS technology: it detects differences in sequences by digesting PCR products. In the absence of restrictases capable of digesting DNA at the point mutation site, restriction sites can be introduced into the primer sequence (derived CAPS). The aim of this study was to propose a system of CAPS-, dCAPS- and STS-markers for identifying alleles of the reduced height genes frequently used in breeding programs. Three CAPS have been developed to identify the Rht-B1b, Rht-D1b, Rht-B1p alleles, as well as two dCAPS for Rht-B1b, Rht-B1e. STS-markers for the insertion-containing alleles Rht-B1c, Rht-B1h and Rht-B1i-1 have been selected from publications. The proposed markers were tested during the genotyping of 11 bread wheat accessions from the VIR collection with the abovementioned mutant alleles and the wild-type Rht-B1a and Rht-D1a. The presence of nonsense mutations was also conf irmed by the results of allele-specif ic PCR. This marker system, along with the existing ones, can be used to identify dwarf ing alleles of the Rht-B1 and Rht-D1 genes in bread wheat for genetic screening of accessions from ex situ collections and/or for marker-assisted selection.
Rht - B1和Rht - D1（矮秆）基因的突变等位基因在面包小麦育种中广泛用于培育紧凑型品种。这些基因及其侧翼区域已被测序，导致无义密码子的点突变（Rht - B1b、Rht - B1e、Rht - B1p和Rht - D1b等位基因）以及与株高变化相关的各种插入（Rht - B1c、Rht - B1h和Rht - B1i - 1）已被描述。基于等位基因特异性PCR的DNA标记已被开发用于鉴定单核苷酸变化。然而，使用这种技术需要严格的PCR条件，并且所得数据并不总是明确的。CAPS技术是一种替代方法：它通过消化PCR产物来检测序列差异。在没有能够在点突变位点消化DNA的限制酶的情况下，可以将限制位点引入引物序列（衍生CAPS）。本研究的目的是提出一个CAPS、dCAPS和STS标记系统，用于鉴定育种计划中常用的矮秆基因的等位基因。已开发出三种CAPS用于鉴定Rht - B1b、Rht - D1b、Rht - B1p等位基因，以及两种用于Rht - B1b、Rht - B1e的dCAPS。从出版物中选择了用于含插入等位基因Rht - B1c、Rht - B1h和Rht - B1i - 1的STS标记。在对VIR种质库中11份具有上述突变等位基因以及野生型Rht - B1a和Rht - D1a的面包小麦种质进行基因分型时，对所提出的标记进行了测试。等位基因特异性PCR的结果也证实了无义突变的存在。这个标记系统与现有的标记系统一起，可用于鉴定面包小麦中Rht - B1和Rht - D1基因的矮化等位基因，用于对异地种质库中的种质进行遗传筛选和/或标记辅助选择。
