An introduction to markers, quantitative trait loci (QTL) mapping and marker-assisted selection for crop improvement: The basic concepts

Recognizing the enormous potential of DNA markers in plant breeding, many agricultural research centers and plant breeding institutes have adopted the capacity for marker development and marker-assisted selection (MAS). However, due to rapid developments in marker technology, statistical methodology for identifying quantitative trait loci (QTLs) and the jargon used by molecular biologists, the utility of DNA markers in plant breeding may not be clearly understood by non-molecular biologists. This review provides an introduction to DNA markers and the concept of polymorphism, linkage analysis and map construction, the principles of QTL analysis and how markers may be applied in breeding programs using MAS. This review has been specifically written for readers who have only a basic knowledge of molecular biology and/or plant genetics. Its format is therefore ideal for conventional plant breeders, physiologists, pathologists, other plant scientists and students.     

大豆籽粒蛋白质含量相关QTL 定位研究进展

籽粒蛋白质含量是大豆品质性状改良的主要目标之一。笔者介绍了大豆遗传图谱的构建与基因组测序发展历程,从基于分离群体的连锁分析和基于自然群体的关联分析两方面阐述了大豆籽粒蛋白质含量QTL定位研究进展,进而讨论了大豆蛋白质含量MAS育种存在的问题,最后展望了大豆蛋白质含量分子遗传改良的研究趋势。以期为大豆高蛋白育种提供参考。     

Use of molecular markers for the development of new cultivars and the evaluation of genetic diversity

Molecular markers bring new information on the determinism of trait variation and the organisation of genetic diversity within plant species of agricultural interest. We review here how this information can be used to increase the efficiency of plant breeding programs, considering both theoretical analyses and recent experimental data. Use of mapping information to assemble alleles of interest is discussed first, considering an increasing complexity in trait determinism and its consequences on the breeding schemes. Experimental data now confirm the efficiency of these approaches. They call however, for (i) a better modelling of phenotype determinism, to better anticipate the final effect of marker assisted selection and (ii) studies that would address trait variation determinism within a broad diversity, to increase the probability to identify alleles of key interest and identify stable marker-trait associations. Recent promising developments in genetic diversity analysis are discussed with respect to these last objectives. This revised version was published online in July 2006 with corrections to the Cover Date.     

Mapping and validation of PCR-based markers associated with a major QTL for seed dormancy in wheat

Seed dormancy is one of the important factors controlling pre-harvest sprouting (PHS) resistance in wheat. We identified a major quantitative trait locus (QTL) for seed dormancy on the long arm of wheat chromosome 4A (4AL) via simple sequence repeat (SSR)-based genetic mapping using doubled haploid lines from a cross between Japanese PHS resistant variety ‘Kitamoe’ and the Alpine non-resistant variety “Münstertaler” (K/M). The QTL explained 43.3% of total phenotypic variation for seed dormancy under greenhouse conditions. SSR markers flanking the QTL were assigned to the chromosome long arm fraction length 0.59–0.66 on the basis of chromosome deletion analysis, suggesting that the gene(s) controlling seed dormancy are probably located within this region. Under greenhouse conditions, the QTL explained 28.5 and 39.0% of total phenotypic variation for seed dormancy in Haruyutaka/Leader (HT/L) and OS21-5/Haruyokoi (O/HK) populations, respectively. However, in field conditions, the effect was relatively low or not significant in both the K/M and HT/L populations. These markers were considered to be widely useful in common with various genetic backgrounds for improvement of seed dormancy through the use of marker-assisted selection. Further detailed research using near isogenic lines will be needed to define how this major QTL interacts with environmental conditions in our area.     

Bulked segregant analysis to detect main effect QTL associated with grain quality parameters in Basmati 370/ASD 16 cross in rice Oryza sativa L) using SSR markers

In the present study a population consisting of 247 F₂ individuals from the cross between Basmati 370, a superior quality basmati variety and ASD16, a non-basmati high-yielding variety was analyzed for their segregation pattern of grain length (GL), grain breadth (GB), cooked grain length (CGL), cooked grain breadth (CGB), and gelatinization temperature (GT). Except GT, all other traits showed normal distribution indicating the polygenic control over the traits. The correlation analysis between traits indicated that GT had positive significant association with GL (0.125), and CGL (0.243). To identify main effect QTL (MQTL) for the above grain quality traits, both the parents were surveyed with 86 primer pairs of simple sequence repeats (SSR). The parental survey revealed 63.95% polymorphism between parents. In order to detect the MQTL associated with grain quality traits, a strategy of combining the DNA pooling from selected segregants and genotyping was adopted. The number of individuals forming the bulk influenced the identification of putative marker(s) for each of the traits. The association of putative markers identified based on DNA pooling from selected segregants was established by Single Marker Analysis (SMA). The results of SMA revealed that SSR markers, RM225 on chromosome #6 and RM247 on chromosome #12 showed significant association with GB and CGB respectively. It is established that molecular marker analysis involving DNA pooling of phenotypic extremes and selective genotyping helps to detect MQTL for complex traits involving early segregating generations. The molecular marker analysis involving the DNA pooling of phenotypic extremes could be a useful strategy to detect the genetic loci with major effects of other complex grain quality traits in rice.     

Genetic analysis of net-like cracking in soybean seed coats

Cracking of seed coats in soybean (Glycine max (L.) Merr.) deteriorates the external appearance of seeds and reduces their commercial value. Two types of cracking have been reported that occur in some cultivars: Type I with irregular cracks and Type II with net-like cracks. This study was conducted to determine the genetic basis of net-like cracking. Genetic analysis was performed using F₁ plants produced by crossing Uzuramame, a Japanese landrace with black seed coats having net-like cracking and a Clark mutant with black seed coats, their F₂ population and F₃ lines. Degree of cracking in individual plants was calculated by averaging cracking index (no cracking: 0 to severe cracking: 4) of total or 100-seed samples (average cracking index, ACI). Uzuramame exhibited intense cracking, whereas the Clark mutant showed slight cracking. Intermediate degree of cracking in F₁ plants suggested incomplete dominance. ACI of F₂ plants was continuously distributed. Gene number involved was estimated to be 1.4 by Wright's method. All F₃ lines derived from F₂ plants with ACI more than 2.8 displayed severe cracking phenotypes. In contrast, F₃ lines derived from F₂ plants with ACI less than 2.8 segregated from low to high ACI (0.5 to 3.2). When F₂ plants were classified as slight (ACI<2.8) or severe (ACI>2.8) cracking, the frequency distribution of the F₂ plants fitted to a 3:1 ratio. Genotypes of SSR marker Satt264 that is closely linked to SoyPRP1 locus for proline-rich cell wall protein had a minor effect on ACI. Further, seed weight was positively associated with ACI (r =0.46^**). Our results suggest that net-like cracking is controlled primarily by a major gene, and SoyPRP1 and gene(s) contributing to seed weight may have minor effects on the intensity of cracking. This revised version was published online in July 2006 with corrections to the Cover Date.     

大豆重组自交系群体NJRIKY遗传图谱的加密及其应用效果

作物基因组研究,包括基因或数量性状位点(QTL)定位、图位克隆以及物理图谱构建等,首先必须建立具有丰富标记信息的高密度遗传连锁图谱。由科丰1号和南农1138-2杂交组合衍生的重组自交系群体NJRIKY已经构建了4张大豆遗传连锁图谱,但由于遗传信息和标记数目不够充分,在基因和QTL作图时仍然存在精确度和准确度问题。为增加NJRIKY图谱密度,本研究在967对SSR引物中获得了401个多态性SSR标记。结合其他分子数据,使用作图软件Mapmaker/Exp3.0b,获得一张含有553个遗传标记,25个连锁群,总长2071.6cM,平均图距3.70cM的新遗传连锁图谱,其中SSR标记316个,RFLP标记197个,EST标记39个,形态标记1个。连锁群上大于20cM的标记间隔由原来42个减少到2个。原图谱的3个SMV抗性基因定位于D1b连锁群末端的开放区间上且仅与一个RFLP标记连锁,利用加密图谱对Rsc-3、Rsc-7、Rsc-9、Rsc-13、Rsa、Rn1和Rn3等7个SMV抗性基因重定位,全部位于D1b连锁群,与相邻分子标记距离均小于6cM,其中Rsc-9、Rn1、Rsa的距离小于1cM,Rsc-13与EST标记GMKF168a共分离。对本群体农艺性状进行QTL重定位,获得8个性状相关的42个主效QTL,其中20个QTL遗传贡献率大于10%,与原图谱比较,新定位的各QTL的标记区间明显缩短,与相邻标记的连锁更加紧密。     

一个水稻落粒性基因SH1的SSR标记定位

以籼稻品种93-11为轮回亲本,与粳稻品种日本晴杂交并回交的高世代分离群体为研究材料,选用104个多态性的SSR标记对水稻的落粒性基因进行定位。结果表明,在BC₄F₂群体中,6个标记的基因型来自于日本晴;在BC₄F₃定位群体中,难落粒植株数与易落粒植株数的分离比例为3:1,落粒性受1对显性基因控制,命名为SH1;分子标记与落粒性共分离分析将SH1定位在SSR标记RM5389和RM1068、RM1387之间,与3个标记的遗传距离分别为0.7cM、5.5cM和13.1cM,此结果为该基因的分子标记辅助选择奠定了基础。     

Quantitative trait loci associated with lodging tolerance in soybean cultivar ‘Toyoharuka’

Lodging tolerance (LT) is an important trait for high yield and combine-harvesting efficiency in soybean [Glycine max (L.) Merr.]. Many previous studies have investigated quantitative trait loci (QTLs) for lodging score (LS) in soybean. Most of the investigated QTLs were located in the proximal region of maturity or growth habit loci. The aim of this study was to identify genetic factors for LT not associated with maturity or growth habit. QTL analysis was performed using a recombinant inbred line (RIL) population derived from a cross between ‘Toyoharuka’ (TH), a lodging-tolerant cultivar, and ‘Toyomusume’ (TM). The genotypes of TH and TM were estimated as both e1e2E3E4 and dt1. The average LS over 4 years was used for QTL analysis, identifying a major and stable QTL, qLS19-1, on chromosome 19. The LS of the near-isogenic line (NIL) with the TH allele at Sat_099, the nearest marker to qLS19-1, was significantly lower than the NIL with the TM allele at that position. The TH allele at Sat_099 rarely had a negative influence on seed yield or other agronomic traits in both NILs and the TM-backcrossed lines. Our results suggest that marker-assisted selection for qLS19-1 is effective for improving LT in breeding programs.     

Experimental evaluation of several cycles of marker-assisted selection in maize

A program was initiated in 1994 to compare the efficiency of marker-assisted selection (MAS) and conventional phenotypic selection. A population of 300 F_3:4 families was generated from the cross between two maize inbred lines F2 and F252 and selected on an index combining grain yield and grain moisture at harvest. This population was characterised for 93 RFLP markers and evaluated as testcrosses in a large range of environments. Three methods of selection were applied (i) two cycles of conventional phenotypic selection; (ii) two cycles of MAS based on an index combining phenotypic values and QTL genetic values and (iii) one cycle of combined MAS followed by two cycles of selection based only on the QTL effects estimated in the first generation. The different populations were characterised for RFLP markers. The evolution of allele frequencies showed that selection on only-markers was very efficient for fixing QTL alleles found favourable in the initial population. This evolution was quite different from that observed for phenotypic selection or combined MAS. Genetic gain was evaluated and found significant for each method of selection. Nevertheless, the difference between phenotypic selection and combined MAS was not significant. The two additional cycles of MAS on only-markers did not improve significantly the genetic value of the population. Moreover, the genetic variance of this population remained high, despite most of the QTL initially detected were almost fixed. The results suggest that the QTL effects estimated in the initial population were not stable due to epistasis and/or QTL by environment interactions. This revised version was published online in July 2006 with corrections to the Cover Date.     

Breeding for Fusarium head blight resistance in wheat—Progress and challenges

Fusarium head blight is among the most extensively studied fungal diseases of wheat and other small grain cereals due to its impact on yield and quality, but particularly due to its potential to produce mycotoxins, which are harmful to humans and animals. Since our last comprehensive review on QTL mapping and marker-assisted selection for FHB resistance in wheat in 2009, numerous studies have been conducted to identify, validate or fine-map resistance QTL. The main aim of this review is to update and summarize findings on FHB resistance breeding of wheat published during the last decade. Furthermore, we compiled a user-friendly table listing FHB resistance QTL data providing a valuable resource for further FHB resistance research. The role of morphological and phenological traits on FHB resistance and possible consequences for resistance breeding are discussed. This review concentrates current knowledge on breeding for FHB resistance and suggests strategies to enhance resistance by deploying molecular breeding methods, including marker-assisted and genomic selection.     

水稻糙米蛋白质含量QTL定位及上位性分析

稻米蛋白质含量是水稻(Oryza sativa L.)营养品质育种的重要内容之一.本研究以华恢3号与中国香稻构建的重组自交系(RILs)群体为材料,构建了含有156个SSR标记的遗传连锁图谱,结合近红外分析仪测定两年(2009年,2010年)的糙米蛋白质含量,进行了QTL定位和遗传基础分析研究.两年共定位到3个控制糙米蛋白含量的QTL和16对上位性互作的位点.其中检测到的3个QTLs,分别位于第4、6、8染色体上,单个QTL解释的表型变异率为3.39％～34.2％,两年解释的总表型变异率分别为41.2％和5.95％,这些QTL中只有位于第8染色体短臂在区间RM310～RM547的qbpc8在2009年和2010年被重复检测到.LOD值分别为22.5和3.5,解释表型变异率分别为34.2％和5.95％.这些QTL的发掘,为分子标记辅助选择改良水稻营养品质奠定了基础.     

Construction and characterization of a BAC library of soybean

We have constructed a soybean (Glycine max (L.) Merrill) bacterial artificial chromosome (BAC) library from green leaf protoplasts of the cultivar, Misuzudaizu. The library contains 53,760 clones with an average insert size of 116 kb. About 2.9% chloroplast DNA origin was revealed by PCR and colony hybridization. Apart from 2.8% clones having no insert, this library represents 5.2 genome equivalents. With this genome coverage, the probability of having any DNA sequence represented in the library is higher than 99.5%. Three-dimensional pools of the BAC library in combination with the use of a high efficiency genome scanning (HEGS) electrophoresis facilitate rapid and efficient PCR-based screening. An average of five positive clones were identified after screening the BAC library with SSR and STS markers. BAC-end walking was performed for three SSR associated BACs. This library will provide a good resource for positional cloning of agronomically and biologically important QTL genes that Misuzudaizu possesses.     

Potential of marker-assisted selection in hemp genetic improvement

Summary The development and applications of molecular markers to hemp breeding are recent, dating back only to the mid-1990s. The main achievements in this field are reviewed. The analysis of Cannabis germplasm by RAPD, AFLP and microsatellites is discussed, with its consequence for the still debated species concept in Cannabis. DNA-based markers have also been exploited in the field of forensic science, in an attempt to discriminate licit from illicit crop. The main applications of the molecular markers to the breeding, however, have been achieved with the development of markers closely linked to the male sex and to some of the most relevant chemotypes. Active research is carried out by several groups in the field of identification and characterization of the genes involved in fiber quality and quantity, and in the determination of monoecy, another very important target of hemp breeding. Besides, markers associated to new, potentially useful chemotypes are being developed, for the marker-assisted breeding of pharmaceutical Cannabis.