标记辅助导入的原理与应用

标记辅助导入是分子遗传信息应用于动物育种的一个重要方面, 其目的是在标记信息的辅助下将一个品种(供体)中的一个或多个优良基因导入另一个品种(受体), 同时还要尽可能地保持受体群体原有的遗传背景。在标记辅助导入过程中, 标记信息既可用于辅助前景选择, 即对目标基因携带者的选择, 以保证目标基因的正确导入, 也可用于辅助背景选择, 即对受体基因组的选择, 以加快受体遗传背景的恢复。本文介绍了标记辅助导入的原理和基本方法, 综述了目前已提出的不同前景选择和背景选择方法以及消除遗传累赘(与目标基因连锁的不利基因)的方法, 同时列举了标记辅助导入在动物上的一些成功应用。     

标记辅助回交育种中所需最小样本容量的近似估计

回交育种是把有利基因从供体亲本向受体亲本转移的一种有效方法,标记辅助选择可加速其进程。为了制定合理的标记辅助选择计划,育种家必须知道所需的后代群体大小。该文提出了一种估算在标记辅助回交育种中同时进行前景选择和背景选择所需群体大小的方法。在假定所需转移的目标基因座与遗传背景之间为相互独立的简化假设下,可以通过将解析方法（针对前景选择）与基于回交亲本图示基因型的模拟方法（针对背景选择）相结合,近似地估计出在每一世代中选到所需基因型的概率,进而估算出在一定概率水平下至少获得一个符合要求的个体所需的最小样本容量,用假想的例子演示了该方法的使用情况。该方法可以很方便地应用于实际的回交育种。     

大豆SSR标记辅助遗传背景选择的效果分析

本研究利用鲁豆4号回交转育的大豆种子脂氧酶缺失株系为材料,用IEF-PAGE鉴定大豆种子脂氧酶缺失基因,SSR标记进行遗传背景分析,通过遗传背景回复率相关分析,探索分子标记辅助遗传背景选择时所需的适宜的标记数和选择方式,期望获得可进一步回的鲁豆4号脂氧酶缺失株系。研究明确了大豆SSR标记辅助背景选择时适宜标记数目和选择方式,即先用少数标记初筛,选出遗传背景回复率较高的材料,再用适宜标记鉴定,随着世代递增,已恢复为轮回亲本的标记住点不再分析,逐代减少选择标记数目。获得了合有脂氧酶缺失基因,遗传背景与鲁豆4号差异较小的株系,可用鲁玉4号进一步回交,从而加速培育鲁豆4号脂氧酶缺失近等基因系。     

回交育种中供体基本组成分的分布及其应用

推导了回交群体中供体基因组成分的条件概率分布,并用平均数预测各个体的供体染色体片段的大小。预测的精度则用有关方差的公式,以供体的实际片断大小（y）与根据标记基因型预测的供体片断大小（y^p)的相关表示,并表达为标记密度的函数。结果表明：虽然在标记密度中等（例如40cM／标记）时即能获得y和y^p的高度相关,但对一个在群体,仍必须通过高密度的标记图（例如10－20cM／标记）才可能鉴别出其中的最佳个体。因此,为了在标记辅助回交育种中充分利用标记信息,应当分步骤鉴定标记基因型和选择具体。这就是：先根据少数标记对所有个体作初步选择,再根据较多标记对少数个体作精细选择。这样,在基因渐渗实验中,就可以既提高对大群体的选择强度又只要鉴定有限数目的标记基因型。这是一种非常有效的方法。     

不同QTL增效基因初始频率下标记辅助选择的效果

采用随机模拟方法模拟了在一个闭锁群体内连续对单个性状选择10个世代的情形。在假定选择性状受一个位于常染色体上的QTL和多基因共同控制的情况下,采用动物模型标记辅助最佳线性无偏预测方法估计个体育种值并据此进行种畜的选留,并在此基础上系统地比较了QTL增效基因初始频率对标记辅助选择效果的影响。结果表明：当群体中QTL增效基因的初始频率较低时,选择所获得的QTL基因型值的进展会更大,标记辅助选择在单位时间内可获得较大的遗传进展;此时,尽管QTL增效基因在群体中固定所需的世代数会更长一些,但其频率上升的速度却更快。而QTL增效基因初始频率的高低对群体近交增量的影响不是很大。     

三基因聚合改良恢复系福恢673的稻瘟病抗性

为了改良水稻优良恢复系福恢673的稻瘟病抗性,以该恢复系为轮回亲本,以携有3个稻瘟病抗性基因(Pi-1、Pi-9和Pi-k~h)的优质恢复系金恢1059为供体亲本,通过回交育种结合分子标记辅助选择,选育出10个导入了这3个抗稻瘟病基因的福恢673近等基因系,其遗传背景恢复率为92.96%–98.59%。抗性鉴定结果表明,这些近等基因系及其与不育系宜香A配制的杂种一代均表现抗稻瘟病,抗性明显强于对照福恢673和宜优673,且半数以上杂种一代基本保留了原组合的主要优点。用近等基因系Line 9配组的杂交稻新组合两优7283和金泰优683在区试中均表现出产量高、稻瘟病抗性强、生育期适中等特点,表明该近等基因系具较好的应用前景。     

回交育种中供体基因组成分的分布及其应用

推导了回交群体中供体基因组成分的条件概率分布,并用平均数预测各个体的供体染色体片段的大小.预测的精确度则用有关方差的公式,以供体的实际片断大小(y)与根据标记基因型预测的供体片断大小(yp)的相关表示,并表达为标记密度的函数.结果表明虽然在标记密度中等(例如40cM/标记)时即能获得y和yp的高度相关,但对一个大群体,仍必须通过高密度的标记图(例如10～20cM/标记)才可能鉴别出其中的最佳个体.因此,为了在标记辅助回交育种中充分利用标记信息,应当分步骤鉴定标记基因型和选择个体.这就是先根据少数标记对所有个体作初步选择,再根据较多标记对少数个体作精细选择.这样,在基因渐渗实验中,就可以既提高对大群体的选择强度又只要鉴定有限数目的标记基因型.这是一种非常有效的方法.     

利用分子标记辅助选择改良珍汕97的稻瘟病抗性

利用回交育种中产生的回交群体,结合前人的研究结果构建了Pil基因区域的局部分子标记连锁图,通过BC1F2家系的接种结果判断其基因型.将Pi1定位在RFLP标记RZ536与SSR标记RM144之间,图距分别为9.7cM、6.8 cM,从而建立了一套完整的以PCR为基础的分子标记辅助选择体系.通过分子标记和抗性验证两种选择方式相结合,经过三代回交将Pi1区段快速导入受体亲本珍汕97B中.在BC3F1中利用15条ISSR引物扩增的167条随机分布在基因组中的多态性带筛选背景,得到4个背景较好的单株.经过纯合筛选及抗性验证后共得到17个带有抗性基因Pi1的改良珍汕97株系.试验表明微卫星标记在正向选择、负向选择及背景选择中都起到极大的作用.     

利用分子标记辅助选择改良珍汕97的稻瘟病抗性

利用回交育种中产生的回交群体，结合前人的研究结果构建了Pi1基因区域的局部分子标记连锁图，通过BC1F2家系的接种结果判断其基因型。将Pi1定位在RFLP标记RZ536与SSR标记RM144之间，图距分别为9．7cM、6．8cM，从而建立了一套完整的以PCR为基础的分子标记辅助选择体系。通过分子标记和抗性验证两种选择方式相结合，经过三代回交将Pi1区段快速导入受体亲本珍汕97B中。在BC3F1中利用15条ISSR引物扩增的167条随机分布在基因组中的多态性带筛选背景，得到4个背景较好的单株。经过纯合筛选及抗性验证后共得到17个带有抗性基因Pi1的改良珍汕97株系。试验表明微卫星标记在正向选择、负向选择及背景选择中都起到极大的作用。     

植物基因组研究与利用的新型工具——异源单体附加系

在高等植物中, 以种间杂交和回交把有益基因从一个物种转移到另一个物种为目的育种项目中, 单个外源染色体常常被附加到含有受体细胞完整一套染色体中, 形成异源单体附加系。这种异源单体附加系是阐明基因组结构和转移基因的有效工具。它可以通过回交形成覆盖整个基因组的渗入系重叠群, 用于建立以受体物种基因组为载体的外源物种基因组文库。另外, 一套完整的异源单体附加系也可看作是一个拥有分散供体基因组成为单个染色体单位的文库, 便于精确高通量地将标记分配到单个供体染色体上, 从而可以比较供体染色体和各自的直向同源受体染色体之间的标记位置和同线性关系。同时, 也便于研究同源染色体的渗入机制和配对状态。文中介绍了异源单体附加系的培育和特性, 并着重阐明了它在遗传育种和基础研究中的应用。     

Comparison of Different Foreground and Background Selection Methods in Marker-Assisted Introgression

Three different methods for foreground selection and four different methods for background selection were compared in terms of the efficiency of marker-assisted introgression of a QTL allele from a donor line into a recipient line and also in terms of the recovery of the recipient genetic background. The results showed that for the introgression of a donor QTL allele, a direct selection on the QTL itself (when the QTL genotype can be directly identified) would ensure that the allele is successfully introgressed and rapidly fixed. However, when a direct selection on the QTL is not feasible, an indirect selection using two closely linked flanking markers can be used, which also shows similar results. For the recovery of the recipient genetic background, if the goal is to recover the whole genetic background of the recipient, genomic similarity selection or marker index selection would be the best choice: Only three generations of backcrosses were required to recover over 98% of the recipient genome. Whereas if the goal is to recover certain background traits of the recipient, MBLUP selection would give the best results, which achieved not only over 99% recovery of the recipient QTL alleles for the background traits after three generations of backcrosses, but also showed the best genetic improvement of these traits.     

Stability over genetic backgrounds, generations and years of quantitative trait locus (QTLs) for organoleptic quality in tomato

The efficiency of marker-assisted backcross for the introgression of a quantitative trait locus (QTL) from a donor line into a recipient line depends on the stability of QTL expression. QTLs for six quality traits in tomato (fruit weight, firmness, locule number, soluble solid content, sugar content and titratable acidity) were studied in order to investigate their individual effect and their stability over years, generations and genetic backgrounds. Five chromosome regions carrying fruit quality QTLs were transferred following a marker-assisted backcross scheme from a cherry tomato line into three modern lines with larger fruits. Three sets of genotypes corresponding to three generations were compared: (1) an RIL population, which contained 50% of each parental genome, (2) three BC3S1 populations which segregated simultaneously for the five regions of interest but were almost fully homozygous for the recipient genome on the eight chromosomes carrying no QTL and (3) three sets of QTL-NILs (BC3S3 lines) which differed from the recipient line only in one of the five regions. QTL detection was performed in each generation, in each genetic background and during 2 successive years for QTL-NILs. About half of the QTLs detected in QTL-NILs were detected in both years. Eight of the ten QTLs detected in RILs were recovered in the QTL-NILs with the genetic background used for the initial QTL mapping experiment, with the exception of two QTLs for fruit firmness. Several new QTLs were detected. In the two other genetic backgrounds, the number of QTLs in common with the RILs was lower, but several new QTLs were also detected in advanced generations.     

Marker-assisted introgression using non-unique marker alleles I: selection on the presence of linked marker alleles

This paper investigates marker-assisted introgression of a major gene into an outbred line, where identification of the introgressed gene is incomplete because marker alleles are not unique to the base populations (the same marker allele can occur in both donor and recipient population). Those markers are used to identify the introgressed allele as well as the background genotype. The effect of using those markers, as if they were completely informative on the retention of the introgressed allele, was examined over five generations of backcrossing by using a single marker or a marker bracket for different starting frequencies of the marker alleles. Results were calculated by using both a deterministic approach, where selection is only for the desired allele, and by a stochastic approach, where selection is also on background genotype. When marker allele frequencies in donor and recipient population diverged from 1 and 0 (using a diallelic marker), the ability to retain the desired allele rapidly declined. Marker brackets performed notably better than single markers. If selection on background marker genotype was applied, the desired allele could be lost even more quickly than expected at random because the chance that the allele, which is common in the donor line, is present on the locus identifying the introgressed allele and is surrounded by alleles common in the recipient line on the background marker loci, will descend from the donor line (double recombination has taken place), is a lot smaller than the chance that this allele will stem from the recipient line (in which the allele occurs in low frequency). Marker brackets again performed better. Preselection against marker homozygotes (producing uninformative gametes) gave a slightly better retention of the introgressed allele.     

Combined detection and introgression of QTL in outbred populations

Background

Detecting a QTL is only the first step in genetic improvement programs. When a QTL with desirable characteristics is found, e.g. in a wild or unimproved population, it may be interesting to introgress the detected QTL into the commercial population. One approach to shorten the time needed for introgression is to combine both QTL identification and introgression, into a single step. This combines the strengths of fine mapping and backcrossing and paves the way for introgression of desirable but unknown QTL into recipient animal and plant lines.

Methods

The method consisting in combining QTL mapping and gene introgression has been extended from inbred to outbred populations in which QTL allele frequencies vary both in recipient and donor lines in different scenarios and for which polygenic effects are included in order to model background genes. The effectiveness of the combined QTL detection and introgression procedure was evaluated by simulation through four backcross generations.

Results

The allele substitution effect is underestimated when the favourable QTL allele is not fixed in the donor line. This underestimation is proportional to the frequency differences of the favourable QTL allele between the lines. In most scenarios, the estimates of the QTL location are unbiased and accurate. The retained donor chromosome segment and linkage drag are similar to expected values from other published studies.

Conclusions

In general, our results show that it is possible to combine QTL detection and introgression even in outbred species. Separating QTL mapping and introgression processes is often thought to be longer and more costly. However, using a combined process saves at least one generation. With respect to the linkage drag and obligatory drag, the results of the combined detection and introgression scheme are very similar to those of traditional introgression schemes.     

Introgression of a major QTL from an inferior into a superior population using genomic selection

Background

Selection schemes aiming at introgressing genetic material from a donor into a recipient line may be performed by backcross-breeding programs combined with selection to preserve the favourable characteristics of the donor population. This stochastic simulation study investigated whether genomic selection can be effective in preserving a major quantitative trait locus (QTL) allele from a donor line during the backcrossing phase.

Methods

In a simulation study, two fish populations were generated: a recipient line selected for a production trait and a donor line characterized by an enhanced level of disease resistance. Both traits were polygenic, but one major QTL affecting disease resistance was segregating only within the donor line. Backcrossing was combined with three types of selection (for total merit index) among the crossbred individuals: classical selection, genomic selection using genome-wide dense marker maps, and gene-assisted genomic selection. It was assumed that production could be observed directly on the selection candidates, while disease resistance had to be inferred from tested sibs of the selection candidates.

Results

Classical selection was inefficient in preserving the target QTL through the backcrossing phase. In contrast, genomic selection (without specific knowledge of the target QTL) was usually effective in preserving the target QTL, and had higher genetic response to selection, especially for disease resistance. Compared with pure genomic selection, gene-assisted selection had an advantage with respect to disease resistance (28–40% increase in genetic gain) and acted as an extra precaution against loss of the target QTL. However, for total merit index the advantage of gene-assisted genomic selection over genomic selection was lower (4–5% increase in genetic gain).

Conclusion

Substantial differences between introgression programs using classical and genomic selection were observed, and the former was generally inferior with respect to both genetic gain and the ability to preserve the target QTL. Combining genomic selection with gene-assisted selection for the target QTL acted as an extra precaution against loss of the target QTL and gave additional genetic gain for disease resistance. However, the effect on total merit index was limited.     

性状遗传力与QTL方差对标记辅助选择效果的影响

在采用动物模型标记辅助最佳线性无偏预测方法对个体育种值进行估计的基础上,模拟了在一个闭锁群体内连续对单个性状选择10个世代的情形,并系统地比较了性状遗传力和QTL方差对标记辅助选择所获得的遗传进展、QTL增效基因频率和群体近交系数变化的影响。结果表明：在对高遗传力和QTL方差较小的性状实施标记辅助选择时,可望获得更大的遗传进展;遗传力越高,QTL方差越大,则QTL增效基因频率的上升速度越快;遗传力较高时,群体近交系数上升的速度较为缓慢,而QTL方差对群体近交系数上升速度的影响则不甚明显。结合前人关于标记辅助选择相对效率的研究结果,可以认为：当选择性状的遗传力和QTL方差为中等水平时,标记辅助选择可望获得理想的效果。     

Evaluation of quantitative trait loci affecting intramuscular fat and reproductive traits in pigs using marker‐assisted introgression

We investigated the effects of previously identified quantitative trait loci (QTL) in an experimental backcross (BC) between Chinese Meishan pigs and commercial Duroc pigs. We performed marker‐assisted introgression of two QTL for intramuscular fat (IMF) content (IMF population) and three QTL for reproductive traits (reproduction population) from a donor Meishan pig into a recipient Duroc pig. At the fourth BC generation of the IMF population and third BC generation of the reproduction population, carrier animals were selected for the production of animals homozygous for the QTL. Our previous studies have shown that the presence of a Meishan allele on the IMF QTL is associated with low IMF values, and the Meishan allele on the reproductive QTL is associated with large litters. In this study, the presence of a Duroc allele at the IMF QTL on SSC9 resulted in a 0.27% increase in IMF (additive effect = 0.27 ± 0.08), whereas the presence of a Meishan allele at the IMF QTL on SSC7 resulted in a 0.34% increase in IMF (additive effect = ?0.34 ± 0.09). The presence of the Meishan allele at the IMF QTL on SSC7 thus had the opposite effect to our previous studies, that is, increased IMF. In the reproduction population, we observed no differences between the genotypes of the three QTL in regard to number of corpora lutea or litter size. Marker‐assisted introgression at these QTL is thus unlikely to result in an associated increase in litter size. These results show that it is possible to introgress alleles from other breeds into a selection population using molecular markers; any unexpected results might be associated with the genetic background.     

Genetic architecture of a selection response in Arabidopsis thaliana

Quantitative trait locus (QTL) mapping has become an established and effective method for studying the genetic architecture of complex traits. In this report, we use a QTL mapping approach in combination with data from a large selection experiment in Arabidopsis thaliana to explore a response to selection of experimental populations with differentiated genetic backgrounds. Experimental populations with genetic backgrounds derived from ecotypes Landsberg and Niederzenz were exposed to multiple generations of fertility and viability selection. This selection resulted in phenotypic shifts in a number of life-history and fitness-related characters including early development time, flowering time, dry biomass, longevity, and fruit production. Quantitative trait loci were mapped for these traits and their positions were compared to previously characterized allele frequency changes in the experimental populations (Ungerer et al. 2003). Quantitative trait locus positions largely colocalized with genomic regions under strong and consistent selection in populations with differentiated genetic backgrounds, suggesting that alleles for these traits were selected similarly in differentiated genetic backgrounds. However, one QTL region exhibited a more variable response; being positively selected on one genetic background but apparently neutral in another. This study demonstrates how QTL mapping approaches can be combined with map-based population genetic data to study how selection acts on standing genetic variation in populations.