CN104830967A - Positioning method of rice selected introgression lines QTL - Google Patents

Abstract

The invention relates to a positioning method of rice selected introgression lines QTL. According to the method, different rice varieties are selected as donors and are hybridized with same recurrent parent, and then are continuously back-crossed with the recurrent parent for two times to obtain random colonies. Through the Markov chain principle, selected introgression line groups, having same target characters, of the random colonies from different sources are combined through genotype. By means of the target character introgression line combined groups from the random colonies sourced from different combinations as an original positioning group, frequency partial separation of all marked sites of the original positioning group is compared with those of a random controlled group in same generation, so that the target character QTL can be positioned through partial separation of the combined group by means of Wald statistic detection. The method is suitable for positioning high-yield and dryness-resistant QTL through the introgression line groups having the target characters of high yield, dryness resistance and the like, and the genes are subjected to assistant-selected pyramiding breeding through molecular markers.

Description

A kind of paddy rice selects the localization method of introgressive line QTL

Technical field

The invention belongs to Rice molecular breeding and molecular genetics field, be specifically related to the localization method that a kind of paddy rice selects introgressive line QTL, particularly relate to a kind of method utilizing paddy rice objective trait to select introgressive line localizing objects proterties QTL.

Background technology

In recent decades, utilize the segregating populations such as the RIL built from parents, single amphiploid, backcross progeny and Single Segment Substitution Lines in Rice to located quantitative trait locus (QTL) that the is a large amount of and complex character such as rice grain output and drought resisting, very large promoter action is played to the Genetic Mechanisms of understanding these complex characters in depth.But following major defect is existed to paddy rice QTL Position Research: one is the hereditary segregating population built from two parents in the past, two allelic quality relatively of parent can only be compared, the allelic diversity of QTL cannot be dissected, thus cannot provide best favorable allels for molecular breeding; Two is interference that the random genetic background cannot getting rid of segregating population is located QTL, causes the QTL of same proterties to show in different genetic background and differs; Three is that QTL target group disconnects mutually with breeding population, and affect by genetic background because QTL expresses, the QTL information utilizing target group to obtain is difficult to instruct the genetic improvement of breeding population; Four is that to comprise the resistance of Rice Drought Resistence relevant for period with vine growth and development, and it is unified that difference breeding time in segregating population between individuality makes the environment stress time be difficult to, thus increases the difficulty that phenotype precisely identifies, finally affect the accuracy that QTL locates.

In order to solve the problem that QTL location disconnects mutually with breeding practice, Tanksley etc. (1996) propose AB-QTL Research Thinking, utilize germ plasm resource Backcross introgression to the basically identical introgressive line colony Mapping of QTL of improved seeds background constructing genetic background, again the QTL favorable allels navigated to is improved recurrent parent kind by marker assisted selection, accomplish that QTL excavates to combine with breeding, on QTL excavates and utilizes, achieve certain success.But the defects such as AB-QTL still exists that mapping population is excessive, Mapping parents allelic variation degree limited (being still parents' in-group).For effectively overcoming the Genetic Background Effects that the complex character QTL such as high yield, drought resisting expresses, germ plasm resource beneficial gene is excavated and organically combines with breed improvement, Li etc. (2005) propose germ plasm resource to be imported improved seeds background and cultivate objective trait selection introgressive line, proterties QTL are located the New Policy combined with breed improvement.

Adopt large (normally 200 individual left and right) random segregating population Mapping of QTL different from tradition, objective trait selects introgressive line to be identify from a random Backcross introgression colony through objective trait, select the extreme strain of phenotype being significantly better than recurrent parent on objective trait to form an objective trait and select introgressive line colony, general groups size is 20-50 strain.These select introgressive line genetic background similar to recurrent parent, but are significantly better than recurrent parent on objective trait, not only may be used for the QTL location of objective trait, and are the desirable breeding materials that realize target QTL is polymerized.Select introgressive line due to colony less, the degree of variation of colony's internal object proterties is little, brings very large impact to objective trait QTL positioning function.For this reason, we have developed the method Mapping of QTL adopting Chi-square statistic full-length genome mark to be partially separated, but still due to single selections colony less than normal, existence navigates to false positive QTL defect on the high side.

Summary of the invention

The object of the invention is to overcome the deficiencies in the prior art, a kind of paddy rice is provided to select the localization method of introgressive line QTL, select introgressive line to carry out genotype merging the objective trait of different donor source under same recurrent parent background, and utilize the colony after merging as the target group of original QTL.

The technical scheme that the present invention solves the problems of the technologies described above is as follows: a kind of paddy rice selects the localization method of introgressive line QTL, and the method comprises the steps:

1) introgressive line informative population is selected

Select different rice varieties to be that donor is hybridized with same recurrent parent respectively, build filial generation, then described filial generation obtains corresponding random population for twice with recurrent parent continuous backcross respectively;

2) introgressive line mass screening is selected

With described recurrent parent for contrast, the objective trait of above-mentioned corresponding random population is identified under specific objective character screening condition, individuality objective trait in each colony being significantly better than recurrent parent is selected in as the selection introgressive line of objective trait, forms corresponding objective trait and selects introgressive line colony;

3) introgressive line colony genotype identification is selected

Adopt CTAB method to extract above-mentioned 2) in corresponding objective trait select the DNA of all individualities of introgressive line colony, carry out SSR marker qualification;

4) introgressive line colony QTL is selected to locate

Adopt Markov chain principle by above-mentioned 3) in the selection introgressive line colony of same target proterties that derives from different random colony merged by genotype, and utilize the colony after merging as the target group of original QTL, comparison and location colony is separated partially with the frequency of all marker sites of random controls colony of identical generation, and after utilizing Wald statistic mixed-state to merge to selecting introgressive line colony, the inclined separation of colony carrys out localizing objects proterties QTL.

Further, for coercing lower qualification drought resistance in drought or identifying productive under the condition of normal paddy field under described specific trait condition.

Technical characterstic of the present invention is as follows:

Because 4 cover drought resistings described in the present invention select the genotype data of introgressive line colony different, simple individuality merges the target group that can not obtain complete set.In order to overcome this difficulty, we adopt the principle of Markov chain, utilize conditional probability to estimate the probable value of deletion segment different genotype in every suit selection introgressive line colony.Markov chain represents with a condition distribution: P (Xn+1|Xn), namely estimates the state in n+1 moment by the state in n moment.Application in molecule marker is filled up is exactly: utilize the genotype information of known site to estimate the genotype information of adjacent sites.Calculation formula is:

$\mathrm{Pr}(B=k|\mathrm{AC})=\frac{J^T{D}_A{T}_{\mathrm{AB}}{D_{\left(k\right)}T}_{\mathrm{BC}}{D}_{C}J}{{\mathrm{\Σ}}_{k=1}^3{J}^T{D}_A{T}_{\mathrm{AB}}{D_{\left(k\right)}T}_{\mathrm{BC}}{D}_{C}J}=\frac{J^T{D}_A{T}_{\mathrm{AB}}{D}_{\left(k\right)}{T}_{\mathrm{BC}}{D}_{C}J}{J^T{D}_A{T}_{\mathrm{AB}}{T}_{\mathrm{BC}}{D}_{C}J}$

Known A, C two point gene type estimate the probability that 3 kinds of genotype of B point may exist.Wherein TAB and TBC is transition matrix, BC ₂f ₂transition matrix is from generation to generation:

R=recombination fraction

We just can calculate each colony deletion segment 3 kinds of genotypic probability like this, thus select the genotype data of introgressive line colonies to merge 4 cover drought resistings, form a relatively large target group.

The beneficial effect of the present invention compared with other technologies is:

Current QTL location mainly adopts F ₂, the random population such as RIL, because the natural population's sample size built is large, so phenotypic evaluation workload is large, genotype data expends height, and disconnect due to most random population and breeding population, QTL expresses exists obvious Genetic Background Effects, and the QTL that therefore random population navigates to is difficult to directly be used in breeding.For overcoming above-mentioned shortcoming, we propose by backcrossing, germ plasm resource favorable variation is imported to improved seeds (as recurrent parent) background, the extreme selection of combining target proterties, the selection introgressive line colony of establishing target proterties, these select introgressive lines to be significantly better than recurrent parent on objective trait, and other proterties and recurrent parent are similar, have Breeding Application value.Adopt the selection introgressive line breeding population Mapping of QTL of objective trait, the QTL navigated to like this can be directly used in breeding practice.Adopted Chi-square statistic single selection colony to carry out the QTL of positioning effects objective trait, because colony's number of individuals causes the variance of character variation little less, QTL positioning result is undesirable in the past.We adopt Markov chain principle, merge different choice colony by the deletion Genotype between expection different groups and expand colony's capacity, utilize stochastic model Wald Test to be partially separated localizing objects proterties QTL, thus improve selection introgressive line colony QTL positioning function.

We suppose that the effect of a certain 4 colonies in site obeys same variance, after estimating the prior variance in each site like this, just can calculate the effect value a in this site of each colony, thus calculate the variance of effect, carry out Wald inspection.The Wald statistic merged after colony is the Wald statistic sum of 4 independent colonies, inapparent site is detected like this in some colonies, after so merging colony, this site just may be detected significantly, so merge detection effect that colony improves QTL.

Embodiment

Be described principle of the present invention and feature below, example, only for explaining the present invention, is not intended to limit scope of the present invention.

Embodiment

(1) select introgressive line colony genotype to merge and carry out non-irrigated QTL location

1. material construction

Utilize the lucky round-grained rice 88 of the japonica rice variety of Jilin Province's spread to hybridize respectively for donor with the rice varieties from 4 country variants, build hybrid F ₁generation, then recurrent parent and F ₁backcross and obtain BC ₁f ₁, recurrent parent again with BC ₁f ₁backcross and obtain BC ₂f ₁, BC ₂f ₁selfing sowing obtains 4 cover BC respectively ₂f ₂with body.

Introgressive line community information is selected in the drought resisting that table 14 overlaps lucky round-grained rice 88 background

N1: original population's number of individuals; N2: first round drought resisting screening number of individuals; N3: offspring verifies remaining drought resisting number of individuals; PS: selection percentage

2. drought resisting screening

By 4 BC ₂f ₂colony's seed is divided into two parts, a is used for drought resisting screening, and another part and drought resisting screens colony and synchronously planted, and miscegenation mixes the random controls colony of receiving as drought resisting selection colony.Drought resisting screening colony is planted in normal paddy field (contrast) and non-irrigated stressful environmental (process) respectively, and 800 individual plants are planted by every colony, with the lucky round-grained rice 88 of recurrent parent for contrast.Paddy field environment carries out normal field management, dry land environment stops pouring water after tillering regularity, make plant experience drought and coerce entering generative growth phase, after this carry out intermittent irrigation mainly through filling with horse race water, non-irrigated stress level controls to and the lucky round-grained rice 88 production loss half of contrast is advisable.After seed fully matured, carry out field seed selection for single plant yield, under non-irrigated stressful environmental, screen the individual plant of single plant yield apparently higher than the lucky round-grained rice 88 of recurrent parent, think that it has good drought resistance.Such 4 colonies screen altogether 135 drought resisting strains.Subsequently in Ningxia and Liang Ge place, Beijing, repetition is arranged respectively to 135 selected drought resisting individual plants, carry out the drought resistance checking of drought resisting offspring strain, finally obtain the drought resisting strain of 106 genetic stabilities.By table 2, we can find out that 4 covers select colony under non-irrigated stress conditions, and single plant yield is significantly higher than recurrent parent, significantly improves the single plant yield under non-irrigated stress conditions.

Under the non-irrigated stress conditions of table 2, the average phenotypic data of colony selected by 4 covers

^*, ^*represent in 0.05 level and 0.01 level remarkable respectively, NX: Ningxia, BJ: Beijing

3. genotype identification

Adopt CTAB method to extract the DNA of all individualities of 4 cover selection introgressive line, carry out SSR marker qualification.4 covers are selected to screen polymorphic mark number respectively in introgressive line colony and are respectively 73,84,77 and 38.4 covers have 133 different polymorphic marker after selecting the merging of introgressive line colony, estimated the probable value of deletion Genotype in each colony by Markov chain conditional probability.Just there have been a set of on all four genotype data in such 4 cover colonies, for merging the QTL location of colony.

4. drought resisting QTL locates

4 drought resisting introgressive lines deriving from various combination are utilized to merge colony as original target group, comparison and location colony is separated partially with the frequency of all marker sites of random controls colony of identical generation, QTL location is separated partially to selection introgressive line colony, 7 QTL site detected altogether, wherein 3 sites (QDTY 2.4, QDTY 6.5 and QDTY 11.1) are identical with forefathers QTL positioning result, 3 anti-drought gene location overlaps of 3 QTL that No. 2, No. 7 and No. 11 chromosomal localizations arrive and forefathers' functional verification.

Colony QTL positioning result selected by table 3

(2) drought resisting QTL verifies

In order to prove the verity navigating to QTL further, the QTL navigated to is carried out phenotype checking by us in random population.By the significance of the individual difference between non-irrigated stress conditions mean yield of the different parent's allelotrope of inspection QTL site, verify the verity that QTL locates.We adopt output increased per-cent simultaneously, namely the percentage ratio of individuality output increased compared with recurrent parent of QTL is carried to weigh the effect of drought resisting QTL, that is: mean yield × 100 of output increased (%)=(carrying the mean yield of the mean yield-recurrent parent of QTL individuality)/recurrent parent.

Through the checking of random population, we find have 4 sites can be verified in random population more than 2 in 7 sites navigated to, and carry between the individuality of QTL and the single plant yield not carrying QTL individuality and reach significant difference (table 5), further demonstrate the feasibility and the reliability that utilize and select colony to be partially separated QTL location.

The QTL navigated to is verified in table 5 random population

The above; be only the present invention's preferably embodiment, but protection scope of the present invention is not limited thereto, is anyly familiar with those skilled in the art in the technical scope that the present invention discloses; the change that can expect easily or replacement, all should be encompassed within protection scope of the present invention.

Claims (2)

1. paddy rice selects a localization method of introgressive line QTL, and it is characterized in that, the method comprises the steps:

1) introgressive line informative population is selected

Select different rice varieties to be that donor is hybridized with same recurrent parent respectively, build filial generation, then described filial generation obtains corresponding random population for twice with recurrent parent continuous backcross respectively;

2) introgressive line mass screening is selected

With described recurrent parent for contrast, the objective trait of above-mentioned corresponding random population is identified under specific objective character screening condition, individuality objective trait in each colony being significantly better than recurrent parent is selected in as the selection introgressive line of objective trait, forms corresponding objective trait and selects introgressive line colony;

3) introgressive line colony genotype identification is selected

Adopt CTAB method to extract above-mentioned 2) in corresponding objective trait select the DNA of all individualities of introgressive line colony, carry out SSR marker qualification;

4) introgressive line colony QTL is selected to locate

Adopt Markov chain principle by above-mentioned 3) in the selection introgressive line colony of same target proterties that derives from different random colony merged by genotype, and utilize the colony after merging as the target group of original QTL, comparison and location colony is separated partially with the frequency of all marker sites of random controls colony of identical generation, and after utilizing Wald statistic mixed-state to merge to selecting introgressive line colony, the inclined separation of colony carrys out localizing objects proterties QTL.

2. a kind of paddy rice according to claim 1 selects the localization method of introgressive line QTL, it is characterized in that, for coercing lower qualification drought resistance in drought or identifying productive under the condition of normal paddy field under described specific objective character screening condition.