Identification for Heat Tolerance in Backcross Recombinant Lines and Screening of Backcross Introgression Lines with Heat Tolerance at Milky Stage in Rice

The present study aimed at breeding new rice germplasms with similar genome but significantly differed in heat tolerance during the grain filling stage.A total of 791 BC1F8 backcross recombinant lines,derived from the cross of Xieqingzao B /N22 //Xieqingzao B,were used as materials.Each rice line was separated evenly into two groups,and the heat tolerance of all rice lines were evaluated at natural high temperature in fields.The rice lines with heat tolerant index higher than 90% or lower than 40% were selected to compare the phenotypic characters and further identify heat tolerance at the early milky stage in a phytotron.Rice lines with similar phenotypic characters but significantly differed in heat tolerance at the milky stage were analyzed by 887 simple sequence repeat markers that were evenly distributed on the 12 rice chromosomes.In the result,12(6 pairs) rice lines with similar phenotypic characters but significantly differed in heat tolerance at the milky stage were obtained.Molecular marker analysis indicated that the genomic polymorphism between 703T and 704S was the smallest in the 6 pairs of rice lines,with only 16 polymorphic sites,including 22 different alleles.The application of these two backcross introgression rice lines for future study on the mechanisms of heat tolerance in rice at the milky stage will be theoretically beneficial in reducing the interference caused by genetic differences from experimental materials.

Analysis on Genomic Structure Changes and Diversity of Introgression Lines in Dongxiang Wild Rice(Oryza rufipogon Griff.)

[Objective] The aim of the study was to make research on genomic struc- ture variation and variety analysis of Dongxiang wild rice. [Method] Introgression groups of BC1F6 were based on donor of Oryza rufipogon Griff. and receptor of O. sativa sp. indica Kate. Strains of 239 in the group were analyzed on Polymor- phism with the help of 25 couples of SSR primers distributed in 12 pairs of chromo- somes. [Result] Gene fragments of O. rufipogon Griff. were found penetrated in the 25 microsatellite sites and most of the groups kept the parents of Xieqinzao B or DNA sequence of O. rufipogon Griff. The average rate of recurrent homozygous bands was 78.13% in the ILs, but the highest was 94.98% （amplified by primer RM131） and the lowest was 60.25% （RM171）. The average rate of donor homozy- gous bands was 13.37%, but the highest was 32.64% （RM171） and the lowest was 2.93% （RM1095）. There were numerous heterozygous sites in the population and the average heterozygosis rate was 5.62%, while the highest was 10.04%（RM401）. Moreover, we found some parental fragments were lost and some novel fragments were not detected in either parent in BC1F6 population. The average rate of lost bands was 2.88%, while the highest was 13.39% （RM311） and the lowest was 0 （RM401）. The average rate of new bands was 1%. The average of Nei＇s gene di- versity （He） and Shannon＇s Information index （I） were 0.276 and 0.457 respectively in high generation of introgression lines. [Conclusion] The study demonstrated that distant hybridization led to extensive genetic and epigenetic variations in high gener- ation of introgression lines, which expanded the base of genetic variation and laid an important foundation for rice improvement and germplasm innovation.

Development and Identification of Introgression Lines from Cross of Oryza sativa and Oryza minuta

Introgression line population is effectively used in mapping quantitative trait loci（QTLs）,identifying favorable genes,discovering hidden genetic variation,evaluating the action or interaction of QTLs in multiple conditions and providing the favorable experimental materials for plant breeding and genetic research.In this study,an advanced backcross and consecutive selfing strategy was used to develop introgression lines（ILs）,which derived from an accession of Oryza minuta（accession No.101133） with BBCC genome,as the donor,and an elite indica cultivar IR24（O.sativa）,as the recipient.Introgression segments from O.minuta were screened using 164 polymorphic simple sequence repeat（SSR） markers in the genome of each IL.Introgressed segments carried by 131 ILs covered the whole O.sativa genome.The average number of homozygous O.minuta segments per introgression line was about 9.99.The average length of introgressed segments was approximate 14.78 cM,and about 79.64% of these segments had sizes less than 20 cM.In the genome of each introgression line,the O.minuta chromosomal segments harbored chromosomal fragments of O.sativa ranging from 1.15% to 27.6%,with an overall average of 8.57%.At each locus,the ratio of substitution of O.minuta alleles had a range of 1.5% 25.2%,with an average of 8.3%.Based on the evaluation of the phenotype of these ILs,a wide range of alterations in morphological and yield-related traits were found.After inoculation,ILs 41,11 and 7 showed high resistance to bacterial blight,brown planthopper and whitebacked planthopper,respectively.These O.minuta-O.sativa ILs will serve as genetic materials for identifying and using favorable genes from O.minuta.

Dissection of heterosis for yield and related traits using populations derived from introgression lines in rice

Despite the great success achieved by the exploitation of heterosis in rice,the genetic basis of heterosis is still not well understood.We adopted an advanced-backcross breeding strategy to dissect the genetic basis of heterosis for yield and eight related traits.Four testcross(TC) populations with 228 testcross F1 combinations were developed by crossing57 introgression lines with four types of widely used male sterile lines using a North Carolina II mating design.Analysis of variance indicated that the effects of testcross F1 combinations and their parents were significant or highly significant for most of the traits in both years,and all interaction effects with year were significant for most of the traits.Positive midparent heterosis(HMP) was observed for most traits in the four TC populations in the two years.The relative HMPlevels for most traits varied from highly negative to highly positive.Sixty-two dominant-effect QTL were identified for HMPof the nine traits in the four TC populations in the two years.Of these,22 QTL were also identified for the performance of testcross F1.Most dominant-effect QTL could individually explain more than 10% of the phenotypic variation.Four QTL clusters were observed including the region surrounding the RM9–RM297 region on chromosome 1,the RM110–RM279–RM8–RM5699–RM452 region on chromosome 2,the RM5463 locus on chromosome 6 and the RM1146–RM147 region on chromosome 10.The identified QTL for heterosis provide valuable information for dissecting the genetic basis of heterosis.

Genetic Dissection of Low Phosphorus Tolerance Related Traits Using Selected Introgression Lines in Rice

To dissect the genetic basis of low phosphorus tolerance （LPT）, 114 BC2F4 introgression lines （ILs） were developed from Shuhui 527 and Minghui 86 （recurrent parents）, and Yetuozai （donor parent）. The progenies were tested for 11 quantitative traits under three treatments including normal fertilization in normal soil （as control）, normal fertilization in barren soil and low phosphorus stress in barren soil in Langfang, Hebei Province, China. Moreover, the ILs were investigated at the seedling stage using nutrient solution culture method in greenhouse in Beijing, China. A total of 49 main-effect quantitative trait loci （QTLs） underlying yield related traits were identified in Langfang, and their contributions to phenotypic variations ranged from 6.7% to 16.5%. Among them, 25 （51.0%） QTLs had favorable alleles from donor parent. A total of 48 main-effect QTLs were identified for LPT-related traits in Beijing, and their contributions to phenotypic variations ranged from 7.7% to 16.6%. Among them, 21 （43.8%） QTLs had favorable alleles from donor parent. About 79.6% of the QTLs can be detected repeatedly under two or more treatments, especially QTLs associated with spikelet number per panicle, spikelet fertility and 1000-grain weight, displaying consistent phenotypic effects. Among all the detected QTLs, eight QTLs were simultaneously identified under low phosphorus stress across two environments. These results can provide useful information for the genetic dissection of LPT in rice.

Molecular characterization of LMW-GS genes from a somatic hybrid introgression line Ⅱ-12 between Triticum aestivum and Agropyron elongatum in relation to quick evolution

In order to exploit the evolution and find novel low-molecular-weight glutenin subunit （LMW-GS） for improvement of common wheat quality, thirteen variants from a somatic hybrid introgression line II-12 between Triticum aestivum cv. Jinan 177 （JN177） and Agropyron elongatum were characterized via genomic PCR. Four clones were pseudogenes because they contained an internal stop codon. The remaining nine variants contained intact open reading frames （ORFs）. Sequence alignment indicates that the proteins deduced from the nine ORFs have similar primary structure with LMW-GS cloned from its parents previously. However, they have some unique modifications in the structures. For example, EU292737 contains not only an extra Cys residue in the C-terminal domain but also a long repetitive domain. Both EU 159511 and EU292738 start their first Cys residue in the N-terminal repetitive domain, but not in the N-conserved domain traditionally. These structural alterations may have positive contributions to wheat flour quality. The results of phylogeny showed that most LMW-GS variances from 11-12 were homologous to those from parent JN177 and other wheat lines. The reason for quick evolution of LMW-GS in 11-12 was discussed.

Preliminary Phenotypic and SNP-Based Molecular Characterization of Maize (<i>Zea mays</i>L.)-Mexicana (<i>Zea mays</i>SSP. <i>Mexicana</i>) Introgression Lines under Inbred Background of 48-2

Wild relatives possess potential genetic diversity for maize (Zea mays L.) improvement. Characterization of maize-mexicana introgression lines (ILs) is of great value to diversify the genetic base and improve the maize germplasm. Four maize-mexicana IL generations, i.e. BC1, BC2, BC3, and RIL, were constructed under the elite inbred background of 48-2, elite inbred line that is widely used in maize breeding in Southwestern China, and were phenotyped in different years and genotyped with 56110 SNPs. The results indicated that 48-2 had higher phenotypic performances than all the characterized ILs on most of the agronomic traits. Compared with other ILs, BC2 individuals exhibited more similar performance to 48-2 on most traits and possessed the highest kernel ratio (66.5%). Population structure and principal component analysis indicated that BC3 individuals gathered closer to 48-2 and exhibited the lowest mexicana-introgression frequency (0.50%), while BC2 (29.06%) and RIL (18.52%) showed higher introgression frequency. The high level of genetic diversity observed in the maize-mexicana ILs demonstrated that Z. mays ssp. mexicana can serve as a potential source for the enrichment of maize germplasm.

Development of Oryza rufipogon and O. sativa Introgression Lines and Assessment for Yield-related Quantitative Trait Loci

Introgression lines population was effectively used in mapping quantitative trait loci （QTLs）, identifying favorable genes, discovering hidden genetic variation, evaluating the action or interaction of QTLs in multiple conditions and providing the favorable experimental materials for plant breeding and genetic research. In this study, an advanced backcross and consecutive selfing strategy was used to develop introgression lines （ILs）, which derived from an accession of Oryza rufipogon Griff. collected from Yuanjiang County, Yunnan Province of China, as the donor, and an elite indica cultivar Teqing （O. sativa L.）, as the recipient. Introgression segments from O. rufipogon were screened using 179 polymorphic simple sequence repeats （SSR） markers in the genome of each IL. Introgressed segments carried by the introgression lines population contained 120 ILs covering the whole O. rufipogon genome. The mean number of homozygous O. rufipogon segments per introgression line was about 3.88. The average length of introgressed segments was approximate 25.5 cM, and about 20.8% of these segments had sizes less than 10 cM. The genome of each IL harbored the chromosomal fragments of O. rufipogon ranging from 0.54% to 23.7%, with an overall average of 5.79%. At each locus, the ratio of substitution of O. rufipogon alleles had a range of 1.67-9.33, with an average of 5.50. A wide range of alterations in morphological and yield-related traits were also found in the introgression lines population. Using single-point analysis, a total of 37 putative QTLs for yield and yield components were detected at two sites with 7%-20% explaining the phenotypic variance. Nineteen QTLs （51.4%） were detected at both sites, and the alleles from O. rufipogon at fifteen loci （40.5%） improved the yield and yield components in the Teqing background. These O. rufipogon-O, sativa introgression lines will serve as genetic materials for identifying and using favorable genes from common wild rice.

Mapping Heterotic Loci for Yield and Agronomic Traits Using Chromosome Segment Introgression Lines in Cotton

In the present study, a set of chromosome segment introgression lines （CSILs） using Gossypium hirsutum L. TM-1 as the recipient parent and G. barbadense Hai7124 as the donor parent were used to explore the genetic basis of heterosis for interspecific hybrids. Two sets of F1 populations individually derived from CSILs crossing with both parents were configured to investigate heterotic loci （HL） and substitution effect loci （SL）. A total of 58 HL and 39 SL were identified in 3 years. One stable HL, hLP-A4-3, could be detected in all 3 years. Three HLs, hBS-A8-1, hLP-D6-1, and hSI-D7-11, could be detected in 2 years. Four SLs, sBSoD7- 1, sLP-A8-1, sLP-D7-1, and sLP-D12-1, could be detected in 2 years. HL and SL tended to be distributed in some HL-rich chromosome segments with close positions. Compared with QTL detected in a former study, HL showed little overlap with QTL, indicating that trait phenotype and heterosis might be controlled by different sets of loci. All three forms of genetic effects （partial-, full-, over-dominant） were identified, while the over-dominant effect made the main contribution to heterosis. These results may help lay the foundation for clarifying the heredity mechanism of heterosis in cotton.

Root and shoot traits responses to phosphorus deficiency and QTL analysis at seedling stage using introgression lines of rice

Phosphorous （P） deficiency is a major restraint factor for crop production and plants have developed several mechanisms to adapt to low P stress. In this study, a set of 271 introgression lines （ILs） were used to characterize the responses of seedlings to low P availability and to identify QTLs for root traits, biomass, and plant height under P-deficiency and P-sufficiency conditions. Plant height, total dry weight, shoot dry weight, and root number were inhibited under P-deficiency, whereas maximum root length （MRL） and root-shoot ratio （RS） were induced by P-deficiency stress. Relative MRL （RMRL, the ratio of MRL under P-deficiency to MRL under P-sufficiency con- dition） and relative RS （RRS） were used to evaluate P-deficiency tolerance at the seedling stage. A total of 24 additive QTLs and 29 pairs of epistatic QTLs were detected, but only qRN4 was detected in both conditions. This suggested that different mechanisms may exist in both P supply levels. QTLs for adaptive traits （RMRL, RRS, RRV, and RRDW） and qRN4 consistently expressed to increase trait stability may contribute to P-deficiency tolerance. Twelve intervals were cluster regions of QTLs for P-deficiency tolerance, and one QTL （qRRSS） showed pleiotropic effects on P-deficiency tolerance and drought tolerance. These interesting QTLs can be used in marker-assisted breeding through the target ILs.

Development of upland rice introgression lines and identification of QTLs for basal root thickness under different water regimes

Introgression lines （ILs） are valuable materials for identifying quantitative trait loci （QTLs）, evaluating genetic interactions, and marker assisted breeding. A set of 430 ILs （BC5F3） containing segments from upland tropical japonica cultivar IRAT109 in a lowland temperate japonica cultivar Yuefu background were developed. One hundred and seventy-six polymorphic markers were used to identify introgressed segments. No segment from IRAT109 was found in 160 lines. Introgressed segments of the other 270 lines covered 99.1% of the donor genome. The mean number of introgressed donor segments per individual was 3.3 with an average length of 14.4 cM. QTL analysis was conducted on basal root thickness （BRT） of the 270 ILs grown under irrigated lowland, upland and hydroponic conditions. A total of 22 QTLs affecting BRT were identified, six QTLs （qBRT3.1, qBRT3.2, qBRT6.1, qBRTS.2, qBRT9.1, and qBRT9.2） were consistently expressed under at least two environments （location and water regime）, and qBRT7.2 was a new BRT QTL identified under lowland conditions. IL255 containing qBRT9.1 showed an increase of 10.09% and 7.07% BRT over cultivar Yuefu when grown under upland and lowland conditions, respectively. Using a population of 304 F2：3 lines derived from the cross IL255 Yuefu, qBRT9.1 was validated and mapped to a 1.2 cM interval between RM24271 and RM566. The presence of qBRT9.1 explained 12% of BRT variation. The results provide upland rice ILs and BRT QTLs for analyzing the genetic basis of drought resistance, detecting favorable genes from upland rice, and rice drought resistance breeding.

Development, identification and utilization of introgression lines using Chinese endemic and synthetic wheat as donors

Chromosome segmental introgression lines（ILs）are an effective way to utilize germplasm resources in crops.To improve agronomic traits of wheat cultivar（Triticum aestivum） Shi 4185, four sets of ILs were developed. The donors were Chinese endemic subspecies accessions Yunnan wheat（T. aestivum ssp. yunnanense） YN3, Tibetan semiwild wheat（T. aestivum ssp. tibetanum） XZ-ZM19450, and Xinjiang wheat（T. aestivum ssp. petropavlovskyi） XJ5, and synthetic wheat HC-XM1620 derived from a cross between T. durum acc. D67.2/P66.270 with Aegilops tauschii acc. 218.Totals of 356, 366, 445 and 457 simple sequence repeat（SSR）markers were polymorphic between Shi 4185 and YN3, XZZM19450, XJ5 and HC-XM1620, respectively. In total, 991 ILs were identified, including 300 derived from YN3, covering 95%of the genome of Shi 4185, 218 from XZ-ZM19450（79%）, 279 from XJ5（97%）, and 194 from HC-ZX1620（84%）. The sizes and locations of each introgression were determined from a consensus SSR linkage map. Using the ILs, 11 putative Resear quantitative trait loci（QTLs） were identified for plant height（PH）, spike length（SL） and grain number per spike（GNS）.Comparative analyses of 24 elite ILs with the parents revealed that the four donor parents could be important resources to improve wheat SL and GNS. Our work offers a case for utilizing endemic landraces for QTL mapping and improvement of wheat cultivars using introgression lines.

Breeding by selective introgression: Theory, practices, and lessons learned from rice

Future demands for increased productivity and resilience to abiotic/biotic stresses of major crops require new technologies of breeding by design(BBD)built on massive information from functional and population genomics research.A novel strategy of breeding by selective introgression(BBSI)has been proposed and practiced for simultaneous improvement,genetic dissection and allele mining of complex traits to realize BBD.BBSI has three phases:a)developing large numbers of trait-specific introgression lines(ILs)using backcross breeding in elite genetic backgrounds as the material platform of BBD;b)efficiently identifying genes or quantitative trait loci(QTL)and mining desirable alleles affecting different target traits from diverse donors as the information platform of BBD;and c)developing superior cultivars by BBD using designed QTL pyramiding or marker-assisted recurrent selection.Phase(a)has been implemented massively in rice by many Chinese research institutions and IRRI,resulting in the development of many new green super rice cultivars plus large numbers of ILs in 30+elite genetic backgrounds.Phase(b)has been demonstrated in a series of proof-of-concept studies of high-efficiency genetic dissection of rice yield and tolerance to abiotic stresses using ILs and DNA markers.Phase(c)has also been implemented by designed QTL pyramiding,resulting in a prototype of BBD in several successful cases.The BBSI strategy can be easily extended for simultaneous trait improvement,efficient gene and QTL discovery and allele mining of complex traits using advanced breeding lines from crosses between a common"backbone"parent and a set of elite parents in conventional pedigree breeding programs.BBSI can be relatively easily adopted by breeding programs with small budgets,but the BBSI-based BBD strategy can be fully and more efficiently implemented by large seed companies with sufficient capacity.

Detection of Drought-Related Loci in Rice at Reproductive Stage Using Selected Introgressed Lines

The present study was carried out to illustrate high-efficient detection of quantitative trait loci （QTLs） with selected introgression lines （ILs） and the existence of ＇hidden genes＇ conferring drought tolerance （DT）. 52 selected DT ILs, derived from BC2Fz population developed by crossing and backcrossing the susceptible recurrent parent （RP） IR64 with the susceptible donor Khazar were planted under irrigation and drought condition. Four important agronomic traits, e.g., grain yield （GY）, heading date （HD）, panicle numbers per plant （PN）, and plant height （PH） were evaluated and 83 SSR polymorphic molecular markers were used for genotypic analysis. Chi-square test based on genetic hitch-hiking and one-way analysis of variances （ANOVA） were used to detect drought-related loci. Nine and 36 loci were detected by chi-square test and one-way ANOVA, respectively. Five common loci were observed by comparing the results of the two methods, among which two QTLs linked with RM7, and RM241 were detected under irrigation condition, both of the favorable alleles were from RP and explained 13% phenotypic variation （PV） for GY and 28% PV for PH, respectively. The other three QTLs linked with RM 163, RM 18, and RM270 were detected under drought condition, the favorable alleles were all from the donor and explained 10, 24, and 19% PV for HD, PH, and PH, respectively. Five common loci were observed by comparing the results of chi-square test and one-way ANOVA including two QTLs （one for GY and one for PH） under irrigation condition and three QTLs （one for HD and two for PH） under drought condition. By combining phenotypic and genotypic analysis, drought escape could be inferred as the main mechanism for drought tolerance in the present study. The results in present study suggested that the selected ILs population analyzed by chi-square test and one-way ANOVA was quite effective for DT QTL detection with low inputs and could also produce useful materials for breeding with wide genetic diversity for drought tolerance.

Fingerprinting Analysis of the Introgressed lines from Gossypium hirsutum L.× G.barbadense L.based on AFLP markers

The main cultivated varieties in the world belong to the species of upland cotton(Gossypium hirsutum L.),and their genetic background is very narrow.However,the wild species and races in

Identification of Introgressed Lines by the SSR Markers and Agronomic Traits

The wild cotton cultivars and species have abundant genetic polymorphisms,and they possess lots of excellent genes,such as drought resistance,insect resistance,fine and strong fiber,and so on.

Isolation of Zizania latifolia Species-specific DNA Sequences and Their Utility in Identification of Z. latifolia DNA Introgressed into Rice

根据两个植物抗病基因N和RPS2中核酸结合位点 (NBS)和富亮氨酸重复区 (LRR)中的保守序列设计了一对特异引物 ,用PCR从具有水稻 (OryzasativaL .)改良所需要的许多优良性状的水稻近缘野生种菰 (Zizanialatifolia(Griseb .)Turcz.exStapf)的基因组DNA中扩增同源片段。PCR产物经克隆后 ,分别以菰和水稻的基因组DNA为探针 ,通过点杂交对所得克隆进行了分析。点杂交结果表明 ,在所分析的 6 0个克隆中有 2个克隆是菰专化的序列 ,即它们与水稻无杂交信号。基因组DNA的Southern杂交进一步证实了这 2个克隆的专化性。为了验证一些可能的“水稻_菰”渐渗杂交系是否确实含有源自供体菰的DNA ,以这 2个克隆为探针 ,与经EcoRⅠ酶切的 5个可能的渐渗杂交系进行了Southern杂交。结果表明 ,这 2个克隆均能检测出其中的一个系含有其同源序列。这一结果为曾经报道的经一种非常规有性杂交方法将菰DNA导入水稻提供了确凿的证据。

Progress on Transferring Elite Genes from Non-AA Genome Wild Rice into Oryza sativa through Interspecific Hybridization

The progress of research on transferring elite genes from non-AA genome wild rice into Oryza sativa through interspecific hybridization are in three respects, that is, breeding monosomic alien addition lines （MAALs）, constructing introgression lines （ILs） and analyzing the heredity of the characters and mapping the related genes. There are serious reproductive barriers, mainly incrossability and hybrid sterility, in the interspecific hybridization of O. sativa with non-AA genome wild rice. These are the ＇bottleneck＇ for transferring elite genes from wild rice to O. sativa. Combining traditional crossing method with biotechnique is a reliable way to overcome the reproductive barriers and to improve the utilizing efficiency of non-AA genome wild rice.

Development and Identification of Novel Rice Blast Resistant Sources and Their Characterization Using Molecular Markers

To develop and characterize introgression lines for leaf and neck blast resistance, 326 introgression lines were developed using various accessions of six different AA genome wild species in the genetic background of elite Indian varieties like PRl14 and Pusa 44 and were screened for blast resistance. Stringent phenotyping coupled with genotyping using gene based markers led to the identification of four resistant introgression lines, which showed promising resistance and do not possess any of the tested genes. Furthermore, multi-location screening confirmed the field resistance of the four introgression lines to both leaf and neck blast. Molecular characterization of these introgression lines using genome-wide simple sequence repeat markers revealed the presence of small percentage of wild Oryza genome introgrssion. So these lines can be used for mapping and identification of novel leaf and neck blast resistance genes. Thus, these four introgression lines can be considered as new genetic resources for blast resistance.

Conversion of the Statistical Combining Ability into a Genetic Concept

Since the combining ability was proposed in 1942, efforts to uncover the genetic basis underlying this phenomenon have been ongoing for nearly 70 yr, with little success. Some breeding strategies based on evaluation of combining ability have been produced, and are still extensively used in hybrid breeding. In this review, the genetic basis underlying these breeding strategies is discussed, and a potential genetic control of general combining ability （GCA） is postulated. We suggested that GCA and the yields of inbred lines might be genetically controlled by different sets of loci on the maize genome that are transmitted into offspring. Different inbred lines might possess different favorable alleles for GCA. In hybrids, loci involved in multiple pathways, which are directly or indirectly associated with yield performance, might be regulated by GCA loci. In addition, a case of GCA mapping using a set of testcross progeny from introgression lines is provided.